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ANDROGEN REGULATES THE EXPRESSION OF CRITICAL HEDGEHOG SIGNALING COMPONENTS IN PROSTATE CANCER CELLS
96
THE JOURNAL OF UROLOGY®
levels of Cdc25A and its interplay with androgen receptor may contribute to increase understanding of the tumor biology of human prostate cancer associated with cell cycle regulation. Source of Funding: None
259 ANDROGEN REGULATES THE EXPRESSION OF CRITICAL HEDGEHOG SIGNALING COMPONENTS IN PROSTATE CANCER CELLS. Mengqian Chen, Matthew Tanner, Albany, NY; Alice C. Levine, New York, NY; Elina Levina, Ralph Buttyan*, Albany, NY INTRODUCTION AND OBJECTIVE: Hedgehog (Hh) is a ligandactivated developmental signaling pathway that is thought to play a role in human cancers including prostate cancer. Although prostate cancer cells express many gene products needed for Hh signaling, these cells were found to be refractory to canonical Hh activating stimuli and the pathway leading to Hh activity in prostate cancer remains enigmatic. Here, we measured the effects of androgen on the expression of Hh ligands and Hh target genes in the androgen-sensitive prostate cancer cell line, LNCaP and in more metastatic variants. METHODS: LNCaP and derivative C4-2 and C4-2B cells were grown for increasing time in androgen-free medium with or without R1881. RNAs were extracted, reverse-transcribed and assayed for relative expression of the three Hh ligands (Sonic [SHH], Indian [IHH] or Desert [DHH] by qPCR and for the relative expression of the Hh receptor and target genes, Patched (Ptch), Gli1 and Gli2. Expression of the later genes was also measured in the presence of the Hh inhibitor, cyclopamine. Expression of Hh ligand protein was measured on western blot of cell extracts and conditioned medium (CM). CM was also assayed for paracrine Hh activity on Hh-sensitive mouse MC3T3-E1 fibroblasts. RESULTS: Androgen strongly suppresses expression of Hh ligands in LNCaP cells and their derivatives and their prolonged maintenance in androgen-free medium upregulated SHH and IHH mRNA and protein levels by up to 30,000-fold. Mature Hh ligand was released into the CM of androgen-deprived cells and this ligand was paracrine-active as shown by the ability of CM to increase Hh target gene (mPtch and mGli) expression in MC3T3-E1 cells. This activity was also accompanied by increased expression of Gli target genes, Ptch and Gli2, in LNCaP cells that was suppressed by cyclopamine. In contrast to the suppressive effects of R1881 on Hh ligand expression, we found that Gli1 expression in LNCaP cells was significantly induced by R1881. CONCLUSIONS: The expression of Hh ligands and the activity of the autocrine Hh signaling pathway are regulated by androgen in LNCaP cells and their derivatives. This finding indicates that androgen deprivation therapy might create a Hh signaling environment around a prostate tumor that could impact on the long term effectiveness of this treatment. Our results also support the idea that some of the putative aberrations in Hh signaling in prostate cancer may be a manifestation of the androgen signaling pathway in the cancer cells rather than the canonical Hh signaling pathway. Source of Funding: NIH CA111618; DOD W81XWH-06; Equinox Foundation
260 INTERLEUKIN-6 ENHANCES INTRATUMORAL ANDROGEN LEVELS BY REGULATING THE EXPRESSION OF GENES MEDIATING ANDROGEN METABOLISM Yae Chun, Joy C Yang, Hsing-Jien Kung, Christopher P Evans, Allen C Gao*, Sacramento, CA INTRODUCTION AND OBJECTIVE: The standard systemic treatment for prostate cancer patients is androgen deprivation therapy. Although serum testosterone concentrations were significantly reduced after androgen deprivation therapy, levels of intraprostatic androgens are reproducibly measured at concentrations sufficient to activate androgen receptor (AR) and stimulate tumor growth. Thus, prostate cancer cells
Vol. 181, No. 4, Supplement, Sunday, April 26, 2009
may survive androgen deprivation therapies by regulating intracrine androgen synthesis within the prostate. However, factors involved in de novo intracrine androgen synthesis have not been identified. Interleukin-6 (IL-6) has been implicated in the modulation of AR activation and growth and differentiation in prostate cancer. In this study, we investigate whether IL-6 associate with intraprostatic androgen synthesis and AR activation in castration-resistant prostate cancer. METHODS: Quantitative reverse transcription-PCR and western blot were performed to detect expression levels of steroidogenic enzymes. To measure intraprostatic androgen levels, IL-6 overexpressing LNCaPIL6+ cells were inoculated orthotopically into the prostate of castrated male nude mice and generated tumors. Intraprostatic androgen levels were measured by an enzyme immunoassay (Testosterone EIA kit) based on the competition between testosterone and a testosteroneacetylcholinesterase (AChE) conjugate (testosterone tracer) for a limited amount of testosterone in the sample. RESULTS: We found that IL-6 increases the expression of genes encoding many steroidogenic enzymes including HSD3B2 and AKR1C1-3 involved in androgen biosynthesis, which suggest that IL-6 may increase the levels of intraprostatic androgen. Down regulation of IL-6 receptor and gp130 expression using specific siRNA abolished IL-6 mediated AKR1C1-3 expression, suggesting that IL-6 signaling is responsible for AKR1C1-3 expression. The tumor testosterone levels were detected at 320 ± 65 pg/g tissue by testosterone EIA kit in tumors generated from IL-6 overexpressing LNCaP-IL6+ cells inoculated orthotopically into the prostate of the castrated male nude mice, such levels of testosterone are found sufficient to activate AR signaling. CONCLUSIONS: These studies suggest that IL-6 increased levels of intratumoral androgens through enhanced expression of genes mediating androgen metabolism during androgen deprivation therapy. Source of Funding: NIH CA90271, CA109441
261 TREATMENT FAILURE IS ASSOCIATED WITH DIFFERENT DOSING REGIMENS OF LEUTINIZING HORMONE RELEASING HORMONE AGONIST THERAPY FOR PROSTATE CANCER Jeremy M Blumberg*, Eric Kwon, T. Craig Cheetham, Fang Niu, Steven J. Jacobsen, Charles E. Shapiro, Stephen G. Williams, Gary W. Chien, Los Angeles, CA INTRODUCTION AND OBJECTIVE: Leutinizing Hormone Releasing Hormone (LHRH) agonist therapy is a mainstay of prostate cancer treatment. Several dosing regimens currently exist: calendar based, intermittent, and a testosterone-based (T-based) regimen we have previously reported. We investigated the relative risk of treatment failure based on dosing regimen. METHODS: We evaluated 1617 patients with prostate cancer who received LHRH agonist therapy in the Kaiser Permanente Southern California Cancer Registry from Jan 2003-December 2006. Patients were grouped according to their respective dosing regimen; calendar based, intermittent dosing, or T-based. Calendar based is defined as patients who are re-dosed with LHRH-agonist every 3 months. Intermittent dosing is defined as patients who are re-dosed with LHRH-agonist based on the patient’s PSA level, irrespective of testosterone levels. T-based is defined as patients who are re-dosed with LHRH-agonist whenever serum testosterone (T) level rises above 50ng/dl. We defined treatment failure as 2 consecutive rises in PSA measurement (with a minimum of 3 PSAs) following the last dose of LHRH-agonist. Cox proportional hazards regression was used to estimate the Hazards Ratio (HR) for failure between the three groups. RESULTS: A total of 692 patients who received an LHRH agonist as primary mono-therapy for prostate cancer fit our study criteria. (Patients with inadequate PSA follow-up and with prior radiation treatment and/or surgery were excluded). Calendar based dosing was employed in 325 patients, 252 received T-based dosing, and 115 underwent intermittent dosing regimens. The relative risk for treatment failure was significantly lower for T-based dosing (HR = 0.65, p= 0.02). Intermittent dosing trended toward lower treatment failure risk (HR = 0.80, p = 0.3).
THE JOURNAL OF UROLOGY®
levels of Cdc25A and its interplay with androgen receptor may contribute to increase understanding of the tumor biology of human prostate cancer associated with cell cycle regulation. Source of Funding: None
259 ANDROGEN REGULATES THE EXPRESSION OF CRITICAL HEDGEHOG SIGNALING COMPONENTS IN PROSTATE CANCER CELLS. Mengqian Chen, Matthew Tanner, Albany, NY; Alice C. Levine, New York, NY; Elina Levina, Ralph Buttyan*, Albany, NY INTRODUCTION AND OBJECTIVE: Hedgehog (Hh) is a ligandactivated developmental signaling pathway that is thought to play a role in human cancers including prostate cancer. Although prostate cancer cells express many gene products needed for Hh signaling, these cells were found to be refractory to canonical Hh activating stimuli and the pathway leading to Hh activity in prostate cancer remains enigmatic. Here, we measured the effects of androgen on the expression of Hh ligands and Hh target genes in the androgen-sensitive prostate cancer cell line, LNCaP and in more metastatic variants. METHODS: LNCaP and derivative C4-2 and C4-2B cells were grown for increasing time in androgen-free medium with or without R1881. RNAs were extracted, reverse-transcribed and assayed for relative expression of the three Hh ligands (Sonic [SHH], Indian [IHH] or Desert [DHH] by qPCR and for the relative expression of the Hh receptor and target genes, Patched (Ptch), Gli1 and Gli2. Expression of the later genes was also measured in the presence of the Hh inhibitor, cyclopamine. Expression of Hh ligand protein was measured on western blot of cell extracts and conditioned medium (CM). CM was also assayed for paracrine Hh activity on Hh-sensitive mouse MC3T3-E1 fibroblasts. RESULTS: Androgen strongly suppresses expression of Hh ligands in LNCaP cells and their derivatives and their prolonged maintenance in androgen-free medium upregulated SHH and IHH mRNA and protein levels by up to 30,000-fold. Mature Hh ligand was released into the CM of androgen-deprived cells and this ligand was paracrine-active as shown by the ability of CM to increase Hh target gene (mPtch and mGli) expression in MC3T3-E1 cells. This activity was also accompanied by increased expression of Gli target genes, Ptch and Gli2, in LNCaP cells that was suppressed by cyclopamine. In contrast to the suppressive effects of R1881 on Hh ligand expression, we found that Gli1 expression in LNCaP cells was significantly induced by R1881. CONCLUSIONS: The expression of Hh ligands and the activity of the autocrine Hh signaling pathway are regulated by androgen in LNCaP cells and their derivatives. This finding indicates that androgen deprivation therapy might create a Hh signaling environment around a prostate tumor that could impact on the long term effectiveness of this treatment. Our results also support the idea that some of the putative aberrations in Hh signaling in prostate cancer may be a manifestation of the androgen signaling pathway in the cancer cells rather than the canonical Hh signaling pathway. Source of Funding: NIH CA111618; DOD W81XWH-06; Equinox Foundation
260 INTERLEUKIN-6 ENHANCES INTRATUMORAL ANDROGEN LEVELS BY REGULATING THE EXPRESSION OF GENES MEDIATING ANDROGEN METABOLISM Yae Chun, Joy C Yang, Hsing-Jien Kung, Christopher P Evans, Allen C Gao*, Sacramento, CA INTRODUCTION AND OBJECTIVE: The standard systemic treatment for prostate cancer patients is androgen deprivation therapy. Although serum testosterone concentrations were significantly reduced after androgen deprivation therapy, levels of intraprostatic androgens are reproducibly measured at concentrations sufficient to activate androgen receptor (AR) and stimulate tumor growth. Thus, prostate cancer cells
Vol. 181, No. 4, Supplement, Sunday, April 26, 2009
may survive androgen deprivation therapies by regulating intracrine androgen synthesis within the prostate. However, factors involved in de novo intracrine androgen synthesis have not been identified. Interleukin-6 (IL-6) has been implicated in the modulation of AR activation and growth and differentiation in prostate cancer. In this study, we investigate whether IL-6 associate with intraprostatic androgen synthesis and AR activation in castration-resistant prostate cancer. METHODS: Quantitative reverse transcription-PCR and western blot were performed to detect expression levels of steroidogenic enzymes. To measure intraprostatic androgen levels, IL-6 overexpressing LNCaPIL6+ cells were inoculated orthotopically into the prostate of castrated male nude mice and generated tumors. Intraprostatic androgen levels were measured by an enzyme immunoassay (Testosterone EIA kit) based on the competition between testosterone and a testosteroneacetylcholinesterase (AChE) conjugate (testosterone tracer) for a limited amount of testosterone in the sample. RESULTS: We found that IL-6 increases the expression of genes encoding many steroidogenic enzymes including HSD3B2 and AKR1C1-3 involved in androgen biosynthesis, which suggest that IL-6 may increase the levels of intraprostatic androgen. Down regulation of IL-6 receptor and gp130 expression using specific siRNA abolished IL-6 mediated AKR1C1-3 expression, suggesting that IL-6 signaling is responsible for AKR1C1-3 expression. The tumor testosterone levels were detected at 320 ± 65 pg/g tissue by testosterone EIA kit in tumors generated from IL-6 overexpressing LNCaP-IL6+ cells inoculated orthotopically into the prostate of the castrated male nude mice, such levels of testosterone are found sufficient to activate AR signaling. CONCLUSIONS: These studies suggest that IL-6 increased levels of intratumoral androgens through enhanced expression of genes mediating androgen metabolism during androgen deprivation therapy. Source of Funding: NIH CA90271, CA109441
261 TREATMENT FAILURE IS ASSOCIATED WITH DIFFERENT DOSING REGIMENS OF LEUTINIZING HORMONE RELEASING HORMONE AGONIST THERAPY FOR PROSTATE CANCER Jeremy M Blumberg*, Eric Kwon, T. Craig Cheetham, Fang Niu, Steven J. Jacobsen, Charles E. Shapiro, Stephen G. Williams, Gary W. Chien, Los Angeles, CA INTRODUCTION AND OBJECTIVE: Leutinizing Hormone Releasing Hormone (LHRH) agonist therapy is a mainstay of prostate cancer treatment. Several dosing regimens currently exist: calendar based, intermittent, and a testosterone-based (T-based) regimen we have previously reported. We investigated the relative risk of treatment failure based on dosing regimen. METHODS: We evaluated 1617 patients with prostate cancer who received LHRH agonist therapy in the Kaiser Permanente Southern California Cancer Registry from Jan 2003-December 2006. Patients were grouped according to their respective dosing regimen; calendar based, intermittent dosing, or T-based. Calendar based is defined as patients who are re-dosed with LHRH-agonist every 3 months. Intermittent dosing is defined as patients who are re-dosed with LHRH-agonist based on the patient’s PSA level, irrespective of testosterone levels. T-based is defined as patients who are re-dosed with LHRH-agonist whenever serum testosterone (T) level rises above 50ng/dl. We defined treatment failure as 2 consecutive rises in PSA measurement (with a minimum of 3 PSAs) following the last dose of LHRH-agonist. Cox proportional hazards regression was used to estimate the Hazards Ratio (HR) for failure between the three groups. RESULTS: A total of 692 patients who received an LHRH agonist as primary mono-therapy for prostate cancer fit our study criteria. (Patients with inadequate PSA follow-up and with prior radiation treatment and/or surgery were excluded). Calendar based dosing was employed in 325 patients, 252 received T-based dosing, and 115 underwent intermittent dosing regimens. The relative risk for treatment failure was significantly lower for T-based dosing (HR = 0.65, p= 0.02). Intermittent dosing trended toward lower treatment failure risk (HR = 0.80, p = 0.3).