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1440 A NON-CANONICAL WNT SIGNALING MEDIATES ANDROGEN-DEPENDENT TUMOR GROWTH IN A MOUSE MODEL OF PROSTATE CANCER
Vol. 185, No. 4S, Supplement, Tuesday, May 17, 2011
Prostate Cancer: Basic Research Moderated Poster 46 Tuesday, May 17, 2011
8:00 AM-10:00 AM
1440 A NON-CANONICAL WNT SIGNALING MEDIATES ANDROGENDEPENDENT TUMOR GROWTH IN A MOUSE MODEL OF PROSTATE CANCER Sayuri Takahashi*, Takahiro Matsumoto, Yukio Homma, Tadaichi Kitamura, Shigeaki Kato, Tokyo, Japan INTRODUCTION AND OBJECTIVES: Prostate cancer development is considered to be associated with hyperactive androgen signaling. However, the molecular link between androgen receptor(AR) function and humoral factors remains elusive. We generated conditional mice which have activated AR only in their prostatic epithelial cells and revealed the molecular link between AR signaling and Wnt signaling. METHODS: AR threonine 877 alanine (T877A) mutation found in LNCaP cell is known to be activated by AR agonists and antagonists in vitro. We applied a Cre-loxP system to generate prostate-specific AR knock-in mice expressing AR-T877A. Floxed AR mice were mated with PSA-Cre AR-T877A mice which activates Cre protein in the prostates by tamoxifen. Thus, this mouse line expressed AR-T877A only in the prostates by tamoxifen. At 8 weeks of age, tamoxifen was injected in AR-T877A mice and control mice. Firstly, ventral prostate (VP) was weighed in 16 weeks of age and the tissue was examined. Next, AR-T877A mice and control mice were mated with prostate cancer model (TRAMP) transgenic mice which develop prostate tumor spontaneously. Whole prostates were weighed on every 3 weeks. Thirdly, RNA expressions of various humoral factors on AR-T877A mice, TRAMP-AR-T877A mice and control mice were analyzed by microarray and real time PCR. Based on the results, TRAMP-AR-T877A mice were mated with several kinds of knock-out mice and the prostates were examined by 48 weeks of age. RESULTS: The average VP of AR-T877A mice was bigger and heavier in 16 weeks of age than that of control mice. Histological examination revealed that formation of tubules was promoted. Next, all TRAMP- AR-T877A mice developed prostate tumor rapidly by 24 weeks of age, whereas all control mice were tumor free at 24 weeks of age. Microarray showed up-regulation and down-regulation of several kinds of genes. The RNA expressions of the genes were examined by real time PCR and four humoral factors; FGF10, STAT3, TAK, Wnt-5a showed enhanced RNA expressions. TRAMP-AR-T877A mice mated with FGF10 knockout mice, or STAT3 knockout mice, or TAK knockout mice did not show any significant difference from control TRAMP-ART877A mice. However, TRAMP-AR-T877A mice mated with Wnt-5a knock-out mice suppressed the growth of prostate cancer. CONCLUSIONS: The prostatic growth and prostatic tumorigenesis were significantly promoted by introduction of AR-T877A mutation in the prostate. Genetic screening of mice identified Wnt-5a as an activator. These findings suggest that a non-canonical Wnt signal stimulates development of prostate cancer with AR hyperfunction. Source of Funding: None
1441 DIHYDROTESTOSTERONE ENHANCES ANDROGEN-INDEPENDENT PROSTATE CANCER CELL SURVIVAL THROUGH STAT5 ACTIVATION VIA GLUCOCORTICOID RECEPTOR PATHWAY Cheryn Song*, Gyeong Eun Min, Yunlim Kim, Hanjong Ahn, Seoul, Korea, Republic of INTRODUCTION AND OBJECTIVES: Paradoxical survival of androgen-independent prostate cancer (AIPC) cells in the presence of
THE JOURNAL OF UROLOGY姞
e577
dihydrotestosterone (DHT) has been explained by androgen receptor (AR) functional mutation, supersensitivity and growth factor bypass. Signal transducer and activator of transcription (STAT)-5 has been demonstrated to be upregulated after becoming androgen independent. While functional interaction between STAT5 and AR has been reported, mechanism in AIPC cells remains unclear. METHODS: Using AIPC cell lines DU145 and PC3, and LNCap cells as controls cell viability assay and Western blot for phosphorylated STAT5 were done after DHT treatment at 0.1, 1, 10 nM concentrations. Endogenous levels of AR, glucocorticoid receptor (GR) and estrogen receptors (ER␣, ER) were identified using real time PCR and Western blot. Cells were pre-treated with GR-antagonist RU486 before repeating cell viability and Western blot to see DHT-induced proliferation and STAT5 activation were affected. Immunofluorescence staining of the DU145 cells with anti-GR Ab before and after DHT treatment was done and visualized. RESULTS: DHT treatment enhanced STAT5 phosphorylation and promoted proliferation of both PC3 and DU145 cell lines dosedependently. Endogenous GR proteins were identified strongly in DU145 cells, weakly in PC3 cells but absent in LNCap cells. Conversely AR proteins were identified only in LNCap cells. After pretreatment with RU486, DHT-induced cell proliferation and STAT5 activation were both abolished in both DU145 and PC3 cell lines but not in LNCap cells. On immunofluorescence and DAPI nuclear staining, activation of GR translocating into the nucleus after DHT treatment was confirmed. CONCLUSIONS: In AIPC cells, DHT enhanced cell proliferation by activating STAT5 pathway. Activated STAT5, by directly binding to GR abundantly present in the AIPC cells promoted cell survival. Source of Funding: None
1442 CONSTITUTIONAL GENETIC VARIATION ASSOCIATED WITH PROSTATE CANCER PROGRESSION Barbara Ercole*, Carolina B. Livi, San Antonio, TX; Doug K. Price, Bethesda, MD; Kathleen C. Torkko, Aurora, CO; J. Michael Drake, Christopher H. Cantrill, Ian M. Thompson, Robin J. Leach, San Antonio, TX INTRODUCTION AND OBJECTIVES: Biomarker discovery for prostate cancer has been an ongoing effort and in an attempt to determine markers of progression the analysis of single nucleotide polymorphisms (SNP) has been employed. Variations in DNA sequences can affect how disease develops and how we respond to different agents. Genetic differences in constitutional DNA of men with metastatic prostate cancer compared to men whose prostate cancer has remained biochemical recurrence free after treatment may hold the key to discovering a marker for prostate cancer progression. We have used a gene candidate approach to select a panel of 96 SNPs genotyped from DNA samples from subjects with metastatic disease or without biochemical recurrence for over 5 years after treatment. METHODS: Constitutional DNA from 85 men who were biochemical recurrence free after treatment of their prostate cancer was used in this study. These men, who were identified from the San Antonio center for Biomarkers Of risk for prostate cancer (SABOR) and PREF, were compared to the constitutional DNA from 252 men with documented metastatic prostate cancer identified from patients treated at the National Cancer Institute, with an additional 11 men with documented metastatic disease from the San Antonio cohort. All men in this study were Caucasian. Ninety-six promising SNP’s were identified from prior studies and from a literature search. Genotyping was performed using a custom 96 -plex Illumina Golden Gate array. RESULTS: Five SNPs showed a statistically significant difference with a p value of ⬍0.05, without adjusting for multiple testing, between men with metastatic disease and those without (Table 1). Two SNPs, rs1544410 and rs2254210, found in the vitamin D receptor gene locus appeared to be protective against metastatic disease. Three SNPs found in genes for MSR1, osteoprotegerin, and ERG (rs3789015, rs2073617, rs2836582; respectively) appeared to increase the risk of metastatic disease.
Prostate Cancer: Basic Research Moderated Poster 46 Tuesday, May 17, 2011
8:00 AM-10:00 AM
1440 A NON-CANONICAL WNT SIGNALING MEDIATES ANDROGENDEPENDENT TUMOR GROWTH IN A MOUSE MODEL OF PROSTATE CANCER Sayuri Takahashi*, Takahiro Matsumoto, Yukio Homma, Tadaichi Kitamura, Shigeaki Kato, Tokyo, Japan INTRODUCTION AND OBJECTIVES: Prostate cancer development is considered to be associated with hyperactive androgen signaling. However, the molecular link between androgen receptor(AR) function and humoral factors remains elusive. We generated conditional mice which have activated AR only in their prostatic epithelial cells and revealed the molecular link between AR signaling and Wnt signaling. METHODS: AR threonine 877 alanine (T877A) mutation found in LNCaP cell is known to be activated by AR agonists and antagonists in vitro. We applied a Cre-loxP system to generate prostate-specific AR knock-in mice expressing AR-T877A. Floxed AR mice were mated with PSA-Cre AR-T877A mice which activates Cre protein in the prostates by tamoxifen. Thus, this mouse line expressed AR-T877A only in the prostates by tamoxifen. At 8 weeks of age, tamoxifen was injected in AR-T877A mice and control mice. Firstly, ventral prostate (VP) was weighed in 16 weeks of age and the tissue was examined. Next, AR-T877A mice and control mice were mated with prostate cancer model (TRAMP) transgenic mice which develop prostate tumor spontaneously. Whole prostates were weighed on every 3 weeks. Thirdly, RNA expressions of various humoral factors on AR-T877A mice, TRAMP-AR-T877A mice and control mice were analyzed by microarray and real time PCR. Based on the results, TRAMP-AR-T877A mice were mated with several kinds of knock-out mice and the prostates were examined by 48 weeks of age. RESULTS: The average VP of AR-T877A mice was bigger and heavier in 16 weeks of age than that of control mice. Histological examination revealed that formation of tubules was promoted. Next, all TRAMP- AR-T877A mice developed prostate tumor rapidly by 24 weeks of age, whereas all control mice were tumor free at 24 weeks of age. Microarray showed up-regulation and down-regulation of several kinds of genes. The RNA expressions of the genes were examined by real time PCR and four humoral factors; FGF10, STAT3, TAK, Wnt-5a showed enhanced RNA expressions. TRAMP-AR-T877A mice mated with FGF10 knockout mice, or STAT3 knockout mice, or TAK knockout mice did not show any significant difference from control TRAMP-ART877A mice. However, TRAMP-AR-T877A mice mated with Wnt-5a knock-out mice suppressed the growth of prostate cancer. CONCLUSIONS: The prostatic growth and prostatic tumorigenesis were significantly promoted by introduction of AR-T877A mutation in the prostate. Genetic screening of mice identified Wnt-5a as an activator. These findings suggest that a non-canonical Wnt signal stimulates development of prostate cancer with AR hyperfunction. Source of Funding: None
1441 DIHYDROTESTOSTERONE ENHANCES ANDROGEN-INDEPENDENT PROSTATE CANCER CELL SURVIVAL THROUGH STAT5 ACTIVATION VIA GLUCOCORTICOID RECEPTOR PATHWAY Cheryn Song*, Gyeong Eun Min, Yunlim Kim, Hanjong Ahn, Seoul, Korea, Republic of INTRODUCTION AND OBJECTIVES: Paradoxical survival of androgen-independent prostate cancer (AIPC) cells in the presence of
THE JOURNAL OF UROLOGY姞
e577
dihydrotestosterone (DHT) has been explained by androgen receptor (AR) functional mutation, supersensitivity and growth factor bypass. Signal transducer and activator of transcription (STAT)-5 has been demonstrated to be upregulated after becoming androgen independent. While functional interaction between STAT5 and AR has been reported, mechanism in AIPC cells remains unclear. METHODS: Using AIPC cell lines DU145 and PC3, and LNCap cells as controls cell viability assay and Western blot for phosphorylated STAT5 were done after DHT treatment at 0.1, 1, 10 nM concentrations. Endogenous levels of AR, glucocorticoid receptor (GR) and estrogen receptors (ER␣, ER) were identified using real time PCR and Western blot. Cells were pre-treated with GR-antagonist RU486 before repeating cell viability and Western blot to see DHT-induced proliferation and STAT5 activation were affected. Immunofluorescence staining of the DU145 cells with anti-GR Ab before and after DHT treatment was done and visualized. RESULTS: DHT treatment enhanced STAT5 phosphorylation and promoted proliferation of both PC3 and DU145 cell lines dosedependently. Endogenous GR proteins were identified strongly in DU145 cells, weakly in PC3 cells but absent in LNCap cells. Conversely AR proteins were identified only in LNCap cells. After pretreatment with RU486, DHT-induced cell proliferation and STAT5 activation were both abolished in both DU145 and PC3 cell lines but not in LNCap cells. On immunofluorescence and DAPI nuclear staining, activation of GR translocating into the nucleus after DHT treatment was confirmed. CONCLUSIONS: In AIPC cells, DHT enhanced cell proliferation by activating STAT5 pathway. Activated STAT5, by directly binding to GR abundantly present in the AIPC cells promoted cell survival. Source of Funding: None
1442 CONSTITUTIONAL GENETIC VARIATION ASSOCIATED WITH PROSTATE CANCER PROGRESSION Barbara Ercole*, Carolina B. Livi, San Antonio, TX; Doug K. Price, Bethesda, MD; Kathleen C. Torkko, Aurora, CO; J. Michael Drake, Christopher H. Cantrill, Ian M. Thompson, Robin J. Leach, San Antonio, TX INTRODUCTION AND OBJECTIVES: Biomarker discovery for prostate cancer has been an ongoing effort and in an attempt to determine markers of progression the analysis of single nucleotide polymorphisms (SNP) has been employed. Variations in DNA sequences can affect how disease develops and how we respond to different agents. Genetic differences in constitutional DNA of men with metastatic prostate cancer compared to men whose prostate cancer has remained biochemical recurrence free after treatment may hold the key to discovering a marker for prostate cancer progression. We have used a gene candidate approach to select a panel of 96 SNPs genotyped from DNA samples from subjects with metastatic disease or without biochemical recurrence for over 5 years after treatment. METHODS: Constitutional DNA from 85 men who were biochemical recurrence free after treatment of their prostate cancer was used in this study. These men, who were identified from the San Antonio center for Biomarkers Of risk for prostate cancer (SABOR) and PREF, were compared to the constitutional DNA from 252 men with documented metastatic prostate cancer identified from patients treated at the National Cancer Institute, with an additional 11 men with documented metastatic disease from the San Antonio cohort. All men in this study were Caucasian. Ninety-six promising SNP’s were identified from prior studies and from a literature search. Genotyping was performed using a custom 96 -plex Illumina Golden Gate array. RESULTS: Five SNPs showed a statistically significant difference with a p value of ⬍0.05, without adjusting for multiple testing, between men with metastatic disease and those without (Table 1). Two SNPs, rs1544410 and rs2254210, found in the vitamin D receptor gene locus appeared to be protective against metastatic disease. Three SNPs found in genes for MSR1, osteoprotegerin, and ERG (rs3789015, rs2073617, rs2836582; respectively) appeared to increase the risk of metastatic disease.