FUNCTIONAL AND MECHANISTIC CHARACTERIZATION OF CRTC1-MAML2 FUSION ONCOGENE IN MUCOEPIDERMOID CARCINOMAS AND LONG NONCODING RNA LINC00473 IN NON-SMALL CELL LUNG CANCER
By
ZIRONG CHEN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2016
© 2016 Zirong Chen
To my wife and my parents!
4
ACKNOWLEDGMENTS
First of all, I sincerely thank my mentor Dr. Lizi Wu who has been providing me
constant support and excellent guidance throughout the years. She always encouraged
me when I had difficulties for my research and has trained me into an independent
scientist that I am today. I also would like to thank to my supervisory committee
members, Drs. Frederic Kaye, Jianrong Lu and Peggy Wallace for their great scientific
guidance and advice for my study.
I also want to thank all the former and current colleagues in Dr. Lizi Wu’ lab,
including Huangxuan Shen, Shuibin Lin, Yumei Gu, Chunxia Cao, Liang Tian, Chengbin
Hu, Ernesto Fernandez, Xianlin Zou, Rongqiang Yang, Wei Ni, Xuehui Li and Xin Zhou.
Their contributions and help allowed me to successfully carry out my research.
I would like to thank Interdisciplinary Program (IDP) in Biomedical Sciences for
offering me the wonderful opportunity and support for my graduate training. Special
thanks go to Ms. Kristyn Minkoff for all her assistance.
Last but not least I would like to thank my wife Xiaohui Wen and my family for
their continued support and encouragement during my PhD studies.
5
TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 9
LIST OF FIGURES ........................................................................................................ 10
LIST OF ABBREVIATIONS ........................................................................................... 13
ABSTRACT ................................................................................................................... 15
CHAPTER
1 RESEARCH BACKGROUND ................................................................................. 17
Salivary Gland Tumor ............................................................................................. 17
Mucoepidermoid Carcinoma ............................................................................. 18 The CRTC1-MAML2 Fusion Oncogene In MEC ............................................... 19
Long Noncoding RNA In Cancer ............................................................................. 20
Molecular Mechanisms Of LncRNAs ................................................................ 20 LncRNAs In Cancer Development And Progression ........................................ 21
LncRNAs As Cancer Biomarkers And Therapeutic Targets ............................. 22
2 GENERATION AND CHARACTERIZATION OF A MOUSE MODEL OF CRTC1-MAML2-INDUCED MUCOEPIDERMOID CARCINOMA ........................... 25
Abstract ................................................................................................................... 25 Rationale ................................................................................................................. 26
Materials And Methods ........................................................................................... 26 Transgenic Construction, Generation Of Transgenic Mice, And Genotyping ... 26
Transgene Copy Number Calculation .............................................................. 27 Transduction Of Mouse Salivary Glands With AAV5-Cre-GFP Virus ............... 27
RNA Isolation And QRT-PCR ........................................................................... 28 Tissue Protein Extract Preparation And Western-Blot Analysis ........................ 28 H&E staining, PAS Staining And Immunohistochemistry ................................. 29 Isolation Of Primary Cells From Mouse Salivary Gland Tumors And Culture
Of Tumor Spheres ......................................................................................... 29
Microarray Gene Expression Profiling .............................................................. 30 Results .................................................................................................................... 30
Three Independent Cre-Inducible CRTC1-MAML2 Transgenic Mouse Lines Were Established. ......................................................................................... 30
Cre-Regulated CRTC1-MAML2 Transgenic Mice Developed Salivary Gland Tumors After Mating With MMTV-Cre Mice. .................................................. 32
Salivary Gland Tumors Developed From MCre-CM(+) Mice Had Histological Feature Of Human MEC................................................................................ 33
6
MCre-CM(+) Mice Developed Ear Cysts And Skin Cysts Phenotypes With CRTC1-MAML2 Fusion Expression. ............................................................. 34
CRTC1-MAML2 Expression Promotes Salisphere Formation .......................... 35
Gene Expression Profiling Analysis Identified Differentially Expressed Genes Associated With Mouse CRTC1-MAML2-Induced MEC .................... 36
Mice With AAV Cre-Induced CRTC1-MAML2 Expression In Salivary Glands Developed MEC-Like Tumors ....................................................................... 37
CCSP-CM(+) Mice Developed Lung Tumors With CRTC1-MAML2 Fusion Expression. ................................................................................................... 38
Discussion .............................................................................................................. 39
3 A NOVEL LONG NONCODING RNA LINC00473 FUNCTIONS AS A MEDIATOR FOR CRTC1-MAML2 FUSION ONCOGENIC ACTIVITY IN MUCOEPIDERMOID CARCINOMA ....................................................................... 53
Abstract ................................................................................................................... 53
Rationale ................................................................................................................. 54 Materials And Methods ........................................................................................... 55
Cell Culture ....................................................................................................... 55 Plasmids ........................................................................................................... 55 Viral Production And Transduction ................................................................... 56
Western Blotting Analysis ................................................................................. 56 Quantitative RT-PCR (qRT-PCR) ..................................................................... 56
Luciferase Reporter Assays ............................................................................. 57 Chromatin Immunoprecipitation (ChIP) ............................................................ 57 RNA-Fluorescence In Situ Hybridization (RNA-FISH) ...................................... 57 RNA In Situ Hybridization (RNA ISH) ............................................................... 58
Microarray Gene Expression Profiling .............................................................. 58
Nanostring Ncounter Assay .............................................................................. 59 Cell Growth And Cell Death Assay ................................................................... 59
Mouse Xenograft Studies ................................................................................. 60 Study Approval ................................................................................................. 60
Statistical Analysis ............................................................................................ 60 Results .................................................................................................................... 60
LINC00473 Expression Is Enhanced In CRTC1-MAML2 Fusion-Positive Human MEC Cell Lines And Primary Tumors. .............................................. 60
LINC00473 Is A Novel Target Gene Induced By CRTC1-MAML2 Fusion Oncoprotein ................................................................................................... 62
CRTC1-MAML2 Fusion Oncoprotein Directly Regulates LINC00473 Expression Through CRTC1-MAML2/CREB Signaling Axis. ........................ 63
Depletion Of LINC00473 Expression In Fusion-Positive MEC Cells Led To Reduce Cancer Cell Growth And Survival Both In Vitro And In Vivo ............. 64
LINC00473 Is Revealed As A Nuclear-Retained Long Noncoding RNA .......... 65
Gene Expression Profiling Identified LINC00473 Regulated Genes In Fusion-Positive MEC ..................................................................................... 66
Discussion .............................................................................................................. 66
7
4 CAMP/CREB-REGULATED LINC00473: POTENTIAL BIOMARKER AND THERAPEUTIC TARGET FOR LKB1-INACTIVATED CANCER ............................ 77
Abstract ................................................................................................................... 77
Rationale ................................................................................................................. 78 Materials And Methods ........................................................................................... 80
Plasmids ........................................................................................................... 80 Cell Culture ....................................................................................................... 81 Rapid Amplification Of CDNA Ends (RACE) .................................................... 81
Coding Potential Prediction .............................................................................. 82 Viral Production And Transduction ................................................................... 82
Western Blotting Analysis ................................................................................. 83 Quantitative RT-PCR (qRT-PCR) ..................................................................... 83 Reporter Assays ............................................................................................... 83 Chromatin Immunoprecipitation ........................................................................ 84 RNA-Fluorescence In Situ Hybridization (RNA-FISH) ...................................... 84
RNA Fractionation And Northern Blotting Analysis ........................................... 85
RNA Immunoprecipitation ................................................................................. 85 Cell Proliferation And Apoptosis Assay ............................................................ 85 Mouse Xenograft Studies ................................................................................. 86
LncRNA Microarray Analysis ............................................................................ 86
Nanostring Ncounter Gene Expression Analysis .............................................. 86 RNAscope In Situ Hybridization (RNA ISH) ...................................................... 87
Proteomic Analysis Of The LINC00473-Associated Protein Complex .............. 88
Analysis Of RNA Sequencing And Clinical Data .............................................. 88 Statistical Analysis ............................................................................................ 88 Study Approval ................................................................................................. 89
Results .................................................................................................................... 89 Genome-Wide LncRNA Profiling Identified LINC00473 As A Top LKB1
Signaling-Regulated LncRNA In NSCLC Cells. ............................................. 89 Elevated LINC00473 Expression Is Tightly Correlated With NSCLC LKB1
Inactivation Status ......................................................................................... 91
Direct RNA Detection In FFPE Specimens Revealed That LINC00473 Expression Is Elevated In A Subset Of NSCLC That Tend To Have Mutations In The LKB1 Gene Coding Regions .............................................. 92
Elevated LINC00473 Expression Is Associated With Tumor LKB1 Mutations And Correlated With Poor Prognosis In TCGA Lung Adenocarcinomas ....... 95
LINC00473 Expression Is Promoted By LKB1-Loss-Induced CRTC/CREB Activation....................................................................................................... 96
In Vitro And In Vivo Approaches Revealed Critical Functions Of LINC00473 In The Growth Of LKB1-Null Lung Cancer Cells. .......................................... 98
LINC00473 Is A Nuclear LncRNA And Functions As A Regulator Of Gene Expression In Part Through Interacting With NONO And Modulating CRTC/CREB Transcription. ........................................................................... 99
Discussion ............................................................................................................ 102
5 CONCLUSION ...................................................................................................... 129
8
LIST OF REFERENCES ............................................................................................. 132
BIOGRAPHICAL SKETCH .......................................................................................... 143
9
LIST OF TABLES
Table page
4-1 Primer and probe sequences used in this study. .............................................. 128
10
LIST OF FIGURES
Figure page
1-1 Diagram depicting the t(11;19) chromosomal translocation that generates a new CRTC1-MAML2 fusion. ............................................................................... 24
2-1 Three Cre-regulated CRTC1-MAML2 transgenic mouse lines were established.. ....................................................................................................... 43
2-2 The Cre-regulated CRTC1-MAML2 transgene mouse model developed salivary gland tumors after crossing with MMTV-Cre mice. ................................ 44
2-3 mCre-CM(+) mice developed salivary gland tumor with human MEC histology. ............................................................................................................ 45
2-4 mCre-CM(+) mice developed ear cysts with CRTC1-MAML2 fusion expression. ......................................................................................................... 46
2-5 Skin cysts were observed in the mCre-CM(+) mice with CRTC1-MAML2 fusion expression. ............................................................................................... 47
2-6 CRTC1-MAML2 expression enhanced salisphere formation .............................. 48
2-7 Gene expression profiling analysis identified genes differentially regulated by CRTC1-MAML2 in salivary gland tumors. .......................................................... 49
2-8 LSL-CM(+) mice with AAV-Cre-induced fusion expression in salivary glands led to MEC-like tumor development and the tumor was transplantable. ............. 50
2-9 LSL-CM(+) mice with Ad5-Cre-induced fusion expression in salivary glands led to MEC-like tumor development and the tumor was transplantable. ............. 51
2-10 CCSP-CM(+) mice developed lung tumors with CRTC1-MAML2 fusion expression. ......................................................................................................... 52
3-1 LINC00473 expression is highly evaluated in human CRTC1-MAML2 fusion-positive MEC cell lines and primary tumors. ....................................................... 69
3-2 LINC00473 expression is directly regulated by CRTC1-MAML2 fusion oncoprotein. ........................................................................................................ 70
3-3 CRTC1-MAML2 fusion activates LINC00473 expression though co-activating the transcription factor CREB.. ........................................................................... 71
3-4 Depletion of LINC00473 expression in CRTC1-MAML2 fusion-positive MEC H3118 cells results in a reduction in cell proliferation and survival in vitro and blocked the growth of human MEC xenografts in vivo. ....................................... 72
11
3-5 Depletion of LINC00473 expression in CRTC1-MAML2 fusion-positive MEC HMC3A cells results in a reduction in cell proliferation and survival in vitro. ...... 73
3-6 Specific nuclear distribution of LINC00473 was revealed in CRTC1-MAML2 fusion positive human MEC cells and primary tumors. ....................................... 74
3-7 Gene expression profiling analysis identified genes differentially regulated by LINC00473 in MEC cells. ................................................................................... 75
3-8 A model for the molecular basis of LINC00473 induction by CRTC1-MAML2 in fusion-positive MEC cells. ............................................................................... 76
4-1 LncRNA profiling revealed that LINC00473 is induced by LKB1 loss in NSCLC cells. .................................................................................................... 108
4-2 LKB1 wt protein, but not LKB1 K78I mutant, was capable of activating AMPK signaling. .......................................................................................................... 109
4-3 RACE and coding potential analyses of LINC00473 transcripts. ...................... 110
4-4 Expression analyses of LINC00473 and SIK1 genes in LKB1-wt and LKB1-mut lung NSCLC cell lines. ............................................................................... 111
4-5 Enhanced LINC00473 expression is highly correlated with human lung adenocarcinoma with LKB1 mutational status and associated with poor survival. ............................................................................................................ 112
4-6 Detection of LINC00473 expression in FFPE specimens by RNA in situ hybridization using RNAscope® 2.0 High Definition (HD) assays. .................... 113
4-7 Human lung adenocarcinomas with high LINC00473 expression were enriched with mutations in the LKB1 gene coding region. ................................ 114
4-8 Box plots show expression levels of LINC00473 (A), SIK1 (B), and LKB1 (C) genes in LKB1 mutant (Mut) and wildtype (Wt) human lung adenocarcinomas. ............................................................................................ 115
4-9 Analysis of the correlation between LINC00473 Expression, the LKB1-Loss Signature, LKB1 Expression, and SIK1 Expression. ........................................ 116
4-10 Kaplan-Meier survival analyses showed that high LINC00473 expression, but not LKB1 mutations in the coding regions, was associated with poor prognosis. ......................................................................................................... 117
4-11 LINC00473 expression is regulated by LKB1-CRTC1-CREB signaling axis. ... 118
4-12 Regulation of LINC00473 expression by the LKB1/CRTC/CREB signaling axis. .................................................................................................................. 119
12
4-13 Depletion of LINC00473 expression in LKB1-null NSCLC cells causes reduced cell growth and survival in vitro and in vivo. ........................................ 120
4-14 Knockdown of LINC00473 expression in LKB1-null human NSCLC H157 resulted in reduced cell growth and survival in vitro and in vivo. ...................... 121
4-15 Overexpression of LINC00473 in LKB1-wt lung human NSCLC cells increased cell proliferation and expression of several CREB target genes. ..... 122
4-16 LINC00473 shows predominantly nuclear localization with distinct nuclear structures. ......................................................................................................... 123
4-17 LINC00473 showed a distinct nuclear localization pattern. .............................. 124
4-18 LINC00473 is associated with NONO protein and stimulates CRTC-NONO interaction. ........................................................................................................ 125
4-19 NEDD9 is a LINC00473-regulated and CREB-regulated target gene, but its expression shows no significant difference between LKB1 wt and LKB1 mut NSCLC cell lines. .............................................................................................. 126
4-20 A model for the molecular basis of LINC00473 induction and the role of sustained LINC00473 expression as a potential biomarker and prognostic marker, therapeutic target, and gene regulator for LKB1-inactivated NSCLC. . 127
5-1 A model for aberrant CRTC-CREB activation in cancer cell. ............................ 131
13
LIST OF ABBREVIATIONS
AAV Adeno-associated virus
ACC Adenoid cystic carcinoma
AREG Amphiregulin
ASO Antisense oligonucleotides
bp Base pair
CCLE Cancer Cell Line Encyclopedia
CCSP Clara Cell Secretory Protein
Cdk6 Cyclin-dependent kinase 6
ChIP Chromatin immunoprecipitation
CIP Calf Intestine Alkaline Phosphatase
CM CRTC1-MAML2
CMV-IE Cytomegalovirus--immediate early
CPC Coding Potential Calculator
CREB cAMP responsive element binding protein
CRPC Castration-resistant prostate cancer
CRTC1 CREB Regulated Transcription Coactivator 1
Ctl Control
DMEM Dulbecco’s modified Eagle’s medium
dnCRTC Dominant negative form of CRTC
EGFR Epidermal growth factor receptor
ER Estrogen receptors
FDA Food and Drug Administration
FFPE Formalin-fixed, paraffin-embedded
GO Gene ontology
H&E Hematoxylin and eosin
HD High definition
HOTAIR HOX transcript antisense RNA
HTA Human transcriptome array
IgG Immunoglobulin G
IHC Immunohistochemistry
IP Immunoprecipitation
K78I Lysine 78 mutated to isoleucine
KEGG Kyoto Encyclopedia of Genes and Genomes
LKB1 Liver kinase B1
LncRNA Long noncoding RNA
LNP Lipid nanoparticles
LSL-CM LoxP-STOP-LoxP-FLAG-tagged CRTC1-MAML2
LUAD Lung adenocarcinoma
14
Luc Luciferase
MALAT1 Metastasis-associated lung adenocarcinoma transcript 1
MAML2 Mastermind like transcriptional coactivator 2
mCre-CM MMTV-Cre;LSL-CM
MEC Mucoepidermoid carcinoma
MMTV Mouse mammary tumor virus
MSG Mouse salivary gland
mut Mutant
NEDD9 Neural precursor cell expressed developmentally down-regulated protein 9
NFAT Nuclear factor of activated T cells
NOD.SCID Nonobese diabetic/severe combined immunodeficiency
NONO Non-POU-domain-containing, octamer binding protein
NRON Non-coding repressor of NFAT
NSCLC Non-small cell lung cancer
nt Nucleotide
PAGE Polyacrylamide
PAS Periodic acid–Schiff
PCA3 Prostate cancer gene antigen 3
PFA Paraformaldehyde-
PPIB Peptidylprolyl isomerase
PRC2 Polycomb repressive complex 2
qRT-PCR Quantitative reverse transcription polymerase chain reaction
RACE Rapid amplification of cDNA ends
RNA-FISH RNA fluorescence in situ hybridization
RNA-ISH RNA in situ hybridization
RNAi RNA interference
RT Room temperature
SD Stable disease
SG Salivary gland
SGT Salivary gland tumor
SIK1 Salt-Inducible Kinase 1
SNV Single-nucleotide variant
TAD Transcriptional activation domain
TAP Tobacco Acid Pyrophosphatase
TCGA The Cancer Genome Atlas
tv Transcript variant
UTR Untranslated region
wt Wild-type
XIST X inactive-specific transcript
15
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
FUNCTIONAL AND MECHANISTIC CHARACTERIZATION OF CRTC1-MAML2 FUSION ONCOGENE IN MUCOEPIDERMOID CARCINOMAS AND LONG NONCODING RNA LINC00473 IN NON-SMALL CELL LUNG CANCER
By
Zirong Chen
August 2016
Chair: Lizi Wu Major: Medical Sciences-Genetics
This research centered on studying aberrant CRTC-CREB signaling in human
mucoepidermoid carcinoma (MEC) and lung cancer. MEC is the most common salivary
gland malignancy. Advanced and metastatic MEC have limited therapeutic options and
poor unpredictable treatment outcomes and therefore a better understanding of the
molecular pathogenesis of MEC is necessary for developing effective treatments. The
majority of MEC cases contain a specific recurrent t(11;19)(q14-21;p12-13)
translocation that generates the CRTC1-MAML2 fusion protein. Existing data support
that CRTC1-MAML2 constitutively activates CREB-mediated transcription and has
oncogenic potential in vitro. However, its critical mediators and in vivo roles in MEC
tumorigenesis remain poorly elucidated. I hypothesized that CRTC1-MAML2 is essential
for MEC carcinogenesis in vivo and drives MEC pathogenesis through the aberrant
activation of downstream target genes. To test this hypothesis, I established a Cre
regulated CRTC1-MAML2 transgenic mouse model and investigated downstream
CRTC1-MAML2-regulated targets. Mice expressing the CRTC1-MAML2 transgene in
salivary glands developed MEC-like tumors. A long noncoding RNA (LINC00473) was
16
identified as a mediator for CRTC1-MAML2 function in MEC growth and survival.
Therefore, my studies supported a causal role for CRTC1-MAML2 in MEC and
established the transgenic mouse as a model of human MEC that is invaluable for
future therapeutic testing. Moreover, LINC00473 is critical for MEC tumorigenesis and
servers as a novel therapeutic target.
CRTC-CREB signaling can also be aberrantly induced by the inactivation of the
tumor suppressor gene LKB1. LKB1 mutational events are common in lung cancer. I
observed that LINC00473 was consistently the most highly induced gene in LKB1-
inactivated human primary lung cancer samples and derived cell lines. Elevated
LINC00473 expression correlated with poor prognosis and sustained LINC00473
expression was required for the growth and survival of LKB1-inactivated NSCLC cells.
Mechanistically, LINC00473 was induced by LKB1 inactivation and subsequent
activation of CRTC-CREB signaling. My data support that LINC00473 expression is a
robust biomarker for tumor LKB1 functional status and a therapeutic target for LKB1-
inactivated lung cancer.
My collective data demonstrated the importance of aberrantly activated CRTC-
CREB signaling in human MEC and lung cancer, which contributed to a better
understanding of oncogenic mechanisms and revealed new therapeutic targets.
17
CHAPTER 1 RESEARCH BACKGROUND
Salivary Gland Tumor
Salivary glands are located in the mouth and produce saliva, which moistens
food to help with chewing and swallowing. There are three main pairs of salivary glands,
including parotid glands, submandibular glands and sublingual glands. Thousands of
minor glands are around the rest of the mouth (1). Salivary glands are the sites of origin
for a wide variety of tumors. The salivary gland tumors are relatively rare with about 2.2
to 2.5 cases per 100 000 people, which constitutes 3% to 6% of all head and neck
cancers (2). About 70% of salivary gland tumors occur in the parotid glands, 8% in the
submandibular glands and 22% in the sublingual and minor salivary glands (3). Salivary
gland tumors are heterogeneous and consist of many different types. Mucoepidermoid
carcinoma (MEC) and adenoid cystic carcinoma (ACC) rank the first and second most
common salivary gland malignancies, respectively. Surgery, radiation therapy and
chemotherapy are the standard treatments for patients with salivary gland cancer.
However, effective treatments for advanced or metastatic salivary gland tumors are
currently not available.
Multiple studies have identified genomic alternations in salivary gland cancer,
and the identification of targeted therapies in salivary gland tumor is crucial for
improving patients’ treatment outcomes (4). Several targeted therapies have been
tested in salivary gland cancers. For instance, the monoclonal antibody against the
HER-2/neu receptor Trastuzumab was used to treat 14 patients with salivary gland
cancers that overexpressed HER-2/neu in a phase II clinical trial. One of three MEC
patients had a partial response with disappearance of some bony lesions and
18
stabilization without new lesion 45 months after treatment (5). Human-murine chimeric
monoclonal antibody against EGFR receptor, Cetuximab (Erbitux) was tested in 30
patients with recurrent and/or metastatic salivary gland tumors in a phase II clinical
study. Stable disease (SD) was observed in 24 patients and 15 patients had lasting
more than 6 months after treatment (6). The small molecule inhibitor Axitinib (AG-
013736) targeting VEGF receptor was tested in a patient with ACC in a phase I study.
Partial response was identified in this patient after three treatment cycles (7). These
novel molecular-targeted therapies provide potential effective treatments for some
salivary gland cancers. The identification of molecular abnormalities in salivary gland
cancer and characterization of their functional roles in tumorigenesis are essential for
the new development of specific targeted therapies.
Mucoepidermoid Carcinoma
Mucoepidermoid carcinoma (MEC) is one of the most frequent salivary gland
malignancies, accounting for 30% to 35% of all malignant neoplasms of the major and
minor salivary glands (8, 9). MEC also develops at distant and atypical sites, including
lung, breast, skin, esophagus, pancreas, thyroid, ovary and cervix (10, 11). MEC tumors
show a typical histological feature consisting of three cell types in varying proportions:
epidermoid cells, mucin-secreting cells, and intermediate cells, often with the presence
of cyst structure (9). Current grading systems are based on the cyst formation,
differentiation of the three cell types and cytomorphologic changes. MEC cases are
histologically classified as low, intermediate and high-grade tumors. Low-grade tumors
are usually removed by surgical resection while high-grade tumors require radiation
therapy and neck dissection. Histologic tumor grade is a useful but not consistent
prognostic indicator for mucoepidermoid carcinomas of the major and minor salivary
19
glands. Currently, patients with locally advanced, nonresectable and metastatic MEC
have limited therapeutic options and poor unpredictable treatment outcomes. The
clinical improvement has been hindered by a lack of understanding of the basic
mechanisms underlying MEC development as well as suitable preclinical models.
The CRTC1-MAML2 Fusion Oncogene In MEC
Recurrent chromosomal rearrangements in MEC were first described by
Nordkyist et al. in 1994 (12). More than 50% of MEC cases contain a specific recurrent
t(11;19)(q14-21;p12-13) translocation, strongly suggesting that this genetic
rearrangement is involved in MEC pathogenesis (13, 14). The t(11;19) (q14-21;p12-13)
translocation involving the CRTC1 and MAML2 genes was cloned and characterized by
Tonon et al. in 2003. This translocation fuses exon 1 of the CRTC1 gene at 19p13 to
exons 2-5 of the MAML2 gene at 11q21, generating a new CRTC1-MAML2 fusion
protein (Figure 1-1) (15, 16). The CRTC1-MAML2 fusion consists of the N terminal of
CRTC1 (42 amino acids) that serves as a CREB binding domain and the C terminal of
MAML2 (981amino acids) that is the transcriptional activation domain (TAD). The
CRTC1-MAML2 fusion exhibits a strong transcription co-activator activity and is capable
of interacting with the transcription factor CREB and activating CREB-mediated
transcription (17, 18). Importantly, ectopic expression of the CRTC1-MAML2 fusion
gene transformed rat RK3E epithelial cells in vitro. The resulting transformed cells were
able to grow tumors in nude mice (17). More recently, we observed that shRNA-
mediated depletion of CRTC1-MAML2 fusion expression reduced cell proliferation and
survival in human MEC cells in vitro and in mouse xenografts (19). All the data strongly
support a role for the CRTC1-MAML2 fusion protein in MEC tumorigenesis and
maintenance. However, the molecular mechanisms and functions of the CRTC1-
20
MAML2 fusion protein in MEC tumor development and progression in vivo have not
been fully elucidated.
It should be noted that less frequently detected MEC-related fusions (about 6%)
involve another member of the CRTC family CRTC3 and MAML2 (20-22). The CRTC3-
MAML2 fusion also consists of the CREB binding domain of CRTC3 and the TAD
domain of MAML2. Therefore, it is predicted that CRTC3-MAML2 has similar functional
activity to that of CRTC1-MAML2. However, further studies are needed to validate that
hypothesis.
Long Noncoding RNA In Cancer
A long noncoding RNA (lncRNA) is defined as a non-protein-coding transcript
that has more than 200 nucleotides in length. LncRNAs are described according to their
locations of gene region, including intergenic lncRNA, antisense transcripts and
enhancer RNA (23). The human genome encodes more than 10,000 lncRNAs and
currently only a handful of lncRNAs have been characterized (24-26). It has become
increasingly clear that many lncRNAs play important roles in cancer.
Molecular Mechanisms Of LncRNAs
LncRNAs have been reported to carry out various cellular functions through
multiple diverse mechanisms. LncRNAs can regulate gene expression through
epigenetic regulation or post-transcriptional modification. For example, lncRNA XIST (X
inactive-specific transcript) interacts with polycomb repressive complex 2 (PRC2), which
results in X chromatin repression and thus leads to transcriptional silencing (27).
Moreover, lncRNA can act as a ‘miRNA sponge’ to interact with microRNA and
influence the miRNA target gene expression. For instance, liver specific lncRNA HULC
interacts with microRNA-372 and reduces translational repression of microRNA-372
21
target gene, PRKACB (28). LncRNAs can also interact with cellular proteins and
modulate protein activity and localization. For instance, lncRNA NRON (non-coding
repressor of NFAT) binds to NFAT (nuclear factor of activated T cells) protein and
regulates the NFAT nuclear trafficking to down-regulate NFAT target gene expression
(29). Furthermore, lncRNAs interact with and guide transcription factors to their target
gene promoters and activate gene expression. For example, lncRNA Evf-2 specifically
cooperates with transcription factor Dlx-2 to increase the transcriptional activity (30).
These findings demonstrate that lncRNAs play important cellular functions through
heterogeneous mechanisms.
LncRNAs In Cancer Development And Progression
LncRNAs contribute to cancer progression and development via modulating
important cancer signaling pathways. Firstly, lncRNA is essential for cancer cell
proliferation. For example, lncRNA HOTAIR (HOX transcript antisense RNA) expression
is sufficient to induce androgen-independent AR activation and drive the AR-mediated
transcriptional program in the absence of androgen in castration-resistant prostate
cancer (CRPC) (31). Secondly, several lncRNAs were identified to play significant roles
in modulating tumor suppressor and cell growth-arrest signaling pathways. For instance,
p53-regulated long noncoding RNA lincRNA-p21 interacts with hnRNPK to regulate
CDKN1A and arrests the cell cycle in a p53-dependent p21 transcription (32). Thirdly,
lncRNA plays a role in the maintenance of genome stability. A recent report showed that
an abundant lncRNA NORAD sequesters PUMILIO protein away from target mRNA to
regulate chromosomal stability (33). Finally, many cancer-associated lncRNAs play a
role in regulating cancer cell invasion and metastases. The depletion of the long
noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1)
22
reduced lung cancer cell migration and decreased tumor nodules in a mouse xenograft
(34). Therefore, lncRNAs have growing importance in various aspects of cancer biology.
LncRNAs As Cancer Biomarkers And Therapeutic Targets
LncRNAs exhibit up-regulated or down-regulated expression in specific type of
cancers, suggesting that lncRNA expression may serve as a biomarker for cancer
diagnosis, prognosis and potential therapeutic targets. One particularly well-
characterized long noncoding RNA prostate cancer gene antigen 3 (PCA3) is a highly
prostate-specific gene. PCA3 is significantly elevated expression in prostate cancer,
suggesting that PCA3 is a specific biomarker of prostate cancer (35). The lncRNA
PCA3 expression-based molecular test was FDA-approved for the prostate cancer
diagnosis in 2012. Therefore, lncRNAs have great potential to serve as excellent cancer
diagnostic markers.
Since lncRNAs are associated with cancer-specific phenotypes, they provide
tremendous opportunities for RNA-based targeting as anti-cancer approaches. Several
therapeutic strategies have been successfully explored to modulate the lncRNA
expression, such as small RNA interference (RNAi), antisense oligonucleotides (ASOs)
and CRISPR/CAS9 system. For instance, HOTAIR is up-expressed in several types of
carcinoma cells. Depletion of HOTAIR expression using RNAi significantly reduced the
renal carcinoma cell proliferation and survival in vitro and in vivo (36). Locked nucleic
acid GapmeRs, a modified version of ASO that targeted lncRNA SAMMON reduced
melanoma cell growth in vitro and in patient-derived xenograft models, demonstrating
potential therapeutic efficacy of targeting this lncRNA (37). ASOs have already
demonstrated success in lncRNA-targeted HOTAIR therapeutics in breast cancer (38).
Recently, Ji-long Liu group reported that the use of CRISPR/CAS9 system with multiple
23
sgRNAs that flank the transcription start site of lncRNA roX gene successfully reduced
the roX gene expression in Drosophila cells, which demonstrated that CRISPR/CAS9
system has potential application in targeting lncRNA as anti-cancer treatment (39).
These studies strongly suggest that lncRNA targeting could offer novel opportunities for
cancer therapy and should be further investigated.
24
Figure 1-1: Diagram depicting the t(11;19) chromosomal translocation that generates a new CRTC1-MAML2 fusion.
25
CHAPTER 2 GENERATION AND CHARACTERIZATION OF A MOUSE MODEL OF CRTC1-
MAML2-INDUCED MUCOEPIDERMOID CARCINOMA
Abstract
In this study, I determined the oncogenic potential of the CRTC1-MAML2 fusion
in vivo by establishing a Cre-regulated CRTC1-MAML2 transgenic mouse model.
Through genetic crossing with MMTV-Cre mice or direct AAV-Cre transduction to
induce expression of the CRTC1-MAML2 transgene in salivary glandular cells, the
transgenic mice developed salivary gland tumors with typical human MEC histological
characteristics. Moreover, isolated tumor cells were capable of forming subcutaneous
tumors in immune-compromised hosts that again recapitulated the MEC histological
feature. Importantly, transcriptome analysis of CRTC1-MAML2-induced tumors showed
that these tumors exhibited a similar gene expression signature to that of human MEC,
strongly supporting that this mouse tumor model molecularly resembles human MEC.
Therefore, this study offers a direct proof for an oncogenic role of CRTC1-MAML2
fusion in vivo and provides the first genetically engineered mouse model for human
MEC. This mouse MEC model will be a promising preclinical model in evaluating
therapeutic strategies.
26
Rationale
Previous studies strongly supported a role for the CRTC1-MAML2 fusion gene in
MEC tumorigenesis and maintenance. A better understanding of the biology of this
fusion oncogene would provide targeting opportunities for blocking MEC. However,
whether the CRTC1-MAML2 fusion is sufficient to induce MEC in vivo remained
unknown and no genetically engineered mice that model MEC development and
progression were available, until this work presented here.
In this study, I aimed to investigate whether the CRTC1-MAML2 fusion has a
causal role in MEC tumorigenesis in vivo. Our lab previously created a mouse model
carrying the CRTC1-MAML2 knock-in allele under the control of the CRTC1 enhancer
and promoter to reproduce the genetic abnormality in human MEC. However, pups
heterozygous for the knock-in allele died immediately after birth. To bypass the problem
of perinatal lethality, I established a Cre-regulated CRTC1-MAML2 transgenic mouse
model to determine whether CRTC1-MAML2 expression in salivary glands induces
MEC development. My study provided direct proof for an oncogenic role of CRTC1-
MAML2 fusion in MEC in vivo. This model represents the first genetic mouse model that
is highly relevant to human MEC, which is invaluable for further understanding of MEC
tumorigenesis and developing new diagnostic and mechanism-based treatment
approaches for human MEC.
Materials And Methods
Transgenic Construction, Generation Of Transgenic Mice, And Genotyping
A ~3.6 kb of NheI-NotI human CRTC1-MAML2 cDNA fragment, consisting of
Kozak-ATG-FLAG epitope sequence in frame with the entire open reading frame of
CRTC1-MAML2 followed by ~400 bp of 3’UTR, was cloned into the pCBR vector to
27
obtain the construct pCBR_LSL-CM. The pCBR_LSL-CM plasmid DNA was then
digested with KpnI to remove plasmid vector backbone, purified and used for
microinjection into the pronucleo of fertilized eggs from FVB/J mice (#001800, Jackson
laboratory) in our transgenic core facility. The transgenic mice were identified by PCR
amplification of tail genomic DNA with the CRTC1-MAML2 primers (Forward, 5’-
TTCGAGGAGGTCATGAAGGA-3’; Reverse, 5’-TTGCTGTTGGCAGGAGATAG-3’).
Three lines of transgenic founder mice (LSL-CM1, CM2 and CM3) were maintained by
crossing with wild-type FVB/J mice. The transgenic mice were crossed with
homozygous transgenic MMTV-Cre mice [Tg(MMTV-cre)4Mam/J, #003553, Jackson
laboratory]. The fusion transgene was identified as described above. Littermates without
carrying the transgene were used as negative controls.
Transgene Copy Number Calculation
Since the haploid content of a mammalian genome is approximately 3 X 109 bp
and the transgenic mice are heterozygous. It is estimated that 100 ng of tail DNA is
equivalent to 0.277 pg of purified LSL-CM fragment from the transgene construct (8.3
kb). The qRT-PCR assays were performed with 0.1, 1, 10, 100 and 1000 copies of
pCBR-CRTC1-MAML2 construct and a standard curve was established. The transgene
copy numbers of three LSL-CM lines were determined by performing qRT-PCR for their
Ct values followed by inferring their gene copy numbers from the established standard
curve.
Transduction Of Mouse Salivary Glands With AAV5-Cre-GFP Virus
The AAV5-Cre-GFP (#7018, Vector Biolab) vector was delivered into
submandibular glands by retrograde ductal administration (40). In brief, LSL-CM(+) mice
(8 to 10 weeks old) were anesthetized with ketamine (60 mg/mL, 1 µL/g body weight,
28
Phoenix Scientific) and xylazine (8 mg/mL, 1 µL/g body weight, Phoenix Scientific) by
intramuscular injection. Then the mice were administered atropine (0.5 mg/kg body
weight, Sigma) by intramuscular injection to reduced salivary flow. After that, 50 µL of
AAV5-Cre-GFP (4.5 X 108 viral particles) in isotonic saline was infused through a
tapered cannula inserted into an orifice of the main excretory duct.
RNA Isolation And QRT-PCR
Tissues or tumors from these transgenic mice were processed for RNA isolation
after snap freezing in liquid nitrogen. Total RNA was extracted using Trizol reagent
(Invitrogen) and purified by RNAeasy Mini Column (QIAGEN). The yield and quality of
RNA were assessed using spectrophotometry at the Agilent 2100 Bioanalyzer (Agilent
Technologies). RNA was transcribed into cDNA using a GeneAmp RNA PCR kit
(Applied Biosystems). Real-time qPCR was performed using the StepOne Real-Time
PCR System (Applied Biosystems) with the SYBR Green PCR Core Reagents Kit
(Applied Biosystems). Gapdh was used as an internal control to normalize gene
expression levels. The following primers were used: CRTC1-MAML2 primers (Forward,
5’-TTCGAGGAGGTCATGAAGGA-3’; Reverse, 5’-TTGCTGTTGGCAGGAGATAG-3’);
and Gapdh primers (Forward, 5’-CAATGACCCCTTCATTGACC-3’; Reverse, 5’-
GACAAGCTTCCCGTTCTCAG-3’).
Tissue Protein Extract Preparation And Western-Blot Analysis
Tissues or tumors were processed for protein isolation after snap freezing in
liquid nitrogen. Whole-cell protein extracts were prepared using RIPA buffer and
subjected to immunoblotting. For immunoblot analysis, protein extracts were
fractionated in SDS-polyacrylamide (PAGE) gels and electrotransferred to nitrocellulose
membranes. The membranes were blocked for 1 hour in a buffer containing 10 mM Tris,
29
pH 8.0, 150 mM NaCl, 0.05% Tween 20, and 5% nonfat dry milk. The membranes were
incubated with the antibodies (anti-MAML2, Cell signaling CST-4618; anti-FLAG,
Thermos PA1-984B, or anti-β-actin, Santa Cruz sc-47778) overnight at 4 °C, then
washed and incubated with a horseradish peroxidase-conjugated secondary antibody
for 1 hour at RT. The protein bands were visualized by enhanced chemiluminescence
(Pierce).
H&E staining, PAS Staining And Immunohistochemistry
Tissues were excised, fixed for 24 hours in 4% Paraformaldehyde (PFA),
transferred to 70% ethanol, processed and embedded in paraffin. Sections were cut at 5
μm and mounted on slides for hematoxylin and eosin (H&E) staining and Periodic acid
Schiff (PAS) staining. For immunohistochemistry, sections were incubated for 30
minutes with the primary antibodies. Antigen–antibody complexes were detected by
using the peroxidase ABC kit (Vector labs) according to the manufacturer’s instructions.
The stained tissue sections were scanned and digitized using the Aperio Imagescope
(Leica).
Isolation Of Primary Cells From Mouse Salivary Gland Tumors And Culture Of Tumor Spheres
Tumors were resected, cut into small pieces, digested with collagenase (23
mg/mL) and hyaluronidase enzyme (40 mg/mL) for 2 hours at 37 ºC, and then filtered
through 100 µM and 50 µM pore-size filters (BD/Falcon). Cell suspensions were
cultured in mouse salivary gland (MSG) culture medium [DMEM:F12 with penicillin (100
I.U./mL), streptomycin (100 μg/mL), glutamax (2 mM), epidermal growth factor-2 (20
ng/mL), fibroblast growth factor-2 (20 ng/mL), N2 supplement (1 %), insulin (10 μg/mL)
and dexamethasone (1 μM)] with ultra-low attachment 6-well plates (41).
30
Microarray Gene Expression Profiling
Total RNAs from the salivary gland tumors, tumor adjacent tissues of mCre-
CM(+) mice or normal salivary glands of mCre-CM(-) mice were extracted using the
RNeasy RNA extraction kit (Qiagen). RNA samples were subjected to expression
analysis using the Affymetrix human transcriptome array 2.0 (HTA 2.0). Genes were
considered differentially expressed in salivary gland tumor tissues compared with tumor
adjacent tissue or normal salivary gland control based on an absolute fold changes of
≥10 and p-value <0.05. Duplicate biological replicates were included in microarray gene
expression analysis.
Results
Three Independent Cre-Inducible CRTC1-MAML2 Transgenic Mouse Lines Were Established.
To study the direct in vivo role of CRTC1-MAML2 in MEC tumorigenesis, I used a
strategy that allows conditional expression of CRTC1-MAML2 in salivary glands in a
Cre-regulated transgenic mouse model (Figure 2-1A). The CRTC1-MAML2 fusion cDNA
was placed downstream of a stop cassette flanked with two loxP sites (LoxP-STOP-
LoxP-FLAG-tagged CRTC1-MAML2, denoted as LSL-CM). The stop cassette can be
deleted upon expression of Cre recombinase, allowing fusion expression. Specifically, I
cloned a ~3.6 kb of NheI-NotI fusion fragment, consisting of Kozak-ATG-FLAG epitope
sequence in frame with the open reading frame of CRTC1-MAML2 followed by ~400 bp
of 3’UTR, into a pCBR vector. The LSL-CM transgene fragment was under control of a
ubiquitous expressed CAG promoter consisting of a 1.7 kb chicken β-actin promoter
combined with the cytomegalovirus-immediate early enhancer (CMV-IE). The N-
terminal FLAG tag had no effect on fusion transforming and co-activator functions (18). I
31
first verified the transgene construct by DNA sequencing. I next tested the induction of
CRTC1-MAML2 expression by Cre-dependent expression in mammalian cells, through
co-transfection of the transgene construct pCBR-LSL-CM with pCAGIG empty vector or
pCAGIG-Cre recombinase expression vector into 293T cells. Through western blotting
analysis, I observed induced expression of CRTC1-MAML2 fusion in the presence, but
not in the absence, of Cre recombinase co-expression (Figure 2-1B), which confirmed
Cre-regulated CRTC1-MAML2 expression.
Subsequently, I purified the 8.3 kb KpnI transgenic fragment for microinjection
after removing the prokaryotic vector sequence (Figure 2-1C). The Mouse Models Core
(University of Florida) performed microinjection of the purified CRTC1-MAML2
transgenic fragment into the pronucleo of more than 200 fertilized FVB/NJ oocytes, and
eventually generated 47 mice. After genotyping of these mice for the presence of the
CRTC1-MAML2 transgene by PCR analysis using the primers that span the
translocation breakpoint, I identified four F0 transgenic founders (2 males and 2
females). Three of these founders (designated as LSL-CM1, 2, and 3) transmitted the
transgene to progeny in a Mendelian fashion when bred to FVB/J mice (Figure 2-1D
and not shown). To determine the copy numbers of transgene in these mice, I
performed qPCR and generated a linear standard curve using the pCBR-CRTC1-
MAML2 plasmid DNAs (Figure 2-1E). I then estimated the copy number of the
transgene in three founders, LSL-CM1, 2 and 3 as 14.66±0.80, 4.19±0.09, and
0.96±0.01, respectively (Figure 2-1F). Moreover, the transgene copy numbers in these
three mouse lines remained similar over three generations in comparison to their
founder mice, indicating that the transgene was stably integrated in mouse genome.
32
Therefore, I successfully established three independent Cre-inducible transgenic
CRTC1-MAML2 mouse lines with different transgene copy numbers.
Cre-Regulated CRTC1-MAML2 Transgenic Mice Developed Salivary Gland Tumors After Mating With MMTV-Cre Mice.
To induce expression of the CRTC1-MAML2 transgene in salivary glands, I
generated a bi-transgenic mouse model (MMTV-Cre; LSL-CM, denoted as mCre-CM)
by crossing hemizygous transgenic mice LSL-CM mice to homozygous MMTV-Cre
transgenic mice [Tg(MMTV-Cre)4Mam/J from Jackson Lab] that express Cre
recombinase under control of the MMTV LTR promoter (Figure 2-2A). The MMTV-Cre
line directs high level of Cre expression that causes Cre-mediated deletion of loxP stop
cassette in salivary glands and other specific tissues such as skin and mammary
glands, and was previously used to induce Cre-regulated gene expression in salivary
glands (42). Since LSL-CM line 1 expressed the fusion transgene at the level equivalent
to that of the endogenous fusion in human MEC and consistently developed salivary
gland tumors, I focused my analysis on this line and “mCre-CM” mice here refers to
“MMTV-Cre; LSL-CM” line 1 mice.
To determine whether expression of CRTC1-MAML2 fusion transgene results in
a tumor phenotype, a cohort of bi-transgenic mice mCre-CM(+) and age-matched fusion
transgene negative littermates mCre-CM(-) were examined at least twice weekly.
I detected palpable masses in the neck region of the mCre-CM(+) mice as early
as the age of 8 to 12 weeks (Figure 2-2B). These mice developed salivary gland
tumors, usually showing one big tumor (Figure 2-2C). Western blotting analysis of
CRTC1-MAML2 fusion transgene expression in multiple organs in mCre-CM(+) mice
showed that the CRTC1-MAML2 transgene protein was highly expressed in salivary
33
glands tumor and skin cysts, with low level in tumor adjacent salivary glands and no
detectable expression in lung, heart, liver, kidney, intestine, spleen, brain and muscle
tissues at age of 3-months (Figure 2-2D). The isolated salivary gland tumor cells from
mCre-CM(+) mice were able to form a tumor mass within two months after
subcutaneous injection to NOD.SCID mice, indicating that these tumor cells were from
fully transformed tumors (Figure 2-2E). I observed that both female and male mCre-
CM(+) mice (n=79) developed salivary gland tumors with different tumor onsets. The
time for half of mice to develop tumors in these mice was 129 days, while no tumor
development occurred in the mCre-CM(-) mice (n=14) (Figure 2-2F). Moreover, the
kinetics of salivary gland tumor occurrence analysis in both female and male showed
that the time for tumor development in of 50% of transgenic mice mCre-CM(+) in male
(n=40) and female (n=39) was 118 days and 131 days, respectively (Figure 2-2G).
These data indicated that there was no significant difference in tumor occurrence
between male and female mCre-CM(+) mice.
Salivary Gland Tumors Developed From MCre-CM(+) Mice Had Histological Feature Of Human MEC
To determine whether salivary gland tumors from the mCre-CM(+) mice were
MEC, I performed the H&E staining for histological analysis, periodic acid-Schiff (PAS)
staining for mucin detection, and IHC for the CRTC1-MAML2 fusion protein expression
using human MAML2 TAD antibodies. The control salivary gland tissues from non-
transgenic mCre-CM(-) mice, salivary gland tissues from transgenic mCre-CM(+) mice
before overt tumor phenotype [4-week-old mCre-CM(+) mice], and salivary gland
tumors developed from mCre-CM(+) mice were used (Figure 2-3A). I observed that
salivary gland tumors developed in the mCre-CM(+) mice contained epidermoid cells,
34
mucin-secreting cells and intermediate cells, with different degrees of cysts as shown by
H&E and PAS staining. The human CRTC1-MAML2 positive MEC cell H292 xenograft
tumor sections were stained with H&E and IHC for fusion expression and used as a
positive MEC histological control (Figure 2-3B). These data indicated that these salivary
gland tumors displayed a histologic feature resembling human MEC. The majority of
cells within the tumors were positive for CRTC1-MAML2 fusion expression.
In contrast, the control salivary glands from the mCre-CM(-) mice showed normal
histology without detectable CRTC1-MAML2 expression (Figure 2-3A). In the salivary
gland tissues from mCre-CM(+) mice before tumor development, I observed
hyperproliferative lesions in salivary gland duct on H&E staining, which showed clusters
of cells with CRTC1-MAML2 expression. The fusion transgene expression was not
uniform in the salivary glands, and the data suggest that the cells expressing the fusion
likely have proliferative advantage that subsequently gives rise to tumors. These data
suggested a progressive step from normal, hyperplasia, carcinoma in situ to
mucoepidermoid carcinoma, which was further supported by the tumor kinetics with
various latency and focal nature of the tumors using mCre-CM(+) mouse model (Figure
2-3C and D). These data indicated that tumor formation is driven by the expression of
CRTC1-MAML2 fusion transgene.
MCre-CM(+) Mice Developed Ear Cysts And Skin Cysts Phenotypes With CRTC1-MAML2 Fusion Expression.
Since the MMTV-Cre line expressed Cre in the skin, it was not surprising that I
also observed skin cysts phenotype in our mCre-CM(+) mice. Skin-related phenotypes
were found in the ear and tail, as cysts mainly developed in these areas as early as 4
weeks old and became more prominent in the size and the number with ages (Figure 2-
35
4A). Histological analysis of the ear cysts from mCre-CM(+) mice showed masses in the
ear when compared to the control ear tissue from mCre-CM(-) mice. The ear cysts had
high expression of the CRTC1-MAML2 fusion protein (Figure 2-4B). Animals from all
transgenic mCre-CM(+) mice had skin cysts in compared with mCre-CM(-) normal
control after birth (Figure 2-5A). At 6-days of age, the skin cysts appeared more
prominently in mCre-CM(+) mice (Figure 2-5B). The skin cysts were found both in back
and front skins from the mCre-CM(+) mice at 3-months of age (Figure 2-5 C to E).
Histologic sections of the skin from mCre-CM(+) mice showed cystic structure within the
dermis, containing a thick cellular lining. These cystic structures showed CRTC1-
MAML2 expression by IHC assay (Figure 2-5 F and G). All mCre-CM(+) mice (n=79),
but no control siblings mCre-CM(-) mice (n=14), showed cysts in skin and developed
tumors during a 180-day observation period.
CRTC1-MAML2 Expression Promotes Salisphere Formation
Our knowledge of cell origins and events for oncogenic transformation leading to
salivary gland tumor is limited, which presents a significant barrier for the understanding
of the complexity of salivary gland tumor. The CRTC1-MAML2 gene fusion was
detected in all three cell types within MEC tumors including epidermoid cells, mucin-
secreting cells, and intermediate cells; this suggests that CRTC1-MAML2 might
transform salivary gland stem/progenitor cells. Therefore, as initial steps to study the
transformation events, I tested whether effect of the CRTC1-MAML2 fusion oncogene
on salivary gland stem/progenitor cells in vitro using a newly developed in vitro
salisphere assay. In brief, I isolated mouse submandibular glands cells from 4-week age
of mCre-CM(-) and mCre-CM(+) mice and cultured them into 6-well ultralow attachment
plate with mouse salivary gland culture medium to form salisphere (Figure 2-6A). The
36
efficiency of sphere formation was higher in the mCre-CM(+) mice than the mCre-CM(-)
mice (Figure 2-6B). Moreover, I analyzed the sphere formation from the salivary gland
tumor cells and cells from tumor adjacent in mCre-CM(+) mice (Figure 2-6C), and found
that tumor cells from the mCre-CM(+) mice had enhanced sphere formation efficiency
when compared with the tumor adjacent cells (Figure 2-6D). These data indicated that
CRTC1-MAML2 expression likely enhances the self-renewal of salivary gland
stem/progenitor cells.
Gene Expression Profiling Analysis Identified Differentially Expressed Genes Associated With Mouse CRTC1-MAML2-Induced MEC
To study the molecular mechanisms of CRTC1-MAML2 in salivary gland
tumorigenesis, I extracted total RNA from salivary gland tumors and adjacent salivary
gland biopsies from mCre-CM(+) mice, including age-matched normal salivary gland
tissues from mCre-CM(-) mice for gene expression profiling analysis. I evaluated the
expression differences between two pairs of salivary gland tumors from mCre-CM(+)
mice and normal salivary glands from mCre-CM(-) mice. Using an absolute fold
changes of at least 10 and p-value less than 0.05, I identified a total of 2221
differentially expressed genes (1409 up-regulated and 812 down-regulated) in salivary
gland tumors compared with normal salivary glands (Figure 2-7A). Moreover, I identified
a total of 1258 differentially expressed genes (696 up-regulated and 562 down-
regulated) in salivary gland tumors compared with tumor-adjacent salivary gland tissues
(Figure 2-7B). Comparing different expression profiles of salivary gland tumors in tumor
adjacent tissues and normal salivary glands, I identified differentially regulated genes
(640 up-regulated and 529 down-regulated) in mouse CRTC1-MAML2-induced tumor
cells (Figure 2-7C and D). To further understand and the functions of these differentially
37
regulated genes in mouse MEC-like tumor, I performed a KEGG pathway analysis on
the 640 up-regulated and 529 down-regulated genes using software David 6.7 (Figure
2-7E and F). This analysis revealed that 17 genes out of 640 up-regulated genes were
highly associated with signaling pathway in cancer according to the GO annotations.
These differentially regulated genes associated with mouse MEC will provide us a
molecular understanding for the role CRTC1-MAML2 in salivary gland tumorigenesis.
Mice With AAV Cre-Induced CRTC1-MAML2 Expression In Salivary Glands Developed MEC-Like Tumors
The MMTV-Cre line expresses Cre in other tissues besides salivary glands, as
shown by skin cysts developed in mCre-CM(+) mice (Figure 2-3A and 2-4A). In addition,
it is unclear whether CRTC1-MAML2 expression exerts any impact during salivary gland
development. In human MEC, the t(11;19) chromosomal translocation is a somatic
event, so postnatal expression of CRTC1-MAML2 will be more physiological to study its
oncogenic effects. Therefore, I took an adeno-associated viral vector (AAV)
transduction approach to induce CRTC1-MAML2 transgene expression during the
postnatal stage and specifically in salivary glands. AAV5 is currently being studied as
vectors for gene therapies in salivary glands because it is nonpathogenic, can efficiently
transduce cells in salivary glands, and maintain long-lived stable expression (40). To
test this in our system, I injected AAV5-Cre-GFP into the salivary glandular ducts of 8-
10 week old LSL-CM(+) mice (Figure 2-8A). These AAV5 viruses express an improved
Cre along with a GFP under control of the CMV promoter. I observed that mice
developed salivary gland tumors at ~3 months after retrograde injection of AAV5-Cre-
GFP (Figure 2-8B). To determine whether CRTC1-MAML2-induced tumors were fully
transformed, I made cell suspensions from CRTC1-MAML2-induced tumors, and
38
injected them to immunocompromised NOD.SCID mice. These allograft tumors reached
the size of 1 cm within one month (Figure 2-8C). These data indicated that CRTC1-
MAML2-induced tumors were transplantable as tumorigenic feature. The induced
tumors and allograft tumor again showed MEC histologic characteristics by H&E
staining (Figure 2-8D and E), further indicating the oncogenic function of CRTC1-
MAML2 in MEC induction. Furthermore, I also tested Ad5-Cre-luc virus injection into the
line 2 LSL-CM(+) mice (Figure 2-9A). Luciferase expression was detected in salivary
glands at 24 hours after virus injection by bioluminescent imaging, indicating that the
Ad5-Cre-luc virus were successfully delivered to salivary gland of LSL-CM(+) mice
(Figure 2-9B). The induced salivary gland tumors were detected at 5-months after virus
injection in LSL-CM(+) mice (Figure 2-9C). The induced-salivary gland tumors were
transplantable into the NOD.SCID mice and were able to grow tumors (Figure 2-9D).
The induced tumors and allograft tumors again showed MEC histologic characteristics
by H&E staining and IHC staining for CRTC1-MAML2 expression (Figure 2-9E to I). The
CRTC1-MAML2 expressions were found in the induced salivary gland tumors and
several passages of allograft tumors. These data strongly demonstrated that CRTC1-
MAML2 fusion expression directly contribute to salivary gland tumorigenesis.
CCSP-CM(+) Mice Developed Lung Tumors With CRTC1-MAML2 Fusion Expression.
The mucoepidermoid carcinoma (MEC) of the lung is a rare malignant neoplasm
(43, 44). The CRTC1-MAML2 fusion protein is also seen MEC arising with the lung and
has been demonstrated independently in several case-reports (45, 46). The causal role
of the CRTC1-MAML2 fusion oncogene in lung carcinoma pathogenesis had not been
demonstrated in vivo. I sought to study the lung MEC using our LSL-CM(+) mouse
39
model. To induce expression of the CRTC1-MAML2 transgene in the lung, I generated
a bitransgenic mouse model (CCSP-ER/Cre; LSL-CM, denoted as CCSP-CM) by
crossing the LSL-CM(+) to the CCSP-ER/Cre mice (#016225, Jackson laboratory)
(Figure 2-10A). The clara cell secretory protein (CCSP) promoter was used to
specifically express the target gene in the lung. The induced CRTC1-MAML2
expression in the lung was detected by qRT-PCR analysis after tamoxifen (75mg/kg)
injection to the CCSP-CM(+) mice (Figure 2-10B). The lung tumor masses were
detectable at 7 months and 10 months after tamoxifen injection in the CCSP-CM(+)
mice. CRTC1-MAML2 expression was detected in these tumors by IHC assay (Figure
2-10C and D). These data indicated that CRTC1-MAML2 fusion expression can induce
lung carcinogenesis in vivo.
Discussion
The CRTC1-MAML2 fusion gene represents a major driver oncogene and an
important therapeutic target for MEC (16). However, a comprehensive understanding of
the mechanisms and roles of the CRTC1-MAML2 fusion oncogene in MEC
development and progression remains limited. No genetically engineered mice (GEM)
that model MEC development and progression were previously available. GEM models
for human MEC are important for the elucidation of MEC tumorigenesis and progression
and for preclinical studies of targeted therapies, because they mimic MEC
tumorigenesis in situ in the host environment with an intact immune system.
In this study, I introduced human MEC-associated CRTC1-MAML2 fusion gene
to the mouse genome and established three independent Cre-regulated CRTC1-
MAML2 conditional transgenic lines. I utilized two strategies to introduce Cre and
subsequent CRTC1-MAML2 fusion expression in salivary glands. The first approach is
40
through crossing with the MMTV-Cre transgenic mice that have been well characterized
with high Cre expression in mammary glandular cells and striated ductal cells in the
salivary gland (42). I observed that conditional expression of the CRTC1-MAML2 fusion
transgene in the salivary glandular cells resulted in the development of salivary gland
tumors that mimic human MEC histology. Tumor development is mainly determined by
specific oncogene and tissue context. I observed lesions in the salivary gland, but not
mammary glands, suggesting that the CRTC1-MAML2 oncogene can transform salivary
gland cells, but not mammary glands. The analogy is that c-MYC can transform
mammary glandular cells, but not salivary gland cells (47). C-Myc does not appear to
predispose to the developments of salivary gland neoplasms, likely reflecting the fact
that biochemical targets necessary for c-Myc action are absent in parotid cells. In
addition, tumors are frequently focal and have latency, suggesting that additional
genetic events are needed to develop overt tumors.
I was able to observe expression of the CRTC1-MAML2 transgene in the induced
tumors at the level comparable to fusion expression in human MEC. Cell-based
detection using immunhistochemical staining revealed a mosaic nature of CRTC1-
MAML2 fusion oncogene expression in salivary gland regions that was co-localized with
hyperproliferative regions and uniform expression in the induced tumors. The mosaic
Cre expression of the MMTV-Cre line was previously reported, which offered a unique
advantage allowing direct comparison of wild-type and overexpressed cells in close
proximity for their cellular behaviors such as cell proliferation, cell fate determination,
cell adhesion and migration. In my model, the mosaic nature of fusion oncogene
expression instead of uniform overexpression likely represents a more realistic condition
41
in human cancer as it mimics the situation in which somatic oncogenic changes occur in
a few somatic cells during cancer development. The co-localization of CRTC1-MAML2
fusion expression to the hyperproliferative region indicates that CRTC1-MAML2 exerts
proliferative effects on salivary glandular cells and drives formation of pre-malignant
lesions. Overt tumors arose stochastically among carrier animals over the 3 to 6 months
observation period. The uniform fusion expression within the induced tumors suggests
that overt tumors were generated from a subset of cells expressing CRTC1-MAML2
fusion. These data strongly indicate a causal role for CRTC1-MAML2 fusion in MEC
development and a multistep of MEC carcinogenesis.
I also utilized direct transduction of glandular cells by retrograde injection of AAV-
Cre, which has been shown to successfully transduce salivary ductal cells (40). Using
this approach, I also observe MEC-like tumor development, again strongly supporting
that CRTC1-MAML2 drives MEC. The derived tumor cells were capable of forming
secondary tumors in immune-compromised mice that again characteristically resembled
human MEC.
The data present here suggests that MEC cancer is derived from stem cells and
our mouse data suggest that the transformation of ductal cells lead to MEC. It will be
important to understand whether and how CRTC1-MAML2 fusion promotes self-renewal
of salivary gland stem/progenitor cells and alters their differentiation.
Molecular analysis of the induced tumors at early and late stages will be useful
for investigating CRTC1-MAML2-induced downstream signaling as well as its
cooperative mutational events in MEC development. It is known that tumor development
requires proliferative signal and anti-apoptotic signal, and additional genetic changes
42
confer tumor malignancy and poor prognosis. The gene expression profiling analysis
was performed in the salivary gland tumors and tumor adjacent tissues or normal
salivary glands. Our analyses of these data are ongoing, and preliminary data indicate
novel gene inactivation and CRTC1-MAML2 regulated genes. In the future, I will
continue study the functions and mechanisms of these genes in salivary gland tumor.
In summary, conditional induction of mucoepidermoid carcinoma in transgenic
mice by expressing the CRTC1-MAML2 fusion oncogene strongly supports the
tumorigenic activity of CRTC1–MAML2 in vivo. My genetic mouse model generates
tumors that histologically and genetically recapitulate the human MEC will be valuable
for further molecular understanding of MEC tumorigenesis. Since this mouse model
generates tumors that histologically and genetically recapitulate the human MEC, this
model will provide an excellent preclinical mouse models for screening of therapeutic
compounds before initiating time consuming and expensive clinical trials.
43
Figure 2-1: Three Cre-regulated CRTC1-MAML2 transgenic mouse lines were established. (A) A schematic representation of the Cre-LoxP-mediated CRTC1-MAML2 fusion transgene constructs (LSL-CM) that was used to make transgenic mice. The relevant restriction sites were also shown. (B) The fusion transgene pCBR-CRTC1-MAML2 construct were co-transfected with pCAGIG-Cre plasmid or empty vector to 293T cells, and CRTC1-MAML2 fusion expression was detected by Western blotting using either anti-Flag antibodies or anti-MAML2 antibodies. Tubulin expression was used as a loading control. (C) The fusion transgene fragment (8.3kb) was isolated after the vector sequence (2.9 kb) was removed from pCBR-CRTC1-MAML2 construct with KpnI digestion. The purified fusion transgenic fragments were used for microinjection into fertilized eggs to make transgenic mice. (D) The F0 founder was bred to FVB mice and the transgene was detected in F1 offspring mice. PCR analysis of tail DNA of one litter of F1 offspring was shown. (E) A standard curve was established to correlate threshold cycle with transgene gene copy number using purified fusion transgene plasmids by qPCR. (F) The qPCR analysis of tail DNAs was performed to determine the transgene copy numbers for three transgenic lines.
44
Figure 2-2: The Cre-regulated CRTC1-MAML2 transgene mouse model developed salivary gland tumors after crossing with MMTV-Cre mice. (A) A schematic diagram shows that LSL-CM transgenic mice were crossed with MMTV-Cre mice to induce transgene expression in salivary glands and observed tumor development. (B) A mCre-CM(+) mouse showed a protruding mass in the neck region. (C) Salivary gland tumors were detected and dissected from mCre-CM(+) mouse. (D) Western blotting analysis of CRTC1-MAML2 expression in salivary gland tumor, tumor adjacent tissue and various tissues from a 3-month-old mCre-CM(+) mouse. (E) Salivary gland tumor cells from mCre-CM(+) mouse were injected to NOD.SCID mice The allograft tumors were formed within two months. (F) Kinetics of salivary gland tumor occurrence in mCre-CM(+) mice. The time of 50% transgenic mice developed SG tumors was 129 days while the control mCre-CM(-) mice did not develop SG tumor. (G) The time for the development of palpable tumor in 50% transgenic mice in male (n=40) and female (n=39) was 118 days and 131 days, respectively.
45
Figure 2-3: mCre-CM(+) mice developed salivary gland tumor with human MEC histology. (A) Salivary gland tissues from a nontransgene control mCre-CM(-) or from mCre-CM(+) mice at age of 4 weeks before overt tumor formation, or tumors developed at age of 8 weeks and 10 weeks were stained with H&E for histological analysis, PAS to detect mucin-expressing cells or IHC with MAML2 TAD antibodies to detect the CRTC1-MAML2 fusion expression. (B) Human H292 MEC cell xenograft tumor was stained with H&E and IHC and used as a MEC positive control. (C and D) The salivary gland tumor and tumor adjacent normal tissue were showed to have different histology and fusion expression.
46
Figure 2-4: mCre-CM(+) mice developed ear cysts with CRTC1-MAML2 fusion
expression. (A) mCre-CM (+) mice developed ear cyst at the age of 4-week old when compared to the mCre-CM(-) mice control. (B) The ears of mCre-CM(-) and mCre-CM(+) mice were stained with H&E for histological analysis, PAS to detect mucin-expressing cells or IHC with MAML2 antibodies to detect the CRTC1-MAML2 fusion expression.
47
Figure 2-5: Skin cysts were observed in the mCre-CM(+) mice with CRTC1-MAML2
fusion expression. (A) The skin cyst was found in the 1-day old mCre-CM(+) pups. (B) The skin cysts in the mCre-CM(+) mice developed more prominently in the day 6 after birth. (C-E) The skin cysts appeared in the back and front skin of mCre-CM (+) mice at 3-months old. (F and G) The skins of mCre-CM(-) and mCre-CM(+) were stained with H&E for histological analysis and IHC with MAML2 antibodies to detect the CRTC1-MAML2 fusion expression.
48
Figure 2-6: CRTC1-MAML2 expression enhanced salisphere formation. (A) The salivary
glandular cells were isolated from the 4-week old mCre-CM(-) and mCre-CM(+) mice and cultured in the salivary gland sphere medium to develop salisphere. (B) The sphere formation efficiency was higher in the mCre-CM(+) mice compared with the mCre-CM(-) mice. (C) The salivary gland tumor cells and tumor adjacent cells from the 3-months old mCre-CM(+) mice were cultured in the salivary gland sphere medium and observed the sphere formation. (D) The tumor cells from the mCre-CM(+) mice showed enhanced sphere formation efficiency when compared with the tumor adjacent cells.
49
Figure 2-7: Gene expression profiling analysis identified genes differentially regulated
by CRTC1-MAML2 in salivary gland tumors. (A) Volcano plot shows differentially expressed genes in mCre-CM(+) salivary gland tumors in compared with mCre-CM(-) normal salivary glands. (B) Volcano plot shows differentially expressed genes in mCre-CM(+) salivary gland tumors in compared with tumor adjacent normal salivary glands. The cutoff criteria were fold-change of ≥10 and p<0.05. (C and D) Venn diagram shows the significantly up-regulated genes (n=640) and down-regulated genes (n=529) in mCre-CM(+) salivary gland tumors when compared with mCre-CM(+) tumor adjacent tissues and mCre-CM(-) normal salivary glands. (E and F) The 640 up-regulated and 529 down-regulated target genes were sorted by KEGG pathway analysis using DAVID software and identified several signaling pathways. The GO enriched pathways were filtered in accordance with p<0.05 and FDR<0.05.
50
Figure 2-8: LSL-CM(+) mice with AAV-Cre-induced fusion expression in salivary glands
led to MEC-like tumor development and the tumor was transplantable. (A) A schematic diagram shows directly delivery AAV5-Cre-GFP to salivary glandular cells of LSL-CM(+) mice to induce expression of CRTC1-MAML2 transgene and observe salivary gland tumor development. (B) The aCre-CM(+) mouse developed salivary gland tumor at -3 months after retrograde injection of AAV5-Cre-GFP. (C) Salivary gland tumor cells from CRTC1-MAML2-induced tumors were injected to immunocompromised NOD.SCID mice. The allograft tumors reached the size of 1 cm within one month. (D and E) The salivary gland tumor from aCre-CM(+) mouse and the allograft tumor were stained with H&E and showed that have the human MEC-like histology.
51
Figure 2-9: LSL-CM(+) mice with Ad5-Cre-induced fusion expression in salivary glands
led to MEC-like tumor development and the tumor was transplantable. (A) A schematic diagram shows directly delivery Ad5-Cre-luc to salivary glandular cells of LSL-CM(+) mice to induce expression of CRTC1-MAML2 transgene and observe salivary gland tumor development. (B) The luciferase expression was detected in salivary glands at 24 hours after Ad5-Cre-luc virus injection. (C) The AdCre-CM(+) mouse developed salivary gland tumor at -5 months after direct injection of Ad5-Cre-luc. (D) Salivary gland tumor cells from CRTC1-MAML2-induced tumors were injected to immunocompromised NOD.SCID mice and the allograft tumors reached the size of 1.5 cm within two months. (E to I) The salivary gland tumor adjacent tissue, salivary gland tumors and allograft tumors were stained with H&E for histological analysis and IHC with MAML2 antibodies to detect the CRTC1-MAML2 fusion expression.
52
Figure 2-10: CCSP-CM(+) mice developed lung tumors with CRTC1-MAML2 fusion
expression. (A) A schematic diagram shows that LSL-CM transgenic mice were crossed with CCSP-Cre/ER mice to attain the CCSP-CM(+) mice. The CCSP-CM(+) mice were treated with Tamoxifen to induce the transgene expression in lung and then observed the lung carcinoma development. (B) The CRTC1-MAML2 fusion expression was detected in the salivary gland and lung of CCSP-CM(+) mice at 10 days after Tamoxifen treatment by qRT-PCR analysis. (C and D) The lung carcinoma was observed at 7 months and 10 months after Tamoxifen treatment in CCSP-CM(+) mice by H&E staining histological analysis. The fusion expression was detected by IHC with MAML2 antibodies.
53
CHAPTER 3 A NOVEL LONG NONCODING RNA LINC00473 FUNCTIONS AS A MEDIATOR FOR
CRTC1-MAML2 FUSION ONCOGENIC ACTIVITY IN MUCOEPIDERMOID CARCINOMA
Abstract
Mucoepidermoid carcinoma (MEC) occurs in many glandular tissues and
accounts for the most common salivary gland malignancies. MEC is specifically
associated with t(11;19) translocation. The resulting CRTC1-MAML2 fusion is a major
oncogenic driver for MEC initiation and maintenance. Currently, it is unknown whether
lncRNAs are involved in CRTC1-MAML2-mediated oncogenic activity. Through gene
expression profiling analysis, we identified a novel lncRNA, LINC00473 as a top target
for the CRTC1-MAML2 fusion in human mucoepidermoid carcinoma (MEC) cells.
In this study, I observed that LINC00473 expression is significantly induced in
human CRTC1-MAML2-positive MEC cell lines and primary tumors, and tightly
correlated with the level of CRTC1-MAML2 expression. LINC00473 transcription is
induced by CRTC1-MAML2 co-activation of the CREB transcription factor. Depletion of
LINC00473 significantly reduces MEC cell proliferation and survival in vitro and blocks
the growth of human MEC xenograft tumors. RNA in situ hybridization results indicated
that LINC00473 has a predominantly nuclear localization pattern in human MECs.
Furthermore, expression profiling of LINC00473-depleted MEC cells revealed that
LINC00473 regulates expression of genes involved in critical cellular processes.
Collectively, my data indicate that LINC00473 is a novel biomarker for CRTC1-MAML2
fusion activity in human MEC and has novel regulatory functions in mediating CRTC1-
MAML2 cancer gene activity.
54
Rationale
The discovery of the CRTC1-MAML2 fusion has important implications for
understanding MEC pathogenesis and developing novel diagnostics and targeted
therapy. However, the molecular mechanisms underlying CRTC1-MAML2 fusion in
MEC remain limited. To understand the genes and pathways downstream of the
CRTC1-MAML2 fusion gene that are critical for tumorigenesis and progression, I
previously performed gene expression profiling and identified differentially expressed
genes after the depletion of CRTC1-MAML2 fusion in human MEC cell lines though
gene expression profiling analysis (48). A long non-coding RNA (lncRNA), LINC00473
(C6orf176) was shown to be a top hit among those significantly down-regulated genes
after the depletion of CRTC1-MAML2 fusion in our microarray analysis. LncRNAs are
an emerging class of genes with regulatory function in gene expression but their
functions and mechanisms remain an understudied area in cancer (49). Studies have
strongly implicated that lncRNAs are specifically associated with cancer, thus they are
presenting promising diagnostic biomarkers and potential therapeutic targets (37, 50,
51). Currently, whether lncRNAs participates in CRTC1-MAML2-mediated oncogenic
activity and contributes to MEC tumorigenesis has not been investigated.
In this study, I interrogated the functional significance of LINC00473 in CRTC1-
MAML2 fusion oncogenic activity in human MEC through studying analyzing the
expression, regulation, and function of LINC00473. My combined findings indicate that
LINC00473 is directly induced by the CRTC1-MAML2 fusion and its expression reflects
CRTC1-MAML2 levels in human MEC. LINC00473 has a novel regulatory function in
mediating CRTC1-MAML2 cancer gene activity and is essential for human MEC cell
growth and survival. Based on my mechanistic studies, LINC00473 is mediated by the
55
CRTC1-MAML2 fusion oncoprotein and CREB activation. LINC00473 is a specific
nuclear lncRNA and functions as a mediator in regulation of gene expression from my
gene expression profiling analysis in fusion-positive MEC. Therefore, CRTC1-MAML2
fusion oncoprotein aberrantly activates expression of target gene LINC00473, which
promotes cell growth and survival, ultimately contributing MEC tumorigenesis and
progression.
Materials And Methods
Cell Culture
Human MEC cell lines (HPA, HMC3A and HMC3B) were obtained from Dr.
Jacques Nör’s lab (52). H3118 and H292 MEC cells were previously described (19). All
the cells were maintained in Dulbecco’s modified Eagle’s medium (Mediatech)
supplemented with 1% antibiotic (Corning Cellgro) and 10% FBS (Life Technologies).
Cells were maintained in the humidified incubator at 37 °C with 5% CO2.
Plasmids
The shRNA targeting LINC00473 gene’s oligonucleotide sequences were cloned
into lentiviral-based pLKO.1 vector (Open Biosystems). These sequences are:
shLINC00473-2(shLnc473-2): 5’-AACTGGATCTTTGCAGACAGG-3’; shLINC00473-
4(shLnc473-4); 5’- AAGAACCCAAGTCATATTCAT-3’. The lentiviral-based pLKO.1
constructs against MAML2 (RHS4533-NM_032427) and CREB (RHS4533-NM_004379)
were purchased from Open Biosystems. The N-terminal FLAG-tagged CRTC1-MAML2
cDNA was cloned into the pCMV vector. The LINC00473 promoter sequences which
contain 2 CRE half sites (from -523 to +88) were subcloned into pGL3 luciferase
reporter construct (Promega) to generate the LINC00473 promoter reporter. The
56
retroviral-base pMSCV-dnCRTC-GFP construct expresses the CREB binding domain of
CRTC1 (1-55 amino acids) fusion protein with GFP and a nuclear localization signal.
Viral Production And Transduction
Viral production and transduction in MEC cells were performed as previously
described (48) with minor modifications. The transduced cells were further used for
western blotting analysis, qRT-PCR, microarray analysis, cell proliferation, cell
apoptosis assay and mouse MEC xenograft model study.
Western Blotting Analysis
Western blotting analysis was performed to detect protein expression as
previously described (53). The following antibodies were used for western blotting
analysis: anti-MAML2 (CST-4618, Cell Signaling Technology, 1:500), anti-CREB (06-
863, Millpore, 1:1000), anti-GFP (SC-8334, Santa Cruz, 1:1000) and anti-β-ACTIN (SC-
47778, Santa Cruz, 1:20000).
Quantitative RT-PCR (qRT-PCR)
The qRT–PCR analysis was performed as previously described (19). The primer
sequences were listed as following: LINC00473 primers (forward, 5’-
AAACGCGAACGTGAGCCCCG-3’, reverse,5’-CGCCATGCTCTGGCGCAGTT-3’),
CRTC1-MAML2 fusion primers (forward, 5′-TTCGAGGAGGTCATGAAGGA-3′; reverse,
5′-TTGCTGTTGGCAGGAGATAG-3′); MAML2 primers (forward, 5′-
TTTCCTTCACCCAACCAAAG-3′; reverse, 5′-GGGCCCATGTTATCATTTTG-3′),
NR4A2 primers (forward, 5’-GTTCAGGCGCAGTATGGGTC-3’, reverse,5’-
AGAGTGGTAACTGTAGCTCTGAG-3’) and GAPDH primers (forward, 5′-
CAATGACCCCTTCATTGACC-3′; reverse, 5′-GACAAGCTTCCCGTTCTCAG-3′).
57
Luciferase Reporter Assays
Transfection of HEK293T cells for luciferase reporter assays was performed as
previously described (54). Briefly, HEK293T cells were seeded in 24-well plates the day
before transfection. On the second day, the cells were transfected with pGL3-
LINC00473 reporter vector and pCMV FLAG-tagged CRTC1-MAML2 or empty pCMV
vector along with internal control Renilla luciferase construct (pEF-RL) using Effectene
transfection reagent (Qiagen). Cells were collected at 48-hours post-transfection and
luminescence was measure using Dual-luciferase reporter assay system (Promega)
according to the manufacture’s protocol.
Chromatin Immunoprecipitation (ChIP)
ChIP was performed as previously described (54) with minor modification. The
CREB antibodies (06-863, Millipore), MAML2 antibodies (CST-4618, Cell Signaling
Technology) or control immunoglobulin G (SC-2027, Santa Cruz) were used to
immunoprecipitate the H292 chromatin complex. The purified DNA was used for the
qRT-PCR analysis with LINC00473 specific primers flanking the two CREB binding half
sites. The primer sequences were used as following: forward, 5’-
AGCAGCCTTGCCAAAGGTC-3’; revers, 5’-TTTCCCTTTAAGCCGGAGAT-3’.
RNA-Fluorescence In Situ Hybridization (RNA-FISH)
LncRNA LINC00473 RNA-FISH was performed using Stellaris® FISH probes
(Biosearch Technologies) according to the manufacturer’s protocol. Twenty-seven FISH
probes labeled with Quasar 570 dyes against LINC00473 were designed using the
Stellaris RNA FISH probe designer. Cells were hybridized with the LINC00473 Stellaris®
FISH probes and visualized the LINC00473 signal using the Leica DM6000B
58
fluorescence microscope. For the negative control, the cells were treated with RNase A
(50 µg/mL) at 37°C for 1 hour before prior to the hybridization step.
RNA In Situ Hybridization (RNA ISH)
RNA ISH was carried out on formalin-fix paraffin-embedded H3118 xenograft
tumors and MEC tumor tissue sections using RNAscope in situ hybridization technology
(Advanced Cell Diagnostics) and custom probe set Hs-LINC00473 as the
manufacturer’s instructions. Briefly, tissue sections were deparaffinized and
permeablized using xylene and 100% Ethanol. The slides were then incubated with
pretreat 1 solution for 10 minutes at room temperature. The tissue sections were boiled
at 95 °C for 15 minutes in pretreat 2 solution and rinsed in water. The pretreat 3 solution
was then applied to the tissue sections for 30 minutes at 40 °C in the HybEZ oven
(ACD). The LINC00473 probes were hybridized to the tissue sections in the HybEZ
oven at 40 °C for 2 hours. At the same time, the RNAscope probes of human
housekeeping gene PPIB were used as a positive control. After hybridizations, the
slides were washed in the provided washing buffer and immediately proceed to the
RNAscope assay using the RNAscope® 2.0 HD Detection Kit (BROWN, Catalog No.
320497 or RED, Catalog No. 320487) for six cycles of hybridization amplifications. The
signal was detected by using a 1:60 ratio of solution B and A. Finally, the Hematoxylin
counterstaining was performed. The stained tissue sections were scanned and digitized
using the Aperio Imagescope (Leica).
Microarray Gene Expression Profiling
H3118 cells were infected with lentiviral-based shLINC00473 or control shRNAs
for 96 hours and total RNAs were extracted using RNeasy RNA extraction kit (Qiagen)
after transduction. RNA samples were subjected to Affymetrix human transcriptome
59
array 2.0 (HTA 2.0). The microarray data have been deposited in the NCBI Gene
Expression Omnibus under accession GSE. Genes were considered differentially
expressed in H3118 cells transduced with shLINC00473 or shRNA control knockdown
using an absolute fold change of ≥2 and p-value < 0.05. Duplicate biological repeats
were set up for microarray gene expression analysis.
Nanostring Ncounter Assay
For the Nanostring gene expression assay, our customized nCounter GX
Codeset was used following the manufacturer’s instructions (Nanostring Technologies).
Briefly, total RNA (100 ng) from cultured cells or RNA (200ng) derived from FFPE MEC
tissues were using as input for nCounter RNA sample preparation reaction. Then the
nCounter Digital Analyzer was used to count and tabulate the individual fluorescent
barcodes for target gene in each RNA samples. The raw data was subjected to gene
expression analysis using nSolver™ Analysis Software. The Heatmap.2 R package was
performed to generate heatmap using the normalized Nanostring gene expression data.
Cell Growth And Cell Death Assay
Cell growth and cell death were performed as previously described (19). In brief,
the cells were transduced with lentiviral-based shLINC00473 or shRNA control for 72
hours and then were collected and cultured at 5 X 105 /well in 6-well plates for 96 hours.
To detect the cell growth, the cell number was counted using the Trypan blue exclusion
assay. To detect the cell death, the cells were stained with Annexin V and PI for 15
minutes using Annexin V-FITC Apoptosis Detection kit (BD Pharmingen) and subjected
to flow cytometric assays. Triplicate assays were set up.
60
Mouse Xenograft Studies
Luciferase-expressing H3118 (H3118-luc) cells after infection with control shRNA
or shLINC00473 for 96 hours were injected subcutaneously into the dorsal flanks of
NOD.SCID mice (Jackson laboratory) at a cell number of 2 X 106. The Dial Caliper was
used to measure the tumor volumes daily after cells injection and bioluminescence
images were performed weekly as previously described (50).
Study Approval
All mouse xenograft studies were performed following a protocol approved by the
Institutional Animal Care and Use Committee at the University of Florida.
Statistical Analysis
Statistical differences were determined using the Student’s t-test to analyze the
data from qRT-PCR, luciferase reporter assay, cell growth, cell death assay and mouse
xenograft studies. A p value of < 0.05 was considered to be statistically significant. The
biological repeats were mentioned in the figure legends for each experiment.
Results
LINC00473 Expression Is Enhanced In CRTC1-MAML2 Fusion-Positive Human MEC Cell Lines And Primary Tumors.
Our previous study showed that LINC00473 was the top target for the CRTC1-
MAML2 fusion in human mucoepidermoid carcinoma (MEC) cells through gene
expression profiling analysis (48). But the role of lncRNA LINC00473 in human MEC is
still unknown. To understand whether the novel long noncoding RNA LINC00473 has a
functional role in human MEC, I first evaluated the expression level of LINC00473 in a
panel of human MEC-derived cell lines and primary MEC tumors. I first confirmed
detectable endogenous CRTC1-MAML2 fusion protein in 4 fusion-positive human MEC
61
cell lines, including HMC3A, HMC3B, H3118 and H292, but not in the fusion-negative
HPA cells through Western blotting analysis (Figure 3-1A). My qRT-PCR analysis
showed enhanced expression of LINC00473 in all the fusion-positive MEC cell lines but
low or undetectable expression in the fusion-negative cells (Figure 3-1B). The
LINC00473 differential expressions in fusion-positive and –negative cell lines were
further confirmed by Nanostring analysis as shown in the heatmap (Figure 3-1C).
Evaluated expression of known CRTC1-MAML2 downstream target genes (AREG,
AGR2, DMBT1 and NR4A1) were also confirmed in these fusion-positive cell lines. To
investigate whether LINC00473 expression is increased in fusion-positive MEC primary
tumors, I performed Nanostring analysis for human tumor RNAs derived from 6 fusion-
positive and 6 fusion-negative MEC specimens. As shown in the heatmap, LINC00473
were highly expressed in these fusion-positive MECs but had low expression in these
fusion-negative tumors (Figure 3-1D). Moreover, to investigate potential correlation
between the expression levels of LINC00473 and CRTC1-MAML2 in human primary
MEC tumors, I performed qRT-PCR analysis for both LINC00473 and CRTC1-MAML2
transcripts in human fusion-positive (n=6) and fusion-negative (n=6) MEC tumors
(Figure 3-1E). I observed a statistically significant positive correlation between the
LINC00473 and CRTC1-MAML2 expression (n=12, r=0.785157) by Pearson’s
correlation analysis (Figure 3-1F). Taken together, these results indicate that
LINC00473 expression was highly elevated in both fusion-positive MEC cell lines and
primary tumors and its expression level is positively correlated with that of CRTC1-
MAML2 fusion expression.
62
LINC00473 Is A Novel Target Gene Induced By CRTC1-MAML2 Fusion Oncoprotein
High LINC00473 expression is positively correlated with CRTC1-MAML2 fusion
expression in human MEC cell lines and primary tumors, suggesting that CRTC1-
MAML2 is responsible for inducing LINC00473 transcription. Therefore, I next
investigated the effect of CRTC1-MAML2 in the regulation of LINC00473 expression.
Depletion of CRTC1-MAML2 expression using two independent lentiviral-based
shMAML2 (shM2-1 and shM2-3) in two fusion-positive MEC cell lines (H3118 and
H292) resulted in significant decrease in LINC00473 expression (Figure 3-2A and B). I
also performed Nanostring analysis to confirm that reduction in the LINC00473
expression was due to the depletion of CRTC1-MAML2 but not the depletion of MAML2
only in H3118 cells (Figure 3-2C). To verify LINC00473 as a CRTC1-MAML2
downstream target gene, I performed a rescue experiment to investigate whether
exogenous CRTC1-MAML2 fusion would restore LINC00473 expression after the
depletion of endogenous fusion. Here I used a lentiviral-based shRNA (shM2-1)
targeting the 3’ UTR of MAML2 and a FLAG-tagged CRTC1-MAML2 expression
construct that did not contain the 3’ UTR of MAML2 (19), thus is resistant to shM2-1
knockdown. Expression of the endogenous fusion was firstly depleted using lentiviral-
based shRNAs (shM2-1) infection and then followed by reintroduction of exogenous
CRTC1-MAML2 expression in fusion-positive H3118 cells. I found that exogenous
CRTC1-MAML2 expression was capable of restoring LINC00473 expression in
endogenous fusion-depleted cells (Figure 3-2D). Moreover, ectopic expression of
FLAG-tagged CRTC1-MAML2 in fusion-negative HEK293T cells that have low
LINC00473 expression led to a significant increased LINC00473 transcript levels
63
(Figure 3-2E), whereas introduction of exogenous MAML2 expression in HEK293T cells
did not enhance LINC00473 expression (Figure 3-2F). Collectively, these data strongly
suggest that CRTC1-MAML2 acts as a positive regulator to induce the LINC00473
expression in fusion-positive MEC.
CRTC1-MAML2 Fusion Oncoprotein Directly Regulates LINC00473 Expression Through CRTC1-MAML2/CREB Signaling Axis.
I next investigated the molecular mechanism underlying CRTC1-MAML2
regulation of LINC00473 expression. Our previous study showed that CRTC1-MAML2 is
associated with transcription factor CREB and constitutively activates CREB-mediated
transcription (18). It was reported that LINC00473 expression was up-regulated in
human ocular ciliary smooth muscle cells (55) and endometrial stromal cells (56) after
cAMP stimulation. Since there are 2 CRE (cAMP-responsive element) half sites in the
LINC00473 proximal promoter region, I explored whether the activation of LINC00473
transcription is via the CRTC1-MAML2 co-activating the transcription factor CREB. First,
I depleted CREB expression by two independent lentiviral-based shRNAs against
CREB in fusion-positive H3118 cells and found that LINC00473 transcript levels were
significantly reduced after CREB depletion (Figure 3-3A and B). To determine whether
the LINC00473 gene promoter is regulated by the CRTC1-MAML2 and CREB
interaction, I used the LINC00473 promoter luciferase reporter containing the proximal
promoter sequences which contains 2 CRE sites (-523 to +88). I found that ectopic
expression of CRTC1-MAML2 fusion remarkably increased the luciferase activities of
this LINC00473 promoter in HEK293T cells (Figure 3-3C). Furthermore, I performed the
chromatin immunoprecipitation (ChIP) assay to determine whether CRTC1-MAML2 and
CREB physically interact with the LINC00473 promoter in human MEC cells. I observed
64
that the transcription factor CREB and CRTC1-MAML2 fusion oncoprotein were greatly
enriched in the LINC00473 promoter region containing 2 CRE binding sites (Figure 3-
3D). Finally, I generated a retrovirus construct that express a dominant negative form of
CRTC1 (1-55 aa) (dnCRTC) which interferes with the CRTC1-MAML2/CREB
interaction. I detected a reduction in LINC00473 expression in the fusion-positive MEC
cells (H3118 and H292) after transduced with dnCRTC retroviruses (Figure 3-3E and
F). The decreased expression of CRTC1-MAML2 fusion downstream target gene
NR4A2 was also confirmed in the dnCRTC expressing fusion-positive cells. These data
strongly indicate that the LINC00473 transcription is directly promoted by the CRTC1-
MAML2/CREB interaction and activation in the fusion-positive MEC cells.
Depletion Of LINC00473 Expression In Fusion-Positive MEC Cells Led To Reduce Cancer Cell Growth And Survival Both In Vitro And In Vivo
Aberrant high LINC00473 expression is significantly and positively correlated
with fusion-positive MEC, suggesting that LINC00473 may play an importantly
functional role in promoting CRTC1-MAML2 tumorigenesis. I next investigated the
functional effect of downregulation of LINC00473 expression on the fusion-positive MEC
cells’ growth and survival. Depletion of LINC00473 using two independent lentiviral-
based shRNAs targeted LINC00473 (shLnc473-2 and -4) was observed at 96 hours
after lentiviral infection in H3118 cells compared to the cells transduced with scramble
shRNA control (Figure 3-4A). The transduced cells were harvested and subsequently
assayed for cell proliferation and survival in vitro. I found that the depletion of
LINC00473 cells resulted in a dramatically decrease in the number of viable cells
(Figure 3-4B) and increase the amount of apoptotic cells (Figure 3-4C). In addition,
similar effects of depletion of LINC00473 transcript levels in reduction of cell
65
proliferation and enhanced the number of apoptotic cells in another fusion-positive MEC
cell line HMC3A were detected (Figure 3-5). I next sought to determine the functional
relevance of LINC00473 depletion on the growth in H3118 MEC xenografts in vivo. The
luciferase-expressing H3118 (H3118-luc) cells were infected with lentiviral-based
LINC00473 shRNAs or scrambled control and then the same number of LINC00473-
depleted or control cells were injected subcutaneously into NOD.SCID mice. The tumor
growth was monitored through bioluminescent imaging and direct measurement after
cancer cell injection. I found that LINC00473 depletion in H3118-luc xenografts
significantly reduced the tumor size and weight, inhibited the tumor growth (Figure 3-
4D-G). These in vitro and in vivo approaches strongly reveal that LINC00473 plays a
critical role in regulating fusion-positive MEC cell growth and survival.
LINC00473 Is Revealed As A Nuclear-Retained Long Noncoding RNA
To study potential mechanisms of LINC00473 in the regulation of human MEC
growth and survival, I first examined the intracellular distribution of LINC00473. I
performed RNA Fluorescence in situ hybridization (RNA-FISH) on fixed human fusion-
positive MEC cells using Stellaris® FISH oligonucleotide probes that were
complementary to LINC00473 RNA sequence and labeled with a single Quasar 570
fluorophore. Positive staining in the nucleus was observed showing striking dot-like
structures and diffuse signals surrounding the dots (Figure 3-6A). NO signals were
detected when RNase treatment was performed before the hybridization indicating the
specific signals were resulted from the presence of RNA. I also performed a newest
RNA in situ hybridization technique called RNAscope which allows direct visualization of
LINC00473 in FFPE tissue sections. The nuclear visualization of LINC00473 was
observed in both H3118 xenograft FFPE tumor section (Figure 3-6B) and primary
66
fusion-positive MEC tumors (Figure 3-6C). The housekeeping gene PPIB (peptidylprolyl
isomerase) was used as a control for good RNA quality and cytoplasmic marker. The
nuclear localization of LINC00473 data suggests that LINC00473 may play a critical role
in nuclear functions involving transcriptional regulation, chromatin interaction and RNA
processing.
Gene Expression Profiling Identified LINC00473 Regulated Genes In Fusion-Positive MEC
In order to identify the putative targets of LINC0473 and to understand the
mechanism of LINC00473 in the role of fusion-positive MEC tumorigenesis, I performed
gene expressing profiling analysis in LINC00473-depleted along with control cells. The
fusion-positive H3118 cells were infected by lentiviral- based shLINC00473 or shRNA
control and then isolated the total RNA at 96 hours after infection for microarray
analysis. I used Affymetrix human transcriptome array 2.0 (HTA 2.0) to identify the
changes in gene expression after the depletion of LINC00473 in fusion-positive MEC
cells. Using an absolute fold-change ≥ 2.0 and a p-value <0.05, I identified a total of 489
down-regulated and 130 up-regulated genes in H3118 cells upon LINC00473 depletion
(Figure 3-7A). Gene ontology (GO) analysis of the 489 down-regulated genes revealed
several pathways that are regulated by LINC00473 (Figure 3-7B).
Discussion
The CRTC1-MAML2 fusion product is a nuclear protein without enzymatic
activity, which is difficult to perform the direct targeting intervention. Therefore, the
promising approaches for CRTC1-MAML2-specific cancers are to direct targeting the
critical CRTC1-MAML2 regulated downstream targets. Previously, we performed the
67
gene expressional profiling in CRTC1-MAML2 fusion-positive MEC and identified
CRTC1-MAML2 fusion target candidates (48).
The lncRNA LINC00473 is the leading target in fusion-positive MEC in our gene
expression analysis, suggesting that LINC00473 is regulated by CRTC1-MAML2 and
may play critical role in MEC tumorigenesis and progression. In this study, I focused on
the functions and mechanisms of LINC00473 in fusion-positive MEC. Firstly, I found that
high expression of LINC00473 in fusion-positive MEC but low or undetectable
expression in fusion-negative MEC, suggesting that LINC00473 may distinguish the
fusion-positive MEC from the fusion-negative MEC. These studies indicated that
LINC00473 may serve as a novel biomarker for CRTC1-MAML2 fusion-positive MEC.
Therefore, I determined the potential clinical application of LINC00473 as a diagnostic
marker for the CRTC1-MAML2 fusion-positive MEC. In my studies, using the Nanostring
analysis to detect the LINC00473 expression in formalin-fixed paraffin-embedded
(FFPE) MEC specimens has more advantages when compared with traditional assay
qRT-PCR. The total RNA derived from the FFPE tissues are easy to degrade into small
fragments after fixation and embedding process and low-quality RNA samples presents
challenges for routine clinical practice using qRT-PCR analysis. The Nanostring
technology used fluorescent barcode probes to directly and specifically detect the small
region of target RNA allowing the individually counted without the need for target
amplification (57). Nanostring approaches were reported to sensitively detect the gene
expression in FFPE tissue samples and have no significant differences in the detection
gene expression between frozen and FFPE samples (58, 59). And my results showed
that LINC00473 expression was increased in the fusion-positive MEC primary tumors
68
but low or undetectable in the fusion-negative MEC primary tumors using the
Nanostring analysis. Meanwhile, RNAscope in situ hybridization technology was
reported to highly sensitively and specifically visualize the gene expression in FFPE
tissue with single cell resolution (60). My studies indicated that RNAscope permits direct
visualization of LINC00473 expression in FFPE fusion-positive MEC samples. Taken
together, both Nanostring and RNAscope technologies present promising approaches
to detect LINC00473 expression as a diagnostic marker to distinguish the fusion-
positive and -negative MEC.
My functional data indicates that the depletion of LINC00473 in fusion-positive
MEC using lentiviral-based shRNA result in reducing MEC cell growth and survival,
strongly suggest that inhibiting lncRNA LINC00473 function by RNA depletion using
different approaches should be further investigated to offer novel opportunities for MEC
therapy.
In summary, I identified a novel lncRNA LINC00473 that is highly associated with
CRTC1-MAML2 fusion oncogenic activity and is directly regulate by CRTC1-MAML2 in
human MEC. LINC00473 is essential for human MEC cell growth and survival both in
vitro and in vivo, supporting that targeting LINC00473 is an effective strategy to human
MEC therapeutic treatment. Finally, I identified a novel function for LINC00473 in
regulating gene expression in human MEC (Figure 3-8). Collectively, these observations
reveal the potential of LINC00473 as a novel biomarker for the malignancy of fusion-
positive MEC and as a highly selective anti-MEC therapeutic target for drug
development and options for MEC clinical treatment.
69
Figure 3-1: LINC00473 expression is highly evaluated in human CRTC1-MAML2 fusion-
positive MEC cell lines and primary tumors. (A) Western blotting analysis of the expression of CRTC1-MAML2 in human MEC cell lines. The HPA cell line was used as a fusion-negative control. Anti-MAML2 TAD antibodies were used to detect the MAML2 and CRTC1-MAML2 fusion protein. Blotting with anti-ß-ACTIN was used as a protein loading control. (B) The qRT-PCR analysis showed the LINC00473 transcripts had higher levels in 4 CRTC1-MAML2 fusion-positive human MEC cell lines (HMC3A, HMC3B, H3118 and H292) than the fusion-negative cells (HPA). (n=3, **p<0.001 and ***p<0.0001. (C) A heatmap showed that the relatively higher expressions of LINC00473 in 4 fusion-positive MEC cell lines (H292, H3118, HMC3A and HMC3B) were detected when compared to fusion-negative MEC cell line HPA in a Nanostring assay. Several fusion target genes (AREG, ARG2, DMBT1 and NR4A1) were also tested. (D) A heatmap showed gene expression levels in RNA samples isolated from 6 fusion-positive and 6 fusion-negative MEC primary tumors by Nanostring assay. All fusion-positive MEC tumors (n=6) had up-regulated LINC00473 expression as compared with fusion-negative MEC tumors (n=6). (E and F) Expression levels of LINC00473 and CRTC1-MAML2 fusion were positively correlated (r=0.785157) in human MEC tumors (n=12) using qRT-PCR analysis. The ∆Ct values (normalized to GAPDH) were subjected to Pearson correlation analysis.
70
Figure 3-2: LINC00473 expression is directly regulated by CRTC1-MAML2 fusion
oncoprotein. (A) qRT-PCR analysis showed that the transcript levels of LINC00473 were significantly reduced in CRTC1-MAML2 fusion-positive H3118 cells after knockdown the expression of fusion by two independent lentiviral shRNAs (shM2-1 and -3) as compared with the scramble shRNA control (shCtl) (n=3, *p<0.05 and **p<0.001). (B) Another fusion-positive MEC cells (H292) also had the same result that the LINC00473 expression was reduced after fusion knockdown (n=3, **p<0.001). (C) A heat map showed the depletion of CRTC1-MAML2 resulted in decrease the LINC00473 expression while depletion of MAML2 only did not change the LINC00473 expression in H3118 cells by Nanostring assay. (D) Exogenous CRTC1-MAML2 expression rescued the adverse effect of the depletion of endogenous fusion gene expression on LINC00473 expression in H3118 cells. (n=3, *p<0.05). (E)The expression of LINC00473 was enhanced in the HEK293T cells after transfection with FLAG-tagged CRTC1-MAML2 expression constructs but not changed when transfection with MAML2 expression constructs (F) (n=3, *p<0.05 and ***p<0.0001).
71
Figure 3-3: CRTC1-MAML2 fusion activates LINC00473 expression though co-
activating the transcription factor CREB. (A) Western blotting analysis showed that CREB expression was knocked down by two independently lentiviral-mediated CREB shRNAs in H3118 cells. (B) The LINC00473 expression was significantly reduced in the CREB-depleted fusion-positive H3118 cells by qRT-PCR assays (n=3, *p<0.05 and **p<0.001). (C) A LINC00473 promoter reporter containing two conserved CRE half binding sites were cloned into pGl3 luciferase reporter plasmid. LINC00473 promoter luciferase activities were increased after ectopic expression of CRTC1-MAML2 fusion in HEK293T cells (n=3, **p<0.001). (D) ChIP analysis indicated that CREB and CRTC1-MAML2 were significantly enriched on the LINC00473 promoter encompassing the two CRE half sites in H292 cells. The ChIP assays were performed in fusion-positive H292 cells by using anti-CREB antibodies or anti-MAML2 antibodies to immune-precipitate the binding LINC00473 promoter. The immunoglobulin G (IgG) was used as a negative control (n=3, *p<0.05). (E, F) Retroviral-mediated expression of a dominant negative form of CRTC in H31118 and H292 cells reduced LINC00473 expression as compared with the GFP vector control by qRT-PCR analysis. A CRTC1-MAML2 fusion target gene NR4A2 was used as a positive control. Western blotting confirmed dnCRTC-GFP expression in H3118 and H292 cells (n=3, *p<0.05).
72
Figure 3-4: Depletion of LINC00473 expression in CRTC1-MAML2 fusion-positive MEC
H3118 cells results in a reduction in cell proliferation and survival in vitro and blocked the growth of human MEC xenografts in vivo. (A) H3118 cells were transduced with two independent lentiviruses expressing shRNAs against LINC00473 (shLnc473-2 and -4) or the scramble shRNA control (shCtl) on 3 consecutive days. Transduced cells were collected 96 hours after first infection. The LINC00474 transcript levels were significantly reduced as compared with shRNA control by qRT-PCR assay (n=3, *p<0.05 and **p<0.001). (B and C) Transduced cells at 96-hours post-transduction were cultured at 5 X 105 per well in 6-well plates for another 96 hours to perform cell proliferation and survival assay. The viable cell number was counted using Trypan blue assay. (B) The apoptotic cells of LINC00473-knockdown and control cells were determined by Annexin V/PI staining followed by FACS analysis (C) (n=3, *p<0.05 and **p<0.001). Approximately 2 X 106 luciferase-expressing H3118 (H3118-luc) cells after transduction with shLINC00473 or shRNA control for 96 hours were implanted subcutaneously to the dorsal flanks of NOD-SCID mice. (D) A representative bioluminescent image showed that mice injected with shCtl (n=5) or shLnc473-2 (n=5), shLnc473-4 (n=4) H3118-luc cells, respectively. The image was taken at day 18 after the transduced H3118-luc cells injection at the end point study. The excised tumors (E) and the tumor weights (F) at the end points were shown. (G) Tumor growth was suppressed in mice injected with LINC00473-knockdown cells as compared with shRNA control (*p<0.05 and ***p<0.0001).
73
Figure 3-5: Depletion of LINC00473 expression in CRTC1-MAML2 fusion-positive MEC
HMC3A cells results in a reduction in cell proliferation and survival in vitro. (A) HMC3A cells were transduced with two independent lentiviruses expressing shRNAs against LINC00473 (shLnc473-2 and -4) or the scramble shRNA control (shCtl) on 3 consecutive days. Transduced cells were collected 96 hours after first infection. The LINC00474 transcript levels were significantly reduced as compared with shRNA control by qRT-PCR assay (n=3, **p<0.001). (B and C) Transduced cells at 96-hours post-transduction were cultured at 5 X105 per well in 6-well plates for another 96 hours to perform cell proliferation and survival assay. The viable cell number was counted using Trypan blue assay (B) The apoptotic cells of LINC00473-knockdown and control cells were determined by Annexin V/PI staining followed by FACS analysis (C) (n=3, *p<0.05 and **p<0.001).
74
Figure 3-6: Specific nuclear distribution of LINC00473 was revealed in CRTC1-MAML2
fusion positive human MEC cells and primary tumors. (A) Dot-like nuclear signals of LINC00473 were detected by RNA-FISH in H3118 cells. LINC00473 RNA-FISH probe sets were labeled with Quasar 570 dyes (red) and nuclei were labeled with the DNA dye DAPI (blue). No LINC00473 RNA-FISH probe and RNase treatment were used as negative control. (B) Predominantly nuclear localization of LINC00473 was detected based on RNAscope detection in H3118 cells. RNA-ISH of the housekeep gene PPIB was performed for sample RNA quality control and cytoplasmic marker control. (C) One CRTC1-MAML2 fusion-positive MEC tumor sample was detected the nuclear localization of LINC00473 based on the RNAscope analysis.
75
Figure 3-7: Gene expression profiling analysis identified genes differentially regulated
by LINC00473 in MEC cells. (A) Volcano plot shows differentially expressed genes in shLnc473-2 knockdown in compared with shRNA control in H3118 cells. (B) Volcano plot shows differentially expressed genes in shLnc473-2 knockdown in compared with shRNA control in H3118 cells. The cutoff criteria were fold-change of ≥2 and p<0.05. (C) Venn diagrams show the significantly up-regulated genes (n=10) and down-regulated genes (n=39) in two independent shRNAs knockdown when compared with the shRNA control in H3118 cells. (D) The up-regulated and down-regulated target genes were sorted by KEGG pathway analysis using DAVID identified several signaling pathways. The GO enriched pathways were filtered in accordance with p<0.05 and FDR<0.05.
76
Figure 3-8: A model for the molecular basis of LINC00473 induction by CRTC1-MAML2
in fusion-positive MEC cells.
77
CHAPTER 4 CAMP/CREB-REGULATED LINC00473: POTENTIAL BIOMARKER AND
THERAPEUTIC TARGET FOR LKB1-INACTIVATED CANCER
Abstract
The LKB1 tumor suppressor gene is frequently mutated and inactivated in non-
small cell lung cancer (NSCLC). Loss of LKB1 promotes cancer progression and
influences therapeutic responses in preclinical studies; however, specific targeted
therapies for lung cancer with LKB1 inactivation are currently unavailable. This study
identifies a lncRNA signature associated with the loss of LKB1 function. I discovered
that LINC00473 is consistently the most highly induced gene in LKB1-inactivated
human primary NSCLC samples and derived cell lines. Elevated LINC00473 expression
is correlated with poor prognosis, and sustained LINC00473 expression is required for
the growth and survival of LKB1-inactivated NSCLC cells. Mechanistically, LINC00473
is induced by LKB1 inactivation and subsequent CRTC-CREB activation. LINC00473 is
a nuclear lncRNA and interacts with NONO, a component of the cAMP-signaling
pathway, thereby facilitating CRTC-CREB-mediated transcription. Collectively, our study
demonstrates that LINC00473 expression potentially serves as a robust biomarker for
tumor LKB1 functional status which can be integrated into clinical trials for patient
selection and treatment evaluation, and implicates LINC00473 as a novel therapeutic
target for LKB1-inactivated NSCLC.
This chapter was reprinted with permission from the Journal of Clinical Investigation (61).
78
Rationale
Lung cancer is the leading cause of cancer-related deaths (62). Approximately
80% of lung cancer cases are non-small cell lung carcinoma (NSCLC). Conventional
cytotoxic therapeutics show limited effectiveness; but recent targeted therapies utilizing
inhibitors targeting specific driver gene activating mutations, such as EGFR and ALK,
have demonstrated promising improved clinical outcomes (63-65). However, effective
targeted therapies for other common somatic mutations such as LKB1 (STK11) in lung
cancer remain an unmet medical need.
The LKB1 gene encodes a serine-threonine kinase that plays critical roles in cell
growth, polarity, and metabolism (66, 67). LKB1 is a tumor suppressor gene whose
mutations cause Peutz-Jeghers syndrome associated with increased cancer risk (68,
69). LKB1 is a target for mutational inactivation in sporadic cancers, especially NSCLC
where it is mutated in ~20% to 30% of cases making it the third most common site of
genetic alterations after TP53 and KRAS (70-73). LKB1 mutations have been linked
with lung cancer progression and differential treatment responses. LKB1 loss was found
to promote lung cancer metastasis in a KRAS mouse model of NSCLC (74).
Importantly, LKB1 inactivation influenced the responsiveness of cancer cells to
treatments in vitro and in vivo. For instance, LKB1 loss sensitized NSCLC responses to
the metabolism drug phenformin and a COX2 inhibitor (75, 76). On the other hand,
LKB1 loss impaired the responses of KRAS-mutant NSCLC to docetaxel monotherapy,
the combination therapy of docetaxel and MEK inhibitor selumetinib, or dual inhibition of
the PI3K and MEK pathways (77, 78). Additionally, LKB1 mutations were associated
with the modulation of immune microenvironments, thus potentially impacting
immunotherapeutic outcomes (79, 80). The current data emphasize the need to
79
determine tumor LKB1 status for patient stratification for evaluating therapeutic
responses and identifying effective treatments. However, detection of LKB1 functional
loss is technically challenging because LKB1 function can be inactivated by multiple
mechanisms such as somatic gene mutations, epigenetic silencing, post-translational
modifications, or alterations in LKB1 pathway components (81-83). Clinical trials for
evaluating treatment efficacy on LKB1-inactivated NSCLC are lacking due to the
absence of reliable assays for accurate determination of tumor LKB1 status. Therefore,
sensitive and specific assays for LKB1 inactivation as well as effective targeted
therapeutics are needed for effective management of LKB1-inactivated NSCLC.
LncRNAs are non-protein coding transcripts longer than 200 nucleotides and
represent a novel class of gene regulators (84). The human genome encodes more
than 10,000 lncRNAs and currently only a handful of lncRNAs have been characterized
(24-26). LncRNA expression is frequently de-regulated in cancer and shows cell- or
tissue-type specificity (85-87), suggesting significant roles of lncRNAs in human
cancers. Though lncRNAs remain an understudied class of genes, they have been
shown to participate in cancer cell proliferation, survival, migration, and invasion, likely
through exerting multiple regulatory functions at the transcriptional, post-transcriptional,
and epigenetic levels (84, 88). Importantly, targeting cancer-associated lncRNAs, such
as HOTAIR and MALAT1, blocked cancer cell growth and survival in vitro and tumor
growth and metastasis in vivo (89, 90). Therefore, lncRNAs are critical for cancer
development and progression and might serve as novel therapeutic targets. Currently,
the molecular mechanisms underlying LKB1 tumor suppression remain incompletely
80
understood (66, 91). Whether lncRNAs have a role in mediating the loss of LKB1 tumor
suppression in lung cancer has not been studied.
In this study, I performed lncRNA transcriptional profiling to identify lncRNAs that
are associated with inactivated LKB1 signaling. I subsequently focused on one novel
LKB1-regulated lncRNA (LINC00473) because it was consistently the most highly
induced target identified with LKB1 loss. The elevated expression of LINC00473 was
associated with poor patient prognosis and tightly correlated with LKB1 inactivation
status in human lung cancer specimens and derived cell lines. My expression,
functional, and mechanistic data demonstrate that LINC00473 is a potentially robust
biomarker to detect lung cancers with inactivated LKB1, a novel therapeutic target, and
a novel gene regulator.
Materials And Methods
Plasmids
The retroviral pBabe-FLAG-LKB1 (Plasmid #8592) and the kinase-dead mutant
pBabe-FLAG- LKB1 (K78I) (Plasmid #8593) were obtained from AddGene (92). Two
oligonucleotide sequences targeting LINC00473 gene were cloned into pLKO.1-based
lentiviral vector (Open Biosystems), which included shLINC00473-2 (5’-
AACTGGATCTTTGCAGACAGG-3’) and shLINC00473-4 (5’-
AAGAACCCAAGTCATATTCAT-3’). The pLKO.1-based lentiviral constructs targeting
LKB1 (RHS4533-EG_6794) and CREB (RHS4533-NM_004379) as well as the full-
length human LINC00473 cDNA (transcript variant 1) expression construct (MHS6278-
20275602) were obtained from Open Bioystems. The LINC00473 antisense cDNA
(~1.5kb after the deletion of 3’ poly A sequence region) was sub-cloned into pBS-KS
plasmid under the control of T7 promoter. The full-length LINC00473 cDNA (~1.8 kb)
81
was cloned into pLNCX-based retrovirus vector. The pMSCV-A-CREB-IRES-GFP,
CMV-FLAG-CRTC1, CMV-FLAG-CRTC1 (S151A), and CMV-GAL4-NONO expression
vector were previously described (54, 93). The LINC00473 promoter reporter was
generated through subcloning LINC00473 promoter sequence (-523 to +88) into the
pGL3 reporter plasmid (Promega). The pCI-MEG3 (plasmid # 59420) (94) was obtained
from AddGene and ~1.8 kb XhoI-NotI MEG3 sequence was subcloned into pBS-SK
plasmid under the control of T7 promoter. The dnCRTC construct was generated
through cloning the CREB binding domain of CRTC1 (1-55aa) fusion with a nuclear
localization signal and GFP into pMSCV-based retrovirus vector.
Cell Culture
Human NSCLC cancer cell lines (A549, H157, H23, H358, H460, H2122, H2126,
H322, A427, H522, H1819, H2009, H2087 and H3123) and HEK293T cells were
cultured in Dulbecco’s modified Eagle’s medium (Mediatech) supplemented with 10%
inactivated fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin
(Corning Cellgro). Cells were grown in a 5% CO2 cell culture incubator at 37 °C.
Rapid Amplification Of CDNA Ends (RACE)
The 5’ and 3’ RACE were performed using the First Choice RLM-RACE Kit
(Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, for the
cap dependent 5’ RACE, 10 μg total RNA was treated with Calf Intestine Alkaline
Phosphatase (CIP), and Tobacco Acid Pyrophosphatase (TAP) to remove the cap
structure. The RNA 5’ Adapter was then ligated to the CIP and TAP treated RNA using
T4 RNA ligase. Reverse transcription was performed with Random Decamers, and
nested PCR was performed to amplify the 5’ end of LINC00473. For the 3’ RACE,
reverse transcript with performed using 3' RACE Adapter and then nested PCR was
82
performed to amplify the 3’ end of LINC00473. Products of RACE reactions were
sequenced.
Coding Potential Prediction
Coding Potential Calculator (CPC) (95) was used to calculate the coding
potential score with the LINC00473 transcript variants identified in the Rapid
Amplification of cDNA Ends (RACE) assays. PhyloCSF scores (96) were viewed in the
UCSC Genome Browser by adding PhyloCSFtracks to “My Hubs” in UCSC Genome
Browser. Known coding genes (ACTB, GAPDH and CRTC1) and non-coding genes
(HOTAIR, XIST) were used as controls.
Viral Production And Transduction
For lentiviral production, pLKO.1 constructs (shLINC00473-2, 4 and control)
together with packing plasmids pMD2.g and psPAX2 were transfected to 293FT cells
using Effectene transfection reagent (Qiagen). Lentiviruses were collected at 72 hours
and 96 hours after transfection. The A549 cells were infected on two consecutive dates
with viruses in medium containing polybrene (8μg/mL, Sigma). At 96 hours after first
infection, cells were collected for RNA using TRIzol.
For retroviral production, the retroviral pBabe-based construct (LKB1, kinase
death mutant LKB1 K78I or empty vector) together with packing plasmid pMD.MLV and
pseudotyped envelope pMD2-VSV-G were transfected to 293T cells using Effectene
transfection reagent (QIAGEN). Retroviruses were collected at 48 hours and 72 hours
post transfection and used to infect the A549 cells on two consecutive dates. Cells were
collected for RNA and proteins at 96 hours post infection.
83
Western Blotting Analysis
Western blot analysis was performed as previously described (19). The following
antibodies were purchased from the following commercial sources: anti-LKB1 (CST-
3050), anti-phospho-AMPKα (Thr172) (CST-2535), anti-AMPKα (CST-5831), anti-
Phospho-Acetyl-CoA Carboxylase (Ser79) (p-ACC) (CST-11818) and anti- Acetyl-CoA
Carboxylase (ACC) (CST-3676) from Cell Signaling Technology; anti-LKB1 (SC-32245),
anti-GFP (SC-8334) and anti-β-actin (SC-47778) from Santa Cruz; anti-NONO (A300-
587A) from Bethyl Laboratories; anti-CRTC1 (600-40193) from Rockland; anti-CREB
(06-863) from Millipore, and anti-GAPDH antibody (2251-1) from Epitomics.
Quantitative RT-PCR (qRT-PCR)
The qRT–PCR analysis was performed as previously described (19). RNA was
extracted using TRIzol (Life Technologies) and RNeasy mini kit (Qiagen) and then
reverse-transcribed into complementary DNA (cDNA) using a GeneAmp RNA PCR kit
(Applied Biosystems). The qPCR analysis was performed with the iQ™ SYBR® Green
supermix (Bio Rad) using the StepOne Real-Time PCR System (Applied Biosystems).
The primer sequences were listed in Table 4-1.
Reporter Assays
Cells were plated in 24-well plates and transfected with pGL3-LINC00473
promoter firefly luciferase vector and various combinations of the expression constructs
along with internal control Renilla luciferase plasmid (pEF-RL) using Effectene
transfection reagent. The luciferase assays were performed 48 hours post-transfection
with the dual-luciferase assay kit (Promega) as described previously (54).
84
Chromatin Immunoprecipitation
Chromatin immunoprecipitation (ChIP) experiments were performed using the
Millipore ChIP protocol as previously described (19). Briefly, A549 cells were fixed with
1% formaldehyde at room temperature for 10 minutes and then lyzed in SDS lysis buffer
(1% SDS, 10mM EDTA, 50mM Tris-HCl, pH8.1), followed by sonication to shear DNA to
the lengths between about 200 bp to 800 bp. The sonicated cell supernatant was diluted
10 folds in ChIP dilution buffer (0.01% SDS, 1.1% Triton X100, 1.2mM EDTA, 200 mM
Tris-HCl pH 8.1, 167 mM NaCl) and then immunoprecipitated with the CRTC1
antibodies (600-401-93, Rockland) and CREB antibodies (06-863, Millipore) or control
immunoglobulin G. The ChIP DNAs were used for the real-time PCR analysis using the
primers flanking the two CREB-binding half sites of the LINC00473 promoter. The
primer sequences were listed in Table 4-1.
RNA-Fluorescence In Situ Hybridization (RNA-FISH)
Cells were hybridized with a mixture of 27 LINC00473 Stellaris® FISH probes
labeled with Quasar 570 (Biosearch Technologies) following the manufacture’s protocol.
Briefly, Cells cultured on cover slips in 12-well plates were washed with PBS twice, fixed
with 3.7% formaldehyde for 10 minutes at RT, and permeabilized with 70% ethanol in
Rnase-free water at 4°C for 1 hour. Cells were then washed with wash buffer (2x SSC
buffer with 10% formamide) followed by hybridization in hybridization buffer (2x SSC
buffer with 10% dextran sulfate and 10% deionized formamide) with LINC00473
Stellaris® FISH probes in a humidified chamber at 37 °C overnight. After washing with
wash buffer for 30 minutes at 37°C, cells were counterstained with DAPI (5 ng/mL) and
mounted with Vectashield mounting medium. Imaging was performed immediately using
Leica DM6000B fluorescence microscope.
85
RNA Fractionation And Northern Blotting Analysis
RNA fractionation was performed as previously described with modifications (97).
Briefly, the cells were lysed in Buffer A (10mM Tris PH 7.4, 10mM NaCl, 3mM MgCl2,
0.25% NP40, 0.5mM DTT) for 10 minutes, and then centrifuged at 10,000rpm for 5
minutes. Supernatant was used for the extraction of cytoplasma RNA by Phenol–
chloroform extraction. The cell pellets were washed with Buffer A for three times and
then used for isolation of nucleus RNA with TRIzol. The tRNA and U6 were used as
markers of cytoplasm RNA and nucleus RNAs, respectively. RNA northern blot was
performed as previously described (98). The probe sequences were listed in Table 4-1.
RNA Immunoprecipitation
A549 cells were crosslinked with UV Then lyzed in RIPA buffer (50 mM Tris HCl
pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplied with
Proteinase and Rnase inhibitors. Cell lysates were collected for immunoprecipitation
overnight at 4°C using anti-NONO antibodies and protein A/G beads. The beads that
bound with protein-RNA complex were then washed with RIPA buffer for 5 times and
digested with Proteinase K at 45 °C for 45 minutes to release the RNA from the beads.
Then RNAs were isolated with TRIzol and analyzed by qRT-PCR using the LINC00473
and ASNS primers with the primer sequences listed in Table 4-1.
Cell Proliferation And Apoptosis Assay
Cell proliferation and apoptosis analyses were performed as previously described
(19). Briefly, at 72 hours post lentivirus infection, transduced cells (control and
LINC00473-depleted cells) were cultured at 2 X 105 /well in 6-well plates for 96 hours.
The cell numbers of control and LINC00473 knockdown cells were determined by direct
cell counting using Trypan blue exclusion assay. The numbers of apoptotic cells were
86
quantified by flow cytometric assays using Annexin V-FITC Apoptosis Detection kit (BD
Pharmingen). Triplicate assays were set up.
Mouse Xenograft Studies
The mouse xenograft studies were performed according to an animal protocol
approved by the institutional animal care and use committee of the University of Florida.
A549 cells stably expressing firefly luciferase (A549-luc) were infected with control
shRNA or shLINC00473 lentiviruses for 96 hours. A total of 1 X 106 cells in 50 µL of
phosphate-buffered saline were mixed with 50 µL Matrigel (BD Biosciences) and the cell
mixture were then injected into the right flanks of 8-12 week-old NOD/SCID mice
(Jackson Laboratory). Tumors were measured for two perpendicular diameters with a
Dial caliper, and tumor volumes were calculated using the following equation: tumor
volume= (width× width× length)/2. The bioluminescence images were taken using a
Xenogen in vivo imaging system (Caliper Life Sciences).
LncRNA Microarray Analysis
Total RNAs were extracted from A549 cells after the transduction of wild-type
LKB1, LKB1 K78I mutant, or control retroviruses for 96 hours and two biological
replicates were set up. RNAs were subjected to human lncRNA expression microarray
(V3.0) analyses (ArrayStar). The microarray data were deposited in NCBI Gene
Expression Omnibus (GEO: GSE73414). Genes with an absolute fold-change of ≥2 and
p-value < 0.05 were considered as significantly differentially expressed.
Nanostring Ncounter Gene Expression Analysis
Nanostring gene expression assay analyses were performed according to the
manufacture’s protocols (Nanostring Technologies) using our customized nCounter GX
CodeSet. In brief, total RNA from cultured cells (100ng) or RNA from FFPE tissues
87
(150-200ng) were hybridized with the specific capture probes and barcoded reporter
probes at 65 °C for 18 hours and then loaded into the nCounter Pre-station for purifying
the hybridized probes. Data collection was performed on the nCounter Digital Analyzer
that counted and tabulated the individual fluorescent barcodes for target RNA molecules
in each samples following the manufacturer’s instructions. The raw data were analyzed
with nSolver™ Analysis Software for gene expression analysis. The heatmap was
generated with Heatmap.2 R package using the normalized Nanostring expression
data.
RNAscope In Situ Hybridization (RNA ISH)
RNA ISH was performed on FFPE xenograft tumors and tissue microarrays
(TMAs) using RNAscope® 2.0 HD Reagent Kit [BROWN 310033 or RED 310034,
Advanced Cell Diagnostics (ACD)]. Briefly, tissue sections were deparaffinized with
xylene and 100% ethanol, and then incubated with pretreat 1 solution for 10 minutes,
pretreat 2 for 15 minutes, and pretreat 3 for 30 minutes (Pretreatment kit 310020, ACD).
The slides were then hybridized with a custom probe Hs-LINC00473-tv1 (targeting 781-
1755 of NR_026860.1) in the HybEZ oven (ACD) at 40°C for 2 hours. The Hs-PPIB
probe for human housekeeping gene PPIB was used as a control to ensure RNA
quality. After hybridizations, slides were subjected to signal amplification using HD 2.0
detection Kit, and hybridization signal was detected using a mixture of solutions A and B
(1:60). After counterstaining with hematoxylin, slides were dried in a 60°C dry oven for
15 minutes and mounted with Ecomount (BioCare Medical, EM897L). The stained
sections were scanned and digitized with Aperio Imagescope (Leica).
88
Proteomic Analysis Of The LINC00473-Associated Protein Complex
LncRNA was first transcribed in vitro using the MEGAscript® T7 Transcription Kit
(AM1333, Life Technologies) according to the manufacturer’s instructions. Identification
of lncRNA interacting protein complexes was performed as previously described with
modifications (99, 100). Briefly, RNAs were covalently linked to adipic acid dihydrazide
agarose beads by periodate oxidation of RNA 3’-OH terminus. The beads bound with
RNAs were incubated with nuclear extracts of A549 cells to pull down the lncRNA-
interacting proteins. After extensive washing, beads were boiled with a loading buffer to
elute the lncRNA-interacting proteins, which were further separated by SDS-PAGE and
subjected to mass spectrometric analysis.
Analysis Of RNA Sequencing And Clinical Data
RNA sequencing data from the lung adenocarcinoma (LUAD) dataset of The
Cancer Genome Atlas (TCGA) sequencing project and the accompanying clinical data
(101) were used for analysis. Normalized read counts (RSEM) for each LUAD sample
aligned to LINC00473 were obtained and used for the survival curve analysis.
Nonparametric tests were performed to examine the association between LINC00473
expression levels and patient survival. All LUAD samples with ≥90th percentile of
LINC00473 expression and those with <90th percentile was considered as high and low
groups, respectively. The overall survival was analyzed by Kaplan-Meier curves and
log-rank test for all LUAD patients. P-values of <0.05 were considered statistically
significant.
Statistical Analysis
Data from real-time PCR, reporter assay, cell proliferation, apoptosis and in vivo
xenograft experiments were analyzed using Student’s t-test. Results were expressed as
89
mean and standard deviation (± s.d.) and a p<0.05 was considered statistically
significant. The biological replicates for each experiment were indicated in the figure
legends.
Study Approval
All mouse experiments were performed in accordance with a protocol approved
by the Institutional Animal Care and Use Committee at the University of Florida.
Results
Genome-Wide LncRNA Profiling Identified LINC00473 As A Top LKB1 Signaling-Regulated LncRNA In NSCLC Cells.
LncRNA involvement in altered LKB1 signaling in lung cancer is currently
unknown. To investigate whether lncRNAs contribute to loss of LKB1 tumor suppression
in lung tumorigenesis and maintenance, I performed genome-wide lncRNA
transcriptional profiling to identify lncRNAs associated with aberrant LKB1 signaling. I
generated three groups of cells by transducing LKB1-null A549 NSCLC cells with
retroviruses harboring wild-type (wt) LKB1, LKB1 kinase-dead mutant (K78I), or vector
control (Ctl). Western blotting analysis confirmed expression and kinase activity of
LKB1-wt and –K78I proteins in an AMPK activation assay under glucose-free culture
conditions (Figures 4-1A, 4-2). I subsequently used Arraystar human lncRNA
expression microarrays to profile changes in expression of ~30,000 lncRNAs in my
three experimental conditions. Using an absolute fold change ≥ 2.0 and a p-value <0.05,
I identified a total of 164 differentially expressed lncRNAs (64 up-regulated and 100
down-regulated) in A549 cells upon LKB1-wt expression compared to control (Figure 4-
1B). I also observed 17 up-regulated and 49 down-regulated lncRNAs associated with
LKB1-K78I mutant expression compared to control (Figure 4-1C). Comparing
90
expression profiles of LKB1-wt- and K78I-expressing cells, I identified 33 up-regulated
and 83 down-regulated lncRNAs, suggesting that their expression is dependent on
LKB1 kinase activity. Furthermore, I profiled and compared lncRNA expression patterns
between 2 groups of cell lines: LKB1-null (A549, H460) and LKB1-wt (H322, H3123),
and observed 1449 up-regulated and 918 down-regulated lncRNAs in LKB1-null cell
lines (Figure 4-1D). Finally, by integrating LKB1-regulated lncRNAs and lncRNAs
differentially expressed in LKB1-null cell lines, I identified a list of LKB1-regulated
lncRNAs (10 up-regulated and 1 down-regulated) that are differentially expressed
between LKB1-null and -wt lung cancer cells.
Notably, this list contained 3 probes for LINC00473 gene (aka. C6orf176,
abbreviated as Lnc473 in the figures), which encodes an intergenic lncRNA from the
chromosome 6q27 locus. LINC00473 consists of two exons and expresses two
transcript isoforms sharing exon 1: transcript variant 1 (tv1; NR_026860, 1822 nt) and
tv2 (NR_026861, 1123 nt) (Figure 4-3A). Our 5’ and 3’ RACE assays identified 3
LINC00473 transcript variants, tv1.1, tv2.1 and tv2.2, but not two annotated transcripts
(Figure 4-3B, C). LINC00473-tv1.1 had a different 5’ end from the annotated tv1, while
tv2.1 and tv2.2 have different 5’ and 3’ ends from the annotated tv2 (Figure 4-3B, C).
Coding potential analysis strongly suggested that LINC00473 is a noncoding RNA
(Figure 4-3D, E). Both tv1 and tv2 transcript variants showed significant activation in
LKB1-null NSCLC cells. LINC00473 tv1 was the top differentially expressed lncRNA
(>10,000 fold-change) comparing the 2 LKB1-null (A549 and H460) and 2 LKB1-
expressing cells (H322 and H3123), while LINC00473 tv2 showed about 40 fold-
change.
91
Elevated LINC00473 Expression Is Tightly Correlated With NSCLC LKB1 Inactivation Status
To validate LKB1-regulated lncRNAs, I utilized a Nanostring-based assay that
allows direct digital detection of multiple RNA molecules of interest using target-specific,
color-coded probe pairs (102). This platform enabled us to simultaneously evaluate
multiple LKB1-regulated mRNA and lncRNA candidates (especially LINC00473).
Hybridizations were performed on RNA samples from a pair of control and LKB1-
expressing A549 cells and a panel of NSCLC cell lines (7 LKB1-wt and 7 LKB1-mut)
using a customized codeset. The codeset included several LKB1-regulated lncRNA
candidates, known LKB1-regulated protein-coding genes (103, 104), as well as three
housekeeping genes (GAPDH, GUSB and TUBB). As shown in the heatmap (Figure 4-
1E, right panel), LKB1 expression in LKB1-null A549 cells caused significant down-
regulation of known protein-coding genes (AVPI1, CTH, CPS1, DUSP4, FGA, NR4A2,
PDE4B, PDE4D, PTGS2, PTP4A1, SIK1, SLC7A2, SNAI1, TESC and TFF1), as well as
three lncRNAs (LINC00473, AL109792, and BX64110). However, when examined
across various cell lines with the confirmed status of cellular LKB1 protein expression
(Figure 4-4A), LINC00473 expression showed the best correlation with LKB1-
inactivated status (Figure 4-1E, left and middle panels). Other LKB1-regulated genes,
including lncRNAs AL109792 and BX64110, were only partially correlated with the
LKB1 status, suggesting cell context-dependent gene regulation.
Both LINC00473 tv1 and tv2 isoforms showed low or absent expression in 7
LKB1-wt NSCLC cell lines but significantly enhanced expression in 7 LKB1-mut cell
lines (Figure 4-1F, G). LINC00473 differential expression in LKB1-wt and LKB1-mut cell
lines was further corroborated by qRT-PCR data (Figure 4-4B). Enhanced expression of
92
a known LKB1 target gene SIK1 was also confirmed, but was not consistent across all
cell lines tested (Figure 4-4C). Moreover, SIK1 has relatively high basal expression.
Additionally, I surveyed LINC00473 expression in 130 NSCLC cell lines using Affymetrix
microarray data from the Cancer Cell Line Encyclopedia (CCLE) (105). These arrays
only contained the probes for LINC00473 tv1. I found that a subset of NSCLC cell lines
showed an outlier LINC00473 tv1 expression (Figure 4-1H). Analysis of cell lines with
annotated LKB1 mutation status revealed that LINC00473 expression was significantly
enhanced in LKB1-mutant NSCLC cell lines (n=32) in comparison to LKB1-wt lines
(n=59) (Figure 4-1I). The enhancement was more significant as compared to SIK1
expression (Figure 4-4D). Furthermore, two LKB1-wt cell lines with high LINC00473
expression, H292 and DV90, were predicted to have LKB1 loss based on a 16-gene
signature score (103). H292 is a lung mucoepidermoid carcinoma cell line that contains
a t(11;19) translocation that lead to the generation of the CRTC1-MAML2 fusion (106)
and subsequent constitutive activation of CREB-mediated transcription, thus mimicking
LKB1 loss. Collectively, these data strongly support that LINC00473 is consistently the
most elevated gene in LKB1-inactivated NSCLC cell lines regardless of other co-
existing gene mutations.
Direct RNA Detection In FFPE Specimens Revealed That LINC00473 Expression Is Elevated In A Subset Of NSCLC That Tend To Have Mutations In The LKB1 Gene Coding Regions
To investigate whether LINC00473 could serve as a potential biomarker for LKB1
status in human NSCLC cancers, I evaluated expression of LINC00473 and several
other LKB1 targets in formalin-fixed paraffin-embedded (FFPE) human lung
adenocarcinoma (LUAD) specimens. Target amplification-based strategies such as RT-
PCR and microarray analyses for measuring RNA levels from fixed tissue is
93
complicated by the fact that the RNA is highly cross-linked and significantly fragmented.
Nanostring-based assays are optimal for gene expression quantification using FFPE
tumor-derived RNA samples since the bar-coded fluorescent probes recognize small
target regions (~100bp) allowing direct single-molecule counting without need for target
amplification. Therefore, I performed Nanostring assays for FFPE tumor-derived RNAs
from 5 LKB1-wt and 5 LKB1-mut human LUAD specimens. The LKB1 gene mutation
status was analyzed by exon sequencing as previously described (80) and showed in
Table S7. LINC00473 expression was consistently found to best correlate with the
tumor LKB1 status among all the genes tested (Figure 4-5A). Expression of LINC00473
tv1, but not tv2, was significantly enhanced in LUAD with LKB1 mutations (Figure 4-5B).
These data suggest that expression of LINC00473 tv1 alone can predict tumor LKB1
status.
To examine whether LINC00473 expression could be directly visualized at
cellular levels in human FFPE tumors, I next performed RNA in situ hybridizations (RNA
ISH) for detection of LINC00473 transcripts using customized LINC00473 probes
(RNAscope). The specificity of LINC00473 probes was first validated by positive
LINC00473 signals in LKB1-null A549 xenograft tumors and negative signals in LKB1-
wt H522 xenograft tumors (Figures 4-6A, B). LINC00473 transcripts were detected as
easily distinguishable nuclear "dots" in A549 xenograft tumors (Figures 4-6A, C). I then
performed RNA ISH on FFPE human LUAD tissue array. This array also included FFPE
cell pellets from LKB1-null A549 and LKB1-positive H322 as controls, which showed
respective positive and negative LINC00473 signals (Figures 4-6D, E). I analyzed only
those tumors that were positive for a housekeeping gene PPIB (peptidylprolyl
94
isomerase), indicative of tumors with good RNA quality. Representative positive and
negative LINC00473 staining results were shown (Figures 4-5C, D). I found that all
normal human lung tissues (n=38) were negative for LINC00473 staining while
exhibiting positive staining for PPIB staining (Figures 4-5E and 4-6F), indicating absent
or low basal LINC00473 expression in normal lung tissues. A total of 9 out of 89 NSCLC
specimens (10.11%) with annotated LKB1 wt and 12 out of 22 lung tumors (54.54%)
with annotated LKB1 mutations were positive for LINC00473 staining (Figures 4-5E and
4-6G-J). LINC00473 expression showed significant positive correlation with LKB1
mutations based on Fisher Exact Test (p=1.93E-05). It is likely that those tumors
carrying the LKB1 wt gene yet showing elevated LINC00473 expression have LKB1
functional inactivation due to other mechanisms besides LKB1 mutations such as
epigenetic silencing, or post-translational modifications, or alteration in LKB1 signaling
components (81-83). On the other hand, it is worth noting that not all mutations found in
LKB1 are inactivating. Tumors carrying the LKB1 gene mutations yet not showing
LINC00473 induction could have intact LKB1 function if the mutations do not impair
LKB1 function. For example, one of the tumors with LKB1 F354L mutation, which was
predicted not to have damaging effects on LKB1 function based on PolyPhen-2
prediction (107), showed undetectable LINC00473 expression. In addition, negative
LINC00473 staining was found in several other cancer types and tissues including
prostate cancer, IDC breast, large cell Lymphoma, hepatocellular carcinoma, colon
adenocarcinoma and osteosarcoma as well as placenta, tonsil, and normal spleen (n=1;
data not shown). These data strongly indicate that LINC00473 is low or undetectable in
normal lung tissues but exhibits elevated expression in the subset of lung NSCLC
95
specifically with functional LKB1 inactivation, indicating that LINC00473 is a potential
robust surrogate biomarker for LKB1 functional status in lung cancer.
Elevated LINC00473 Expression Is Associated With Tumor LKB1 Mutations And Correlated With Poor Prognosis In TCGA Lung Adenocarcinomas
To investigate any potential association between LINC00473 expression, LKB1
mutation status, and clinical data of lung cancers, I examined a lung adenocarcinoma
(LUAD) RNAseq dataset from The Cancer Genome Atlas (TCGA) (101). I observed a
subset of lung cancers with outlier LINC00473 expression (90 percentile rank) and a
significant difference in LINC00473 expression between tumors (either paired n=57 or
unpaired n=454) and normal tissues (n=57) (Figure 4-5F). LUAD samples with high
LINC00473 expression were enriched with LKB1 gene-level non-silent mutations and
somatic mutation SNPs and small INDEL within the LKB1 gene coding regions (Figures
S5). LINC00473 expression was not associated with KRAS and TP53 gene mutations
(Figures 4-7). Moreover, the difference in LINC00473 expression between LKB1 wt and
LKB1 mutant populations was more significant compared to SIK1 or LKB1 expression
(Figure 4-8). LINC00473 expression was positively correlated with LKB1-loss gene
signature (103) and inversely correlated with LKB1 expression, and such correlations
were more significant in comparison to SIK expression and LKB1 expression (Figures 4-
9). These data support a strong association of LINC00473 expression with the LKB1
inactivation in LUAD samples.
Kaplan-Meier survival analysis showed highly significant difference in overall
survival between high expression (n=48) and low expression (n=421) groups (p<0.001,
Figure 4-5F). The elevated LINC00473 expression significantly correlated with a shorter
survival time in LUAD patients (< 50 months). When analyzing those tumors with the
96
available data on LINC00473 expression, LKB1 mutations, and clinical information, I
observed that LKB1 mutation status was not significantly associated with survival, but
high LINC00473 expression was associated with a poor survival within both LKB1-wt
and mutant groups (Figure 4-10). These data indicate that LINC00473 has a prognostic
value and may play an important role in cancer progression.
LINC00473 Expression Is Promoted By LKB1-Loss-Induced CRTC/CREB Activation
High LINC00473 expression has a positive correlation with LKB1 functional
inactivation in NSCLC cell lines and human primary tumors, suggesting that LKB1
inactivation leads to increased expression of LINC00473. To further examine LKB1
regulation of LINC00473 expression, I first tested whether cellular LKB1 levels directly
impacted LINC00473 expression. I observed that introduction of exogenous LKB1 in
LKB1-null cancer cell lines (H157, A549) resulted in a significant decrease in
LINC00473 transcript level (Figure 4-11A), whereas shRNA-mediated depletion of
endogenous LKB1 expression in LKB1-positive cells (H3123, H322) led to an increase
in LINC00473 level (Figure 4-11B). Therefore, modulating cellular LKB1 protein
expression affects LINC00473 expression in NSCLC cells.
LKB1 regulates multiple downstream AMPK family members, influencing multiple
signaling pathways (66). Our lab previously showed that loss of LKB1 expression
resulted in dephosphorylation and nuclear entry of CRTC transcriptional co-activators
and subsequent CREB-mediated transcriptional activation in both lung and esophageal
cancer cells (54, 108). LINC00473 was transiently up-regulated in response to cAMP
signaling in human ocular ciliary smooth muscle cells (109). The LINC00473 gene
contains 2 CRE (cAMP-responsive element) half sites within the proximal promoter
97
region. To test whether the loss of LKB1 induces CRTC-CREB activation and promotes
LINC00473 expression, I first depleted CREB using lentiviral-mediated shRNAs or
expressed a dominant negative form of CRTC (dnCRTC) that interferes with CRTC-
CREB interaction. I detected a reduction in LINC00473 transcript level in CREB-
depleted or dnCRTC-expressing A549 cells (Figures 4-11C, 4-12). LKB1 over-
expression further blocked LINC00473 expression (Figure 4-12). These data
demonstrate that LINC00473 is regulated by LKB1 loss and CRTC/CREB activation.
Next, I cloned the proximal LINC00473 promoter sequence (−523 to +88)
encompassing 2 CRE sites into the upstream region of a luciferase reporter (pGL3
basic) (Figure 4-11D) and then determined LINC00473 promoter activity by modulating
LKB1–CRTC-CREB signaling. I observed that the LINC00473 promoter reporter was
significantly repressed by over-expression of LKB1, but not LKB1 kinase-dead mutant
K78I when transfected in LKB1-deficient A549 cells (Figure 4-11E). Moreover, promoter
activity was significantly inhibited by expression of A-CREB (Figure 4-11F), a dominant-
negative mutant that specifically blocked CREB binding to DNA (110, 111). Also, the
LINC00473 promoter reporter was activated by over-expression of CRTC1 and to a
larger extent, by constitutively activated form of CRTC1 (S151A) (Figure 4-11G). Finally,
chromatin immunoprecipitation (ChIP) assays demonstrated that CRTC1 and CREB
were enriched in the LINC00473 promoter region spanning the CRE sites (Figure 4-
11H). Overall, these data suggest that LINC00473 transcription is directly induced by
CRTC-CREB activation in LKB1-inactivated NSCLC cells.
98
In Vitro And In Vivo Approaches Revealed Critical Functions Of LINC00473 In The Growth Of LKB1-Null Lung Cancer Cells.
High LINC00473 expression correlated with poor survival of lung cancer patients
(Figure 4-5G), indicating a role of LINC00473 in cancer progression. Therefore, I
investigated functional significance of sustained LINC00473 expression in LKB1-
inactivated NSCLC cells. I first determined the functional impact of LINC00473 depletion
on cell growth and survival using two independent lentiviral pLKO.1-based shRNAs
targeting exon 2 of LINC00473 tv1 (shLINC00473-2 and -4). I found that two
shLINC00473 caused approximately 90% reduction in LINC00473 transcript levels in
A549 cells at 96 hours after lentiviral infection (Figure 4-13A). The shLINC00473–
expressing as well as scrambled shRNA control (shCtl)-expressing cells were
subsequently assayed for cell growth and survival. LINC00473 knockdown reduced cell
proliferation and enhanced apoptosis in LKB1-null A549 cells (Figure 4-13B and C).
Similar effects of LINC00473 depletion on LKB1-null NSCLC cell line H157 were also
observed (Figure 4-14). Conversely, exogenous LINC00473 over-expression via
retroviral transduction in LKB1-wt H522 lung cancer cells resulted in a moderate, yet
significant increase in cell proliferation (Figure 4-15). These data demonstrate that
LINC00473 is essential for maintaining LKB1-inactivated lung cancer cell growth and
survival.
I next determined the effect of LINC00473 depletion on the growth of NSCLC
xenografts. The luciferase-expressing A549 (A549-luc) cells were transduced with
lentiviral-based shLINC00473 or scrambled shRNA control for 72 hours, and then equal
numbers of LINC00473-depleted and control cells were implanted to NOD.SCID mice
by subcutaneous injection. I observed that LINC00473 depletion significantly reduced
99
the tumor size and weight, and blocked the growth of A549-luc xenografts over time
(Figure 4-13D-F). IHC analysis showed that LINC473-depleted xenograft tumors
contained a reduced number of cells that were positive for the cell proliferation marker
Ki-67 (Figure 4-13G). Similarly, deletion of LINC0473 expression decreased the growth
of H157 xenografts in NOD.SCID mice (Figure 4-14). Therefore, both in vitro and in vivo
evidence supports critical functions of LINC00473 in regulating lung cancer growth and
survival.
LINC00473 Is A Nuclear LncRNA And Functions As A Regulator Of Gene Expression In Part Through Interacting With NONO And Modulating CRTC/CREB Transcription.
The molecular mechanisms underlying LINC00473 functions are unknown.
Studies have indicated that lncRNAs may be involved in various processes, including
transcription, splicing, post-transcriptional regulation, organization of protein complexes,
cell-cell signaling, and allosteric regulation of proteins (112). Knowledge of subcellular
localization for lncRNAs can provide a clue for lncRNA functions. I observed nuclear
localization of LINC00473 transcripts in FFPE human lung cancer specimens using
customized LINC00473 probes in RNAscope RNA-ISH assays (Figure 4-5C). To
validate LINC00473 as a nuclear lncRNA, we performed subcellular fractionation assay
and RNA fluorescence in situ hybridization (RNA-FISH). For fractionation assay, we
prepared cytoplasmic and nuclear fractions and determined LINC00473 transcript levels
in both fractions. By comparing with the respective cytoplasmic (tRNA) and nuclear (U6)
controls, we confirmed that LINC00473 is enriched in the nuclear fraction (Figures 4-
16A and B). For RNA-FISH, I hybridized fixed cells with a mixture of 27 oligonucleotide
probes (20-mer) targeting LINC00473, with each probe linked with a single Quasar 570
fluorophore. I observed positive nuclear signals with 1-2 distinctive dot-like structures as
100
well as less intense, diffuse signals outside the dots (Figure 4-16C). LINC00473 signals
were undetectable when hybridizations were performed in the presence of RNase
(Figure 4-17A), indicating that the signal detection was RNA-dependent. Moreover,
exogenous LINC00473 showed similar nuclear localization when over-expressed in
LKB1-wt H522 cells (Figure 4-17C). These data strongly support that LINC00473 has
distinct nuclear localization and likely participates in nuclear functions.
To investigate LINC00473-interacting proteins, we performed a RNA pull-down
assay followed by a proteomic analysis of the LINC00473-associated protein complex in
A549 cells. We incubated the in vitro transcribed LINC00473 bound to beads with A549
nuclear extracts to purify RNA-protein complex. The LINC00473-associated protein
complex components were separated by SDS-PAGE (Figure 4-18A) and the protein
identity was revealed by mass spectrometry (MS). A notable protein was a known
CRTC interacting protein, NONO (93), with 115 peptides detected in this MS analysis
(Table 4-8). To validate the physical interaction between LINC00473 and NONO, we
performed RNA pull-down followed by Western blotting with NONO antibodies. NONO
was readily detected in LINC00473 RNA pull-down complex but not in the control
samples including LINC00473 antisense RNA (AS), lncRNA MEG3 and beads only
(Figure 4-18B). We also performed a RNA immune-precipitation (RNA-IP) for the RNA-
NONO complex using NONO antibodies and measured the amount of LINC00473
associated with NONO immune-precipitates. The immune-precipitated NONO protein
levels were confirmed by Western blotting (Figure 4-18C). The qRT-PCR results
showed significant enrichment of LINC00473, but not the negative control ASNS in
101
NONO immune-precipitates (Figure 4-18D). These data indicate that NONO is an
Lnc473-associated protein.
NONO interacts with CRTC co-activators upon cAMP stimulation, which is
essential for CREB-mediated transcription (93). Since LINC00473 physically interacted
with NONO, I hypothesize that LINC00473 regulates NONO recruitment to CRTCs and
promotes subsequent activation of CREB target gene transcription. To test this
hypothesis, I determined the interaction of NONO and CRTC when LINC00473 was
either depleted or over-expressed using mammalian two-hybrid assays. I observed that
depletion of LINC00473 caused a reduced interaction of gal4-NONO and CRTC1, which
was indicated by reduced activation of a Gal4 promoter reporter (Figure 4-18E).
Conversely, enhanced LINC00473 expression promoted NONO-CRTC1 interaction
(Figure 4-18F). These data suggest that LINC00473 facilitates the recruitment of NONO
to CRTC and subsequently promotes CREB-mediated transcription. To further examine
whether LINC00473 regulates endogenous CRTC/CREB target genes, I performed
gene expression analysis in scrambled shRNA control and LINC00473-depleted A549
cells as well as in vector control and LKB1-expressing A549 cells in Nanostring assays.
LINC00473 depletion impaired expression of several known and potential CRTC/CREB
targets including CTH, CPS1, DUSP4, FGA, NR4A2, PDE4B, PDE4D, PTGS2,
PTP4A1, SIK1, SLC7A2, TESC and TFF1, whose expression were induced by LKB1
loss (Figure 4-18G). Moreover, NEDD9, a CRTC-CREB target gene implicated in
regulating cell proliferation and metastasis (113) was down-regulated in LINC00473-
depeleted A549 cells, although its expression was not significantly different between
LKB1-mut and –wt cell lines (Figure 4-19). Furthermore, quantitative RT-PCR analysis
102
showed that expression of exogenous LINC00473 in LKB1-wt H522 significantly
promoted transcription of CREB target genes such as CPS1, PDE4B, PTGS2 and
PDE4D (Figure 4-17C). All these data support a model where LINC00473 acts as a co-
activator with CRTC/CREB in a positive feedback mechanism to maintain high steady-
state levels and induce expression of other LKB1-regulated targets (Figure 4-20).
Discussion
Lung cancer is the leading cause of cancer death. Progress in lung cancer
treatment improvements will require improved predictive biomarkers so that cancer
patients can be provided with the most effective treatments available as well as a larger
repertoire of therapeutic targets. In this study, I discovered a novel lncRNA (LINC00473)
whose elevated expression was highly associated with loss of the tumor suppressor
LKB1 gene function, one of the most common mutational events in lung cancer. Our
gene expression and functional data strongly support the potential utility of LINC00473
as a biomarker and as a therapeutic target for lung cancers with impaired LKB1
signaling. Moreover, I provide mechanistic insights into LINC00473 as a critical nuclear
regulator of gene expression in lung cancer.
Recent studies reveals that specific lncRNA expression is associated with
disease state (85, 114), strongly supporting the utility of lncRNAs in clinical diagnosis
and prognosis. Loss of LKB1 function in NSCLC cells caused differential responses to
therapeutic agents in vitro and in animal studies. LKB1 inactivation sensitized NSCLC
cells to the metabolism drug phenformin and a COX2 inhibitor while conferred
resistance to PI3K/Akt and MEK inhibitors (75-78). However, no specific effective
treatments are currently available for patients with LKB1-deficient lung cancer. One big
barrier is the lack of reliable assessment for tumor LKB1 inactivation that can be used in
103
clinical trials for patient selection and treatment evaluation. Current clinical LKB1
analysis includes evaluation of LKB1 mutations through sequencing 9 coding exons and
flanking region of the LKB1 gene as well as immunohistochemistry (IHC) assay of LKB1
protein expression. However, LKB1 functional inactivation could result from mutations
across the entire LKB1 gene, epigenetic silencing, or post-translational inactivation (81,
83, 115), posing a great challenge to detect LKB1 functional loss through direct
genomic sequencing. Also, not all LKB1 mutations impair LKB1 functions. LKB1 IHC
assay was shown for specific detection of LKB1 protein loss (116); however, those
LKB1 antibodies used were not highly specific. Recently, a 16-gene signature was
reported to be capable of predicting tumor cells with LKB1 inactivation (103), yet
expression of those individual genes is not completely correlated with the tumor LKB1
status and combined scoring of multiple genes for individual tumors require complicated
analysis.
In this study, I observed that LINC00473 is consistently the most highly induced
gene in LKB1-inactivated primary NSCLC samples and derived cell lines, supporting
that LINC00473 expression could be used for predicting LKB1 functional status in
clinically relevant FFPE tumor specimens. There are advantages of using LINC00473
as a surrogate marker for tumor LKB1 functional inactivation. First, LINC00473
expression is a functional readout for LKB1 inactivation; thus LINC00473-based
detection will be advantageous over direct sequencing of the entire LKB1 gene or IHC
analysis of LKB1 protein detection. I observed a subset of human lung tumors without
detectable mutations in the coding region of the LKB1 gene showed positive staining for
LINC00473. These tumors likely reflect scenarios where functional LKB1 inactivation is
104
caused by epigenetic affects such as promoter hypermethylation or functional
suppression by other mechanisms, including alterations in other components of the
LKB1 pathway. For example, BRAF activating mutations were shown to suppress LKB1
kinase activity and thus inactivated LKB1 signaling (81). On the other hand, a subset of
tumors with LKB1 mutations had non-detectable LINC00473 expression. One possibility
is that these tumors have LKB1 mutations yet such mutations have no significant impact
on LKB1 functions. Also, tumor cells having the mutation(s) on one allele of the LKB1
gene may express sufficient level of functional LKB1 proteins. Second, a significant
number of LKB1-wt stromal cells within tumors can obscure detection of LKB1-null
tumor cells in LKB1 IHC studies. Since LINC00473 normally expresses at a low or
undetectable level but at a significantly high level in LKB1-inactivated cells, detection of
up-regulated LINC00473 expression will not be affected by the presence of stromal
cells. Third, LINC00473 has sufficiently high expression in LKB1-inactivated lung cancer
and can be detected in biopsy specimens in the clinic. We present “proof of concept”
data showing that both Nanostring-based expression assay and RNAscope-based RNA
ISH can be used for LINC00473 detection in clinically relevant FFPE tumors. The
Nanostring-based assay is rapid and reproducible in clinical laboratories, and provides
quantitative expression scores. The RNAscope-based assays, while requiring subjective
scoring due to variable levels of staining signals, can allow colorimetric detection of
LINC00473-expressing tumor cells to infer LKB1 functional status. Such transcript-
based tests, capable of detecting pathway activity, are of special importance for tumor
suppressor gene detection, as histologically clinical diagnosis based on LKB1 IHC and
DNA mutation assays are more difficult as compared to activated oncogene detection.
105
Our data suggest that LINC00473 expression-based test could be developed for scoring
tumors with LKB1 functional loss, which can be integrated with clinical trial design for
patient selection and treatment decision. In the future, it will be important to identify the
specific mechanisms underlying LKB1 silencing in the subset of “LKB1-wt, LINC00473-
positive” tumors or intact functional LKB1 signaling in “LKB1-mutant, LINC00473-
negative” tumors. Due to the limited size and complexity of working with stored primary
tumors with heterogeneous stromal contamination, this effort will require extensive
follow-up studies using fresh samples at the genomic, epigenomic, and expressional
levels.
Patients with high LINC00473-expressing lung cancers had worse survival,
suggesting that LINC00473 likely confers lung cancer cells with aggressive behaviors.
Interestingly, a significant number of lncRNAs in human genome seem to have arisen
within the primate lineage based on sequence conservation (117) and LINC00473
belongs to this group. It is unclear whether LINC00473 may contribute to any unique
features associated with human cancers. The functional data strongly indicate an
essential role for LINC00473 in maintaining human NSCLC cell growth and survival.
LINC00473 has low or undetectable expression in normal tissues; therefore, targeting
LINC00473 expression is an attractive approach of specifically blocking lung cancer
without significantly affecting normal tissues. Currently, anticancer drugs mainly target
DNA or proteins in tumor cells. Therapeutic development for RNA-based targeting is in
the infancy but various new approaches are being explored such as antisense
oligonucleotides and RNA interference (RNAi). Progress has been made to improve
delivery and specificity. For example, first proof of principle RNAi therapeutics based on
106
RNAi and lipid nanoparticles (LNP) has been tested in humans (118), supporting further
development of RNA targeting drugs in treating cancers. Therefore, approaches for
targeting LINC00473 expression for blocking NSCLC should be further investigated.
I identified an upstream LKB1-CRTC-CREB signaling axis directly controlling
LINC00473 transcription. LINC00473 expression is strongly tied with the LKB1 signaling
in different cellular context, as I observed that up-regulation of LINC00473 is tightly
linked with the LKB1 functional status in spite of many other gene mutations co-existing
in cancer cells. Future detailed analysis of the LINC00473 promoter/enhancer is
necessary to gain insights into the tight, specific regulation by the LKB1 pathway.
LINC00473 is a nuclear lncRNA. The distinct nuclear pattern of LINC00473 is intriguing.
Mechanistically, LINC00473 promotes the recruitment of NONO to CRTCs and
enhances transcriptional activation of CREB-mediated transcription. Our data thus
identified one novel function for LINC00473 in assisting with the recruitment of critical
factors to assemble active CRTC-CREB transcription complexes and promote
transcription, elucidating a potential positive feedback mechanism for LINC00473 in
regulating gene expression. These data are consistent with the finding that LINC00473
depletion attenuated a set of genes that are induced by LKB1 loss, suggesting that
LINC00473 likely mediates certain effects of the loss of LKB1 tumor suppression. It
should be noted that LINC00473 appeared to have a negative feedback regulation in
normal cells after cAMP stimulation, as LINC00473 RNAi caused moderate
transcriptional upregulation of cAMP signaling-responsive genes in response to an EP2
agonist (109). The molecular action of LINC00473 in regulating cAMP/CREB signaling
should be further investigated and compared between normal cells with low if not
107
undetectable basal LINC00473 expression and cancer cells with sustained high
LINC00473 expression. Finally, LINC00473 most likely has other mechanisms that
contribute to its regulatory functions. Elucidation of the endogenous chromatin-
associated LINC00473 binding sites as well as downstream target genes will provide
mechanistic insights into the roles of this lncRNA in lung tumorigenesis.
In summary, I identified a novel nuclear lncRNA LINC00473 whose expression
has the potential utility in classifying the LKB1 functional status in human NSCLC and is
associated with poor patient survival. Sustained LINC00473 expression is essential for
maintaining NSCLC cell growth and survival, supporting that targeting LINC00473 is an
effective strategy to specifically block NSCLC growth. Finally, I identified a novel
function for Lnc473 in regulating gene expression. Collectively, this study reveals
mechanistic insights into the roles of Lnc473 in lung cancers and supports Lnc473 a
biomarker and a therapeutic target for LKB1-inactivated lung cancer.
108
Figure 4-1: LncRNA profiling revealed that LINC00473 is induced by LKB1 loss in NSCLC cells. (A) Western blotting analysis of expression of LKB1 wild-type (wt) or kinase-dead K78I mutant in transduced LKB1-null lung adenocarcinoma A549 cells. A549 cells infected with pBabe vector retroviruses were used as controls (Ctl). (B, C) Volcano plots show differentially expressed lncRNAs after expression of LKB1 wt (B) or LKB1 K78I mutant (C) in A549 cells by lncRNA microarray (V3.0) analysis. The cutoff criteria were fold-change of ≥2 and p<0.05. (D) A volcano plot shows differentially expressed lncRNAs in two LKB1-null cell lines (A549 and H460) as compared with two LKB1-wt cell lines (H322 and H3123). (E) A heatmap shows expression levels of several LKB1-regulated protein-coding and non-coding genes measured in Nanostring assays. (F and G) Two LINC00473 transcript variants (tv1: NR_026860 and tv2: NR_026861) show significantly elevated expression in LKB1-mutant groups as compared to LKB1-wt groups based on the Nanostring assays. (H) CCLE data analysis identified NSCLC cell lines (n=130) with outlier LINC00473 expression levels. (I) Enhanced LINC00473 expression was significantly associated with LKB1 mutations in CCLE NSCLC cell lines.
109
Figure 4-2: LKB1 wt protein, but not LKB1 K78I mutant, was capable of activating AMPK signaling. LKB1-null A549 cells transduced with pBabe empty vector (Ctl), LKB1-K78I, or LKB1-wt retroviruses were cultured in glucose-free culture medium for 2 hours. Cell lysates were harvested for immunoblotting to assess the levels of p-AMPK [Phospho-AMPKα (Thr172)] and p-ACC [Phospho-Acetyl-CoA Carboxylase (Ser79)] and their respective total proteins. Enhanced p-AMPK and p-ACC, indicative of AMPK activation, were only observed for A549 cells expressing LKB1-wt protein, but not control or K78I mutant under no glucose conditions. These data indicate that LKB1 K78I is a kinase-dead mutant.
110
Figure 4-3: RACE and coding potential analyses of LINC00473 transcripts. (A) Schematic representation of LINC00473 gene. (B) The fragments amplified in 5’ and 3’ RACE reactions from RNA samples from two LKB1-null A549 and HeLa cells were run on the agarose gels. (C) RACE assays identified 3 LINC00473 transcript variants, tv1.1, tv2.1 and tv2.2, but not two annotated transcripts. (D) Coding Potential Calculator (CPC) analysis suggested that LINC00473 is a non-coding RNA. (E) PhyloCSF predicted that LINC00473 has no protein coding potential.
111
Figure 4-4: Expression analyses of LINC00473 and SIK1 genes in LKB1-wt and LKB1-
mut lung NSCLC cell lines. (A) Western blotting analysis of LKB1 protein expression in a panel of human NSCLC cell lines. The mouse monoclonal anti-LKB1 antibody (CST-3050) was used. The β-Actin protein was used as a loading control. (B) The qRT-PCR assays showed significantly increased LINC00473 tv1 expression in 7 LKB1-null (mut) cell lines as compared with 7 LKB1-wildtype (wt) cell lines (p<0.001). (C) The qRT-PCR assays showed significantly increased SIK1 expression in 7 LKB1-null (mut) cell lines as compared with 7 LKB1-wildtype (wt) cell lines (p<0.01). However, two LKB1- null cell lines (H23 and H157) had relatively low expression of SIK1. (D) SIK1 expression was significantly enhanced in LKB1-mutant lung NSC cell lines (n=32) compared to LKB1-wt cells (n=59) based on CCLE data analysis.
112
Figure 4-5: Enhanced LINC00473 expression is highly correlated with human lung adenocarcinoma with LKB1 mutational status and associated with poor survival. (A) A heatmap shows gene expression levels in RNA samples isolated from 5 LKB1-wt and 5 LKB1-mut FFPE LUAD samples in Nanostring assays. (B) Expression of LINC00473 transcript variant (tv1), but not tv2, was significantly different between LKB1-wt and -mut groups. (C, D) Representative images for tumors with LINC00473 positive (C) and negative (D) signals based on RNAscope detection on FFPE LUAD sections. (E) Survey of human LUAD arrays indicated that 0% of normal lung tissues, 10.11% of NSCLC tumors with annotated wild-type LKB1, and 55.54% of NSCLC tumors with annotated LKB1 mutations were positive for LINC00473 expression. (F) TCGA-LUAD dataset showed outlier LINC00473 expression in matched tumor (T) (n=57) compared to adjacent normal tissues (N) (n=57) as well as unpaired tumor (UT) (n=454). (G) Kaplan-Meier survival analysis of high LINC00473 expression (n=48) and low LINC00473 expression (n=421) in lung cancer patients (p<0.001).
113
Figure 4-6: Detection of LINC00473 expression in FFPE specimens by RNA in situ hybridization using RNAscope® 2.0 High Definition (HD) assays. (A, B) Positive LINC00473 signals were detected in LKB1-null A549 xenograft (A), but not in LKB1-wt H522 xenograft (B) using RNAscope HD brown assays. (C) Positive LINC00473 signals were detected in LKB1-null A549 xenograft tumor using RNAscope HD red assay (Left panel). Positive signals for the housekeeping PPIB gene indicated tissues with good RNA quality (Right panel). (D, E) Positive LINC00473 signals were detected in LKB1-null A549 cell pellet, but not in LKB1-expressing H322 cell pellet. Both cell pellets showed positive signals for the housekeeping PPIB gene expression. (F) A representative case of normal human lung tissues (n=38) showing negative LINC00473 signals. (G, H) Representative cases of LKB1 wildtype (wt) lung adenocarcinomas (n=89) showed negative (n=80) (G) or positive LINC00473 signals (n=9) (H). (I, J) Representative cases of LKB1 mutated (mut) lung adenocarcinomas (n=22) showed negative (n=10) (I) or positive LINC00473 (n=12) signals (J). All cases (F-J) were positive for expression of the housekeeping gene PPIB.
114
Figure 4-7: Human lung adenocarcinomas with high LINC00473 expression were
enriched with mutations in the LKB1 gene coding region. (A) Expression patterns of LINC00473 and other genes in TCGA LUAD samples. Data was sorted by normalized expression value. Samples with high expression are colored red, samples with low expression are colored green, and samples with no expression data are colored grey. (B) LKB1 gene-level non-silent mutations in LUAD samples. Red means that a non-silent mutation was found in the gene. White means that no such mutation was found. Gray means that the sample has no data. (C) Somatic mutation SNPs and small INDELs in LKB1, KRAS and TP53 genes in LUAD samples. Each colored dot shows a mutation along the transcript with each line being its own sample. Red indicates that the mutation is likely to prevent a functional protein from being made (nonsense mutations, frame shift, etc). Blue indicates that the protein is likely to be made, but may have an altered function (missense mutation, etc). Green indicates that the protein is unlikely to be affected (such as synonymous).
115
Figure 4-8: Box plots show expression levels of LINC00473 (A), SIK1 (B), and LKB1 (C) genes in LKB1 mutant (Mut) and wildtype (Wt) human lung adenocarcinomas. The LUAD level 3 RNAseqv2 normalized data were downloaded from TCGA along with the Meta-Data for each patient. The RNA-seq gene expression data were combined with DNA-sequencing data to compare gene expression to LKB1 mutational status. There were 479 patients with DNA-sequencing information: 402 were WT and the other 77 were LKB1 mutant. The p-value was generated by a two tailed, independent t-test. The data indicate that LINC00473 expression is a more significantly variant between LKB1 WT and Mutant than SIK1 or LKB1.
116
Figure 4-9: Analysis of the correlation between LINC00473 Expression, the LKB1-Loss
Signature, LKB1 Expression, and SIK1 Expression. (A) Correlation of LINC00473 Gene Expression to LKB1-Loss Signature; (B) Correlation of LINC00473 Gene Expression to LKB1 Gene Expression; (C) Correlation of SIK1 Gene Expression to LKB1-Loss Signature; (D) Correlation of LKB1 Gene Expression to SIK1 expression; (E) Correlation of LKB1 Gene Expression to LKB1 Loss Signature. The data indicate that LINC00473 expression is more positively correlated with the LKB1-loss signature than SIK1 expression. The data also show that LKB1 expression is more significantly inversely correlated with LINC00473 expression than that of SIK1 expression and LKB1 expression is significantly inversely correlated with the LKB1 loss signature.
117
Figure 4-10: Kaplan-Meier survival analyses showed that high LINC00473 expression, but not LKB1 mutations in the coding regions, was associated with poor prognosis. (A) LKB1 mutation status was not significantly associated with survival. The tumors in TCGA-LUAD dataset with the available data on LKB1 mutations and clinical information were analyzed. (B, C) High LINC00473 expression was associated with a poor survival in both LKB1 wt and mutant groups. The tumors in TCGA-LUAD dataset with the available data on LINC00473 expression, LKB1 mutations, and clinical information were analyzed.
118
Figure 4-11: LINC00473 expression is regulated by LKB1-CRTC1-CREB signaling axis.
(A) The qRT-PCR analysis showed that LINC00473 expression was significantly reduced in two LKB1-null NSCLC cell lines (H157 and A549) after the transduction with LKB1 retroviruses (LKB1) for 96 hours, with cells transduced with vector retroviruses (Ctl) as controls. Western blotting confirmed LKB1 expression (n=3, **p<0.001). (B) LINC00473 expression was enhanced upon LKB1 shRNA lentiviral infection in two LKB1-wt NSCLC cell lines (H3123 and H322) (n=3, *p<0.05 and ***p<0.0001). (C) LINC00473 expression was significantly reduced in A549 cells after the transduction with 2 independent CREB shRNAs (n=3, *p<0.05). (D) A schematic representation of the LINC00473 promoter reporter was shown. (E) Expression of LKB1, but not the kinase-dead K78I mutant in LKB1-null A549 cells caused significant repression in LINC00473 promoter reporter activity (n=3, *p<0.05). (F) Expression of A-CREB in A549 cells significantly inhibited the LINC00473 promoter activity (n=3, *p<0.05). (G) Expression of wt or constitutively activated CRTC1 (S151A) increased the LINC00473 promoter activity in LKB1-expressing H322 cells (n=3, **p<0.001 and ***p<0.0001). (H) ChIP analysis indicated that CREB and CRTC1 were significantly enriched on the LINC00473 promoter encompassing the CRE half sites in A549 cells (n=3, *p<0.05 and ***p<0.0001).
119
Figure 4-12: Regulation of LINC00473 expression by the LKB1/CRTC/CREB signaling
axis. (A) CREB knockdown via two lentiviral-mediated shRNAs in A549 cells reduced LINC00473 expression, and LKB1 over-expression further blocked LINC00473 expression by qRT-PCR analysis. (n=3, *p<0.05, **p<0.001 and ***p<0.0001). (B) Western blotting confirmed CREB knockdown and LKB1 expression. (C) Retroviral-mediated expression of a dominant negative form of CRTC (dnCRTC-GFP: CRTC1 1-55aa fused to nls-GFP cloned in the retroviral pMSCV vector) in A549 cells reduced LINC00473 expression, and LKB1 over-expression further blocked LINC00473 expression. (n=3, *p<0.05 and ***p<0.0001) (D) Western blotting confirmed dnCRTC-GFP expression and LKB1 expression in A549 cells.
120
Figure 4-13: Depletion of LINC00473 expression in LKB1-null NSCLC cells causes
reduced cell growth and survival in vitro and in vivo. (A) Luciferase-expressing A549 cells were infected with two independent lentiviral-based LINC00473 shRNAs and the scrambled shRNA control (shCtl), respectively. Transduced cells were harvested 96 hours later, and LINC00473 expression was quantified by qRT–PCR (n=3, *p<0.05). (B, C) Transduced cells at 96 hours post-transduction were cultured at 2×105 per well in 6-well plates for another 96 hours and viable cell number was measured using Trypan blue assay (B) and apoptotic cells were detected by Annexin V/PI staining (C) (n=3, *p<0.05 and **p<0.001). (D, E, F) A total of 1×106 A549-luc cells after transduction with shLINC00473 or shCtl for 72 hours were injected subcutaneously to the dorsal flanks of NOD-SCID mice. Representative bioluminescent images of mice injected with shCtl or shLINC00473 A549-luc cells (D) and the weights of excised tumors (E) at the end points were shown. Tumor growth was shown at different days after tumor cell injection (F). (*p<0.05, **p<0.001 and ***p<0.0001). (G) Immunohistochemical staining of A549-control and A549-shLINC00473 xenograft tumor sections with Ki-67 antibody.
121
Figure 4-14: Knockdown of LINC00473 expression in LKB1-null human NSCLC H157
resulted in reduced cell growth and survival in vitro and in vivo. (A) LKB1-null human NSCLC H157 cells were infected with lentiviral-based LINC00473 shRNAs and the scrambled shRNA control (shCtl), respectively. Transduced cells were harvested 96 hours later, and LINC00473 expression was quantified by qRT–PCR (n=3, *p<0.05). (B, C) Transduced cells at 96-hours post-transduction were cultured at 5×105 per well in 6-well plates for another 96 hours and viable cell number was measured using Trypan blue assay (B) and apoptotic cells were detected by Annexin V/PI staining (C) (n=3, *p<0.05). (D, E, F) A total of 1×106 H157 cells after transduction with shLINC00473 or shRNA control for 72 hours were injected subcutaneously to the dorsal flanks of NOD-SCID mice (control shCtl n=5 and shLINC00473 n=6). The excised tumor (D) and the tumor weights (E) were shown at the end points were shown. Tumor growth was measured at different days after tumor cell injection (F) (**p<0.001).
122
Figure 4-15: Overexpression of LINC00473 in LKB1-wt lung human NSCLC cells increased cell proliferation and expression of several CREB target genes. (A) LKB1-wt human lung NSCLC cells (H522) were transduced with pLNCX empty vector or LINC00473 retroviruses and LINC00473 expression was confirmed by qRT-PCR (n=3, **p<0.001). (B) LINC00473 expression resulted in a moderate yet significant increase cell proliferation (n=3, *p<0.05). (C) The qRT-PCR analysis showed that LINC00473 expression enhanced expression of several CREB target genes. (n=3, *p<0.05 and **p<0.001).
123
Figure 4-16: LINC00473 shows predominantly nuclear localization with distinct nuclear structures. (A) The transcript levels of LINC00473 and U6 (a nuclear marker) in the nuclear (Nuc) and cytoplasmic (Cyt) fractions obtained from A549 cells were quantified by qRT-PCR assays. (B) LINC00473 transcripts were enriched in nuclear compartment when compared with nuclear marker U6 and cytoplasmic marker tRNA by Northern blotting analysis in three separate nuclear (N) and cytoplasmic (C) fractions obtained from A549 cells. (C) Nuclear localization of LINC00473 was detected by RNA-FISH in A549 cells. LINC00473 RNA-FISH probe sets were labeled with Quasar 570 dyes (red) and nuclei were labeled with the DNA dye DAPI (blue).
124
Figure 4-17: LINC00473 showed a distinct nuclear localization pattern. (A) The signal of LINC00473 was not detected by RNA-FISH in A549 cells after RNase treatment. A549 cells were treated with RNase A (50 µg/mL) at 37°C for 1 hour before prior to the hybridization step. The RNA-FISH probe set labeled with Quasar 570 dyes (red) targets the LINC00473 RNAs and the DNA dye DAPI (blue) stains the nuclei. (B) No signal was detected in LKB1–wt H522 cells transduced with pLNCX vector retroviruses for 96 hours. (C) The signal of LINC00473 was detected by RNA-FISH in H522 cells transduced with LINC00473 retroviruses for 96 hours.
125
Figure 4-18: LINC00473 is associated with NONO protein and stimulates CRTC-NONO
interaction. (A) Coomassie blue staining of the LINC00473-associated proteins by RNA pull-down in A549 cells. (B) Specific association of LINC00473 RNA with NONO protein was validated through RNA pull-down followed by Western blotting analysis. LINC00473 antisense and MEG RNA were used as controls. (C) Immunoprecipitation of endogenous NONO protein was validated via Western blotting (HC, heavy chain). (D) LINC00473 was significantly enriched in NONO immune-precipitates relative to the IgG control by qRT-PCR assay. ASNS was used as a negative control. (n=3, ***p<0.0001). (E) Depletion of LINC00473 caused reduced CRTC1-NONO interaction. A549 cells, after the transduction with LINC00473 shRNA (shLINC00473-2 and 4) or the scramble shRNA lentiviruses (Ctl) for 72 hours, were co-transfected with Gal4-NONO, pSG5-luc (a firefly luciferase reporter containing GAL4-binding sites), pEF-RL (Renilla luciferase) as well as vector control or CRTC1. The luciferase activity was measured 24 hours after transfection (n=3, *p<0.05 and **p<0.001). (F) Overexpression of LINC00473 enhanced CRTC1-NONO interaction. HEK293T cells were transfected with GAL4-NONO, CRTC1, pSG5-luc, pEF-RL in the presence of vector or LINC00473 construct. The luciferase activity was determined at 24 hours after transfection. (n=3, **p<0.001 and ***p<0.0001). (G) Relative expression levels of LKB1-regulated genes in shLINC00473- vs. shCtl- A549 cells and in LKB1 vs vector control (Ctl) A549 cells showed that LINC00473 depletion attenuated some common target gene expression induced by LKB1 loss.
126
Figure 4-19: NEDD9 is a LINC00473-regulated and CREB-regulated target gene, but its expression shows no significant difference between LKB1 wt and LKB1 mut NSCLC cell lines. (A) NEDD9 knockdown resulted in reduced NEDD9 expression in A549 cells. (B) CREB knockdown led to reduced NEDD9 expression in A549 cells. (C) qRT-PCR assays showed no significant difference (ns) in NEDD9 expression between LKB1-null (mut) and 7 LKB1-wildtype (wt) cell lines (see Figure S3 for cell lines). (ns: not significant). (D) There was no significant difference in NEDD9 expression levels between LKB1-mutant lung NSC cell lines (n=32) compared to LKB1-wt cells (n=59) based on CCLE data analysis.
127
Figure 4-20: A model for the molecular basis of LINC00473 induction and the role of
sustained LINC00473 expression as a potential biomarker and prognostic marker, therapeutic target, and gene regulator for LKB1-inactivated NSCLC.
128
Table 4-1: Primer and probe sequences used in this study.
Gene Forward primer Reverse primer
Amplicon
(bp)
Lnc473 5' AAACGCGAACGTGAGCCCCG 3' 5' CGCCATGCTCTGGCGCAGTT 3' 134
CREB 5' AGCAGCCACTCAGCCGGGTA 3'
5' ACGTCTCCAGAGGCAGCTTGAA
3' 111
ASNS 5' TGGCTGCCTTTTATCAGGGG 3' 5' TCTGCCACCTTTCTAGCAGC 3' 153
GAPDH 5' CAATGACCCCTTCATTGACC 3' 5' GACAAGCTTCCCGTTCTCAG 3' 106
CPS1 5'GGAAATGTAGTTGCTTTCTTAACCT 3' 5' TTGATGATTTGTGGCATGGGC 3' 73
PDE4B 5' CCGATCGCATTCAGGTCCTTCGC 3' 5' TGCGGTCTGTCCATTGCCGA 3' 96
PTGS2 5' GTTCCCACCCATGTCAAAAC 3' 5' CCGGTGTTGAGCAGTTTTCT 3' 108
PDE4D 5' CTCCTACGCGGTGGAGACC 3' 5' CATCAAAACGCCTGAGTCCC 3' 92
SLC7A2 5' CAGTTGCTGCCACGTTGAC 3' 5' GGCTGGTACCTGAGGATGAG 3' 148
NEDD9 5' GCTGCCGAAATGAAGTATAAGAATC 3' 5' CTTCCAGTCCCCCTGTGTTC 3' 133
Lnc473
promoter 5' AGCAGCCTTGCCAAAGGTC 3' 5' TTTCCCTTTAAGCCGGAGAT 3' 163
129
CHAPTER 5 CONCLUSION
In my PhD studies, I investigated the functions and mechanisms of CRTC1-
MAML2 fusion oncogene in human mucoepidermoid carcinoma and long noncoding
RNA LINC00473 in non-small cell lung cancer with LKB1 inactivation. These studies
centered on the aberrant CRTC-CREB signaling in human cancer.
I established a Cre regulated CRTC1-MAML2 transgenic mouse model and
induced the CRTC1-MAML2 fusion expression in the salivary gland to cause salivary
gland tumor development. Importantly, the salivary gland tumors showed typical human
MEC histological characteristics and exhibited a similar gene expression signature to
that of human MEC, suggesting that this mouse model resembles human MEC. This is
the first proof that CRTC1-MAML2 fusion protein plays a direct in vivo role in MEC
carcinogenesis. Our genetically engineered mouse model for human MEC will be a
promising preclinical model in evaluating therapeutic strategies.
Moreover, I studied the gene expression, functions and mechanisms of a novel
long noncoding RNA LINC00473 in human MEC. LINC00473 was identified as the
CRTC1-MAML2 fusion directly regulated target gene. LINC00473 expression was highly
correlated with CRTC1-MAML2 fusion expression in human MEC primary tumors and
MEC-derived cell lines. In addition, LINC00473 expression is essential for fusion-
positive MEC cell growth and survival in vitro and in vivo. These studies indicated that
LINC00473 may serve as a novel biomarker and potential therapeutic target for CRTC1-
MAML2 fusion-positive MEC.
I also studied the aberrant CRTC-CREB signaling in non-small cell lung cancer
especially focused on the gene expression, functions and mechanisms of long
130
noncoding RNA LINC00473. I identified LINC00473 is highly associated with LKB1
inactivation in human NSCLC primary samples and derived cell lines. Elevated
LINC00473 expression is correlated with poor survival in patients and is critical for the
growth and survival of LKB1-inactivated NSCLC cells in vitro and in vivo.
Mechanistically, LINC00473 is induced by LKB1 inactivation and then activates CRTC-
CREB signaling. Moreover, LINC00473 is a nuclear lncRNA and interacts with NONO
proteins to enhance CRTC-CREB-mediated transcription. These studies strongly
indicated that LINC00473 expression potentially serves as a robust biomarker for LKB1
functional status prediction in NSCLC and also serves as a novel therapeutic target for
LKB1-inactivated NSCLC.
In summary, my studies demonstrate the important roles of aberrantly activated
CRTC-CREB signaling in human MEC and NSCLC with LKB1 inactivation (Figure 5-1).
My studies help us better understanding of oncogenic mechanisms in cancer and
revealed novel therapeutic targets in human MEC and NSCLC.
131
Figure 5-1: A model for aberrant CRTC-CREB activation in cancer cell.
132
LIST OF REFERENCES
1. 2002. Salivary Gland Cancer Treatment (PDQ(R)): Health Professional Version. In PDQ Cancer Information Summaries. Bethesda (MD).
2. Carlson, J., Licitra, L., Locati, L., Raben, D., Persson, F., and Stenman, G. 2013. Salivary gland cancer: an update on present and emerging therapies. American Society of Clinical Oncology educational book / ASCO. American Society of Clinical Oncology. Meeting:257-263.
3. Gillespie, M.B., Albergotti, W.G., and Eisele, D.W. 2012. Recurrent salivary gland cancer. Current treatment options in oncology 13:58-70.
4. Bell, D., and Hanna, E.Y. 2012. Salivary gland cancers: biology and molecular targets for therapy. Current oncology reports 14:166-174.
5. Haddad, R., Colevas, A.D., Krane, J.F., Cooper, D., Glisson, B., Amrein, P.C., Weeks, L., Costello, R., and Posner, M. 2003. Herceptin in patients with advanced or metastatic salivary gland carcinomas. A phase II study. Oral oncology 39:724-727.
6. Locati, L.D., Bossi, P., Perrone, F., Potepan, P., Crippa, F., Mariani, L., Casieri, P., Orsenigo, M., Losa, M., Bergamini, C., et al. 2009. Cetuximab in recurrent and/or metastatic salivary gland carcinomas: A phase II study. Oral oncology 45:574-578.
7. Rugo, H.S., Herbst, R.S., Liu, G., Park, J.W., Kies, M.S., Steinfeldt, H.M., Pithavala, Y.K., Reich, S.D., Freddo, J.L., and Wilding, G. 2005. Phase I trial of the oral antiangiogenesis agent AG-013736 in patients with advanced solid tumors: pharmacokinetic and clinical results. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 23:5474-5483.
8. Guzzo, M., Andreola, S., Sirizzotti, G., and Cantu, G. 2002. Mucoepidermoid carcinoma of the salivary glands: clinicopathologic review of 108 patients treated at the National Cancer Institute of Milan. Annals of surgical oncology 9:688-695.
9. Devaraju, R., Gantala, R., Aitha, H., and Gotoor, S.G. 2014. Mucoepidermoid carcinoma. BMJ case reports 2014:bcr-2013-202776.
10. Turk, E., Karagulle, E., Erinanc, O.H., Soy, E.A., and Moray, G. 2013. Mucoepidermoid carcinoma of the breast. The breast journal 19:206-208.
11. Martellucci, S., Pagliuca, G., de Vincentiis, M., Rosato, C., Scaini, E., Gallipoli, C., and Gallo, A. 2013. Mucoepidermoid carcinoma of the tongue base mimicking an ectopic thyroid. Case reports in otolaryngology 2013:925630.
133
12. Nordkvist, A., Gustafsson, H., Juberg-Ode, M., and Stenman, G. 1994. Recurrent rearrangements of 11q14-22 in mucoepidermoid carcinoma. Cancer genetics and cytogenetics 74:77-83.
13. Horsman, D.E., Berean, K., and Durham, J.S. 1995. Translocation (11;19)(q21;p13.1) in mucoepidermoid carcinoma of salivary gland. Cancer genetics and cytogenetics 80:165-166.
14. Okabe, M., Miyabe, S., Nagatsuka, H., Terada, A., Hanai, N., Yokoi, M., Shimozato, K., Eimoto, T., Nakamura, S., Nagai, N., et al. 2006. MECT1-MAML2 fusion transcript defines a favorable subset of mucoepidermoid carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research 12:3902-3907.
15. Tonon, G., Modi, S., Wu, L., Kubo, A., Coxon, A.B., Komiya, T., O'Neil, K., Stover, K., El-Naggar, A., Griffin, J.D., et al. 2003. t(11;19)(q21;p13) translocation in mucoepidermoid carcinoma creates a novel fusion product that disrupts a Notch signaling pathway. Nature genetics 33:208-213.
16. Winnes, M., Enlund, F., Mark, J., and Stenman, G. 2006. The MECT1-MAML2 gene fusion and benign Warthin's tumor: is the MECT1-MAML2 gene fusion specific to mucuepidermoid carcinoma? The Journal of molecular diagnostics : JMD 8:394-395; author reply 395-396.
17. Coxon, A., Rozenblum, E., Park, Y.S., Joshi, N., Tsurutani, J., Dennis, P.A., Kirsch, I.R., and Kaye, F.J. 2005. Mect1-Maml2 fusion oncogene linked to the aberrant activation of cyclic AMP/CREB regulated genes. Cancer research 65:7137-7144.
18. Wu, L., Liu, J., Gao, P., Nakamura, M., Cao, Y., Shen, H., and Griffin, J.D. 2005. Transforming activity of MECT1-MAML2 fusion oncoprotein is mediated by constitutive CREB activation. The EMBO journal 24:2391-2402.
19. Chen, Z., Chen, J., Gu, Y., Hu, C., Li, J.L., Lin, S., Shen, H., Cao, C., Gao, R., Li, J., et al. 2014. Aberrantly activated AREG-EGFR signaling is required for the growth and survival of CRTC1-MAML2 fusion-positive mucoepidermoid carcinoma cells. Oncogene 33:3869-3877.
20. Nakayama, T., Miyabe, S., Okabe, M., Sakuma, H., Ijichi, K., Hasegawa, Y., Nagatsuka, H., Shimozato, K., and Inagaki, H. 2009. Clinicopathological significance of the CRTC3-MAML2 fusion transcript in mucoepidermoid carcinoma. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 22:1575-1581.
21. Chan, E.S., Tong, J.H., and To, K.F. 2013. CRTC3-MAML2 fusion transcript in a bronchial mucoepidermoid carcinoma. Pathology 45:698-701.
134
22. Prieto-Granada, C.N., Inagaki, H., and Mueller, J. 2015. Thymic Mucoepidermoid Carcinoma: Report of a Case With CTRC1/3-MALM2 Molecular Studies. International journal of surgical pathology 23:277-283.
23. Clark, M.B., Johnston, R.L., Inostroza-Ponta, M., Fox, A.H., Fortini, E., Moscato, P., Dinger, M.E., and Mattick, J.S. 2012. Genome-wide analysis of long noncoding RNA stability. Genome research 22:885-898.
24. Iyer, M.K., Niknafs, Y.S., Malik, R., Singhal, U., Sahu, A., Hosono, Y., Barrette, T.R., Prensner, J.R., Evans, J.R., Zhao, S., et al. 2015. The landscape of long noncoding RNAs in the human transcriptome. Nat Genet 47:199-208.
25. Ulitsky, I., and Bartel, D.P. 2013. lincRNAs: genomics, evolution, and mechanisms. Cell 154:26-46.
26. Djebali, S., Davis, C.A., Merkel, A., Dobin, A., Lassmann, T., Mortazavi, A., Tanzer, A., Lagarde, J., Lin, W., Schlesinger, F., et al. 2012. Landscape of transcription in human cells. Nature 489:101-108.
27. de Napoles, M., Mermoud, J.E., Wakao, R., Tang, Y.A., Endoh, M., Appanah, R., Nesterova, T.B., Silva, J., Otte, A.P., Vidal, M., et al. 2004. Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Developmental cell 7:663-676.
28. Wang, J., Liu, X., Wu, H., Ni, P., Gu, Z., Qiao, Y., Chen, N., Sun, F., and Fan, Q. 2010. CREB up-regulates long non-coding RNA, HULC expression through interaction with microRNA-372 in liver cancer. Nucleic acids research 38:5366-5383.
29. Willingham, A.T., Orth, A.P., Batalov, S., Peters, E.C., Wen, B.G., Aza-Blanc, P., Hogenesch, J.B., and Schultz, P.G. 2005. A strategy for probing the function of noncoding RNAs finds a repressor of NFAT. Science 309:1570-1573.
30. Feng, J., Bi, C., Clark, B.S., Mady, R., Shah, P., and Kohtz, J.D. 2006. The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator. Genes & development 20:1470-1484.
31. Zhang, A., Zhao, J.C., Kim, J., Fong, K.W., Yang, Y.A., Chakravarti, D., Mo, Y.Y., and Yu, J. 2015. LncRNA HOTAIR Enhances the Androgen-Receptor-Mediated Transcriptional Program and Drives Castration-Resistant Prostate Cancer. Cell reports 13:209-221.
32. Dimitrova, N., Zamudio, J.R., Jong, R.M., Soukup, D., Resnick, R., Sarma, K., Ward, A.J., Raj, A., Lee, J.T., Sharp, P.A., et al. 2014. LincRNA-p21 activates p21 in cis to promote Polycomb target gene expression and to enforce the G1/S checkpoint. Molecular cell 54:777-790.
135
33. Lee, S., Kopp, F., Chang, T.C., Sataluri, A., Chen, B., Sivakumar, S., Yu, H., Xie, Y., and Mendell, J.T. 2016. Noncoding RNA NORAD Regulates Genomic Stability by Sequestering PUMILIO Proteins. Cell 164:69-80.
34. Gutschner, T., Hammerle, M., Eissmann, M., Hsu, J., Kim, Y., Hung, G., Revenko, A., Arun, G., Stentrup, M., Gross, M., et al. 2013. The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer research 73:1180-1189.
35. Mouraviev, V., Lee, B., Patel, V., Albala, D., Johansen, T.E., Partin, A., Ross, A., and Perera, R.J. 2016. Clinical prospects of long noncoding RNAs as novel biomarkers and therapeutic targets in prostate cancer. Prostate cancer and prostatic diseases 19:14-20.
36. Wu, Y., Liu, J., Zheng, Y., You, L., Kuang, D., and Liu, T. 2014. Suppressed expression of long non-coding RNA HOTAIR inhibits proliferation and tumourigenicity of renal carcinoma cells. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 35:11887-11894.
37. Leucci, E., Vendramin, R., Spinazzi, M., Laurette, P., Fiers, M., Wouters, J., Radaelli, E., Eyckerman, S., Leonelli, C., Vanderheyden, K., et al. 2016. Melanoma addiction to the long non-coding RNA SAMMSON. Nature 531:518-522.
38. Ling, H., Fabbri, M., and Calin, G.A. 2013. MicroRNAs and other non-coding RNAs as targets for anticancer drug development. Nature reviews. Drug discovery 12:847-865.
39. Ghosh, S., Tibbit, C., and Liu, J.L. 2016. Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference. Nucleic acids research 44:e84.
40. Katano, H., Kok, M.R., Cotrim, A.P., Yamano, S., Schmidt, M., Afione, S., Baum, B.J., and Chiorini, J.A. 2006. Enhanced transduction of mouse salivary glands with AAV5-based vectors. Gene therapy 13:594-601.
41. Pringle, S., Nanduri, L.S., van der Zwaag, M., van Os, R., and Coppes, R.P. 2011. Isolation of mouse salivary gland stem cells. Journal of visualized experiments : JoVE.
42. Sinn, E., Muller, W., Pattengale, P., Tepler, I., Wallace, R., and Leder, P. 1987. Coexpression of MMTV/v-Ha-ras and MMTV/c-myc genes in transgenic mice: synergistic action of oncogenes in vivo. Cell 49:465-475.
43. Tanvetyanon, T., Ratanatharathorn, V., and Leopairat, J. 2004. Mucoepidermoid carcinoma of the lung presenting as a cavitary lesion. Journal of the Medical Association of Thailand = Chotmaihet thangphaet 87:988-991.
136
44. Khadilkar, U.N., Kumar, S., Prabhu, P.P., and Kamath, M. 2007. Mucoepidermoid carcinoma of lung: a case report. Indian journal of pathology & microbiology 50:560-562.
45. Serra, A., Schackert, H.K., Mohr, B., Weise, A., Liehr, T., and Fitze, G. 2007. t(11;19)(q21;p12~p13.11) and MECT1-MAML2 fusion transcript expression as a prognostic marker in infantile lung mucoepidermoid carcinoma. Journal of pediatric surgery 42:E23-29.
46. O'Neill, I.D. 2009. Gefitinib as targeted therapy for mucoepidermoid carcinoma of the lung: possible significance of CRTC1-MAML2 oncogene. Lung cancer 64:129-130.
47. Stewart, T.A., Pattengale, P.K., and Leder, P. 1984. Spontaneous mammary adenocarcinomas in transgenic mice that carry and express MTV/myc fusion genes. Cell 38:627-637.
48. Chen, J., Li, J.L., Chen, Z., Griffin, J.D., and Wu, L. 2015. Gene expression profiling analysis of CRTC1-MAML2 fusion oncogene-induced transcriptional program in human mucoepidermoid carcinoma cells. BMC cancer 15:803.
49. Schmitt, A.M., and Chang, H.Y. 2016. Long Noncoding RNAs in Cancer Pathways. Cancer cell 29:452-463.
50. Dickson, I. 2016. Hepatocellular carcinoma: A role for lncRNA in liver cancer. Nature reviews. Gastroenterology & hepatology 13:122-123.
51. Lin, A., Li, C., Xing, Z., Hu, Q., Liang, K., Han, L., Wang, C., Hawke, D.H., Wang, S., Zhang, Y., et al. 2016. The LINK-A lncRNA activates normoxic HIF1alpha signalling in triple-negative breast cancer. Nature cell biology 18:213-224.
52. Warner, K.A., Adams, A., Bernardi, L., Nor, C., Finkel, K.A., Zhang, Z., McLean, S.A., Helman, J., Wolf, G.T., Divi, V., et al. 2013. Characterization of tumorigenic cell lines from the recurrence and lymph node metastasis of a human salivary mucoepidermoid carcinoma. Oral oncology 49:1059-1066.
53. Wang, L.N., Wang, Y., Lu, Y., Yin, Z.F., Zhang, Y.H., Aslanidi, G.V., Srivastava, A., Ling, C.Q., and Ling, C. 2014. Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo. Journal of integrative medicine 12:20-34.
54. Gu, Y., Lin, S., Li, J.L., Nakagawa, H., Chen, Z., Jin, B., Tian, L., Ucar, D.A., Shen, H., Lu, J., et al. 2012. Altered LKB1/CREB-regulated transcription co-activator (CRTC) signaling axis promotes esophageal cancer cell migration and invasion. Oncogene 31:469-479.
137
55. Reitmair, A., Sachs, G., Im, W.B., and Wheeler, L. 2012. C6orf176: a novel possible regulator of cAMP-mediated gene expression. Physiological genomics 44:152-161.
56. Liang, X.H., Deng, W.B., Liu, Y.F., Liang, Y.X., Fan, Z.M., Gu, X.W., Liu, J.L., Sha, A.G., Diao, H.L., and Yang, Z.M. 2016. Non-coding RNA LINC00473 mediates decidualization of human endometrial stromal cells in response to cAMP signaling. Scientific reports 6:22744.
57. Geiss, G.K., Bumgarner, R.E., Birditt, B., Dahl, T., Dowidar, N., Dunaway, D.L., Fell, H.P., Ferree, S., George, R.D., Grogan, T., et al. 2008. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nature biotechnology 26:317-325.
58. Veldman-Jones, M.H., Brant, R., Rooney, C., Geh, C., Emery, H., Harbron, C.G., Wappett, M., Sharpe, A., Dymond, M., Barrett, J.C., et al. 2015. Evaluating Robustness and Sensitivity of the NanoString Technologies nCounter Platform to Enable Multiplexed Gene Expression Analysis of Clinical Samples. Cancer research 75:2587-2593.
59. Chitikova, Z., Pusztaszeri, M., Makhlouf, A.M., Berczy, M., Delucinge-Vivier, C., Triponez, F., Meyer, P., Philippe, J., and Dibner, C. 2015. Identification of new biomarkers for human papillary thyroid carcinoma employing NanoString analysis. Oncotarget 6:10978-10993.
60. Wang, H., Wang, M.X., Su, N., Wang, L.C., Wu, X., Bui, S., Nielsen, A., Vo, H.T., Nguyen, N., Luo, Y., et al. 2014. RNAscope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma. Journal of visualized experiments : JoVE.
61. Chen, Z., Li, J.L., Lin, S., Cao, C., Gimbrone, N.T., Yang, R., Fu, D.A., Carper, M.B., Haura, E.B., Schabath, M.B., et al. 2016. cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth. The Journal of clinical investigation 126:2267-2279.
62. Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., and Forman, D. 2011. Global cancer statistics. CA Cancer J Clin 61:69-90.
63. Janku, F., Stewart, D.J., and Kurzrock, R. 2010. Targeted therapy in non-small-cell lung cancer--is it becoming a reality? Nat Rev Clin Oncol 7:401-414.
64. Koivunen, J.P., Mermel, C., Zejnullahu, K., Murphy, C., Lifshits, E., Holmes, A.J., Choi, H.G., Kim, J., Chiang, D., Thomas, R., et al. 2008. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res 14:4275-4283.
65. Paez, J.G., Janne, P.A., Lee, J.C., Tracy, S., Greulich, H., Gabriel, S., Herman, P., Kaye, F.J., Lindeman, N., Boggon, T.J., et al. 2004. EGFR mutations in lung
138
cancer: correlation with clinical response to gefitinib therapy. Science 304:1497-1500.
66. Shackelford, D.B., and Shaw, R.J. 2009. The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat Rev Cancer 9:563-575.
67. Alessi, D.R., Sakamoto, K., and Bayascas, J.R. 2006. LKB1-dependent signaling pathways. Annu Rev Biochem 75:137-163.
68. Hemminki, A., Markie, D., Tomlinson, I., Avizienyte, E., Roth, S., Loukola, A., Bignell, G., Warren, W., Aminoff, M., Hoglund, P., et al. 1998. A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 391:184-187.
69. Giardiello, F.M., Welsh, S.B., Hamilton, S.R., Offerhaus, G.J., Gittelsohn, A.M., Booker, S.V., Krush, A.J., Yardley, J.H., and Luk, G.D. 1987. Increased risk of cancer in the Peutz-Jeghers syndrome. N Engl J Med 316:1511-1514.
70. Beroukhim, R., Mermel, C.H., Porter, D., Wei, G., Raychaudhuri, S., Donovan, J., Barretina, J., Boehm, J.S., Dobson, J., Urashima, M., et al. 2010. The landscape of somatic copy-number alteration across human cancers. Nature 463:899-905.
71. Ding, L., Getz, G., Wheeler, D.A., Mardis, E.R., McLellan, M.D., Cibulskis, K., Sougnez, C., Greulich, H., Muzny, D.M., Morgan, M.B., et al. 2008. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455:1069-1075.
72. Matsumoto, S., Iwakawa, R., Takahashi, K., Kohno, T., Nakanishi, Y., Matsuno, Y., Suzuki, K., Nakamoto, M., Shimizu, E., Minna, J.D., et al. 2007. Prevalence and specificity of LKB1 genetic alterations in lung cancers. Oncogene 26:5911-5918.
73. Sanchez-Cespedes, M., Parrella, P., Esteller, M., Nomoto, S., Trink, B., Engles, J.M., Westra, W.H., Herman, J.G., and Sidransky, D. 2002. Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung. Cancer Res 62:3659-3662.
74. Ji, H., Ramsey, M.R., Hayes, D.N., Fan, C., McNamara, K., Kozlowski, P., Torrice, C., Wu, M.C., Shimamura, T., Perera, S.A., et al. 2007. LKB1 modulates lung cancer differentiation and metastasis. Nature 448:807-810.
75. Cao, C., Gao, R., Zhang, M., Amelio, A.L., Fallahi, M., Chen, Z., Gu, Y., Hu, C., Welsh, E.A., Engel, B.E., et al. 2015. Role of LKB1-CRTC1 on glycosylated COX-2 and response to COX-2 inhibition in lung cancer. J Natl Cancer Inst 107:358.
76. Shackelford, D.B., Abt, E., Gerken, L., Vasquez, D.S., Seki, A., Leblanc, M., Wei, L., Fishbein, M.C., Czernin, J., Mischel, P.S., et al. 2013. LKB1 inactivation dictates therapeutic response of non-small cell lung cancer to the metabolism drug phenformin. Cancer Cell 23:143-158.
139
77. Chen, Z., Cheng, K., Walton, Z., Wang, Y., Ebi, H., Shimamura, T., Liu, Y., Tupper, T., Ouyang, J., Li, J., et al. 2012. A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response. Nature 483:613-617.
78. Carretero, J., Shimamura, T., Rikova, K., Jackson, A.L., Wilkerson, M.D., Borgman, C.L., Buttarazzi, M.S., Sanofsky, B.A., McNamara, K.L., Brandstetter, K.A., et al. 2010. Integrative genomic and proteomic analyses identify targets for Lkb1-deficient metastatic lung tumors. Cancer Cell 17:547-559.
79. Koyama, S., Akbay, E.A., Li, Y.Y., Aref, A.R., Skoulidis, F., Herter-Sprie, G.S., Buczkowski, K.A., Liu, Y., Awad, M.M., Denning, W.L., et al. 2016. STK11/LKB1 deficiency promotes neutrophil recruitment and proinflammatory cytokine production to suppress T cell activity in the lung tumor microenvironment. Cancer Res.
80. Schabath, M.B., Welsh, E.A., Fulp, W.J., Chen, L., Teer, J.K., Thompson, Z.J., Engel, B.E., Xie, M., Berglund, A.E., Creelan, B.C., et al. 2015. Differential association of STK11 and TP53 with KRAS mutation-associated gene expression, proliferation and immune surveillance in lung adenocarcinoma. Oncogene.
81. Zheng, B., Jeong, J.H., Asara, J.M., Yuan, Y.Y., Granter, S.R., Chin, L., and Cantley, L.C. 2009. Oncogenic B-RAF negatively regulates the tumor suppressor LKB1 to promote melanoma cell proliferation. Mol Cell 33:237-247.
82. Boudeau, J., Baas, A.F., Deak, M., Morrice, N.A., Kieloch, A., Schutkowski, M., Prescott, A.R., Clevers, H.C., and Alessi, D.R. 2003. MO25alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm. EMBO J 22:5102-5114.
83. Esteller, M., Avizienyte, E., Corn, P.G., Lothe, R.A., Baylin, S.B., Aaltonen, L.A., and Herman, J.G. 2000. Epigenetic inactivation of LKB1 in primary tumors associated with the Peutz-Jeghers syndrome. Oncogene 19:164-168.
84. Rinn, J.L., and Chang, H.Y. 2012. Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145-166.
85. Cabanski, C.R., White, N.M., Dang, H.X., Silva-Fisher, J.M., Rauck, C.E., Cicka, D., and Maher, C.A. 2015. Pan-cancer transcriptome analysis reveals long noncoding RNAs with conserved function. RNA Biol 12:628-642.
86. Du, Z., Fei, T., Verhaak, R.G., Su, Z., Zhang, Y., Brown, M., Chen, Y., and Liu, X.S. 2013. Integrative genomic analyses reveal clinically relevant long noncoding RNAs in human cancer. Nat Struct Mol Biol 20:908-913.
87. Gibb, E.A., Vucic, E.A., Enfield, K.S., Stewart, G.L., Lonergan, K.M., Kennett, J.Y., Becker-Santos, D.D., MacAulay, C.E., Lam, S., Brown, C.J., et al. 2011. Human cancer long non-coding RNA transcriptomes. PLoS One 6:e25915.
140
88. Hacisuleyman, E., Goff, L.A., Trapnell, C., Williams, A., Henao-Mejia, J., Sun, L., McClanahan, P., Hendrickson, D.G., Sauvageau, M., Kelley, D.R., et al. 2014. Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre. Nat Struct Mol Biol 21:198-206.
89. Gutschner, T., Hammerle, M., Eissmann, M., Hsu, J., Kim, Y., Hung, G., Revenko, A., Arun, G., Stentrup, M., Gross, M., et al. 2013. The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Res 73:1180-1189.
90. Gupta, R.A., Shah, N., Wang, K.C., Kim, J., Horlings, H.M., Wong, D.J., Tsai, M.C., Hung, T., Argani, P., Rinn, J.L., et al. 2010. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464:1071-1076.
91. Vaahtomeri, K., and Makela, T.P. 2011. Molecular mechanisms of tumor suppression by LKB1. FEBS Lett 585:944-951.
92. Shaw, R.J., Kosmatka, M., Bardeesy, N., Hurley, R.L., Witters, L.A., DePinho, R.A., and Cantley, L.C. 2004. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A 101:3329-3335.
93. Amelio, A.L., Miraglia, L.J., Conkright, J.J., Mercer, B.A., Batalov, S., Cavett, V., Orth, A.P., Busby, J., Hogenesch, J.B., and Conkright, M.D. 2007. A coactivator trap identifies NONO (p54nrb) as a component of the cAMP-signaling pathway. Proc Natl Acad Sci U S A 104:20314-20319.
94. Zhou, Y., Zhong, Y., Wang, Y., Zhang, X., Batista, D.L., Gejman, R., Ansell, P.J., Zhao, J., Weng, C., and Klibanski, A. 2007. Activation of p53 by MEG3 non-coding RNA. J Biol Chem 282:24731-24742.
95. Kong, L., Zhang, Y., Ye, Z.Q., Liu, X.Q., Zhao, S.Q., Wei, L., and Gao, G. 2007. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res 35:W345-349.
96. Lin, M.F., Jungreis, I., and Kellis, M. 2011. PhyloCSF: a comparative genomics method to distinguish protein coding and non-coding regions. Bioinformatics 27:i275-282.
97. Dignam, J.D., Lebovitz, R.M., and Roeder, R.G. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475-1489.
98. Aravin, A., Gaidatzis, D., Pfeffer, S., Lagos-Quintana, M., Landgraf, P., Iovino, N., Morris, P., Brownstein, M.J., Kuramochi-Miyagawa, S., Nakano, T., et al. 2006. A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442:203-207.
141
99. Caputi, M., Mayeda, A., Krainer, A.R., and Zahler, A.M. 1999. hnRNP A/B proteins are required for inhibition of HIV-1 pre-mRNA splicing. EMBO J 18:4060-4067.
100. Lin, S., Shen, H., Li, J.L., Tang, S., Gu, Y., Chen, Z., Hu, C., Rice, J.C., Lu, J., and Wu, L. 2013. Proteomic and functional analyses reveal the role of chromatin reader SFMBT1 in regulating epigenetic silencing and the myogenic gene program. J Biol Chem 288:6238-6247.
101. Cancer Genome Atlas Research, N. 2014. Comprehensive molecular profiling of lung adenocarcinoma. Nature 511:543-550.
102. Geiss, G.K., Bumgarner, R.E., Birditt, B., Dahl, T., Dowidar, N., Dunaway, D.L., Fell, H.P., Ferree, S., George, R.D., Grogan, T., et al. 2008. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 26:317-325.
103. Kaufman, J.M., Amann, J.M., Park, K., Arasada, R.R., Li, H., Shyr, Y., and Carbone, D.P. 2014. LKB1 Loss induces characteristic patterns of gene expression in human tumors associated with NRF2 activation and attenuation of PI3K-AKT. J Thorac Oncol 9:794-804.
104. Goodwin, J.M., Svensson, R.U., Lou, H.J., Winslow, M.M., Turk, B.E., and Shaw, R.J. 2014. An AMPK-independent signaling pathway downstream of the LKB1 tumor suppressor controls Snail1 and metastatic potential. Mol Cell 55:436-450.
105. Barretina, J., Caponigro, G., Stransky, N., Venkatesan, K., Margolin, A.A., Kim, S., Wilson, C.J., Lehar, J., Kryukov, G.V., Sonkin, D., et al. 2012. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483:603-607.
106. Tonon, G., Modi, S., Wu, L., Kubo, A., Coxon, A.B., Komiya, T., O'Neil, K., Stover, K., El-Naggar, A., Griffin, J.D., et al. 2003. t(11;19)(q21;p13) translocation in mucoepidermoid carcinoma creates a novel fusion product that disrupts a Notch signaling pathway. Nat Genet 33:208-213.
107. Adzhubei, I.A., Schmidt, S., Peshkin, L., Ramensky, V.E., Gerasimova, A., Bork, P., Kondrashov, A.S., and Sunyaev, S.R. 2010. A method and server for predicting damaging missense mutations. Nat Methods 7:248-249.
108. Komiya, T., Coxon, A., Park, Y., Chen, W.D., Zajac-Kaye, M., Meltzer, P., Karpova, T., and Kaye, F.J. 2009. Enhanced activity of the CREB co-activator Crtc1 in LKB1 null lung cancer. Oncogene.
109. Reitmair, A., Sachs, G., Im, W.B., and Wheeler, L. 2012. C6orf176: a novel possible regulator of cAMP-mediated gene expression. Physiol Genomics 44:152-161.
142
110. Ahn, S., Olive, M., Aggarwal, S., Krylov, D., Ginty, D.D., and Vinson, C. 1998. A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos. Mol Cell Biol 18:967-977.
111. Wu, L., Liu, J., Gao, P., Nakamura, M., Cao, Y., Shen, H., and Griffin, J.D. 2005. Transforming activity of MECT1-MAML2 fusion oncoprotein is mediated by constitutive CREB activation. Embo J 24:2391-2402.
112. Geisler, S., and Coller, J. 2013. RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts. Nat Rev Mol Cell Biol.
113. Feng, Y., Wang, Y., Wang, Z., Fang, Z., Li, F., Gao, Y., Liu, H., Xiao, T., Li, F., Zhou, Y., et al. 2012. The CRTC1-NEDD9 signaling axis mediates lung cancer progression caused by LKB1 loss. Cancer Res 72:6502-6511.
114. Wapinski, O., and Chang, H.Y. 2011. Long noncoding RNAs and human disease. Trends Cell Biol 21:354-361.
115. Sanchez-Cespedes, M. 2011. The role of LKB1 in lung cancer. Fam Cancer 10:447-453.
116. Calles, A., Sholl, L.M., Rodig, S.J., Pelton, A.K., Hornick, J.L., Butaney, M., Lydon, C., Dahlberg, S.E., Oxnard, G.R., Jackman, D.M., et al. 2015. Immunohistochemical loss of LKB1 is a biomarker for more aggressive biology in KRAS mutant lung adenocarcinoma. Clin Cancer Res.
117. Derrien, T., Johnson, R., Bussotti, G., Tanzer, A., Djebali, S., Tilgner, H., Guernec, G., Martin, D., Merkel, A., Knowles, D.G., et al. 2012. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res 22:1775-1789.
118. Tabernero, J., Shapiro, G.I., LoRusso, P.M., Cervantes, A., Schwartz, G.K., Weiss, G.J., Paz-Ares, L., Cho, D.C., Infante, J.R., Alsina, M., et al. 2013. First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discov 3:406-417.
143
BIOGRAPHICAL SKETCH
Zirong Chen obtained his Bachelor of Science degree in biology engineering
from the South China University of Technology in China (2002-2006). He worked as a
technician in Daan Gene Co. Ltd, a biotechnology company in China from 2006 to 2008.
He joined the masters’ program in Genetics at Sun Yat-sen University in China in 2008,
under the supervision of Dr. Huangxuan Shen. He completed his thesis and received
his M.S. degree in 2012. He transitioned by becoming a laboratory technician in Dr. Lizi
Wu’s lab at the University of Florida in 2009. He was accepted into the Interdisciplinary
Program (IDP) in Biomedical Sciences, at the University of Florida in 2012, under the
mentorship of Dr. Lizi Wu. He passed his qualifying exam and became an official Ph.D.
candidate in October 2014. He received his Ph.D. degree in August 2016.
