Epigenetic Suppression of SERPINB1 Promotes Inflammation ... · 1 1 Epigenetic Suppression of SERPINB1 Promotes Inflammation-Mediated Prostate Cancer 2 Progression 3 4 Irina Lerman1,

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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

[email protected] 21

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COI: The authors declare no potential conflicts of interest 23

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on October 5, 2020. © 2019 American Association for Cancer Research. mcr.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 January 4, 2019; DOI: 10.1158/1541-7786.MCR-18-0638

http://mcr.aacrjournals.org/

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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

129

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

136

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

146

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

151

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

155

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

162

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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http://www.betastasis.com/


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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

296

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

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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




17

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




18

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




19

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




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




21

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597

598




26

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




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




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




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




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




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




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




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




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




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




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




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




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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