miRNA-491-5p and GIT1 serve as modulators and biomarkers ......Human oral cancer cell lines CGHNC9,...

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miRNA-491-5p and GIT1 serve as modulators and biomarkers for oral

squamous cell carcinoma (OSCC) invasion and metastasis

Wei-Chieh Huang1,3, Shih-Hsuan Chan1, Te-Hsuan Jang1, Jer-Wei Chang1, Ying-Chin

Ko4, Tzu-Chen Yen6, Shang-Lun Chiang4, Wei-Fan Chiang8, Tien-Yu Shieh9,

Chun-Ta Liao7, Jyh-Lyh Juang1, Hsueh-Chun Wang2, Ann-Joy Cheng5, Ya-Ching Lu5,

and Lu-Hai Wang1,3

Running title: miRNA-491-5p suppresses OSCC metastasis.

1. Institute of Molecular and Genomic Medicine, National Health Research

Institute, Miaoli, Taiwan.

2. Division of Environmental Health and Occupational Medicine, National Health

Research Institute, Miaoli, Taiwan.

3. Department of Life Sciences, National Central University, Jhongli, Taiwan.

4. Environment-Omics-Disease Research Center, China Medical University

Hospital, Taichung, Taiwan.

5. Departments of Medical Biotechnology, Chang Gung University, Taoyuan,

Taiwan.

6. Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial

on April 17, 2020. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 12, 2013; DOI: 10.1158/0008-5472.CAN-13-1297

http://cancerres.aacrjournals.org/

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Hospital, Taoyuan, Taiwan.

7. Head and Neck Oncology Group, Chang Gung Memorial Hospital, Taoyuan,

Taiwan.

8. Department of Oral and Maxillofacial Section, Chi-Mei Medical Center,

Liouying, Taiwan.

9. Department of Oral Hygiene, Kaohsiung Medical University, Kaohsiung,

Taiwan.

Keywords: miR-491-5p, GIT1, Oral cancer invasion, metastasis, integrin signaling

Corresponding author:

Lu-Hai Wang, Ph.D.

Address: No. 35, Keyan Road, Zhunan, Miaoli County 35053, Taiwan.

Phone #: 886-37-246166 ex 31010

Fax #: 886-37-585242

E-mail: [email protected]

Wei-Chieh Huang and Shih-Hsuan Chan contributed equally to this work and are

co-first authors.

Word count: 4956

Total number of figures and tables: 7

The authors disclose no potential conflicts of interest.




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Abstract:

microRNAs offer tools to identify and treat invasive cancers. Using highly invasive

isogenic oral squamous cell carcinoma (OSCC) cells established using in vitro and in

vivo selection protocols from poorly invasive parental cell populations, we used

microarray expression analysis to identify a relative and specific decrease in

miR-491-5p in invasive cells. Lower expression of miR-491-5p correlated with poor

overall survival of OSCC patients. miR-491-5p overexpression in invasive OSCC

cells suppressed their migratory behavior in vitro and lung metastatic behavior in vivo.

We defined the G protein-coupled receptor kinase-interacting protein 1 (GIT1) as a

direct target gene for miR-491-5p control. GIT1 overexpression was sufficient to

rescue miR-491-5p-mediated inhibition of migration/invasion and lung metastasis.

Conversely, GIT1 silencing phenocopied the ability of miR-491-5p to inhibit

migration/invasion and metastasis of OSCC cells. Mechanistic investigations

indicated that miR-491-5p overexpression or GIT1 attenuation reduced focal

adhesions, with a concurrent decrease in steady-state levels of paxillin,

phospho-paxillin, phospho-FAK, EGF/EGFR-mediated ERK1/2 activation and

MMP2/9 levels and activities. In clinical specimens of OSCC, GIT1 levels were

elevated relative to paired normal tissues and were correlated with lymph node




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metastasis, with expression levels of miR-491-5p and GIT1 correlated inversely in

OSCC where they informed tumor grade. Together, our findings identify a functional

axis for OSCC invasion that suggests miR-491-5p and GIT1 as biomarkers for

prognosis in this cancer.




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Introduction:

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide,

and accounts for more than 95% of all head and neck cancer (1). The incidence of oral

cancer in Taiwan increased by 30% in the last five years, mortality rate grew by 25%

with 30 to 49 years old men having the highest rate. More than forty-four thousand

patients annually require sustained medical treatment (2, 3). Unlike other types of

solid tumors, the metastasis of OSCC mostly involves local invasion and often is

restricted to the head and neck area. Cervical lymph node metastasis is a critical

prognostic factor for OSCC, the patients without such metastasis usually have higher

survival rates (4-6). Like other cancers, oral cancer metastasis requires an extensive

remodeling and degradation of extracellular matrix (ECM) in part via increased

expression of matrix metalloproteinases (MMPs) (7). However, the molecular

mechanism regulating the invasion and metastasis of OSCC is still largely unclear.

MicroRNAs (miRNAs) are an evolutionarily conserved group of small RNAs of

18-24 nucleotides that inhibit or stimulate gene expression. microRNAs have been

known to play important roles in various cancer progression and metastasis (8, 9),

however, study of their roles in OSCC metastasis is relatively scarce (10).

GIT1 is a multi-functional scaffold protein found to be associated with paxillin and




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capable of stimulating lamellipodia formation and spreading of cells (11-13).

Although the GIT1-paxillin complex is known to play a role in regulating focal

adhesion formation and cell migration (14), the precise role of GIT1 in metastasis of

cancer especially in OSCC is unclear. Focal adhesion kinase (FAK) is a ubiquitously

expressed non-receptor protein tyrosine kinase (PTK). Previous studies have indicated

that FAK functions as a positive regulator of tumor invasion, and is overexpressed in

various cancers, including breast, head and neck and ovarian cancers (15, 16).

FAK-paxillin interaction controls cell motility in part through Erk1/2 activation (17).

The epidermal growth factor receptor (EGFR) is a ubiquitous receptor tyrosine kinase

that is often upregulated in head and neck cancer (18). Activation of EGFR signaling

is known to promote cancer cells proliferation and metastasis (19, 20). A previous

report showed that GIT1 linked EGFR-mediated activation of ERK1/2 in 293 cells

(21). However, the role of EGFR/GIT1/FAK signaling in oral cancer is unknown.

In this study, we found that miR-491-5p was substantially down regulated in five

highly invasive OSCC lines and was also widely attenuated in OSCC compared to

paired normal tissues. Our data show that miR-491-5p targets GIT1 to inhibit OSCC

cell focal adhesion formation, invasion and metastasis via regulation of FAK, paxillin,

ERK1/2 and MMP2/9. Thus, this study suggests that miR-491-5p is a metastasis

suppressor and that miR-491-5p and GIT1 are significant biomarkers for OSCC




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metastasis.




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Materials and Methods

Tissue samples acquisition.

All clinical samples were acquired via protocols approved by the respective

Institutional Review Boards. Thirty three oral normal and tumor tissue pairs were

obtained from the Oral and Maxillofacial Surgery Unit, Chi Mei Medical Center,

Tainan, Taiwan, and Department of Public Health and Environmental Medicine,

School of Medicine, College of Medicine, Kaohsiung Medical University, Taiwan.

Tumor and normal oral tissue samples from surgical resection were snap-forzen in

liquid nitrogen or embedded in RNAlater. The clinical characteristics of these 33 N/T

pairs are shown in supplementary Table S1. 48 oral cancer samples for cDNA

microarray analysis (22) and 189 OSCC tissues array slides were obtained from

Nuclear Medicine, Chang Gung Memorial Hospital at Linkou Taiwan. Commercial

tissue array slides containing 134 OSCC tissues were purchased from US Biomax Inc.

Cell culture, DNA and RNA transfections and stable cell line generation.

Human oral cancer cell lines CGHNC9, SAS, SCC25, OECM-1 and OC-3 have been

described (23). CGHNC9, OECM-1 and SAS cells were grown in Dulbecco's

Modified Eagle's Medium (DMEM; Invitrogen) with 10% FBS (Biological Industries).




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SCC25 cells were grown in DMEM/HAM F12 (Invitrogen) supplemented with 10%

FBS, and hydrocortisone (0.4 ug/ml, Sigma-Aldrich). OC-3 cells were grown in 1:1

mixture of DMEM and KSFM (Invitrogen) with 10% FBS. All cells were incubated at

37oC in 5% CO2. Cells were transfected using the LipofectAMINE 2000 (Invitrogen)

and LipofectAMINE RNAiMAX (Invitrogen). The miR-491-5p cell lines stably

expressing pcDNA6.2-EmGFP-miRcon or pcDNA6.2-EmGFP-miR-491-5p were

established by transfection with the respective plasmids followed by selection with

Blasticidin (Sigma-Aldrich). The miR-491-5p and GIT1 cell lines stably expressing

pcDNA6.2-GFP-miR-491-5p or pcDNA-flag-GIT1 were established by transfection

with the respective plasmids followed by selection with neomycin (Sigma-Aldrich).

Vectors, antibodies and reagents.

The miR-491-5p was constructed in pcDNA6.2-EmGFP. GIT1 coding sequence was

cloned in pCMV-HA- and pcDNA3-flag. The luciferase-3’UTR-wt reporter or

luciferase-3’UTR-mutant (mt) plasmids were prepared by inserting the

GIT1-3’-UTR-wt carrying a putative miR-491-5p binding site or its mutant sequence

into the pGL3-control plamid (sequences are shown in Supplementary Table S2).

N-terminally (1-676 amino acid) truncated FAK (dominant-negative FAK) and

dominant-negative MEK (K97A mutant) were constructed in pcDNA3. Antibodies for




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western blotting and immunohistochemistry are mouse anti-GIT1 from BD

Transduction Laboratories and Bethyl Laboratories, Inc. The primers (for plasmid

construction), antibodies and reagents used are described in the Supplementary

Material and Table S2.

3'UTR reporter assays.

pGL3-GIT1-3’UTR-wt or pGL3-GIT1-3’UTR-mt was cotransfected with

pre-miR-491-5p or pre-anti-miR-491-5p into OSCC cells. Luciferase assay was

performed using an assay kit (Promega). Renilla luciferase was co-transfected as a

control for normalization.

Reverse transcriptase (RT)-PCR and qRT-PCR.

RT-PCR and qRT-PCR were used to detect the miR-491-5p and mRNA expression.

We designed a stem-loop RT primer specifically hybridizing with miR-491-5p or

RNU6B respectively, the latter was used for normalization (24). The detailed

conditions and primers used for mRNA expression are listed in the Supplementary

Material and Table S2.

Cell chemotatic migration and invasion assay.




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Chemotatic migration and invasion ability of OSCC cells was assessed using the

Falcon Cell Culture Inserts with or without Matrigel (BD Biosciences) coating as

described (25). The detailed conditions are described in the Supplementary Material.

In vivo metastasis assays.

CB17-SCID mice were used for injection of OSCC cells orthotopically at oral buccal

mucosa or via tail vein and monitored for 21 to 42 days before sacrifice. Lung tissue

were removed, fixed, paraffin-embedded, serially sectioned, and subjected to

hematoxylin and eosin (H&E) staining. The detailed conditions are described in the

Supplementary Material.

Focal adhesion assay and Immunofluorescence microscopy.

Immunostaining was performed to detect the vinculin level and focal adhesions (FA)

in C9-lung-IV2 cells. The detailed conditions are described in the Supplementary

Material.

Western blotting

Detailed procedure was described in the Supplementary Materials and Methods.




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Paxillin degradation assays

C9-lung-IV2 cells were transfected with negative control (NC) or GIT1-siRNA-3 and

incubated for 48h, and MG132 at 10 �mol/L was added to the cells for 8h.

C9-lung-IV2 cells were transfected with negative control or GIT1-siRNA, and

cycloheximide (Sigma-Aldrich) at 20 �g/ml was added to the cells for 0h, 4h and 12h.

Immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH).

IHC was performed to detect GIT1 expression from paraffin-embedded oral cancer

specimens. The slides were stained with primary antibody using an automatic slide

stainer BenchMark XT (Ventana Medical Systems). FISH was performed using the

tyramide signal amplification (TSA) technology and the 5’biotin-labeled miR-491-5p

probe, or a negative control probe (25nM/L; GeneDireX, Inc) as previously described

(26). The IHC score of GIT1 and FISH score of miR-491-5p for each specimen was

graded as follows: +, weak; ++, moderate; +++, strong.

Gelatin zymography

Gelatin zymography was used to detect MMP2/9 activity in C9-lung-IV2 cells using

conditions detailed in the Supplementary Material.




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Statistical analysis.

Survival data was analyzed using the Kaplan-Meier method. Differences between

experimental groups were calculated with the Mann–Whitney U test. Differences with

P values of <0.05 are considered to be statistically significant. The linear correlation

coefficient (Spearman's �) was utilized to estimate the relationship between

miR-491-5p and GIT1 levels in the oral tumor specimens.

Results:

Establishment of isogenic pairs of high and low invasive OSCC lines.

Recent evidence has shown that microRNAs play a role in regulating cancer

metastasis (8). To identify relevant microRNAs associated with invasive phenotypes

of oral cancer, we established isogenic pairs of high and low invasive oral cancer lines

through in vitro and in vivo selections. Using Boyden chamber invasion assay (in vitro

selection), the oral cancer cell lines OC-3, OECM-1, SAS and SCC25 were selected

five to eight cycles to derive the highly invasive sublines OC-3-I5, OECM-1-I8,




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SAS-I5 and SCC25-I6 in which the number following I denotes the cycles of

selection. OC-3 and CGHNC9 lines were selected one to two cycles by in vivo

injection of the respective cells into tail vein of CB17-SCID mice (in vivo selection),

followed by isolation of tumor cells grown from lymph node metastases or lung

metastases to obtain OC-3-I5-Lymph-node-IV1, OC-3-I5-Lymph-node-IV2,

OC-3-lung-IV1, OC-3-I5-lung-IV2 and CGHNC9-lung-IV2 (C9-lung-IV2) where the

number following IV denotes the cycle selection. After invasion selection, we tested

those lines for their migratory and invasive ability by performing Boyden chamber

migration/invasion assays. As shown in supplementary Fig. S1, the migratory- and

invasive ability of the selected invasive lines was dramatically increased.

Down-regulation of miR-491-5p was found in the highly invasive OSCC cells and

was correlated with poor survival of the OSCC patient.

Using microRNA array, we attempted to identify microRNAs associated with the

invasive phenotype of the five selected highly invasive OSCC lines (SAS-I5,

OECM-1-I8, SCC25-I6, OC-3-I5- and OC-3-I5-lung-IV2) compared with their

corresponding parentals. A series of microRNAs with 2-fold differential expression

between the low and high invasive isogenic lines were identified. The differentially

expressed microRNAs of each isogenic pair as shown in each circle and the




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microRNA common among all 5 isogenic pairs are displayed by the Venn diagrams

(Fig. 1A). The miR-491-5p has a greater than two-fold lower expression in all five in

vitro and in vivo selected invasive lines (Fig. 1A). The reduced expression of

miR-491-5p was verified by RT-PCR, the in vivo selected invasive lines displayed a

greater decrease of the microRNA compared with their parentals (Fig. 1B and C).

Furthermore, we examined 33 pairs of oral normal and cancer tissues for the

miR-491-5p expression. The miR-491-5p was underexpressed in 29 of 33 OSCC

samples compared with their matched normal tissues (Fig. 1D).

Kaplan-Meier survival analysis of 189 cases showed that OSCC samples with low

miR-491-5p expression correlated with a significantly poorer survival than those with

high miR-491-5p expression (Fig. 1E, top). The miR-491-5p low expression also had

a correlation with lymph node metastasis (Fig. 1E, bottom). The results suggest that

miR-491-5p may play a role in suppressing OSCC lymph node metastasis and could

serve as a prognostic marker for OSCC.

miR-491-5p inhibits migration, invasion and lung metastasis of OSCC cells.

Next, we tested whether miR-491-5p was able to suppress migration and invasion of

oral cancer cells by Boyden chamber assay. As expected, miR-491-5p significantly

inhibited migration and invasion of OSCC cells, which could be partially or fully




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rescued by anti-miR-491-5p (Fig. 2A). To evaluate whether miR-491-5p inhibited

cancer cell metastasis in vivo, we employed two experimental metastasis models,

namely tail vein injection and orthotopic implantation of cells in SCID mice. In the

tail vein injection model, expression of miR-491-5p resulted in greatly decreased

targeting of the C9-lung-IV2 cells to lung 24h after injection (Supplementary Fig. S2).

As shown in Fig. 2B, the percentage of lung tumor area in the miR-491-5p transiently

transfected group was reduced to 45% of the control. Similarly, in the orthotopic

model (Fig. 2C), stable overexpression of miR-491-5p significantly suppressed lung

metastasis of C9-lung-IV2 OSCC cells. miR-491-5p resulted in fewer metastatic foci

and small nodules in lung. These results indicate that miR-491-5p is able to potently

inhibit migration, invasion and metastasis of OSCC cells.

GIT1 is a direct target of miR-491-5p.

To understand the mechanism of the miR-491-5p regulated OSCC cell invasion and

metastasis, we attempted to identify its target genes by using two computational

softwares, TargetScan and DIANA-microT. Those methods identified 17 candidate

genes to be potential targets of miR-491-5p (Supplementary Table S3). We were

interested in those involved in regulating focal adhesion complexes since they had

been implicated in cancer cell invasion and metastasis (15, 27). Using Gene Ontology




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(GO) analysis, we found a putative target of miR-491-5p, GIT1, which belongs to the

focal adhesion related gene family. To determine whether GIT1 is a direct target of

miR-491-5p, we constructed Luc-GIT1-UTR-wild-type (wt) and its 3'UTR mutant

(mt) plasmids. Luciferase reporter assays showed that miR-491-5p suppressed more

than 50% transcriptional activity of the Luc-GIT1-3'UTR-wt reporter compared with

the control, whereas the GIT1-3’UTR-mt where the putative miR-491-5p binding site

had been mutated was refractile to the inhibition (Fig. 3A). We also found that

miR-491-5p significantly inhibited the GIT1 mRNA expression in CGHNC9 and

SCC25 cells (Fig. 3B), which could be partially reversed by anti-miR-491-5p. As

expected, western blotting demonstrated that overexpression of miR-491-5p

dramatically suppressed GIT1 protein expression in CGHNC9 and SCC25 cells and

the inhibition was substantially reverted by anti-miR-491-5p (Fig. 3C). Consistently,

the in vivo selected lines with lower miR-491-5p expression showed elevated GIT1

mRNA levels (Supplementary Fig. S3). Those results suggest that miR-491-5p

directly targets GIT1 resulting in down regulation of its mRNA and protein.

Suppression of GIT1 by siRNA inhibits OSCC cell migration, invasion and

metastasis.

GIT1 is a multidomain scaffold protein that is usually found to be associated with




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focal adhesions and is able to promotes cell motility (28). We found that GIT1 was

upregulated in the highly invasive oral cancer lines compared with their parental lines

(Supplementary Fig. S4). To assess whether GIT1 level correlated with metastasis, we

analyzed cDNA microarray data of a series of 48 OSCC samples. We first determined

the median GIT1 expression level from the samples and then divided them into two

groups, i.e. the higher or the lower expression than the median level. We found that

the higher GIT1 expression was significantly associated with the development of

lymph node metastasis (Fig. 4A) since the increased level of GIT1 expression

appeared to be correlated with cases having higher number of positive lymph nodes

involvement (Fig. 4B). Next, we examined whether GIT1 could affect cell migration,

invasion and metastasis. Three sets of GIT1-siRNAs were tested for their inhibitory

efficacy by western blot. GIT1-siRNA-3 had the highest knockdown effect (Fig. 4C,

left) and was used in the subsequent experiments. Knockdown of GIT1 significantly

inhibited OSCC cell migration and invasion (Fig. 4C, right). GIT1 was able to rescue

miR-491-5p-mediated inhibition of OSCC cell invasion (Fig. 4D). To further assess

the role of GIT1 in regulating metastasis, we injected GIT1-siRNA expressing or the

control plasmid-transfected C9-lung-IV2 cells into SCID mice via tail vein.

Knockdown of GIT1 resulted in about 5-fold reduction in the lung metastasis (Fig.

4E).




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Re-expression of GIT1 significantly reverses miR-491-5p-mediated suppression of

invasion and metastasis of C9-lung-IV2 cells.

Ectopic expression of GIT1 could partially rescue cell invasion inhibited by

miR-491-5p in vitro (Fig. 4D). Using the tail vein injection assay, we also found that

ectopic expression of GIT1 rescued miR-491-5p-mediated inhibition of lung

metastasis about 3 folds (Fig. 4F). The above results support our hypothesis that

miR-491-5p targets GIT1 to inhibit OSCC invasion and metastasis.

Inhibtion of GIT1 by miR-491-5p enhances degradation of paxillin and impaires

focal adhesion signaling in OSCC cells.

Previous studies indicated that GIT1 interacted with focal adhesion related proteins

including paxillin and FAK (29, 30). To investigate the detailed mechanism through

which the miR-491-5p targeted GIT1 to inhibit cell migration and invasion, we first

examined the effect on FAK and paxillin activation upon GIT1 depletion in OSCC

cells. We hypothesized that suppression of GIT1 could lead to inhibition of focal

adhesion signaling and expression of molecules involved in cancer cell invasion.

Indeed, suppression of GIT1 by miR-491-5p reduced the level of p-FAK, its

interactive protein paxillin and phospho-paxillin (Fig. 5A) while the mRNA of




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paxillin remained unaffected (Supplementary Fig. S5). GIT1 has been shown to bind

and regulate activation of paxillin through FAK (13, 30). Since the paxillin mRNA

was not affected by GIT1 knockdown, we checked whether the decreased paxillin was

due to enhanced proteasome degradation. The level of paxillin in GIT1-depleted cells

with or without addition of MG132 (a proteasome inhibitor) was compared. The result

showed that treatment of MG132 reversed the effect of GIT1 depletion on paxillin

level (Fig. 5B) suggesting that knockdown of GIT1 increased paxillin degradation via

proteasome pathway in oral cancer cells. To further confirm depletion of GIT1

resulting in decreased paxillin stability, we performed protein degradation experiment

in cells treated with cycloheximide (Cyclohex). Paxillin was stable over a course of

12 hours in the control cells. In contrast, paxillin stability was significantly decreased

in the GIT1 depleted cells (Fig. 5C). We further tested whether GIT1 depletion could

affect OSCC cell focal adhesion and lamellipodia formation. As shown in Fig 5D,

vinculin level and focal adhesions were significantly decreased in the miR-491-5p

transfected C9-lung-IV2 cells, which was reversed by overexpression of GIT1.

Likewise, the focal adhesions were disrupted in the si-GIT1 transfected C9-lung-IV2

cells (Fig. 5D). These results suggest that miR-491-5p-mediated decrease of GIT1

results in reduced FAK activation, and increased paxillin degradation leading to

reduced focal adhesion formation of OSCC cells.




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Dominant-negative form of FAK (DN-FAK) partially inhibited OSCC cells

invasiveness which could be rescued by GIT1.

We further determined the role of FAK in miR-491-5p-targeted GIT1 down

regulation and inhibition of migration and invasion. Our data showed that GIT1

promoted cell invasion could be reversed by dominant-negative FAK (DN-FAK)

(Supplementary Fig. S6B). Western blot analysis confirmed the downregulation of

p-FAK by DN-FAK in C9-lung-IV2 cells overexpressing GIT1 (Supplementary Fig.

S6A). Those data suggested that down regulation of GIT1 by miR-491-5p could

suppress migration, invasion and metastasis of OSCC cells through degradation of

paxillin and reduced focal adhesion signaling including FAK and ERK1/2 activation.

miR-491-5p targets GIT1 to regulate expression level and activity of Matrix

metalloproteinases2/9 (MMP2/9) via EGFR/ERK1/2 signaling pathway .

MMP2/9 have been implicated in tumor cell invasion and metastasis (31, 32). We

observed that overexpression of miR-491-5p or knockdown of GIT1 decreased

MMP2/9 mRNA (Fig. 6A) and activity (Fig. 6B) in OSCC cells. Previous studies

indicated that the expression level and activity of MMP2/9 were regulated by ERK1/2

in HEK293 and monocytic cells (33, 34). Our data also showed that the expression




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level of MMP2/9 were reduced by PD98059 (a MEK inhibitor) in OSCC cells

(Supplementary Fig. S7). GIT1 was shown to link the EGFR to ERK1/2 activation in

HEK293 cells (21). However, the role of GIT1 in the regulation of MMPs in oral

cancer cells is unknown. To check whether the miR-491-5p-GIT1 pathway could

regulate ERK1/2 signaling, which in turn affected MMP2/9 expression, we examined

the effect of miR-491-5p and GIT1 on EGF-induced ERK1/2 activation. CGHNC9

cells were transfected with control-siRNA, miR-491-5p or GIT1-siRNA, serum

starved for 24h and then stimulated with EGF. In the control cells, EGF rapidly

stimulated robust activation of ERK1/2. By contrast, miR-491-5p or GIT1-siRNA

treated cells showed significantly reduced activation of ERK1/2 (Fig. 6C). Thus,

reduced GIT1 expression is associated with dramatic inhibition of EGF-stimulated

ERK1/2 activation. Consistently, overexpression of miR-491-5p resulted in

significantly inhibition of the growth beyond 72h especially upon EGF stimulation

(Supplementary Fig. S8B). In addition, our data showed that GIT1 increased MMP2/9

mRNA expression could be reduced by dominant-negative MEK or PD98059

(Supplementary Fig. S9B). Western blot analysis confirmed the downregulation of

p-ERK1/2 by DN-MEK and PD98059 in C9-lung-IV2 cells overexpressing GIT1

(Supplementary Fig. S9A). These results suggest that miR-491-5p targets GIT1 to

suppress MMP2/9 expression and activity, and this is likely to be due to inhibition of




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ERK1/2 activation in OSCC cells.

Correlation of miR-491-5p and GIT1 expression with grades of OSCC and inverse

correlation between miR-491-5p and GIT1 .

There have been no studies reporting the role of GIT1 in oral cancer so far. We

examined GIT1 expression in the matched normal and cancerous oral tissue

specimens to assess its clinical connection. We found that in 26 of 33 cases of OSCC

samples the GIT1 level was 1.5 to 23 fold higher compared with their matched normal

tissues (Fig. 7A), and the GIT1 level was inversely correlated with miR-491-5p

expression (Fig. 7B). Furthermore, 189 OSCC samples showed that 51 patients with

grade 1 had longer survival time than 138 patients with grade 2 and 3 (Fig. 7C). We

next determined whether tumor grade was correlated with GIT1 expression by IHC

staining of oral cancer tissue arrays. For each section, staining was measured as weak

(+), moderate (++), and strong (+++). We observed that GIT1 level was higher in the

moderately (grade 2) and poorly differentiated (grade 3) than in the well differentiated

tumors (grade 1) (Fig. 7D). About 79% of cases with grade 2-3 had moderate to

strong expression of GIT1, whereas 79% of cases with grade 1 had weak expression

of GIT1 (Fig. 7D, table). Thus the GIT1 level appeared to correlate with the

aggressiveness of oral cancer. Furthermore, miR-491-5p signals were relatively weak




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in moderately and poorly differentiated tumor samples (Fig. 7D). 77% of cases with

grade 2-3 had weak to moderate expression of miR-491-5p, whereas 89% of cases

with grade 1 showed a moderate to strong expression of miR-491-5p (Fig. 7D, table).

The results indicate that miR-491-5p and GIT1 expression levels are inversely

correlated and lower miR-491-5p together with higher GIT1 correlates with the

advanced grades of the disease.

Discussion:

OSCC has a high incidence of neck metastasis and often metastasizes

contralaterally (35). It is widely known that the lymph node metastases is an

important prognosticator predicting survival of OSCC patients (36-38). Despite

accumulating evidences pointing to the regulatory role of microRNAs in cancer

progression and invasion, its regulatory role in oral cancer metastasis is still poorly

understood. This study aimed to identify relevant microRNAs and their downstream

target genes involved in the regulation of cancer metastasis in OSCCs. For the first




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time, we establish the miR-491-5p-GIT1-FAK-paxillin and

miR-491-5p-GIT1-ERK1/2 pathways in the regulation of OSCC cell invasion and

metastasis. We also show that low expression of miR-491-5p and high expression of

GIT1 correlates with aggressiveness and lymph node metastasis of OSCC.

Furthermore, we showed that the level of miR-491-5p is significantly correlated with

overall survival of OSCC. Interestingly, in a separate study we found that GIT1 also

played an important role in lymph node metastasis of breast cancer (Chan SH, et al.

unpublished).

We found that miR-491-5p level decreased significantly in the highly invasive

OSCC lines compared with their corresponding less invasive parental cells, and that

miR-491-5p suppressed migration, invasion and metastasis of oral cancer cells mainly

via targeting GIT1 to affect FAK and ERK1/2 signaling. A recent study reported the

inhibitory effect of miR-491-5p on expression of MMP9 and invasion of glioblastoma

multiforme cells (39). Our data also show that MMP9 mRNA expression level and

activity are decreased in the OSCC cells overexpressing miR-491-5p most likely via

inhibition of ERK1/2 activation (Fig. 6). In another study it was shown that

overexpression of CD44 3'UTR interacted with and trapped endogenous miR-491-5p,

leading to increased levels of fibronectin and collagen resulting in enhanced cell




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motility and invasion in human breast cancer cells (40). Our finding in OSCC is in

agreement with those studies suggesting miR-491-5p functions as an invasion

suppressor in glioma cells and breast cancer cells. However, we show here that

miR-491-5p inhibits OSCC invasion and metastasis via targeting GIT1, which is

distinct from those two studies. As shown in Supplementary Fig. S8A, miR-491-5p

although had no significant effect on proliferation of OSCC cells before 72h time

point, did affect the growth beyond that time point especially upon EGF stimulation

likely due to inhibition of ERK1/2 activation (Supplementary Fig. S8B). Our data also

showed that expression of miR-491-5p in C9-lung-IV2 cells resulted in a significant

decrease of lung metastasis compared to the control in SCID mice at 24h time point

(Supplementary Fig. S2). It appears that miR-491-5p targets and regulates distinct

pathways to manifest its biological function in different types of cancer cells. Our

study identified miR-491-5p as a novel metastasis suppressor in oral cancer.

The differential expression level of miR-491-5p and difference in migration/invasion

ability is difficult to equate quantitatively. We think at least there are three possible

explanations, first, a given miRNA could have multiple targets involved in regulating

migration/invasion, second, a two folds difference in miRNA could lead to a greater

decrease in the target resulting in more than two folds difference in




27

migration/invasion, and third, the same effector gene could be targeted by miRNAs

other than miRNA-491-5p.

As a scaffold protein associated with integrin, GIT1 plays a functional role in cell

migration (11, 12), focal adhesion formation (28) and angiogenesis (41). For example,

GIT1 promotes migration in A431 cells by interacting with the MYO18A-PAK2-�pix

complex (11). GIT1 is also an important regulator for VEGF-induced endothelial cell

podosome formation (41). Moreover, several previous studies showed that GIT1 was

associated with p-ERK1/2 in focal adhesions after epidermal growth factor (EGF)

stimulation in HeLa cells (42, 43), and affected focal adhesion formation in

osteoblasts following platelet-derived growth factor (PDGF) stimulation (28).

Although GIT1 has been implicated in the regulation of cell motility, its role in

invasion and metastasis of OSCC cells has not been reported before. For the first time,

our study indicates that GIT1 plays an important role in invasion and metastasis of

oral cancer cells and it is targeted and down regulated by miR-491-5p in the highly

invasive and poorly differentiated tumor cells.

Regulation of focal adhesion complexes involving FAK and paxillin are known to be

important in cell invasion (44). Our data show that knockdown of GIT1 led to




28

decreased FAK activity (Fig 5A) and increase of proteasome-mediated degradation of

paxillin (Fig 5B and 5C). Together, these findings suggest that miR-491-5p inhibits

invasion and metastasis of OSCC cells through depletion of GIT1, leading to

disruption of focal adhesion complexes. Although GIT1 is often implicated in the

regulation of cell focal adhesion, information about its role and regulatory mechanism

for the invasion and metastasis in oral cancer has not been explored previously.

Interaction between FAK and paxillin plays an important role in focal adhesion

signaling and regulation of cell motility (45) in part via Erk1/2 activation (17).

Furthermore, a previous study indicated that paxillin-/- MEF cells showed several

phenotypical changes including reduced cell migration (46). In addition, paxillin-/-

MEF still have focal adhesion (with abnormal shapes). Our data also showed that

GIT1-depleted OSCC cells had decreased level and activity of paxillin protein and

reduced cell migration. Depletion of FAK inhibited the phosphorylation of paxillin as

expected since paxillin is a substrate of FAK and knockdown of paxillin also inhibited

phospohrylation of FAK (Supplementary Fig. S10). These results suggested that

depletion of GIT1 reduced paxillin stability leading to decreased FAK

phosphorylation in agreement with the report of Hagel et al. (46). Therefore, the lost

of focal adhesions upon miR-491-5p overexpression or knockdown of GIT1 may not

be attributed to the reduction of paxillin, but mainly to the reduction of GIT1. Among




29

the potential upstream activation of ERK1/2 is EGFR, which has been previously

implicated in oral cancer development and progression (47, 48). Our data show that

depletion of GIT1 significantly reduces EGF-induced ERK1/2 activation in OSCC

cells (Fig. 6C). This is in agreement with a previous study indicating that GIT1 links

the EGFR to ERK1/2 activation by associating with MEK1 (21). Thus, we propose a

regulatory signaling pathway of OSCC metastasis consisting of miR-491-5p and its

downstream target, GIT1, which functions as a scaffold for focal adhesion signaling

and EGF-induced ERK1/2 activation.

We showed that transient transfection with miR-491-5p or knockdown of GIT1

dramatically inhibited MMP2/9 mRNA expression and activity (Fig 6A and 6B). We

also showed that treatment of dominant-negative MEK or PD98059 (Supplementary

Fig. S7) reduced the MMP2/9 expression level. It was shown that ERK1/2 pathway

enhanced the invasion of lung cancer cells by increasing MMP2/9 expression and

activity (33). Thus the inhibition of MMP2/9 by miR-491-5p and knockdown of GIT1

is likely in part due to decreased ERK1/2 activation in OSCC cells.

Our analysis of clinical samples revealed a low miR-491-5p and high GIT1

expression pattern in the advanced OSCC and they appeared to exist an inverse




30

relationship between miR-491-5p and GIT1 expression. The miR-491-5p seems to

have no effect on OSCC cell growth. Thus miR-491-5p expression is less likely to be

involved in tumor initiation or growth, rather, it is more likely to be involved in the

invasive progression when it needs to be down regulated to allow overexpression of

GIT1 for cancer cell motility and MMPs production/activation. It would be interesting

to explore the upstream regulator for miR-491-5p down regulation during the

metastatic progression of OSCC.

In conclusion, our study has identified miR-491-5p as a novel metastatic suppressor

through regulation of its downstream target, GIT1, resulting in perturbation of

FAK/paxillin and ERK1/2 signaling as well as MMPs expression and activity in oral

cancer cells (Fig. 7E). The miR-491-5p and GIT1 could potentially serve as metastatic

prognosis biomarkers and targets for intervention of OSCC metastasis.

Disclosure of Potential Conflicts of Interest

No potential conflict of interest need to be disclosed.

Acknowledgments

We thank the Pathology and Microarray Core Laboratories of the National Health




31

Research Institutes for H&E and immunohistochemical staining and microRNA array

analysis respectively. We thank Hsin-Lei Yao for discussion. This work was supported

by grants from National Science Council, Taiwan (01D2-MMNSC16) and the

Department of Health, Taiwan (DOH100-TD-111-004).

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Figure 1. The miR-491-5p expression is downregulated in highly invasive OSCC lines

and oral cancers. A, Decreased miR-491-5p expression is shared by 5 highly invasive

OSCC lines. Venn diagram shows number of differentially expressed miRNAs of each

isogenic pair and the single down refulated miRNA common to all 5 pairs. The

expression of miRNAs for each highly invasive OSCC line was normalized to the

corresponding parental cell line. B and C, The expression level of miR-491-5p in the

in vitro and two series of in vivo selected highly invasive cell lines was measured by

RT-PCR. The U6 small nuclear RNA was used as an internal control. D, QRT-PCR

reveals downregulation of miR-491-5p in oral tumors compared with their matched




40

normal oral tissues. The relative amount of miR-491-5p to small nuclear RNU6B

RNA was calculated using the equation 2–�Ct where �CT= (CT miR-491-5P – CT RNU6B

RNA). E, top, kaplan-Meier survival curves of 189 OSCC patients divided by

miR-491-5p expression. Bottom, association between miR-491-5p expression level

and lymph node metastasis status of 189 OSCC patients as evaluated by spearman's �.

The level of miR-491-5p was determined by fluorescence in situ hybridization.

Tissues expressing miR-491-5p at levels lower than median level were assigned to the

low and those above the median level were assigned to the high expression group. **,

P < 0.01.

Figure 2. The miR-491-5p inhibits migration, invasion and metastasis of OSCC cells.

A, Transient transfection of miR-491-5p significantly inhibited migration and

invasion of CGHNC9, OC-3-I5-lung-IV2 and SCC25-I6 cells, which was rescued by

anti-miR-491-5p. B, Lung metastasis via tail vein injection of C9-lung-IV2 cells into

SCID mice was significantly reduced in the cells transiently transfected with

miR-491-5p. The transfection efficiency as judged by fluorescence of FAM-labeled

miR-491-5p was over 90% (Supplementary Fig. S11). C, Lung metastasis via

orthotopic implantation of C9-lung-IV2 cells at oral buccal mocosa was dramatically

reduced in the cells stably expressing miR-491-5p precursor. The images show H&E




41

staining of the lung metastatic tumors (X200). In tail vein injection model lung

metastasis was detected in all mice in 3-4 weeks whereas in orthotopic model, on the

average from three independent experiments 80% of mice developed lung metastasis

in 6-8 weeks. Lung metastasis index was calculated as the following: Metastatic

tumor areas/total lung areas. T indicates metastatic tumor areas. Histograms represent

means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01.

Figure 3. Identification of GIT1 as the direct target of miR-491-5p. A, Effect of

miR-491-5p on GIT1 3’-UTR-wt (GIT1 3’UTR-wt-luc) and GIT1 3’-UTR-mutant

luciferase reporters (GIT1 3’UTR-mt-luc). Top, The GIT1 3’-UTR-wt sequence and

the GIT1 3’-UTR-mt in which the sequence in bold was mutagenized to abolish the

miR-491-5p binding. Bottom, Luciferase assays showing decreased activity after

cotransfection of GIT1 3’-UTR-wt with miR-491-5p in CGHNC9 (left) and SCC25

(right) OSCC cells. The GIT1 3’-UTR-mt reporter was not affected by miR-491-5p. B,

The expression of GIT1 mRNA in CGHNC9 and SCC25 cells was measured by

qRT-PCR from RNAs extracted from cells transfected with miR-491-5p alone or

together with anti-miR-491-5p (anti-491-5p). GAPDH was used as an internal control.

C, The expression of GIT1 protein was detected by western blot of total proteins from

cells transfected with miR-491-5p alone or together with anti-miR-491-5p. -actin




42

was used as an internal loading control. Histograms represent means ± SD from three

independent experiments. *, P < 0.05.

Figure 4. GIT1 expression correlates with lymph node metastasis and knockdown of

GIT1 inhibits OSCC cell migration, invasion and metastasis. A and B, Analysis of

cDNA microarry data from 48 OSCC tumor samples revealed an association between

high number of lymph nodes involvement and increased GIT1 expression determined

from the array analysis. Spearman's � analysis was used to evaluate the relationship of

GIT1 expression and lymph node metastasis. C, left, Western blot of GIT1 in

CGHNC9 cells transfected with the GIT1-siRNA or negative control (NC). Right, A

dramatic decrease in migration and invasion ability was observed in CGHNC9 cells

transfected with GIT1-siRNA compared to the NC. D, left, Western blot of GIT1 from

CGHNC9 cells transfected with the GIT1 plasmid or vector control. Right,

Overexpression of GIT1 significantly rescued CGHNC9 cell invasiveness inhibited

by miR-491-5p. E, Lung metastasis following tail vein injection of C9-lung-IV2 cells

transiently transfected with GIT1-siRNA was dramatically reduced. F, Lung

metastasis index shows that transient transfection of GIT1 plasmid into C9-lung-IV2

cells expressing miR-491-5p significantly rescued their lung metastasis generated by

tail vein injection. All images show H&E staining of the lung metastases (X200).




43

Histograms represent means ± SD from three independent experiments. *, P < 0.05;

**, P < 0.01.

Figure 5. GIT1 depletion enhances degradation of paxillin and reduces FAK

phosphorylation and decreases focal adhesion formation. A, Western blot analysis

showing expression of miR-491-5p or knockdown of GIT1 decreased phosphorylation

of paxillin, FAK and reduced level of paxillin protein. B, Knockdown of GIT1

decreased paxillin protein, which was blocked by MG132, while reduced

phosphorylation of FAK was not. Treatment with the MG132 was at 10 �mol/L for 8h.

C, Knockdown of GIT1 significantly decreased paxillin stability in OSCC cells. The

paxillin levels in control and GIT1-depleted C9-lung-IV2 cells treated with

cycloheximide (20 �g/ml) for the indicated times show a faster decay in the GIT1

depleted cells. D, C9-lung-IV2 cells expressing the indicated plasmids were seeded in

dishes coated with fibronectin for 120 min (top) and analyzed for total focal adhesion

(FA) area per cell (bottom). The focal adhesions and stress fibers were revealed with

anti-vinculin (green) and rhodamine (red), respectively. Histograms represent means

± SD from three independent experiments. *, P < 0.05.

Figure 6. Effect of GIT1 on activation and expression of MMP2/9 and on




44

EGF-induced ERK1/2 phosphorylation. A, Relative MMP2/9 mRNA levels in

CGHNC9 cells transfected with the indicated plasmids. B, top, Western blot of GIT1

expression in CGHNC9 cells transfected with miR-491-5p, GIT1-siRNA or negative

control (NC). Bottom, CGHNC9 cells were pre-incubated in serum-free for 24h, and

conditioned media were extracted and analyzed by gelatin zymography. C, CGHNC9

cells were transfected with the indicated plasmids for 8h, serum starved for 24h and

then treated with EGF 10ng/ml for the indicated times. Histograms represent means ±

SD from three independent experiments. **, P < 0.01.

Figure 7. miR-491-5p and GIT1 expression levels exhibits an inverse relationship in

OSCC specimens. A, QRT-PCR shows upregulation of GIT1 mRNA in OSCC tumors

compared with matched normal oral tissues. B, A negative correlation between

miR-491-5p and GIT1 RNA levels in 33 OSCC tumor specimens. Spearman's �

analysis was used. C, Overall survival time was significantly associated with low

grade (grade 1) compared to high grades (grade 2 and 3) in 189 OSCC samples. D, A

typical example of the expression levels of miR-491-5p and GIT1 protein as

determined by fluorescence in situ hybridization (for miR-491-5p) and

immunohistochemistry (for GIT1) of oral cancer tissue prepared from 134

paraffin-fixed carcinoma (grade 1-3) specimens. E, A proposed scheme of the




45

negative regulation of the GIT1/FAK/paxillin and EGF signaling pathways by

miR-491-5p in OSCC cells. **, P < 0.01.

























Published OnlineFirst December 12, 2013.Cancer Res Lu-Hai Wang, Wei-Chieh Huang, Shih-Hsuan Chan, et al. metastasisfor oral squamous cell carcinoma (OSCC) invasion and miRNA-491-5p and GIT1 serve as modulators and biomarkers

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