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HDAC6 SILENCING INHIBITS ANDROGEN RECEPTOR ACTIVITY AND TUMOR GROWTH IN A XENOGRAFT MODEL OF HUMAN PROSTATE CANCER
THE JOURNAL OF UROLOGY®
Vol. 181, No. 4, Supplement, Sunday, April 26, 2009
RESULTS: The mean age of the patients was 71.6 ± 2.9 years. The mean body weight significantly increased by 1.5 ± 0.5 kg for 12 months. The mean volume of fat significantly increased by 2.1 ± 0.4 kg and that of skeletal muscle significantly decreased by 0.6 ± 0.2 kg for 12 months. The mean abdominal length and visceral fat area significantly increased for 12 months by 2.1 ± 0.5 cm and 17.0 ± 3.1 cm2, respectively. The increase of fat significantly correlated with that of visceral fat area (r2=0.8368, p<0.0001). The mean levels of total cholesterol and LDL cholesterol significantly increased from 201.8 ± 5.1 mg/dl and 118.6 ± 4.1 mg/dl to 215.8 ± 5.2 mg/ dl and 126.4 ± 5.4 mg/dl, respectively for the first 6 months. Triglyceride and HDL cholesterol did not change significantly. CONCLUSIONS:The administration of LH-RH agonists increased the volume of fat and decreased that of skeletal muscle in a majority of patients with prostate cancer. The increase of fat was caused by that of visceral fat, not by subcutaneous fat. The administration also increased LDL cholesterol which can signal medical problems like cardiovascular disease. It was demonstrated that androgen deprivation therapy for prostate cancer affects body composition and lipid metabolism and may cause metabolic syndrome. Source of Funding: None
256 FINASTERIDE INDUCES HYPOXIA-INDUCIBLE FACTOR 1-ALPHA IN THE PROSTATE OF SPRAGUE-DAWLEY RATS Janmejai K Srivastava, Sanjeev Shukla, Cherry Kamel, Gregory T MacLennan, Allen D Seftel, Sanjay Gupta*, Cleveland, OH INTRODUCTION AND OBJECTIVE: The Prostate Cancer Prevention Trial (PCPT) demonstrated a significant delay in the onset of prostate cancer in males exposed to finasteride, but an increased incidence of high-grade prostate tumors in a subset of the finasteride treatment group. Activation of hypoxia-inducible factor (HIF)-1alpha with activated signaling pathways is implicated in tumor progression. HIF-1alpha is the inducible subunit of the HIF-1 transcription factor and is critical for both physiological and pathological processes. HIF1alpha is activated and targets genes that promote survival through their critical roles in angiogenesis and in metabolic adaptation to low oxygen environments. We hypothesized that finasteride might induce HIF-1alpha as a potential mechanism for survival of prostate epithelial cells, and that this process might favor carcinogenesis in the prostate. METHODS: We initiated studies mimicking the PCPT trial by providing Sprague Dawley rats with doses of 4 (equivalent to 7 mg per day PO) and 40 ppm ( equivalent to 70 mg per day PO) finasteride in their drinking water for 10 weeks, starting at 16 weeks of age. Blood was collected at various time intervals for determining plasma T, DHT, NO and VEGF levels. The animals were sacrificed and major organs including the prostate and seminal vesicles were excised and processed for pathologic evaluation and for estimation of various parameters related to HIF-1alpha. RESULTS: Finasteride intake caused significant decrease in T and DHT levels in the serum and in the prostates compared to the control group without change in body weight. These results correlated with a decrease in the wet weights of seminal vesicles and prostates recorded at the study termination. Finasteride intake caused atrophy in the dorso-lateral and ventral prostates: glands appeared smaller, with more irregular wrinkled gland outlines, and with more pronounced papillary infolding of the lining glandular epithelium. There was no appreciable difference in these findings between the low-dose and highdose groups. Furthermore, increased levels of plasma VEGF and NO were observed in the finasteride-administered group, and levels were increased in a dose-dependent fashion. In the prostate, HIF-1alpha and VEGF expression were upregulated in the finasteride group compared to the control group. CONCLUSIONS: Finasteride activates HIF-1alpha in the prostate and suggest a mechanism through which it may contribute to the development of more aggressive prostate tumors in a subset of men exposed to this medication. Source of Funding: None
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257 HDAC6 SILENCING INHIBITS ANDROGEN RECEPTOR ACTIVITY AND TUMOR GROWTH IN A XENOGRAFT MODEL OF HUMAN PROSTATE CANCER Junkui Ai*, Yujuan Wang, Javid A Dar, Lingqi Liu, Joel B Nelson, Zhou Wang, Pittsburgh, PA INTRODUCTION AND OBJECTIVE: Our previous studies have demonstrated that knockdown of histone deacetylase 6 (HDAC6) inhibited expression of prostate-specific antigen (PSA) expression and growth of castration-resistant C4-2 prostate cancer cells in culture. In the present study, we investigated the effects of HDAC6 silencing on AR function and prostate cancer growth in vivo. METHODS: C4-2 cells with stable HDAC6 knockdown (C4-2/ siHDAC6) or shRNA control (C4-2/siCntrl) were established using lentiviral shRNA expression system. Nude mice 6-8 weeks old were randomized into 2 groups (n=12) and castration or sham castration was performed. 1×106 of C4-2/siHDAC6 or C4-2/siCntrl cells was injected subcutaneously into the flanks of nude mice. Tumor take rate was calculated 12 weeks after injection and tumors were measured until the diameter reached 2 cm. AR transcriptional activity in C4-2 xenograft tumor tissues was assayed by measurement of PSA using Western Blot. RESULTS: As expected, C4-2/siCntrl cells showed a tumor take rate at 33% in castrated mice. In contrast, no tumor take was observed for C4-2/siHDAC6 cells in castrated mice. Furthermore, we showed that silencing of HDAC6 by siRNA decreased tumor volume by 54% in intact mice as compared to C4-2/siCntrl tumors 6 weeks after injection. The inhibition of xenograft tumor growth corresponded to a marked decrease in PSA expression levels in tumor tissues. CONCLUSIONS: Our results provide evidence that HDAC6 can regulate AR signaling and HDAC6 may be a potential target to treat both androgen-dependent and castration-resistant prostate cancer. Source of Funding: NIH grants R01 CA 108675, 5 R01 DK51993, 1 P50 CA90386
258 OVEREXPRESSION OF CDC25A, AN ANDROGEN RECEPTOR COACTIVATOR, IN HUMAN PROSTATE CANCER Yoshihiro Hashimoto*, Keiichi Tozawa, Yutaro Hayashi, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVE: Cdc25A is a potent tyrosine phosphatase that catalyzes specific dephosphorylation of cyclin/cyclindependent kinase (cdk) complexes to regulate G1 to S-phase cell cycle progression. Cdc25A, being an oncogene, has been shown to be overexpressed in a variety of human malignancies. To study the role of Cdc25A in prostate cancer, we investigated the expression of Cdc25A in human prostate cancer patients and human prostate cancer cell lines. METHODS: We examined the Cdc25A expression level of 70 prostate cancer patient samples and human prostate cancer cell lines by immunohistochemistry and Western blot analysis, respectively. In addition, we established stable LNCaP cell lines overexpressing Cdc25A. These cell lines were used to investigate the transcriptional activity of androgen receptor and the effect of Cdc25A overexpression in prostate cancer cell growth. RESULTS: We found that 52.9% of human prostate cancer samples (N=70) overexpresses Cdc25A in the tumor area but not in the adjacent normal area by immunohistochemistry. In general, the overexpression of Cdc25A correlated with patient characteristics such as Gleason score, stage and recurrence. Subsequently, we demonstrated that Cdc25A enhances the transcriptional activity of androgen receptor in a hormone-dependent manner. This coactivator function, surprisingly, is independent of its cell cycle functions. Western analysis indicated that more aggressive prostate cancer cells, DU145 and PC3, have a higher expression of Cdc25A than less aggressive prostate cancer cells, LNCaP. We also observed a significant increase in cell growth of LNCaP with overexpression of Cdc25A. CONCLUSIONS: These findings suggest that the expression
Vol. 181, No. 4, Supplement, Sunday, April 26, 2009
RESULTS: The mean age of the patients was 71.6 ± 2.9 years. The mean body weight significantly increased by 1.5 ± 0.5 kg for 12 months. The mean volume of fat significantly increased by 2.1 ± 0.4 kg and that of skeletal muscle significantly decreased by 0.6 ± 0.2 kg for 12 months. The mean abdominal length and visceral fat area significantly increased for 12 months by 2.1 ± 0.5 cm and 17.0 ± 3.1 cm2, respectively. The increase of fat significantly correlated with that of visceral fat area (r2=0.8368, p<0.0001). The mean levels of total cholesterol and LDL cholesterol significantly increased from 201.8 ± 5.1 mg/dl and 118.6 ± 4.1 mg/dl to 215.8 ± 5.2 mg/ dl and 126.4 ± 5.4 mg/dl, respectively for the first 6 months. Triglyceride and HDL cholesterol did not change significantly. CONCLUSIONS:The administration of LH-RH agonists increased the volume of fat and decreased that of skeletal muscle in a majority of patients with prostate cancer. The increase of fat was caused by that of visceral fat, not by subcutaneous fat. The administration also increased LDL cholesterol which can signal medical problems like cardiovascular disease. It was demonstrated that androgen deprivation therapy for prostate cancer affects body composition and lipid metabolism and may cause metabolic syndrome. Source of Funding: None
256 FINASTERIDE INDUCES HYPOXIA-INDUCIBLE FACTOR 1-ALPHA IN THE PROSTATE OF SPRAGUE-DAWLEY RATS Janmejai K Srivastava, Sanjeev Shukla, Cherry Kamel, Gregory T MacLennan, Allen D Seftel, Sanjay Gupta*, Cleveland, OH INTRODUCTION AND OBJECTIVE: The Prostate Cancer Prevention Trial (PCPT) demonstrated a significant delay in the onset of prostate cancer in males exposed to finasteride, but an increased incidence of high-grade prostate tumors in a subset of the finasteride treatment group. Activation of hypoxia-inducible factor (HIF)-1alpha with activated signaling pathways is implicated in tumor progression. HIF-1alpha is the inducible subunit of the HIF-1 transcription factor and is critical for both physiological and pathological processes. HIF1alpha is activated and targets genes that promote survival through their critical roles in angiogenesis and in metabolic adaptation to low oxygen environments. We hypothesized that finasteride might induce HIF-1alpha as a potential mechanism for survival of prostate epithelial cells, and that this process might favor carcinogenesis in the prostate. METHODS: We initiated studies mimicking the PCPT trial by providing Sprague Dawley rats with doses of 4 (equivalent to 7 mg per day PO) and 40 ppm ( equivalent to 70 mg per day PO) finasteride in their drinking water for 10 weeks, starting at 16 weeks of age. Blood was collected at various time intervals for determining plasma T, DHT, NO and VEGF levels. The animals were sacrificed and major organs including the prostate and seminal vesicles were excised and processed for pathologic evaluation and for estimation of various parameters related to HIF-1alpha. RESULTS: Finasteride intake caused significant decrease in T and DHT levels in the serum and in the prostates compared to the control group without change in body weight. These results correlated with a decrease in the wet weights of seminal vesicles and prostates recorded at the study termination. Finasteride intake caused atrophy in the dorso-lateral and ventral prostates: glands appeared smaller, with more irregular wrinkled gland outlines, and with more pronounced papillary infolding of the lining glandular epithelium. There was no appreciable difference in these findings between the low-dose and highdose groups. Furthermore, increased levels of plasma VEGF and NO were observed in the finasteride-administered group, and levels were increased in a dose-dependent fashion. In the prostate, HIF-1alpha and VEGF expression were upregulated in the finasteride group compared to the control group. CONCLUSIONS: Finasteride activates HIF-1alpha in the prostate and suggest a mechanism through which it may contribute to the development of more aggressive prostate tumors in a subset of men exposed to this medication. Source of Funding: None
95
257 HDAC6 SILENCING INHIBITS ANDROGEN RECEPTOR ACTIVITY AND TUMOR GROWTH IN A XENOGRAFT MODEL OF HUMAN PROSTATE CANCER Junkui Ai*, Yujuan Wang, Javid A Dar, Lingqi Liu, Joel B Nelson, Zhou Wang, Pittsburgh, PA INTRODUCTION AND OBJECTIVE: Our previous studies have demonstrated that knockdown of histone deacetylase 6 (HDAC6) inhibited expression of prostate-specific antigen (PSA) expression and growth of castration-resistant C4-2 prostate cancer cells in culture. In the present study, we investigated the effects of HDAC6 silencing on AR function and prostate cancer growth in vivo. METHODS: C4-2 cells with stable HDAC6 knockdown (C4-2/ siHDAC6) or shRNA control (C4-2/siCntrl) were established using lentiviral shRNA expression system. Nude mice 6-8 weeks old were randomized into 2 groups (n=12) and castration or sham castration was performed. 1×106 of C4-2/siHDAC6 or C4-2/siCntrl cells was injected subcutaneously into the flanks of nude mice. Tumor take rate was calculated 12 weeks after injection and tumors were measured until the diameter reached 2 cm. AR transcriptional activity in C4-2 xenograft tumor tissues was assayed by measurement of PSA using Western Blot. RESULTS: As expected, C4-2/siCntrl cells showed a tumor take rate at 33% in castrated mice. In contrast, no tumor take was observed for C4-2/siHDAC6 cells in castrated mice. Furthermore, we showed that silencing of HDAC6 by siRNA decreased tumor volume by 54% in intact mice as compared to C4-2/siCntrl tumors 6 weeks after injection. The inhibition of xenograft tumor growth corresponded to a marked decrease in PSA expression levels in tumor tissues. CONCLUSIONS: Our results provide evidence that HDAC6 can regulate AR signaling and HDAC6 may be a potential target to treat both androgen-dependent and castration-resistant prostate cancer. Source of Funding: NIH grants R01 CA 108675, 5 R01 DK51993, 1 P50 CA90386
258 OVEREXPRESSION OF CDC25A, AN ANDROGEN RECEPTOR COACTIVATOR, IN HUMAN PROSTATE CANCER Yoshihiro Hashimoto*, Keiichi Tozawa, Yutaro Hayashi, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVE: Cdc25A is a potent tyrosine phosphatase that catalyzes specific dephosphorylation of cyclin/cyclindependent kinase (cdk) complexes to regulate G1 to S-phase cell cycle progression. Cdc25A, being an oncogene, has been shown to be overexpressed in a variety of human malignancies. To study the role of Cdc25A in prostate cancer, we investigated the expression of Cdc25A in human prostate cancer patients and human prostate cancer cell lines. METHODS: We examined the Cdc25A expression level of 70 prostate cancer patient samples and human prostate cancer cell lines by immunohistochemistry and Western blot analysis, respectively. In addition, we established stable LNCaP cell lines overexpressing Cdc25A. These cell lines were used to investigate the transcriptional activity of androgen receptor and the effect of Cdc25A overexpression in prostate cancer cell growth. RESULTS: We found that 52.9% of human prostate cancer samples (N=70) overexpresses Cdc25A in the tumor area but not in the adjacent normal area by immunohistochemistry. In general, the overexpression of Cdc25A correlated with patient characteristics such as Gleason score, stage and recurrence. Subsequently, we demonstrated that Cdc25A enhances the transcriptional activity of androgen receptor in a hormone-dependent manner. This coactivator function, surprisingly, is independent of its cell cycle functions. Western analysis indicated that more aggressive prostate cancer cells, DU145 and PC3, have a higher expression of Cdc25A than less aggressive prostate cancer cells, LNCaP. We also observed a significant increase in cell growth of LNCaP with overexpression of Cdc25A. CONCLUSIONS: These findings suggest that the expression