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Epigenetic Suppression of SERPINB1 Promotes Inflammation-Mediated Prostate Cancer 1
Progression 2
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Irina Lerman1, Xiaoting Ma1, Christina Seger1, Aerken Maolake2, Maria de la Luz Garcia-Hernandez3, 4
Javier Rangel-Moreno3, Jessica Ackerman4, Kent L. Nastiuk2, Martha Susiarjo5, Stephen R. Hammes1 5
6 1Department of Medicine, Division of Endocrinology and Metabolism, University of Rochester Medical 7
Center, Rochester, NY. 2Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY. 8
3Department of Medicine, Division of Allergy/Immunology and Rheumatology, University of Rochester 9
Medical Center, Rochester, NY. 4Department of Pathology, University of Rochester Medical Center, 10
Rochester, NY. 5Department of Environmental Medicine, University of Rochester Medical Center, 11
Rochester, NY. 12
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Running Title: SERPINB1 in prostate cancer 14
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Abbreviations: myeloid derived suppressor cell (MDSC), neutrophil elastase (NE) 16
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Corresponding Author: Stephen R. Hammes, MD, PhD, Department of Medicine, Division of 18
Endocrinology and Metabolism, University of Rochester School of Medicine, 601 Elmwood Ave., 19
Rochester, NY 14642. Phone: 585-275-2901; Fax: 585-273-1288; email: 20
stephen_hammes@urmc.rochester.edu 21
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COI: The authors declare no potential conflicts of interest 23
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Abstract 25
Granulocytic myeloid infiltration and resultant enhanced neutrophil elastase (NE) activity is 26
associated with poor outcomes in numerous malignancies. We recently showed that NE expression and 27
activity from infiltrating myeloid cells was high in human prostate cancer xenografts and mouse Pten-null 28
prostate tumors. We further demonstrated that NE directly stimulated human prostate cancer cells to 29
proliferate, migrate, and invade, and inhibition of NE in vivo attenuated xenograft growth. Interestingly, 30
reduced expression of SERPINB1, an endogenous NE inhibitor, also correlates with diminished survival 31
in some cancers. Therefore, we sought to characterize the role of SERPINB1 in prostate cancer. We find 32
that SERPINB1 expression is reduced in human metastatic and locally advanced disease and predicts poor 33
outcome. SERPINB1 is also reduced in Pten-null mouse prostate tumors compared to wild type prostates, 34
and treatment with sivelestat (SERPINB1 pharmacomimetic) attenuates tumor growth. Knockdown of 35
highly expressed SERPINB1 in non-malignant prostatic epithelial cells (RWPE-1) increases proliferation, 36
decreases apoptosis, and stimulates expression of epithelial-mesenchymal transition markers. In contrast, 37
stable SERPINB1 expression in normally low-expressing prostate cancer cells (C4-2) reduces xenograft 38
growth in vivo. Finally, EZH2-mediated histone (H3K27me3) methylation and DNMT-mediated DNA 39
methylation suppress SERPINB1 expression in prostate cancer cells. Human TCGA analysis and 40
pyrosequencing demonstrate hypermethylation of the SERPINB1 promoter in prostate cancer compared to 41
normal tissue, and the extent of promoter methylation negatively correlates with SERPINB1 mRNA 42
expression. 43
Implications: Our findings suggest that the balance between SERPINB1 and NE is physiologically 44
important within the prostate and may serve as a biomarker and therapeutic target in prostate cancer. 45
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Introduction 51
Prostate cancer is the most common non-cutaneous cancer in men in the United States, with more than 52
160,000 new cases diagnosed each year. While most localized prostate cancers have an indolent course, it 53
is essential to accurately assess which tumors will behave more aggressively [1]. The development of 54
biomarkers has revolutionized oncology; yet few useful prostate cancer biomarkers have emerged in 55
recent years. Biomarkers under investigation in prostate cancer include TMPRSS2:ERG rearrangement, 56
PC antigen 3 (PCA3), and the androgen receptor splice variant-7 (ARv7) [2]. Moreover, several genomic, 57
proteomic, and epigenetic tests such as the OncotypeDx, ProMark, and ConfirmMDx evaluate a panel of 58
biomarkers and attempt to stratify patients by risk [2]. At this time however, clinical stage, Gleason grade, 59
and serum prostate-specific antigen (PSA) remains the primary prognostic indicators used by clinicians to 60
guide therapeutic decisions [2]. 61
Growing evidence suggests that the tumor microenvironment contributes to cancer development and 62
progression. Thus, understanding the complex interactions between tumor cells and surrounding reactive 63
stroma may uncover novel biomarkers and therapeutic targets [3, 4]. While prostate cancer is defined as 64
an adenocarcinoma, tumors in fact have a significant non-malignant cellular compartment consisting of 65
fibroblasts, smooth muscle cells, endothelium, and immune cells [3, 4]. The tumor microenvironment is 66
also rich in non-cellular components such as extracellular matrix (ECM), cytokines, growth factors, and 67
proteases, which form fertile ground for cancer cell growth, migration, and invasion [5]. The cellular and 68
non-cellular components evolve over the natural course of cancer. Specifically, tumor and stromal cells 69
undergo genotypic and phenotypic changes, inflammatory cells infiltrate tumors, and the ECM becomes 70
remodeled [3, 4]. Despite progress in other solid tumors, much remains to be learned about 71
microenvironment changes in prostate cancer. 72
We recently showed that granulocytic myeloid cells (likely myeloid-derived suppressor cells or 73
MDSCs) accumulate peripherally in blood and locally in tumors using a xenograft mouse model of 74
human prostate cancer. These granulocytic myeloid cells directly promote tumor growth in the absence of 75
adaptive immunity, since tumor size is significantly reduced upon sustained myeloid cell depletion in 76
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athymic mice [6]. Furthermore, we demonstrated the importance of myeloid-derived neutrophil elastase 77
(NE) as a pro-tumorigenic factor for human prostate cancer cells, as NE promoted human prostate cancer 78
cell line growth, migration, and invasion, while NE inhibition with the drug sivelestat attenuated human 79
xenograft progression [6]. 80
Although accumulation of NE-producing myeloid cells may be sufficient to explain enhanced NE 81
activity within the tumor microenvironment, loss of endogenous NE inhibitors in tumor cells may further 82
shift the balance [5]. There are several endogenous NE inhibitors that are expressed by numerous tissue 83
types, including elafin (PI3), secretory leukocyte peptidase inhibitor (SLPI), and SERPINB1 [7-10]. We 84
chose to focus our investigation on the most recently identified NE-specific inhibitor, SERPINB1. 85
SERPINB1 is a 42-kD member of the Clade B family of serpins (serine protease inhibitors) that potently 86
inhibits NE and neutrophil extracellular trap (NET) formation similar in mechanism to sivelestat [10-12]. 87
SERPINB1 regulates neutrophil homeostasis within the bone marrow and promotes neutrophil survival 88
via intracellular inhibition of neutrophil serine proteases [13-15]. Interestingly, SERPINB1 is expressed 89
by many cell types beyond immune cells and detected both intra- and extra-cellularly [12, 16, 17]. While 90
SERPINB1 lacks a classical signal peptide, it is secreted via an unconventional caspase-1 dependent 91
secretory pathway that also mediates IL-1β and IL-18 secretion [17, 18]. The role of SERPINB1 in 92
epithelial cells and cancer is not clear; however, limited screening studies demonstrate reduced expression 93
in hepatocellular carcinoma, glioma, and melanoma compared to normal tissue counterparts, as well as 94
possible roles in suppressing apoptosis and promoting tumor migration [19-21]. Microarray analysis of 95
laser captured micro-dissected human prostate cancer specimens suggest that SERPINB1 mRNA 96
expression is reduced early in the development of prostatic intraepithelial neoplasia (PIN) and remains 97
low in prostate cancer [22]. 2D DIGE/MS proteomic analysis comparing prostate cancer to benign 98
prostatic hyperplasia (BPH) also suggests lower SERPINB1 expression in tumors [23]. Finally, meta-99
analysis of epithelial-to-mesenchymal transition (EMT) encompassing several cancers, including prostate, 100
identifies SERPINB1 as a commonly down-regulated gene [24]. However, all of the aforementioned 101
studies simply classify SERPINB1 as a gene on a long list of other dysregulated genes, and neither a 102
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functional role for SERPINB1 loss nor mechanisms for its repression have been elucidated in prostate 103
cancer. 104
Here we report that SERPINB1 expression is reduced in mouse Pten-null prostate tumors and treatment 105
with the NE inhibitor sivelestat (SERPINB1 pharmacomimetic) attenuates tumor growth. We confirm that 106
SERPINB1 is expressed in normal human prostatic epithelium but down-regulated in prostate cancer. We 107
demonstrate that SERPINB1 is secreted by non-malignant human prostate cells and its expression is 108
ERK1/2-dependent. Functionally, we show that SERPINB1 loss in non-malignant human prostate cells 109
induces EMT and promotes a proliferative phenotype. In contrast, SERPINB1 overexpression in human 110
prostate cancer cells suppresses xenograft progression. Finally, we find that SERPINB1 is epigenetically 111
repressed in human prostate cancer cells by EZH2-mediated histone methylation (H3K27me) and DNMT-112
mediated DNA methylation. Overall, our data suggest a novel inhibitory role for SERPINB1 in prostate 113
cancer progression, with potential application to both biomarker and therapeutic development. 114
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Materials and Methods 116
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Cell culture. RWPE-1 cells (ATCC) were cultured in keratinocyte serum free media (K-SFM; Gibco) 118
supplemented with 25 μg/mL bovine pituitary extract (BPE; Gibco), 5 ng/mL epidermal growth factor 119
(EGF; Gibco), and 1% penicillin-streptomycin (P-S; Gibco). C4-2 (Ganesh Raj, UTSW), PC3 (ATCC), 120
and LNCaP (ATCC) cells were cultured in RPMI-1640 media (Gibco) supplemented with 10% fetal 121
bovine serum (FBS; Seradigm) and 1% P-S. BPH-1 cells (Donald DeFranco, UPitt) were cultured in 5% 122
FBS and 1% P-S in RPMI-1640. VCaP, DU145, and CWR22Rv1 cells (Kent Nastiuk, UBuffalo) were 123
cultured in 10% FBS and 1% P-S in DMEM (Gibco). LAPC-4 cells (Kent Nastiuk, UBuffalo) were 124
cultured in 10% FBS and 1% P-S in RPMI-1640, 10 mM HEPES pH 7.4 (Gibco). Pten-Cap8 cells (Kent 125
Nastiuk, UBuffalo) were cultured in 10% FBS, 25 μg/mL BPE, 6 ng/mL EGF, 5 μg/mL human 126
recombinant insulin (Sigma), and 1% P-S in DMEM. All cells were maintained below passage 30 at 127
37oC, 95% air, 5% CO2. 128
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Cell treatments and inhibitors. Cells were serum starved for 24 hours and stimulated with 20 ng/mL EGF 130
(Corning) in serum free media for 4 or 24 hours before subsequent lysis and analysis by quantitative PCR 131
or Western, respectively. For inhibitor studies, cells were pre-incubated with 0.25 μM MEK inhibitor 132
PD0325901 (Selleckchem) or 0.25 μM PI3K inhibitor LY294002 (Sigma) for 30 minutes before the 133
addition of EGF. Cells were treated with 10 μM DNMT inhibitor 5-aza-2-deoxycytidine (Sigma) and 10 134
μM EZH2 inhibitor GSK343 (Selleckchem) in complete media for 72 hours. 135
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SERPINB1 knockdown. RWPE-1 cells were transfected with 25 nM Silencer Select Negative Control #1 137
siRNA (#4390843, Ambion) or 25 nM Silencer Select SERPINB1 siRNA (#4392420, Ambion) using 138
jetPRIME (Polyplus). Knockdown was carried out for 72-96 hours as specified. 139
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SERPINB1 cloning and stable overexpression. Human SERPINB1 was cloned via nested PCR. 141
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SERPINB1 cDNA was inserted into pcDNA3.1/Hygro(+) vector (Invitrogen). C4-2 cells were transfected 142
with SERPINB1-pcDNA3.1/Hygro(+) or empty vector using X-tremeGENE 9 DNA Transfection 143
Reagent (Sigma) and selected with 100 μg/mL hygromycin (Sigma). Monoclonal cell lines were 144
established and maintained in 10% FBS and 1% P-S in RMPI-1640 containing 25 μg/mL hygromycin. 145
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Conditioned media. RWPE-1 and C4-2 cells were grown to 80% confluence in respective complete 147
medias and then cultured without supplements for an additional 48 hours. Conditioned media were 148
collected, centrifuged at 2000 rpm for 10 minutes at 4oC, and concentrated 5-fold using Amicon Ultra 0.5 149
mL 3 kDa centrifugal filter units (Millipore). 150
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Gelatin zymography. Conditioned media samples were diluted 1:1 with 2X non-reducing, non-denaturing 152
sample buffer and separated on a 10% gel containing 1 mg/mL gelatin (Sigma). Zymograms were 153
performed and analyzed as previously described [25, 26]. 154
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Western blot. Cells were lysed in mammalian cell lysis buffer (Abcam) supplemented with 1X Halt 156
protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). Lysates or conditioned media were 157
diluted 1:1 with 2X sample buffer containing 2-mercaptoethanol (Sigma) and denatured. Samples were 158
processed for gel electrophoresis and blotted with mouse anti-SERPINB1 (1:2000, #TA800093, Origene), 159
rabbit anti-GAPDH (1:2000, #2118, Cell Signaling), rabbit anti-phospho-ERK1/2 (1:1000, #9101, Cell 160
Signaling), and rabbit anti-total-ERK1/2 (1:1000, #9102, Cell Signaling). 161
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Quantitative PCR. RNA was extracted using the E.Z.N.A. kit (Omega). Quantitative PCR (qPCR) was 163
performed using the qScript XLT 1-Step RT-qPCR ToughMix kit (QuantaBio) and TaqMan primers 164
(Applied Biosystems) for: human SERPINB1 (Hs00961948_m1), MMP9 (Hs00234579_m1), SNAI1 165
(Hs00195591_m1), TWIST1 (Hs01675818_s1), PI3 (Hs00160066_m1), SLPI (Hs00268204_m1), 166
GAPDH (Hs03929097_g1). Expression was normalized to GAPDH using the ∆∆Ct method. 167
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Chromatin Immunoprecipitation (ChIP) 168
ChIP for SERPINB1 was performed using the MAGnify Chromatin Immunoprecipitation System 169
(Invitrogen) [27]. Briefly, chromatin fragments were immunoprecipitated with mouse monoclonal anti-170
H3K27me3 ChIP-grade antibody (#6002, Abcam) or mouse IgG. Quantitative PCR was performed using 171
PerfeCTa SYBR Green SuperMix Reagent (QuantaBio) with primers directed against two regions of the 172
human SERPINB1 promoter (P1-forward-5’ CGT GCG ATT CTA GAG ACG ATT T 3’, P1-reverse-5’ 173
CGA GGA CAG GCA AAG AAG AA 3’; P2-primer – 5’ TCT GAG AGT GGA GAT CGA GAT G 3’, 174
P2-primer-5’ GGT GTA GGA TGT GCC AGT TT 3’). Results were normalized by the fold enrichment 175
method. 176
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Apoptosis. RWPE-1 cells were transfected with control and SERPINB1 siRNA for 72 hours. Cell pellets 178
were collected with 0.05% trypsin-EDTA (Gibco), neutralized with 2% FBS in PBS, and washed with 179
cold PBS. Apoptosis was assessed using the FITC Annexin V Apoptosis Detection Kit I (BD 180
Pharmingen). Briefly, cells were resuspended in 1X Annexin Binding Buffer at 1x106 cells/mL, stained 181
with PI and FITC Annexin V, collected on a LSRII flow cytometer (BD Biosciences), and analyzed with 182
FCS Express 6 software. 183
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Pyrosequencing. Cells were treated with 10 μM 5-aza-2'-deoxycytidine for 72 hours and harvested for 185
genomic DNA isolation with Blood and Cell Culture DNA Mini kit (Qiagen). 2 μg genomic DNA from 186
each sample was used for bisulfite conversion with EpiTect Fast DNA Bisulfite kit (Qiagen). PyroMark 187
PCR kit (Qiagen) was used for subsequent PCR reactions. Three pairs of primers amplifying three 188
regions, each containing 2 CpG sites within 1500bp upstream of TSS, were designed with Qiagen 189
PyroMark Assay Design Software 2.0. PyroMark Q24 sequencer (Qiagen) was used to analyze amplified 190
PCR products, and methylation of CpG sites was determined with PyroMark Q24 Advanced software. 191
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Proliferation assay. RWPE-1 cells were transfected with control and SERPINB1 siRNA for 72 hours. 193
Proliferation was assessed using the BrdU Cell Proliferation Assay Kit (Cell Signaling) [6]. 194
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Animal studies. Experiments were performed in accordance with the guidelines for the Care and Use of 196
Laboratory Animals and approved by the University Committee on Animal Resources at the University of 197
Rochester. 6-8 week old male athymic nude mice were subcutaneously injected with 2x106 C4-2 cells 198
(expressing SERPINB1 or vector) in 0.1 mL of a 1:1 mixture of Matrigel (Corning) and PBS. Tumor 199
growth was monitored over 12 weeks. Once largest tumors reached end point (~2000 mm3), all tumors 200
were harvested. Work with prostate-specific PbCre4/Ptenfl/fl mice in C57/BL6 background [28] was 201
approved by the Roswell Park IACUC. Tumor volume was monitored using 3D ultrasound [29]. Mice 202
bearing tumors of 300-500 mm3 were blindly randomized to vehicle (PBS) or sivelestat (Tocris; 5 mg/kg 203
in PBS) treatment daily via intraperitoneal injection. Tumor volume was monitored weekly via 204
ultrasound. 205
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Flow cytometry for MDSCs. Blood was collected from retro-orbital sinuses at indicated times and 207
processed for CD11b, Ly6G, and Ly6C staining as previously described [6]. 208
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Immunohistochemistry. Sections were de-paraffinized and rehydrated [6, 25]. Heat-mediated antigen 210
retrieval was performed in 0.01 M Citrate pH 6 at 95oC. For human tissues, mouse anti-human 211
SERPINB1 (#TA800093, Origene) was diluted 1:200 in antibody diluent (Thermo Scientific) and 212
incubated overnight at 4oC. Biotinylated horse anti-mouse IgG (#BA-2000, Vector Laboratories) was 213
diluted 1:200 in blocking serum (2.5% normal horse serum in PBS, Vector Laboratories). For mouse 214
tissues, rabbit anti-mouse SERPINB1 (#TA340203, Origene) was diluted 1:200 in antibody diluent and 215
incubated overnight at 4 oC. Biotinylated goat anti-rabbit IgG (#BA-1000, Vector Laboratories) was 216
diluted 1:200 in blocking serum (2.5% normal goat serum in PBS, Vector Laboratories). 217
Immunoreactivity was detected using the Vectastain Elite ABC and DAB peroxidase substrate kits 218
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(Vector Laboratories). Sections were counterstained with hematoxylin and mounted using Cytoseal 60 219
(ThermoFisher Scientific). 220
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Immunofluorescence. Primary antibodies were: biotin rat anti-mouse Ly6G (1:50, #127604, BioLegend) 222
and goat anti-mouse proliferating cell nuclear antigen (PCNA; 1:50, #sc-9857, Santa Cruz 223
Biotechnology). Primary antibodies were detected using streptavidin-Alexa 488 (1:200, #S11223, 224
ThermoFisher Scientific), donkey anti-rat Alexa Fluor 488 (1:200, #A21208, ThermoFisher Scientific), 225
and donkey anti-goat Alexa Fluor 568 (1:200, #A11057, ThermoFisher Scientific). Sections were 226
counterstained and mounted using VECTASHIELD antifade mounting media with DAPI (Vector 227
Laboratories). 228
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NE imaging. Mice received 4 nanomoles of Neutrophil Elastase 680 FAST probe (Perkin Elmer) in 0.1 230
mL PBS via tail-vein injection. Activity was measured on excised tumors using fluorescent microscopy 231
[6, 25] and intensity was analyzed using ImageJ v1.48 software. 232
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Gene expression profiling. Microarray datasets of prostate adenocarcinoma and normal tissue were 234
accessed through the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus 235
(GEO) and queried for SERPINB1 expression. Chandran et al (GEO accession number: GSE6919 [30]), 236
Varambally et al (GEO accession number: GSE3325 [31]), Arredouani et al (GEO accession number: 237
GSE55945 [32]), and Taylor et al (GEO accession number: GSE21036 [33]) expression datasets were 238
analyzed. Kaplan–Meier analysis for recurrence-free survival was performed using the Taylor et al dataset 239
through the open web interface Project Betastasis (http://www.betastasis.com). Analysis for DNA 240
methylation at the SERPINB1 promoter and SERPINB1 expression in cancer and normal tissue was 241
performed using The Cancer Genome Atlas (TCGA) dataset through MethHC 242
(http://methhc.mbc.nctu.edu.tw/php/index.php) [34]. 243
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Statistical analysis. Data are presented as mean ± standard error of the mean (SEM). Comparison between 245
two groups was performed using two-tailed t-test, unless otherwise indicated. Comparison between more 246
than two groups was performed using one-way ANOVA with appropriate post-hoc testing. Statistical 247
analyses were performed using GraphPad Prism 7.0 software, and significance defined as p<0.05. 248
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Results 251
Neutrophil elastase (NE) inhibition reduces prostate tumor growth in immunocompetent Pten-null mice 252
We recently demonstrated that NE inhibition with sivelestat reduces human prostate cancer xenograft 253
growth in athymic mice. Furthermore, as in our xenograft models, we showed that granulocytic myeloid 254
cells (likely myeloid-derived suppressor cells or MDSCs) are elevated in peripheral blood and infiltrate 255
prostate tumors of immunocompetent probasin-driven Pten-null mice. Accordingly, Pten-null prostate 256
tumors exhibit enhanced NE activity [6]. 257
To determine whether NE is a pro-tumorigenic factor in Pten-null mice, we treated mice with 258
sivelestat or vehicle and monitored tumor growth over eight weeks (Fig. 1A). Moreover, we assessed 259
dynamics of peripheral blood MDSC accumulation via flow cytometry at zero, four, and eight weeks of 260
treatment (Fig. 1A). We examined prostates by ultrasound to measure tumor volumes and initiated 261
treatment at ~400 mm3. Tumors were smaller in sivelestat versus vehicle treated mice throughout the 262
study, reaching statistical significance by the final two time points (Fig. 1B), suggesting that NE indeed 263
promotes tumor growth in a prostate cancer mouse model with an intact immune system. Interestingly, we 264
observed time dependent accumulation of MDSCs (CD11b+/Ly6G+/Ly6C+) in the blood of both 265
sivelestat and vehicle treated animals (Fig. 1C), confirming that NE inhibition does not mitigate tumor 266
induced MDSC production. Examination of MDSC infiltration within tumors via Ly6G 267
immunofluorescence revealed that, while there was no difference between sivelestat and vehicle treated 268
tumors (not shown), there was a strong positive correlation between infiltrating MDSCs and expression of 269
proliferating cell nuclear antigen (PCNA) in prostate cells (Fig. 1D & E), supporting the hypothesis that 270
MDSCs contribute to tumor proliferation. 271
Since sivelestat is a pharmacomimetic of the endogenous NE inhibitor SERPINB1 [35], we next 272
examined SERPINB1 expression via immunohistochemistry in untreated Pten-null tumors and wild type 273
mouse prostates. SERPINB1 strongly stained glandular epithelium of normal prostates but was reduced in 274
the overgrown epithelium of Pten-null tumors (Fig. 1F & Supp. Fig. 1). We confirmed by Western blot 275
that SERPINB1 was reduced in Pten-null tumors and the Pten-null prostate cancer cell line Pten-CaP8 276
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compared to wild type prostate (Fig. 1G), suggesting that epithelial down-regulation of SERPINB1, along 277
with increased MDSC infiltration, may contribute to the observed enhanced NE activity in Pten-null 278
prostate tumors [6]. 279
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SERPINB1 mRNA expression is reduced and predicts poor prognosis in human prostate cancer 281
To determine whether SERPINB1 down-regulation occurs in human prostate cancer, we queried 282
public datasets of prostate adenocarcinoma and normal tissue. Interestingly, SERPINB1 mRNA 283
expression was reduced in prostate cancer compared to normal tissue in all four datasets, though the 284
extent and pattern of reduction varied (Fig. 2A-D). Furthermore, SERPINB1 mRNA expression was 285
lowest in metastatic prostate cancer (Fig. 2A, 2B, & 2D). Accordingly, using the Taylor et al dataset that 286
contains clinical patient parameters, we found that low SERPINB1 mRNA expression significantly 287
correlated with diminished recurrence free survival using both 50% and 25% cut-off thresholds (Fig. 2E). 288
To begin elucidating the functional significance of SERPINB1, we performed Western blots for 289
SERPINB1 in human non-malignant and malignant cell lines. The most commonly used non-malignant 290
prostatic epithelial cell line RWPE-1 exhibited strong SERPINB1 expression (Fig. 2F). The benign 291
prostatic hyperplasia cell line BPH-1 expressed lesser amount of SERPINB1. Of the seven prostate cancer 292
cell lines tested, four did not express detectable SERPINB1 (LNCaP, C4-2, VCaP, and LAPC-4), two 293
expressed low levels of SERPINB1 (DU-145 and PC3), and one expressed equal amount of SERPINB1 294
(22Rv1), relative to RWPE-1. 295
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SERPINB1 loss stimulates epithelial-to-mesenchymal (EMT) transition and proliferative phenotype in 297
non-malignant prostate cells 298
Since non-malignant RWPE-1 cells express abundant SERPINB1, we knocked down both cellular 299
and secreted SERPINB1 expression by siRNA to determine how gene expression and physiology would 300
be altered (Fig. 3A). Similar to gene arrays in other cancers [19, 20, 24], we found that mRNA expression 301
of EMT-related genes MMP9, TWIST1, and SNAI1 were induced by SERPINB1 knockdown (Fig. 3B). 302
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Gelatin zymography on conditioned media collected from SERPINB1 and NSP siRNA treated RWPE-1 303
cells confirmed increased expression of pro-MMP9, but not pro-MMP2, activity (Fig. 3C, quantified in 304
Fig. 3D). Since SERPINB1 has also been implicated in cell viability [12], we assessed effects of 305
SERPINB1 loss on apoptosis via flow cytometry for PI and Annexin V. Apoptotic cells (Annexin V+/PI-) 306
were significantly reduced by ~36% in response to SERPINB1 knockdown (Fig. 3E, quantified in Fig. 307
3F). Furthermore, proliferation by BrdU was increased by ~39% with SERPINB1 knockdown (Fig. 3G). 308
Together, these data suggest that SERPINB1 loss contributes to EMT, enhanced proliferation, and 309
reduced apoptosis in normal prostatic epithelial cells and may consequently drive malignant 310
transformation. 311
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SERPINB1 expression is induced via ERK1/2 signaling in normal prostate but not prostate cancer cells 313
To determine how SERPINB1 is repressed in prostate cancer, we first examined regulation of 314
SERPINB1 expression in non-malignant prostatic epithelial cells. We focused on epidermal growth factor 315
(EGF) induced pathways, hypothesizing that enhanced growth factor signaling as seen in cancers might 316
suppress SERPINB1 expression. Unexpectedly, EGF significantly induced SERPINB1 as measured by 317
qPCR and Western blot in RWPE-1 cells (Fig. 4A & 4B). EGF induced expression of mRNAs encoding 318
other endogenous NE inhibitors, PI3 (elafin) and SLPI (secretory leukocyte protease inhibitor), though 319
less dramatically (Fig. 4A). In contrast, EGF did not increase SERPINB1 mRNA or SERPINB1 protein 320
expression in PC3 (Fig. 4C & 4D) or DU145 prostate cancer cells (Supp. Fig. 2A & 2B), which express 321
low but detectable SERPINB1 at baseline (Fig. 2F). Moreover, EGF was unable to induce SERPINB1 322
mRNA in LNCaP prostate cancer cells that do not express endogenous SERPINB1 (Supp. Fig. 2C). In 323
RWPE-1 cells, MEK-inhibition by PD0325901 abrogated EGF-induced SERPINB1 transcription whereas 324
the PI3K inhibitor had no effect (Fig. 4E), with similar but less dramatic results seen when examining PI3 325
and SLPI transcription (Fig. 4E). Thus, SERPINB1 expression in non-malignant RWPE-1 cells may be 326
regulated in part by ERK1/2 signaling. 327
328
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SERPINB1 is repressed by EZH2-mediated histone methylation and DNMT-mediated DNA methylation 329
in prostate cancer 330
If EGF was not promoting SERPINB1 expression in tumor cells, then we hypothesized that some 331
signal was preventing the EGF effects. Tumor suppressors are frequently turned off via epigenetic 332
alterations such as histone and DNA methylation in cancers. Because EZH2 is up-regulated in and 333
considered a driver of prostate cancer, we performed chromatin immunoprecipitation (ChIP) for histone 3 334
lysine 27 tri-methylation (H3K27me3) on the SERPINB1 promoter in non-malignant RWPE-1 versus 335
castration resistant prostate cancer C4-2 cells. We observed marked enrichment of H3K27me3 in C4-2 336
compared to RWPE-1 cells (Fig. 5A). 72-hour treatment of C4-2 cells with an EZH2 inhibitor (GSK343) 337
modestly induced expression of SERPINB1 mRNA, with minimal induction of PI3 and SLPI mRNA (Fig. 338
5B). Since this effect was relatively small, we examined the effects of DNA methyltransferase (DNMT) 339
inhibition on SERPINB1 expression. 72-hour treatment of C4-2 cells with 5-aza-2-deoxycytidine (5-AZA) 340
enhanced SERPINB1 mRNA expression by over 2,000-fold, comparable to transcript levels detected in 341
RWPE-1 cells (Fig. 5C). Moreover, 5-AZA, but not GSK343, enhanced SERPINB1 protein expression in 342
C4-2 cells (Fig. 5D). Similar effects on mRNA levels were observed in LNCaP prostate cancer cells 343
(Supp. Fig. 3A & 3B). 344
Accordingly, pyrosequencing analysis on the SERPINB1 promoters revealed that CpG1, CpG2, and 345
CpG3, the islands closest to the transcription start site, had markedly elevated DNA methylation in C4-2 346
cells compared to non-malignant RWPE-1 cells (Fig. 5E). 22Rv1 cancer cells that expressed comparable 347
level of SERPINB1 as RWPE-1 also exhibited a similar pattern of CpG methylation as RWPE-1 (Fig. 348
5E). Conversely, LNCaP cancer cells that lacked endogenous SERPINB1 had a methylation pattern that 349
closely resembled C4-2 cells (Fig. 5E). Furthermore, in assessing DNA methylation at the SERPINB1 350
promoter in various cancers using TCGA data available through the open web interface MethHC, only 351
prostate adenocarcinoma (PRAD) exhibited increased methylation relative to adjacent normal tissue (Fig. 352
5G). Similar to individual prostate cancer expression datasets (Fig. 2A-D), SERPINB1 mRNA expression 353
was significantly reduced in tumor compared to normal samples using the PRAD TCGA (Fig. 5F), with 354
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SERPINB1 promoter methylation negatively correlating with SERPINB1 mRNA expression in tumors 355
(Fig. 5H). These data suggest that DNA methylation may silence SERPINB1 expression specifically in 356
human prostate cancer versus other cancers. 357
358
SERPINB1 overexpression reduces prostate cancer xenograft growth in vivo 359
We next determined whether rescuing SERPINB1 expression in normally low-expressing prostate 360
cancer cells would diminish tumor burden. We therefore generated two stable monoclonal cell lines 361
overexpressing SERPINB1 or vector control in C4-2 cells (Fig. 6A). Notably, these cells expressed and 362
secreted SERPINB1 just like RWPE-1 (Fig. 6A). Xenografts expressing SERPINB1 were markedly 363
smaller than vector control xenografts after 12 weeks of growth in athymic nude mice (Fig. 6B & D). 364
Stable SERPINB1 overexpression was verified in tumors of both clonal cell lines collected at the end of 365
the experiment (Fig. 6C & E). Using a NE activity probe [6], we observed reduced NE activity in 366
SERPINB1 overexpressing tumors compared to vector controls (Fig. 6F and 6G, quantified), suggesting 367
that SERPINB1 inhibits local NE activity to attenuate prostate cancer xenograft growth. 368
369
SERPINB1 is expressed by normal human prostate glands but reduced in prostate cancer 370
Finally, we confirmed loss of SERPINB1 expression in prostate cancer by performing 371
immunohistochemistry on prostatectomy and core biopsy specimens that contain regions of both normal 372
and malignant tissue. As expected, SERPINB1 was localized to cells lining normal prostate glands and 373
exhibited diffuse stromal staining, both of which were largely absent in cancer (Fig. 7). Scattered 374
SERPINB1 positive cells present in cancer regions were likely infiltrating immune cells expressing 375
SERPINB1. Therefore, epithelial and/or stromal down-regulation of SERPINB1 may serve as a novel 376
biomarker for prostate cancer progression. 377
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Discussion 378
Granulocytic myeloid cells or MDSCs are increased in the circulation, peripheral organs, and primary 379
tumors in mouse models of numerous malignancies, including prostate cancer [36, 37]. MDSCs 380
contribute to cancer progression and metastasis indirectly via T cell immunosuppression and directly via 381
release of pro-inflammatory cytokines, growth factors, and proteases such as matrix metalloproteinases 382
(MMPs) and neutrophil elastase (NE). Consequently, targeting MDSC production and recruitment leads 383
to decelerated tumor growth and reduced metastases [5, 36]. We reported that myeloid-derived NE 384
directly promotes human prostate cancer growth in athymic mice, and that sivelestat (NE inhibitor) 385
reduces tumor burden to the same extent as anti-Gr-1 MDSC depletion [6]. We now demonstrate that 386
targeting NE activity with sivelestat in the immunocompetent Pten-null mouse model of prostate cancer 387
similarly attenuates tumor progression without altering levels of circulating or infiltrating MDSCs. 388
Therefore, tumors continue to recruit pro-tumorigenic MDSCs despite reduced NE activity, which likely 389
explains why the tumor inhibitory effect of sivelestat is somewhat diminished by the later time points 390
when the MDSC burden is too great. MDSC depletion via antibodies against CXCL5 and Gr-1 or 391
inhibitors targeting CXCR2 similarly result in modestly decelerated prostate tumor growth in Pten-null 392
mouse models [38-40]. Unfortunately, MDSC levels rebound after pro-longed antibody-mediated 393
depletion, thus the translational potential of such therapy is uncertain [36]. Therefore, we postulate that 394
perhaps dual targeting of MDSCs and NE activity will lead to synergistic growth inhibitory effects. 395
Local MDSC recruitment likely drives the preponderance of immune-derived proteases in prostate 396
and other cancers, and in fact we find that, in Pten-null mice, prostate cancer cell proliferation directly 397
correlates with the density of surrounding MDSCs. Alternatively, while inflammatory protease levels are 398
increasing in the tumor microenvironment, expression of endogenous protease inhibitors by epithelial 399
cells is often suppressed, favoring enhanced proteolytic activity and resultant cancer cell proliferation, 400
migration, and invasion [41]. For instance, loss of Timp3, an endogenous MMP inhibitor, accelerates 401
tumor growth and invasion of Pten-null prostate tumors via up-regulated MMP activity [42]. Here, we are 402
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first to demonstrate that SERPINB1, an endogenous NE inhibitor, is reduced in Pten-null mouse tumors, 403
which might further explain their heightened intra-tumoral NE activity [6]. 404
SERPINB1 is a potent regulator of neutrophil serine proteases and neutrophil extracellular trap (NET) 405
formation [13]. Consequently, SERPINB1 contributes to the resolution of both acute infections and 406
chronic inflammatory diseases, similar to the therapeutic effects of its pharmacomimetic sivelestat [12]. 407
SERPINB1 is widely expressed in various cell types, and several studies suggest a role in cancer 408
progression separate from its NE inhibitory function. In hepatocellular carcinoma (HCC), glioma, and 409
melanoma, SERPINB1 down-regulation in tumors is associated with poor patient prognosis [19-21]. 410
Intriguingly, two exploratory studies identify SERPINB1 as a down-regulated gene, among many others, 411
in prostate cancer compared to non-malignant prostate [22, 23]. We now demonstrate that SERPINB1 is 412
in fact reduced in human prostate cancer compared to normal prostatic epithelium using a variety of 413
transcriptional and translational approaches. SERPINB1 is further reduced in metastatic disease, and low 414
expression predicts diminished recurrence free survival in patients. Immunohistochemistry staining 415
reveals SERPINB1 is strongly expressed by the basal layer in normal prostate glands, which is largely 416
absent in regions of cancer. Interestingly, there is strong evidence for the basal progenitor as the cell type 417
of origin in both mouse models and human prostate cancer [43, 44]. Moreover, loss of tumor suppressor 418
Pten in basal cells promotes basal-to-luminal differentiation and development of invasive prostate cancer 419
in mice [45]. Accordingly, we observe decreased SERPINB1 expression in mouse Pten-null prostates as 420
well as a Pten-null prostate cell line. It is therefore possible that SERPINB1 loss also plays a critical role 421
during prostate tumorigenesis, though this remains to be carefully dissected. 422
The functional role of SERPINB1 in cancer at this time is unclear, though limited studies suggest an 423
involvement in regulation of cell motility. For instance, SERPINB1 reduction in hepatocellular carcinoma 424
(HCC) and glioma cells enhances migration and invasion in vitro, potentially through increased MMP-2 425
expression [19, 20]. Conversely, SERPINB1 overexpression in lung and breast cancer cells decreases 426
migratory and invasive capacity [46]. Here we demonstrate that SERPINB1 loss in non-malignant 427
prostatic epithelial cells induces expression of EMT markers, including MMP-9, TWIST1, and SNAI1. 428
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EMT is the process by which polarized epithelial cells acquire a mesenchymal, pro-migratory, and pro-429
invasive phenotype, and activation of an EMT program is considered a critical step in malignant 430
transformation [47, 48]. Therefore, as mentioned above, loss of SERPINB1 may be an early event during 431
tumorigenesis. Accordingly, a meta-analysis of 18 independent gene expression datasets of EMT 432
identified SERPINB1 as a commonly down-regulated gene in various cancers [24]. 433
In addition to EMT, two hallmarks of cancer are the ability of tumor cells to resist cell death and sustain 434
proliferative signaling [48]. Here, we report that SERPINB1 loss impedes apoptosis and stimulates 435
proliferation in normal prostatic epithelial cells. SERPINB1 is proposed to control cell viability via 436
interaction with several proteins involved in apoptosis, including PARP-1, BCL-2, and apoptosis inducing 437
factor (AIF), among others [12]. Moreover, SERPINB1 loss induces proliferation in keratinocytes by 438
promoting G1/S cell cycle transition. [49]. Nonetheless, the effect of ectopic SERPINB1 overexpression 439
on tumor growth in vivo has not been previously examined. Here, we demonstrate that stable ectopic 440
expression of SERPINB1 in prostate cancer cells with low endogenous SERPINB1 expression reduces 441
xenograft growth and NE activity in vivo. We also report SERPINB1 secretion by both normal prostatic 442
epithelial cells and ectopically expressing SERPINB1 prostate cancer cells. Together, these data suggest 443
that extracellular SERPINB1 may act as a functional NE inhibitor in vivo. Consequently, down-regulation 444
of SERPINB1 by cancer cells may permit NE activation within the tumor microenvironment to enhance 445
tumor progression. 446
Still, how SERPINB1 is regulated in normal tissues and cancer is not well understood. In this study, 447
we demonstrate that SERPINB1 is strongly induced by epidermal growth factor (EGF) signaling through 448
the ERK1/2 pathway in non-malignant but not malignant prostatic epithelial cells. Furthermore, we 449
demonstrate a novel mechanism of SERPINB1 repression in prostate cancer cells via EZH2-mediated 450
histone methylation (H3K27me) and DNA methyltransferase (DNMT)-mediated DNA methylation. 451
While EZH2 inhibition modestly induces SERPINB1 expression, DNMT inhibition induces SERPINB1 452
expression by over 2,000-fold and rescues protein expression from previously undetectable levels. EZH2 453
may facilitate gene silencing directly through histone methylation and indirectly by serving as a platform 454
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20
for DNMT complex recruitment [50]. Thus, the two epigenetic repression systems may be functionally 455
linked, and both are likely responsible for SERPINB1 silencing during the complex process of tumor 456
initiation and progression. Accordingly, EZH2 and various DNMTs (i.e. DNMT1, DNMT3A, DNMT3B) 457
are overexpressed in mouse and human prostate cancers [51, 52]. 458
Remarkably, SERPINB1 promoter methylation appears to be specific to prostate cancer, as no other 459
human cancers examined through the TCGA exhibit such a stark difference compared to their normal 460
tissue counterparts. Not surprisingly, we observe that promoter methylation and SERPINB1 expression 461
are tightly correlated in prostate cancer. Therefore, promoter methylation may serve as a surrogate marker 462
of SERPINB1 expression and have diagnostic and prognostic potential. Furthermore, SERPINB1 down-463
regulation, combined with elevated markers of inflammation such as neutrophil to lymphocyte ratio 464
(NLR), may identify prostate cancer patients who will best respond to MDSC or NE-targeting therapy. 465
Better mechanistic understanding of the methyltransferases responsible for SERPINB1 repression will be 466
advantageous for future drug and diagnostic test development. Analogously, GSTP1 is a tumor suppressor 467
silenced via promoter methylation in prostate cancer, and this epigenetic alteration may be evaluated 468
clinically through the ConfirmMDx assay [53]. 469
In summary, our findings demonstrate that SERPINB1 is a novel tumor suppressor that is 470
epigenetically silenced in prostate cancer. While SERPINB1 loss may permit NE-driven prostate cancer 471
progression, it also exhibits significant NE-independent tumor promoting effects. Importantly, 472
SERPINB1 expression and promoter methylation status may serve as a potential biomarker to guide 473
therapeutic decisions. 474
475
Acknowledgements 476
S. Hammes was supported by NIH grants R01GM101709 and R01CA193583-01A1. I. Lerman was 477
supported by NIH grant F30CA203517. 478
479
480
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Figure Legends 599
600
Figure 1. Neutrophil elastase (NE) inhibition reduces prostate tumor growth in immunocompetent Pten-601
null mice. A. Schematic illustrating important time points during the Pten-null experiment. Mice bearing 602
tumors 300 - 500mm3 were randomized and treated daily with vehicle (PBS) or sivelestat (5mg/kg in 603
PBS) via intraperitoneal (IP) injection. B. Ultrasound measurements were performed biweekly and are 604
presented as percent volume increase from week 0. Tumor volume was compared using ANOVA with 605
post-hoc Tukey HSD (n = 8 for vehicle, n = 8 for sivelestat; * p < 0.05). C. Peripheral blood MDSCs 606
were assessed using flow cytometry at week 0, 4, and 8. The percent of Ly6G+/Ly6C+ cells at week 8 607
was compared to week 4 using ANOVA with post-hoc Tukey HSD (n = 8 for vehicle, n = 8 for sivelestat; 608
*** p < 0.001 for vehicle ## p < 0.01 for sivelestat). D. Representative immunofluorescence stain for 609
Ly6G positive infiltrating granulocytic MDSCs and PCNA positive proliferating epithelium in Pten-null 610
prostates is shown. E. The number of PCNA positive cells versus the number of Ly6G positive cells per 611
field of view (FOV) were plotted to examine correlation using two-tailed Pearson correlation analysis (n 612
= 20; r = 0.7724, p < 0.0001). F. Representative immunohistochemistry stain for SERPINB1 in wildtype 613
(WT) and Pten-null prostates is shown (n = 3 with identical results). G. Western blot for SERPBIN in 614
wild-type prostate (WT), Pten-null prostates, and the Pten-null mouse derived CaP8 cell line. 615
616
Figure 2. SERPINB1 expression is reduced and predicts poor prognosis in human prostate cancer. 617
Human prostate cancer SERPINB1 mRNA expression data were obtained from (A) Varambally et al 618
(Cancer Cell, 2005 [31]), (B) Chandran et al (BMC Cancer, 2007 [30]), (C) Arredouani et al (Clin Cancer 619
Res, 2009 [32]), and (D) Taylor et al (Cancer Cell, 2010 [33]) through NCBI GEO. Differences between 620
groups were assessed using ANOVA with post-hoc Holm-Sidak in A, B, and D. In C, difference was 621
assessed using Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. E. Kaplan-Meier plots for patients 622
expressing SERPINB1 above (high) and below (low) the first quartile (left panel) and median (right panel) 623
were constructed with Taylor et al data via the web interface Betastasis (http://www.betastasis.com ). 624
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27
Differences in recurrence free survival were assessed using the log-rank (Mantel-Cox) test. F. SERPINB1 625
expression in normal and prostate cancer cell lines were examined via Western blot; 1: RWPE-1, 2: BPH-626
1, 3: LNCaP, 4: C4-2, 5: DU145, 6: PC3, 7: 22Rv1, 8: VCaP, 9: LAPC-4. 627
628
Figure 3. SERPINB1 loss stimulates EMT and a proliferative phenotype in normal prostate cells. A. 629
RWPE-1 cells were transfected with non-specific (NSP) and SERPINB1-specific siRNA, and knockdown 630
was verified using Western blot. GAPDH was used as a loading control. A representative blot is shown. 631
WCL=whole cell lystate, CM=complete media (secreted SERPBINB1). B. mRNA expression of 632
SERPINB1 and EMT markers MMP9, TWIST1, SNAI1 were determined in RWPE-1 cells after 633
SERPINB1 knockdown using quantitative PCR and normalized to GAPDH. Data were normalized to 634
NSP treated samples, and differences were assessed using Student’s t-test (n = 7; * p < 0.05, **** p < 635
0.0001). C. Expression and activity of MMP2 and MMP9 were determined in RWPE-1 cells after 636
SERPINB1 knockdown using gelatin zymography. A representative gel is shown. D. Band densitometry 637
of MMP9 and MMP2 was performed using ImageJ, and normalized differences were assessed using 638
Student’s t-test (n = 3; ** p < 0.01, n.s. = not significant). E. Apoptosis was assessed in RWPE-1 cells 639
after transfection with NSP and SERPINB1 siRNA via PI and Annexin V staining. Representative plots 640
are shown. F. Difference in apoptosis (% Annexin V+/PI- cells) was determined using Student’s t-test (n 641
= 3; **** p < 0.0001). G. Proliferation was assessed in RWPE-1 cells after transfection with NSP and 642
SERPINB1 siRNA via colorimetric BrdU incorporation assay. Data were normalized to NSP treated 643
samples, and difference was assessed using Student’s t-test (n = 3; ** p < 0.01). 644
645
Figure 4. SERPINB1 expression is induced via ERK1/2 signaling in normal prostate but not prostate 646
cancer cells. A. RWPE-1 cells were serum starved and treated with epidermal growth factor (EGF; 20 647
ng/mL) for 4 hours. mRNA expression of SERPINB1, PI3, and SLPI were determined using quantitative 648
PCR and normalized to GAPDH. Data were normalized to untreated samples, and differences were 649
assessed using Student’s t-test (n = 3; ** p < 0.01, *** p < 0.001). B. RWPE-1 cells were serum starved 650
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28
and left untreated (UT) or treated with EGF (20 ng/mL) for 24 hours. SERPINB1 expression was 651
determined using Western blot. GAPDH was used as a loading control. A representative blot with two of 652
each treatment condition is shown. C. PC3 cells were serum starved and left untreated or treated with 653
EGF (20 ng/mL) for 4 hours. mRNA expression of SERPINB1, PI3, and SLPI were determined using 654
quantitative PCR and normalized to GAPDH. Data were normalized to untreated samples, and differences 655
were assessed using Student’s t-test (n = 3, n.s. = not significant). D. PC3 cells were serum starved and 656
treated with EGF (20 ng/mL) for 24 hours. SERPINB1 expression was determined using Western blot. 657
GAPDH was used as a loading control. A representative blot with two of each treatment is shown. E. 658
RWPE-1 cells were serum starved and treated with EGF (20 ng/mL) in the presence of MEK inhibitor 659
PD0325901 (0.25 μM) or PI3K inhibitor LY294002 (0.25 μM) for 4 hours. mRNA expression of 660
SERPINB1, PI3, and SLPI were determined using quantitative PCR and normalized to GAPDH. Data 661
were normalized to untreated samples, and differences were assessed using ANOVA with post-hoc Tukey 662
(**** p < 0.0001, n.s. = not significant). F. RWPE-1 cells were serum starved and treated with EGF (20 663
ng/mL) in the presence of PD0325901 (0.25 μM) or LY294002 (0.25 μM) for 30 minutes. pERK1/2 and 664
tERK1/2 levels were examined via Western blot. 665
666
Figure 5. SERPINB1 is epigenetically repressed by EZH2-mediated histone methylation and DNMT-667
mediated DNA methylation in prostate cancer. A. Chromatin immunoprecipitation for H3K27me3 on the 668
SERPINB1 promoter was performed in RWPE-1 and C4-2 cells. Data are presented as fold enrichment 669
over IgG controls, and the average of two experiments is shown. B. C4-2 cells were treated with EZH2 670
inhibitor GSK343 (10 μM) for 72 hours. mRNA expression of SERPINB1, PI3, and SLPI were 671
determined using quantitative PCR and normalized to GAPDH. Data were normalized to untreated 672
samples, and differences were assessed using Student’s t-test (n = 3; * p < 0.05, ** p < 0.01, *** p 673
<0.001). C. C4-2 cells were treated with DNMT inhibitor 5-aza-2-deoxycytidine (5-AZA, 10 μM) for 72 674
hours. mRNA expression of SERPINB1, PI3, and SLPI were determined using quantitative PCR and 675
normalized to GAPDH. Data were normalized to untreated samples, and differences were assessed using 676
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29
Student’s t-test (n = 3; *** p < 0.001, **** p < 0.0001). D. C4-2 cells were treated with GSK343 and 5-677
AZA (10 μM) for 72 hours. SERPINB1 expression was determined using Western blot. GAPDH was 678
used as a loading control. A representative blot is shown E. Promoter methylation at six CpG sites in C4-679
2, LNCaP, 22Rv1, and RWPE-1 cells was measured using pyrosequencing (CpG1 is the island closest to 680
the transcription start site), and differences were determined using Student’s t-test (n = 4, *** p < 0.001). 681
F. SERPINB1 expression in TCGA PRAD was assessed, and difference was determined using Student’s t-682
test (**** p < 0.0001). G. Analysis for DNA methylation at the SERPINB1 promoter in normal and 683
cancer samples of various malignancies was performed using The Cancer Genome Atlas (TCGA) dataset 684
through the open web interface MethHC (http://methhc.mbc.nctu.edu.tw/php/index.php) [34]. PRAD - 685
prostate adenocarcinoma, BRCA - breast invasive carcinoma, LUAD - lung adenocarcinoma, COAD - 686
colon adenocarcinoma, BLCA – bladder urothelial carcinoma, and PAAD - pancreatic adenocarcinoma. 687
Differences were assessed using Student’s t-test (**** p < 0.0001, n.s. = not significant). H. SERPINB1 688
methylation versus expression were plotted to examine correlation using two-tailed Pearson correlation 689
analysis (r = -0.673, p < 0.0001). 690
691
Figure 6. SERPINB1 overexpression reduces prostate cancer xenograft growth in vivo. A. SERPINB1 692
was detected in whole cell lysates (WCL) and concentrated conditioned media (CM) collected over 24 693
hours in C4-2 cells stably expressing SERPINB1 (SERPINB1 clones B2 and B4) and vector controls 694
(Vector clones A3 and B3). GAPDH was used as a loading control. Representative blots are shown. B. 695
Tumor weight was compared between Vector A3 and SERPINB1 B2 xenografts injected subcutaneously 696
into the flanks of athymic nude mice using Student’s t-test (n = 10 for Vector, n = 3 for SERPINB1; * p < 697
0.05). C. SERPINB1 was detected in harvested tumors at the end of the experiment using Western blot. A 698
representative blot is shown. D. Tumor weight was compared between Vector B3 and SERPINB1 B4 699
xenografts injected subcutaneously into the flanks of athymic nude mice using Student’s t-test (n = 10 for 700
Vector, n = 11 for SERPINB1; p = 0.149). SERPINB1 was detected in harvested tumors at the end of the 701
experiment using Western blot. A representative blot is shown. F. Intra-tumoral neutrophil elastase 702
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30
activity was measured ex vivo using an NE specific optical probe. G. Intra-tumoral NE activity was 703
quantified using ImageJ and normalized to Vector controls. Difference was assessed using Student’s t-test 704
(n = 8 for Vector, n = 4 for SERPINB1; * p < 0.05). 705
706
Figure 7. SERPINB1 is expressed by normal human prostate glands but reduced in prostate cancer. A. 707
Representative immunohistochemistry stains for SERPINB1 in human prostatectomy specimens, showing 708
regions of normal, normal and cancer, and cancer only. Dotted red lines outline cancer foci adjacent to 709
normal prostate glands. B. Representative immunohistochemistry stains for SERPINB1 in human 710
prostatectomy and core biopsy specimens, showing regions of normal and cancer. 711
712
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Wild type (WT) Pten-null
D.
Randomize
(tumor: 300-500mm3)
Initiate
treatment MDSC flow
Week 0 Week 4 Week 8 Week -2
MDSC flow
z
Monitor tumor growth via US
B. C.
E. Low PMN-MDSC High PMN-MDSC
PCNA
Ly6G
F.
0 20 40 60 80 1000
50
100
150
200
250
300
Ly6G+ Cells/FOV
PC
NA
+ C
ells/F
OV
r = 0.7724p < 0.0001
A.
SERPINB1 IHC
0 2 4 6 875
100
125
150
175
200
225
Weeks
Tu
mo
r V
olu
me (
%)
Vehicle
Sivelestat
**
0 2 4 6 850
60
70
80
90
100
Weeks
% L
y6G
+/L
y6C
+ o
f C
D11b
+
Vehicle
Sivelestat
***
##
SERPINB1
GAPDH
G.
Figure 1
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Normal ProstateCancer
MetastaticProstateCancer
0
50
100
150
200
250
Rela
tive
SE
RP
INB
1 E
xp
ressio
n
*
*
Normal Prostate Cancer0
100
200
300
400
500
Rela
tive
SE
RP
INB
1 E
xp
ressio
n **
Normal Normal Adjacentto Tumor
Primary Tumor
Metastatic0
25
50
75
100
125
Rela
tive
SE
RP
INB
1 E
xp
ressio
n
**
p = 0.097
BenignProstate
LocalizedProstateCancer
MetastaticProstateCancer
0
200
400
600
800
1000
Rela
tive
SE
RP
INB
1 E
xp
ressio
n
***
B.
D.
E.
1 2 3 4 5 6 7 8 9
SERPINB1
GAPDH
Normal Prostate Cancer
F.
C.
A.
0 30 60 90 120 1500
25
50
75
100
Time (months)
Recu
rren
ce F
ree (
%)
Low
High
p = 0.0123
0 30 60 90 120 1500
25
50
75
100
Time (months)
Recu
rren
ce F
ree (
%)
Low
High
p < 0.0001
25% threshold Median threshold
Varambally et al Chandran et al
Arredouani et al Taylor et al
Figure 2
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Annexin V FITC
PI
NSP SERPINB1
Annexin V FITC
PI
NSP SERPINB10.0
0.5
1.0
1.5
2.0
2.5
3.0
No
rmalized
MM
P9 A
cti
vit
y
**
B.
Pro-
MMP9
Pro-
MMP2
NSP SERPINB1
NSP SERPINB1
SERPINB1
SERPINB1
GAPDH
WCL
CM
C.
E. F.
G.
A.
NSP SERPINB10.00
0.25
0.50
0.75
1.00
1.25
No
rmalized
MM
P2 A
cti
vit
y
n.s.
SERPINB1 MMP9 TWIST1 SNAI10.0
0.5
1.0
1.5
2.0
2.5
No
rmalized
mR
NA
Exp
ressio
n
NSP
SERPINB1
****
****
p = 0.057
*
NSP SERPINB10
25
50
75
100
125
150
% P
rolife
rati
on
**
NSP SERPINB10123456789
10
% A
po
pto
tic C
ells
(An
nexin
V+
/PI-
)
****
D.
Figure 3
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B. RWPE-1
C. PC3
UT PD LY UT PD LY
EGF
pERK
tERK
A. RWPE-1
D. PC3
E. RWPE-1 F. RWPE-1
SERPINB1 PI3 SLPI0
5
10
15
Rela
tive m
RN
A E
xp
ressio
n
Untreated
EGF
n.s. n.s. n.s.
SERPINB1
GAPDH
UT EGF
SERPINB1
GAPDH
UT EGF
SERPINB1 PI3 SLPI0
1
2
3
4
5
6
Rela
tive m
RN
A E
xp
ressio
n
Untreated
PD0325901
LY294002
EGF
EGF+PD0325901
EGF+LY294002
****
n.s.
****
n.s.
SERPINB1 PI3 SLPI0
5
10
15
Rela
tive m
RN
A E
xp
ressio
n
Untreated
EGF****
**
**
Figure 4
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PRAD BRCA LUAD COAD BLCA PAAD0.0
0.2
0.4
0.6
0.8
1.0
SERPINB1 P
rom
ote
r M
eth
yla
tio
n (
Beta
valu
e)
Normal
Tumor****
n.s. n.s. n.s. n.s. n.s.
Normal Tumor0
200
400
600
800
SERPINB1 E
xp
ressio
n(R
PK
M)
****
SERPINB1
GAPDH
UT 5-AZA GSK343
B.
C. D.
E.
A.
F.
G.
RWPE-1 C4-20
20
40
60
80
100
Fo
ld E
nri
ch
men
t IgG
H3K27me3
PRAD TCGA
PRAD TCGA
SERPINB1 promoter
SERPINB1 PI3 SLPI0
2
4
6
8
mR
NA
Fo
ld In
du
cti
on Untreated
GSK343
***
***
4 5 6 7 8 9 10 110.0
0.2
0.4
0.6
0.8
1.0
Tumor SERPINB1 Expression(log2 RPKM)
Tu
mo
r SERPINB1 P
rom
ote
rM
eth
yla
tio
n (
Beta
valu
e)
PRAD Expression vs Methylation
r = -0.673p < 0.0001
H.
SERPINB1 PI3 SLPI0
5
10
50
1001500
2000
2500
mR
NA
Fo
ld In
du
cti
on Untreated
5-AZA
****
***
***
CpG1 CpG2 CpG3 CpG4 CpG5 CpG60
20
40
60
80
100
120
Meth
yla
tio
n (
%)
RWPE-1
22Rv1
****C4-2
LNCaP
****
****
Figure 5
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B.
A.
D.
Vector SERPINB1
E.
C.
F. G.
Vec A3 Vec B3
SERPINB1
SERPINB1
GAPDH
WCL
CM
SERPINB1
GAPDH
SERPINB1 B4 Vec B3 E.
C.
SERPINB1
GAPDH
SERPINB1 B2 Vec A3
Vector SERPINB10
25
50
75
100
125
No
rmalized
Pix
el In
ten
sit
y
*
Vector A3 SERPINB1 B20.0
0.2
0.4
0.6
Tu
mo
r W
eig
ht
(g)
*
Vector B3 SERPINB1 B40.0
0.5
1.0
1.5
2.0
Tu
mo
r W
eig
ht
(g)
p = 0.149
Figure 6
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Normal Normal / Cancer Cancer
Patient 1
Prostatectomy
Patient 2
Prostatectomy
A.
Patient 3
Prostatectomy
Patient 4
Prostatectomy
Normal Cancer
Patient 5
Core Biopsy
B.
Figure 7
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Published OnlineFirst January 4, 2019.Mol Cancer Res Irina Lerman, Xiaoting Ma, Christina Seger, et al. Inflammation-Mediated Prostate Cancer ProgressionEpigenetic Suppression of SERPINB1 Promotes
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