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MP37-05 ERP46 MEDIATES PROSTATE CANCER TUMORIGENESIS
THE JOURNAL OF UROLOGYâ
Vol. 193, No. 4S, Supplement, Sunday, May 17, 2015
MP37-04 ANDROGEN RECEPTOR POSITIVE STROMAL CELLS REGULATE PROSTATE CANCER PROLIFERATION THROUGH NONCANONICAL WNT SIGNALING Sayuri Takahashi*, Ichiro Takada, Tokyo, Japan; Naoki Terada, Kyoto, Japan; Yukio Homma, Tokyo, Japan; Robert H. Getzenberg, Memphis, TN INTRODUCTION AND OBJECTIVES: The Wnt signaling pathway (canonical and non-canonical) regulates crucial aspects of embryonic development. Previously, we have reported that non-canonical Wnt members promote proliferation of cancer cells by increasing secretion of growth factors and expression of cancer related genes. We have also demonstrated that Wnt5a is predominantly expressed in prostatic stromal cells and plays a key role in stromalepithelial communication and in the development of prostate cancer. The purpose of this study is to reveal the relation between the noncanonical Wnt signaling and AR signaling on prostate. METHODS: Prostate tissue samples were obtained from patients undergoing radical prostatectomy or cystectomy for prostate and bladder cancer respectively. Cells, representing the cancer, stromal from around the cancer, as well as epithelium and stromal from pathologically normal tissues were isolated by laser capture microdissection Stromal cells (WPMY1 and PrSC) were cultured with DHT ligand and protein. The mRNA levels of Wnt5a, AR, and PSA were determined in both sample types. Wnt5a-knockdown in WPMY1 cells (shWnt5a) along with a vector control (shCTL) was performed and effects on growth evaluated by MTT assay. The cells were co-cultured with PC3 cells, and the mRNA expression of AR was evaluated. A DNA fragment upstream of the Wnt5a gene was inserted into pGL4.17 a vector and the luciferase activity representing Wnt5a inductionby DHT was measured. RESULTS: Wnt5a mRNA expression was prediminanly in the stromal cells of the human prostate samples and the expression levels observed were in proportion to PSA mRNA expression levels. DHT increased production of AR as well as PSA and Wnt5a mRNA expression in both WPMY1 and PrSC cells. Wnt5 knockdown decreased the proliferation of WPMY1cells by 50% compared to shCTR. Co-culture of PC3 cells and WPMY1shWnt5a formed fewer and smaller sized colonies than co-culture of PC3 cells and WPMY1shCTR. AR and PSA mRNA expression levels were remarkably lower in WPMY1shWnt5a than in the control. DHT ligand dramatically increased the luciferase activity of Wnt5a promoter in WPMY1 cells. CONCLUSIONS: The AR is expressed not only in prostatic epithelium but also prostatic stromal as measured by their response to DHT. In stromal cells, The studies presented here demonstrated an interplay between AR signaling and Wnt5a that appears to modulate the proliferation of prostate cancer cells providing further support for the importance of Wnt5a in the regulation of prostate cancer. Source of Funding: none
MP37-05 ERP46 MEDIATES PROSTATE CANCER TUMORIGENESIS Wilhelmina Duivenvoorden, Stephanie Federov, Sarah Hopmans, Jehonathan Pinthus*, Hamilton, Canada INTRODUCTION AND OBJECTIVES: We have recently demonstrated that endoplasmic reticulum protein ERp46, a member of the protein disulfide isomerase family of oxidoreductases, is overexpressed in metastatic kidney cancer. The expression and function of ERp46 in prostate cancer (PC) has not been studied. We explored the suitability of ERp46 as a potential therapeutic target in PC. METHODS: Tissue microarray containing normal prostate epithelium (n¼9) and prostate cancer specimens from 57 patients was stained for ERp46. Staining intensity (H-score) was determined (ImageScope). Human prostate adenocarcinoma 22Rv1 cells which are androgen-responsive and produce PSA were used to generate
e439
gain- and loss-of-function models by stable ERp46 shRNA knockdown and ERp46 overexpression, respectively. In vitro, the doubling time and PSA production were determined. In vivo, xenografts of each subclone were established in nude mice (n¼10/group) to determine the longitudinal tumor growth and serum PSA values. Gene expression profiling of RNA isolated from 22Rv1 xenografts was performed using human whole genome HT-12 V4 BeadChip array (Illumina). RESULTS: Human PC samples of Gleason scores (GS) 7 showed strong cytoplasmic ERp46 staining which was significantly increased compared to normal prostatic tissue (p¼0.02). ERp46 staining in PC samples of GS6, however, was not different compared to normal prostate tissue. The stably transfected human prostate carcinoma 22Rv1 cells expressed either 89% knockdown of ERp46 protein expression (shERp46) or a 4-fold increase in ERp46 protein expression (ERp46þ) compared to the respective control cells. In vitro, shERp46 cells proliferated slower, whereas ERp46þ cells exhibited accelerated growth compared to corresponding control cells (p<0.05). Similarly, the tumor volume of subcutaneously growing shERp46 cells in nude mice led to significantly slower tumor growth (p<0.0005, ANOVA). Vice versa, tumors from ERp46þ cells were significantly larger than the tumor volume of shControl-cell injected mice (p¼0.02, ANOVA). Gene expression analysis confirmed the downregulation and upregulation of ERp46 in the corresponding xenografts and showed several candidate genes, including NAAA, SH3BP4 and ID1 that were reciprocally up-and downregulated. CONCLUSIONS: This is the first report to suggest a role for ERp46 as an oncogenic protein and potential therapeutic target in PC given its effects on PC cell growth. Because ERp46 expression is upregulated in significant PC (Gleason score 7) and not in Gleason score 6 tumors further investigation of its role as a potential marker and target of clinicaly significant PC is warranted. Source of Funding: Prostate Cancer Canada
MP37-06 TARGETED SEQUENCING OF PROSTATE CANCER CIRCULATING TUMOR CELLS AND COMPARISON WITH MATCHED CELL FREE DNA AND PROSTATE TUMORS Brian Hu*, Los Angeles, CA; Stephen Liu, Washington, DC; Yucheng Xu, Los Angeles, CA; Paul Dempsey, William Strauss, Westlake Village, CA; Kristopher Wentzel, Tong Xu, Jacek Pinski, Tanya Dorff, Timothy Triche, Los Angeles, CA; Jessamine Winer Jones, Westlake Village, CA; Inderbir Gill, David Quinn, Amir Goldkorn, Los Angeles, CA INTRODUCTION AND OBJECTIVES: Precision oncology care is predicated upon detecting actionable genomic aberrations. Obtaining tissue that accurately reflects the genomic landscape of prostate cancer (PCa) is a challenge, though circulating tumor cells (CTCs) represent a promising tissue source. We demonstrate the feasibility of high throughput targeted sequencing of CTCs across multiple PCa disease states and compare the genomic variation among different cancer substrates (CTCs, cell free DNA [cfDNA], primary tumors, metastasis) in matched patient samples. METHODS: Under IRB approval, 7.5ml of peripheral blood was obtained from healthy controls and patients with PCa. CTCs were enriched using LiquidBiopsyâ (Cynvenio Biosystems), a CLIA-certified immunoaffinity-based microfluidic platform. CTCs were defined as EpCAMþCKþDAPIþCD45. Amplicon libraries covering 50 cancer genes were generated (AmpliSeq 2.0, Life Technology) and sequenced on an Ion Proton System. Somatic single nucleotide variants (SSNV), occurring in >1% of DNA in a sample, were identified based on their presence in CTCs and absence from WBC. When available, matched cfDNA, primary tumors and metastatic tissue were sequenced in parallel. RESULTS: Peripheral blood was collected from healthy controls (n¼31) and PCa patients (n¼26) at various disease states:
Vol. 193, No. 4S, Supplement, Sunday, May 17, 2015
MP37-04 ANDROGEN RECEPTOR POSITIVE STROMAL CELLS REGULATE PROSTATE CANCER PROLIFERATION THROUGH NONCANONICAL WNT SIGNALING Sayuri Takahashi*, Ichiro Takada, Tokyo, Japan; Naoki Terada, Kyoto, Japan; Yukio Homma, Tokyo, Japan; Robert H. Getzenberg, Memphis, TN INTRODUCTION AND OBJECTIVES: The Wnt signaling pathway (canonical and non-canonical) regulates crucial aspects of embryonic development. Previously, we have reported that non-canonical Wnt members promote proliferation of cancer cells by increasing secretion of growth factors and expression of cancer related genes. We have also demonstrated that Wnt5a is predominantly expressed in prostatic stromal cells and plays a key role in stromalepithelial communication and in the development of prostate cancer. The purpose of this study is to reveal the relation between the noncanonical Wnt signaling and AR signaling on prostate. METHODS: Prostate tissue samples were obtained from patients undergoing radical prostatectomy or cystectomy for prostate and bladder cancer respectively. Cells, representing the cancer, stromal from around the cancer, as well as epithelium and stromal from pathologically normal tissues were isolated by laser capture microdissection Stromal cells (WPMY1 and PrSC) were cultured with DHT ligand and protein. The mRNA levels of Wnt5a, AR, and PSA were determined in both sample types. Wnt5a-knockdown in WPMY1 cells (shWnt5a) along with a vector control (shCTL) was performed and effects on growth evaluated by MTT assay. The cells were co-cultured with PC3 cells, and the mRNA expression of AR was evaluated. A DNA fragment upstream of the Wnt5a gene was inserted into pGL4.17 a vector and the luciferase activity representing Wnt5a inductionby DHT was measured. RESULTS: Wnt5a mRNA expression was prediminanly in the stromal cells of the human prostate samples and the expression levels observed were in proportion to PSA mRNA expression levels. DHT increased production of AR as well as PSA and Wnt5a mRNA expression in both WPMY1 and PrSC cells. Wnt5 knockdown decreased the proliferation of WPMY1cells by 50% compared to shCTR. Co-culture of PC3 cells and WPMY1shWnt5a formed fewer and smaller sized colonies than co-culture of PC3 cells and WPMY1shCTR. AR and PSA mRNA expression levels were remarkably lower in WPMY1shWnt5a than in the control. DHT ligand dramatically increased the luciferase activity of Wnt5a promoter in WPMY1 cells. CONCLUSIONS: The AR is expressed not only in prostatic epithelium but also prostatic stromal as measured by their response to DHT. In stromal cells, The studies presented here demonstrated an interplay between AR signaling and Wnt5a that appears to modulate the proliferation of prostate cancer cells providing further support for the importance of Wnt5a in the regulation of prostate cancer. Source of Funding: none
MP37-05 ERP46 MEDIATES PROSTATE CANCER TUMORIGENESIS Wilhelmina Duivenvoorden, Stephanie Federov, Sarah Hopmans, Jehonathan Pinthus*, Hamilton, Canada INTRODUCTION AND OBJECTIVES: We have recently demonstrated that endoplasmic reticulum protein ERp46, a member of the protein disulfide isomerase family of oxidoreductases, is overexpressed in metastatic kidney cancer. The expression and function of ERp46 in prostate cancer (PC) has not been studied. We explored the suitability of ERp46 as a potential therapeutic target in PC. METHODS: Tissue microarray containing normal prostate epithelium (n¼9) and prostate cancer specimens from 57 patients was stained for ERp46. Staining intensity (H-score) was determined (ImageScope). Human prostate adenocarcinoma 22Rv1 cells which are androgen-responsive and produce PSA were used to generate
e439
gain- and loss-of-function models by stable ERp46 shRNA knockdown and ERp46 overexpression, respectively. In vitro, the doubling time and PSA production were determined. In vivo, xenografts of each subclone were established in nude mice (n¼10/group) to determine the longitudinal tumor growth and serum PSA values. Gene expression profiling of RNA isolated from 22Rv1 xenografts was performed using human whole genome HT-12 V4 BeadChip array (Illumina). RESULTS: Human PC samples of Gleason scores (GS) 7 showed strong cytoplasmic ERp46 staining which was significantly increased compared to normal prostatic tissue (p¼0.02). ERp46 staining in PC samples of GS6, however, was not different compared to normal prostate tissue. The stably transfected human prostate carcinoma 22Rv1 cells expressed either 89% knockdown of ERp46 protein expression (shERp46) or a 4-fold increase in ERp46 protein expression (ERp46þ) compared to the respective control cells. In vitro, shERp46 cells proliferated slower, whereas ERp46þ cells exhibited accelerated growth compared to corresponding control cells (p<0.05). Similarly, the tumor volume of subcutaneously growing shERp46 cells in nude mice led to significantly slower tumor growth (p<0.0005, ANOVA). Vice versa, tumors from ERp46þ cells were significantly larger than the tumor volume of shControl-cell injected mice (p¼0.02, ANOVA). Gene expression analysis confirmed the downregulation and upregulation of ERp46 in the corresponding xenografts and showed several candidate genes, including NAAA, SH3BP4 and ID1 that were reciprocally up-and downregulated. CONCLUSIONS: This is the first report to suggest a role for ERp46 as an oncogenic protein and potential therapeutic target in PC given its effects on PC cell growth. Because ERp46 expression is upregulated in significant PC (Gleason score 7) and not in Gleason score 6 tumors further investigation of its role as a potential marker and target of clinicaly significant PC is warranted. Source of Funding: Prostate Cancer Canada
MP37-06 TARGETED SEQUENCING OF PROSTATE CANCER CIRCULATING TUMOR CELLS AND COMPARISON WITH MATCHED CELL FREE DNA AND PROSTATE TUMORS Brian Hu*, Los Angeles, CA; Stephen Liu, Washington, DC; Yucheng Xu, Los Angeles, CA; Paul Dempsey, William Strauss, Westlake Village, CA; Kristopher Wentzel, Tong Xu, Jacek Pinski, Tanya Dorff, Timothy Triche, Los Angeles, CA; Jessamine Winer Jones, Westlake Village, CA; Inderbir Gill, David Quinn, Amir Goldkorn, Los Angeles, CA INTRODUCTION AND OBJECTIVES: Precision oncology care is predicated upon detecting actionable genomic aberrations. Obtaining tissue that accurately reflects the genomic landscape of prostate cancer (PCa) is a challenge, though circulating tumor cells (CTCs) represent a promising tissue source. We demonstrate the feasibility of high throughput targeted sequencing of CTCs across multiple PCa disease states and compare the genomic variation among different cancer substrates (CTCs, cell free DNA [cfDNA], primary tumors, metastasis) in matched patient samples. METHODS: Under IRB approval, 7.5ml of peripheral blood was obtained from healthy controls and patients with PCa. CTCs were enriched using LiquidBiopsyâ (Cynvenio Biosystems), a CLIA-certified immunoaffinity-based microfluidic platform. CTCs were defined as EpCAMþCKþDAPIþCD45. Amplicon libraries covering 50 cancer genes were generated (AmpliSeq 2.0, Life Technology) and sequenced on an Ion Proton System. Somatic single nucleotide variants (SSNV), occurring in >1% of DNA in a sample, were identified based on their presence in CTCs and absence from WBC. When available, matched cfDNA, primary tumors and metastatic tissue were sequenced in parallel. RESULTS: Peripheral blood was collected from healthy controls (n¼31) and PCa patients (n¼26) at various disease states: