Androgen-independent C4-2 Cells Research Articles

A Systematic Comparison of Antiandrogens Identifies Androgen Receptor Protein Stability as an Indicator for Treatment Response.

Antiandrogen therapy is a primary treatment for patients with metastasized prostate cancer. Whilst the biologic mechanisms of antiandrogens have been extensively studied, the operating protocols used for the characterization of these drugs were not identical, limiting their comparison. Here, the antiandrogens Bicalutamide, Enzalutamide, Apalutamide, and Darolutamide were systematically compared using identical experimental setups. Androgen-dependent LNCaP and LAPC4 cells as well as androgen-independent C4-2 cells were treated with distinct concentrations of antiandrogens. Androgen receptor (AR)-mediated gene transactivation was determined using qPCR. Cell viability was measured by WST1 assay. Protein stability and AR localization were determined using western blot. Response to the tested antiandrogens across cellular backgrounds differed primarily in AR-mediated gene transactivation and cell viability. Antiandrogen treatment in LNCaP and LAPC4 cells resulted in AR protein level reduction, whereas in C4-2 cells marginal decreased AR protein was observed after treatment. In addition, AR downregulation was already detectable after 4 h, whereas reduced AR-mediated gene transactivation was not observed before 6 h. None of the tested antiandrogens displayed an advantage on the tested parameters within one cell line as opposed to the cellular background, which seems to be the primary influence on antiandrogen efficacy. Moreover, the results revealed a prominent role in AR protein stability. It is one of the first events triggered by antiandrogens and correlated with antiandrogen efficiency. Therefore, AR stability may surrogate antiandrogen response and may be a possible target to reverse antiandrogen resistance.

Enzalutamide inhibits testosterone-induced growth of human prostate cancer xenografts in zebrafish and can induce bradycardia

The zebrafish has become a popular human tumour xenograft model, particularly for solid tumours including prostate cancer (PCa). To date PCa xenotransplantation studies in zebrafish have not been performed in the presence of testosterone, even when employing androgen-dependent cell models, such as the LNCaP cell line. Thus, with the goal of more faithfully modelling the hormonal milieu in which PCa develops in humans, we sought to determine the effects of exogenous testosterone on the growth of LNCaP, or androgen-independent C4-2 cells xenografted into zebrafish embryos. Testosterone significantly increased engrafted LNCaP proliferation compared to control xenografts, which could be inhibited by co-administration of the anti-androgen receptor drug, enzalutamide. By contrast, C4-2 cell growth was not affected by either testosterone or enzalutamide. Enzalutamide also induced bradycardia and death in zebrafish embryos in a dose-dependent manner and strongly synergized with the potassium-channel blocking agent, terfenadine, known to induce long QT syndrome and cardiac arrhythmia. Together, these data not only indicate that testosterone administration should be considered in all PCa xenograft studies in zebrafish but also highlights the unique opportunity of this preclinical platform to simultaneously evaluate efficacy and toxicity of novel therapies and/or protective agents towards developing safer and more effective PCa treatments.

FOXM1 promotes the progression of prostate cancer by regulating PSA gene transcription.

Androgen/AR is the primary contributor to prostate cancer (PCa) progression by regulating Prostate Specific Antigen (PSA) gene transcription. The disease inevitably evolves to androgen-independent (AI) status. Other mechanisms by which PSA is regulated and develops to AI have not yet been fully determined. FOXM1 is a cell proliferation-specific transcription factor highly expressed in PCa cells compared to non-malignant prostate epithelial cells, suggesting that the aberrant overexpression of FOXM1 contributes to PCa development. In addition to regulating AR gene transcription and cell cycle-regulatory genes, FOXM1 selectively regulates the gene transcription of KLK2 and PSA, typical androgen responsive genes. Screening the potential FOXM1-binding sites by ChIP-PCR, we found that FOXM1 directly binds to the FHK binding motifs in the PSA promoter/enhancer regions. AI C4-2 cells have more FOXM1 binding sites than androgen dependent LNCaP cells. The depletion of FOXM1 by small molecular inhibitors significantly improves the suppression of PSA gene transcription by the anti-AR agent Cadosax. This is the first report showing that FOXM1 promotes PCa progression by regulating PSA gene transcription, particularly in AI PCa cells. The combination of anti-AR agents and FOXM1 inhibitors has the potential to greatly improve therapy for late-stage PCa patients by suppressing PSA levels.

Synergistic co-targeting of prostate-specific membrane antigen and androgen receptor in prostate cancer.

Antibody-drug conjugates (ADCs) are an emerging class of cancer therapies that have demonstrated favorable activity both as single agents and as components of combination regimens. Phase 2 testing of an ADC targeting prostate-specific membrane antigen (PSMA) in advanced prostate cancer has shown antitumor activity. The present study examined PSMA ADC used in combination with potent antiandrogens (enzalutamide and abiraterone) and other compounds. Antiproliferative activity and expression of PSMA, prostate-specific antigen and androgen receptor were evaluated in the prostate cancer cell lines LNCaP and C4-2. Cells were tested for susceptibility to antiandrogens or other inhibitors, used alone and in combination with PSMA ADC. Potential drug synergy or antagonism was evaluated using the Bliss independence method. Enzalutamide and abiraterone demonstrated robust, statistically significant synergy when combined with PSMA ADC. Largely additive activity was observed between the antiandrogens and the individual components of the ADC (free drug and unmodified antibody). Rapamycin also synergized with PSMA ADC in certain settings. Synergy was linked in part to upregulation of PSMA expression. In androgen-dependent LNCaP cells, enzalutamide and abiraterone each inhibited proliferation, upregulated PSMA expression, and synergized with PSMA ADC. In androgen-independent C4-2 cells, enzalutamide and abiraterone showed no measurable antiproliferative activity on their own but increased PSMA expression and synergized with PSMA ADC nonetheless. PSMA expression increased progressively over 3 weeks with enzalutamide and returned to baseline levels 1 week after enzalutamide removal. The findings support exploration of clinical treatment regimens that combine potent antiandrogens and PSMA-targeted therapies for prostate cancer.

Abstract 798: Androgen receptor, PI3K/mTOR and PSMA: Exploring and exploiting the interplay between therapeutic targets in prostate cancer

Abstract Introduction: Androgen receptor (AR), the PI3K/mTOR signaling pathway and prostate-specific membrane antigen (PSMA) represent significant potential targets for prostate cancer therapy. The present study examined the cross-regulation and co-targeting of these molecular pathways. Methods: Cytotoxicity and the kinetics of antigen expression were evaluated in prostate cancer cell lines (LNCaP and C4-2) that vary according to androgen dependence. Expression of PSMA, prostate-specific antigen (PSA), and AR was evaluated in cells cultured in rapamycin and AR inhibitors. Cells were tested for susceptibility to PI3K/mTOR inhibitors used alone and in combination with PSMA ADC, a fully human PSMA monoclonal antibody conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE). Potential drug synergy or antagonism was evaluated using the Bliss independence method. Results: In androgen-dependent LNCaP cells, rapamycin exerted antiproliferative effects that were accompanied by an increase in AR expression and signaling in the absence of any significant effect on PSMA expression. In androgen-independent C4-2 cells, rapamycin increased AR expression/signaling and PSMA expression in the absence of any significant anti-proliferative effect. AR inhibitors synergized with PSMA ADC in LNCaP and C4-2 cells, but PI3K/mTOR pathway inhibitors synergized with PSMA ADC in C4-2 cells only. More modest synergy was observed by combining non-targeted microtubule inhibitors with inhibitors of AR or mTOR. Compared with rapamycin, GDC0941 (PI3K inhibitor) and MK-2206 (Akt inhibitor), both upstream of mTOR, synergized less strongly with PSMA ADC in C4-2 cells. Conclusions: In this study, AR and PI3K/mTOR pathways exhibited cross-regulation impacting PSMA expression. PSMA ADC synergized with AR and mTOR inhibitors via a multimodal mechanism involving increased PSMA expression and disruption of microtubule function. The findings support clinical exploration of regimens combining PSMA-targeted therapies with inhibitors of the AR and/or PI3K/mTOR signaling pathways. Citation Format: Jose D. Murga, Wells W. Magargal, Sameer M. Moorji, Vincent A. DiPippo, William C. Olson. Androgen receptor, PI3K/mTOR and PSMA: Exploring and exploiting the interplay between therapeutic targets in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 798. doi:10.1158/1538-7445.AM2014-798

Abstract A262: Antiandrogen modulation of prostate-specific membrane antigen (PSMA): Dynamics and synergy with PSMA-targeted therapy.

Abstract Introduction: Recent approvals of new prostate cancer (PCa) drugs and a growing number of pipeline agents have created opportunities for designing rational drug combinations. Potent antiandrogens such as enzalutamide and abiraterone affect expression of numerous androgen-regulated molecules, including PSMA, a well-characterized cell-surface target for PCa therapy. Methods: Cytotoxicity and the kinetics of antigen expression were evaluated in PCa cell lines (LNCaP, C4-2 and 22Rv1) that vary according to androgen dependence and level of PSMA expression. Expression of PSMA and prostate-specific antigen (PSA) was evaluated over time in cells cultured in enzalutamide or abiraterone. Reversibility was examined following drug washout. Cells were tested for susceptibility to antiandrogens used alone and in combination with PSMA ADC, which is a fully human PSMA monoclonal antibody conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE). Potential drug synergy or antagonism was evaluated using the Combination Index and Bliss independence methods. Results: In androgen-dependent LNCaP cells, enzalutamide and abiraterone exerted antiproliferative effects that were accompanied by a 2- to 3-fold increase in PSMA expression and a decrease in PSA expression. PSMA expression reached maximum levels after four weeks’ culture in enzalutamide and returned to baseline levels within days following enzalutamide removal. In androgen-independent C4-2 cells, enzalutamide and abiraterone increased PSMA expression in the absence of any significant anti-proliferative effect. PSMA ADC exhibited synergistic antitumor activity with both enzalutamide and abiraterone in LNCaP and C4-2 cells. Lesser effects were observed using 22Rv1 cells, an androgen-independent cell line with low basal expression of PSMA. Conclusions: Enzalutamide and abiraterone significantly and reversibly augmented PSMA expression and potentiated the activity of PSMA ADC in androgen-dependent and -independent PCa cell lines in vitro. In androgen-independent cells, the effects on PSMA expression and PSMA ADC activity were uncoupled from any anti-proliferative effect of the antiandrogens. The findings support clinical exploration of regimens that combine potent antiandrogens and PSMA-targeted therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A262. Citation Format: Jose D. Murga, Wells W. Magargal, Sameer M. Moorji, Vincent A. DiPippo, William C. Olson. Antiandrogen modulation of prostate-specific membrane antigen (PSMA): Dynamics and synergy with PSMA-targeted therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A262.

Abstract 5387: Integrative analysis of transcriptomic and metabolomic data reveals a critical role for aminosugar metabolism in prostate cancer.

Abstract Prostate Cancer is the second most common cause of cancer-related death in men in the US. Like other tumors, prostate cancer development and progression is dictated by multiple molecular events that include changes in levels of genes, transcripts, proteins and metabolites. To better understand the biology of prostate cancer development it is essential to integrate these disparate yet related datasets. Our laboratory has identified matched transcriptomic, proteomic and metabolomic changes in localized prostate cancer relative to adjacent benign tissue as well in metastatic disease compared to organ-confined tumor. To study this using a System's Biology approach we have recently embarked on a pilot study aimed at integrating transcriptomic and metabolomic data to obtain biochemical pathways that key to prostate cancer development. Using an in-house network-based enrichment strategy, amino sugar metabolism was found to be significantly enriched in organ-confined prostate cancer but not in metastatic disease. Amino sugar metabolism describes the utilization of glucose-derived carbon and amino acid (mostly glutamine)-derive nitrogen to produce glucosamines. These amino sugars participate in synthesis of immune modulatory compounds as well as in glycosylation cascades. In this study, we describe the molecular analyses of Glucosamine-6 phosphate-N-acetyl Transferase (GNPNAT1), a key enzyme that converts D-glucosamine 6-phosphate to N-acetyl-D-glucosamine 6-phosphate, in prostate cancer. Our results indicate upregulation of GNPNAT1 in organ-confined prostate cancer as compared to benign adjacent tissue as well as metastatic tissue and regulation of the pathway by androgen. Stable knockdown of GNPNAT1 in androgen dependent LNCap cells leads to diminished cell growth and cell cycle arrest. In contrast, growth and cell cycle are not affected by the knockdown of GNPNAT1 in androgen independent C4-2 cells. Knockdown of GNPNAT1 in C4-2 cells enhances invasiveness which is not observed in LNCap knockdown cells. In this work we show that GNPNAT1 is linked to androgen receptor (AR) action and thus is a critical enzyme for the survival of androgen dependent prostate cancer cells. Citation Format: Katrin Panzitt, Ali Shojaie, Nagireddy Putluri, Sumanta Basu, Vasanta Putluri, Susmita Samanta, Michael Ittmann, Ismael Vergara, George Michailidis, Ganesh Palapattu, Arun Sreekumar. Integrative analysis of transcriptomic and metabolomic data reveals a critical role for aminosugar metabolism in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5387. doi:10.1158/1538-7445.AM2013-5387

Abstract 1319: Regulation of AR transcriptional activity and prostate cancer cellular proliferation by DACH1/Eya1/Six1 pathway.

Abstract Prostate cancer progression from androgen responsive to androgen-depletion therapy resistance is a major cause of treatment failures. Novel therapies targeting this patient are a major clinical challenge. We previously reported that the cell fate determination factor DACH1 inhibits androgen signaling though binding the AR and enhancing co-repressor recruitment in the context of local chromatin at AR responsive genes. The repression of AR function of by DACH1 was partly rescued by the co-activators p300 and SRC. The repression was enhanced by ectopic expression of HDACs. We had previously shown that DACH1 inhibited proliferation of androgen-dependent LnCAP cells. To further explore the significance of DACH1 in human prostate cancer, we established stable cell lines expressing DACH1 or its DS domain deleted mutant (ΔDS) in androgen-independent C4-2 cells and AR-negative PC-3 cells by retrovirus infection. Stable expression of DACH1 inhibited cellular proliferation in both cell lines, requiring the DS domain. Ectopic expression of DACH1 inhibited colony formation of the androgen-independent 22RV1 cells. DACH1 inhibited migration, invasion and tumor growth of AR-negative PC-3 cells. Since DACH1, Eya1 and Six1 function together as a network to control cellular differentiation and organ formation during development, we examined whether other components of RDGN pathway, could regulate androgen signaling. In hormone responsive LnCAP cells, ligand stimulation (DHT, 50nM) induced 17 folds activation of MMTV Luc, co-expression of Eya1 reduced AR transactivation, and the repression required the EYA phophatase domain. Expressions of the AR co-repressors, HDAC1, HDAC3 or SirT1 inhibited the ligand-induced AR activity. Eya1 enhanced co-repressor function at the PSA and MMTV promoters. Consistent with our previous publication, DACH1 repressed ligand-induced activity >70%, Eya1 modified DACH1 repression of AR activity. Together, our studies demonstrated that the RDGN regulates both androgen dependent and independent prostate cancer cellular proliferation and tumorigenesis. Citation Format: Jing Wang, Shaoxin Cai, Ke Chen, Sean Li, Richard Pestell, Kongming Wu. Regulation of AR transcriptional activity and prostate cancer cellular proliferation by DACH1/Eya1/Six1 pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1319. doi:10.1158/1538-7445.AM2013-1319

Snail negatively regulates cell adhesion to extracellular matrix and integrin expression via the MAPK pathway in prostate cancer cells

Snail transcription factor induces epithelial-mesenchymal transition (EMT) in which the epithelial cells downregulate cell-cell adhesion genes such as E-Cadherin and upregulate mesenchymal genes such as vimentin, leading to increased invasion and migration. Very little is known about the role of Snail in cellular adhesion to the extracellular matrix. We hypothesized that Snail will lead to decreased cellular adhesion to fibronectin and collagen I matrix through integrin regulation, concomitant with increased cell migration. Androgen-independent C4-2 cells, an aggressive subline of androgen-dependent LNCaP cells, exhibited decreased cell adhesion and increased cell migration on fibronectin and collagen I as compared to LNCaP cells, which was reversed by Snail knock down in C4-2 cells. ARCaP and LNCaP prostate cancer cells stably transfected with Snail displayed decreased adhesion and increased cell migration on fibronectin and collagen I as compared to control Neo-transfected cells, which was reversed by Snail knockdown. Flow cytometry analysis revealed a decrease in a5, a2 and b1 integrin expression in ARCaP Snail-transfected cells that was reversed in Snail knock down cells. We also observed an increase in ERK phosphorylation in ARCaP Snail-transfected cells as compared to control ARCaP-Neo cells, and inhibition of the MAPK pathway with UO126 inhibitor in ARCaP Snail-transfected cells abrogated Snail-mediated decrease in cell adhesion and reinduced a5, a2 and b1 integrin expression. Collectively, these studies define a new role for Snail transcription factor in cell adhesion to the ECM, which may be mediated by MAPK signaling, and may be crucial for cell detachment and subsequent metastasis.

Modulation of Protein Phosphatase 2A Activity Alters Androgen-Independent Growth of Prostate Cancer Cells: Therapeutic Implications

Earlier we identified PPP2CA, which encodes for the α-isoform of protein phosphatase 2A (PP2A) catalytic subunit, as one of the downregulated genes in androgen-independent prostate cancer. PP2A is a serine/threonine phosphatase and a potent tumor suppressor involved in broad cellular functions; however, its role in prostate cancer has not yet been determined. Here, we have investigated the effect of PP2A activity modulation on the androgen-independent growth of prostate cancer cells. Our data show that the PPP2CA expression and PP2A activity is downregulated in androgen-independent (C4-2) prostate cancer cells as compared with androgen-dependent (LNCaP) cells. Downregulation of PP2A activity by pharmacologic inhibition or short interfering RNA-mediated PPP2CA silencing sustains the growth of LNCaP cells under an androgen-deprived condition by relieving the androgen deprivation-induced cell-cycle arrest and preventing apoptosis. Immunoblot analyses reveal enhanced phosphorylation of Akt, extracellular signal-regulated kinase (ERK), BAD, increased expression of cyclins (A1/D1), and decreased expression of cyclin inhibitor (p27) on PP2A downregulation. Furthermore, our data show that androgen receptor (AR) signaling is partially maintained in PP2A-inhibited cells through increased AR expression and ligand-independent phosphorylation. Pharmacologic inhibition of Akt, ERK, and AR suggest a role of these signaling pathways in facilitating the androgen-independent growth of LNCaP cells. These observations are supported by the effect of ceramide, a PP2A activator, on androgen-independent C4-2 cells. Ceramide inhibited the growth of C4-2 cells on androgen deprivation, an effect that could be abrogated by PP2A downregulation. Altogether, our findings suggest that modulation of PP2A activity may represent an alternative therapeutic approach for the treatment of advanced androgen-independent prostate cancer.

Active sonic hedgehog signaling between androgen independent human prostate cancer cells and normal/benign but not cancer‐associated prostate stromal cells

Sonic hedgehog (Shh) signaling plays a pivotal role in stromal-epithelial interaction during normal development but its role in tumor-stromal interaction during carcinogenic progression is less well defined. Since hormone refractory prostate cancer with bone metastasis is difficult to treat, it is crucial to investigate how androgen independent (AI) human prostate cancer cells communicate with their associated stroma. Shh and its target transcription factor, Gli1 mRNA, were assessed by RT-PCR and/or quantitative RT-PCR in co-cultured cell recombinants comprised of AI C4-2 either with NPF (prostate fibroblasts from normal/benign prostate gland) or CPF (cancer-associated stromal fibroblasts) under Shh/cyclopamine (a hedgehog signaling inhibitor) treatment. Human bone marrow stromal (HS27A) cells were used as controls. In vivo investigation was performed by checking serum PSA and immunohistochemical staining for the apoptosis-associated M30 gene in mice bearing chimeric C4-2/NPF tumors. We found that (1) Shh has minimal growth-stimulating effects on prostate cancer cells, but it stimulated the growth of NPF but not CPF; (2) active Shh signaling was found between AI C4-2 cells and NPF but not CPF; and (3) osteonectin (ON) is a Gli1 target gene in NPF and not in CPF, and ON up-regulation in NPF can be blocked by cyclopamine Based on co-culture and chimeric tumor models, active Shh-mediated signaling was demonstrated between AI prostate cancer and NPF in a paracrine- and tumor progression-dependent manner. Our study suggests that drugs like cyclopamine that interfere with Shh signaling could be beneficial in preventing AI progression in prostate cancer cells.

Survival advantage of AMPK activation to androgen-independent prostate cancer cells during energy stress

Androgen-independent prostate cancer usually develops as a relapse following androgen ablation therapy. Removing androgen systemically causes vascular degeneration and nutrient depletion of the prostate tumor tissue. The fact that the malignancy later evolves to androgen-independence suggests that some cancer cells are able to survive the challenge of energy/nutrient deprivation. AMP-activated protein kinase (AMPK) is an important manager of energy stress. The present study was designed to investigate the role of AMPK in contributing to the survival of the androgen-independent phenotype. Most of the experiments were carried out in the androgen-dependent LNCaP cells and the androgen-independent C4-2 cells. These two cell lines have the same genetic background, since the C4-2 line is derived from the LNCaP line. Glucose deprivation (GD) was instituted to model energy stress encountered by these cells. The key findings are as follows. First, the activation of AMPK by GD was much stronger in C4-2 cells than in LNCaP cells, and the robustness of AMPK activation was correlated favorably with cell viability. Second, the response of AMPK was specific to energy deficiency rather than to amino acid deficiency. The activation of AMPK by GD was functional, as demonstrated by appropriate phosphorylation changes of mTOR and mTOR downstream substrates. Third, blocking AMPK activation by chemical inhibitor or dominant negative AMPK led to increased apoptotic cell death. The observation that similar results were found in other androgen-independent prostate cancer cell lines, including CW22Rv1 abd VCaP, provided further assurance that AMPK is a facilitator on the road to androgen-independence of prostate cancer cells.

Abstract 574: 1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibits mammalian target of rapamycin complex 2 (mTORC2) signaling in prostate cancer cells

Abstract The lack of treatment for worried-well patients with high-grade prostatic intraepithelial neoplasia combined with issues of recurrence and hormone resistance in prostate cancer survivors remains a major public health obstacle. The long latency of prostate cancer development provides an opportunity to intervene with mechanistically-based agents at various stages of disease progression. Preclinical studies and immunohistochemical analyses of human prostate adenocarcinoma tissues have implicated the mammalian target of rapamycin (mTOR) signaling pathway in the progression of prostate cancer and its transition to androgen independence, suggesting mTOR as a potential target for new therapies. Preclinical studies in human prostate cancer cells also show that there is cross-talk between mTOR and androgen receptor signaling, a process critical for prostate cancer growth and survival. We have previously shown that the synthetic organoselenium compound p-XSC effectively inhibits cell viability and down-regulates androgen receptor expression and transcriptional activity in androgen responsive (AR) and androgen independent (AI) human prostate cancer cells; the naturally occurring selenomethionine was either ineffective or weakly active at non-physiological levels. Therefore, we focused our study on the effects of p-XSC and we hypothesized that it inhibits prostate cancer cell growth by interfering with mTOR signaling and that inhibition of this pathway may be linked to down-regulation of androgen receptor signaling in these cells. Here we show that p-XSC inhibits phosphorylation of mTOR in both AR LNCaP and AI LNCaP C4-2 human prostate cancer cells. This inhibition is attenuated by stimulation with androgens in AR LNCaP but not AI C4-2 cells, emphasizing a cross-talk between the two pathways. p-XSC also decreases mTOR binding to mTOR complex 2 (mTORC2) protein Rictor in AR LNCaP and AI C4-2 cells and inhibits the phosphorylation of mTORC2 downstream target Akt. Since p-XSC appears to be inhibiting mTORC2 as well as androgen signaling, we further hypothesized that the combination of p-XSC with rapamycin, which primarily targets mTORC1, may be a superior approach to inhibit prostate cancer cell growth than either agent alone. Our data clearly support this hypothesis. Validation of mTOR as a target of selenium in prostate cancer will provide evidence for its potential role against advanced and hormone refractory prostate disease either individually or in combination with therapies that target other signaling pathways. Support: NIH CA111842, DOD W81XWH-08-1-0297 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 574.

Abstract 3780: Plumbagin a non-toxic natural agent, induces apoptosis and inhibits the growth of prostate cancer LNCaP and C4-2 cells via blocking of both androgen and non-androgen activation of androgen receptor signaling

Abstract Prostate cancer (PCa) is one of the most commonly diagnosed cancers and leading cause of cancer related death in men in Western countries including the Unites States. Androgen receptor (AR)-mediated signaling plays a critical role in the development and progression of PCa. Most androgen independent (AI) prostate tumors continue to express AR as well as its target gene PSA, which indicates that these tumor cells maintain functional AR signaling. Evidence suggests that AR signaling is activated through androgen and androgen bypass mechanism. We believe that identification of non-toxic natural agents capable of blocking both androgen and non-androgen activation of AR signaling could be effective for the treatment of PCa. We have previously shown that a novel plant derived napthoquinone plumbagin (PL) induces apoptosis and inhibits growth of PCa cells in in vitro as well as in in vivo model systems (Cancer Research 2008, PMID: 18974148). We present here that PL inhibits both androgen and non-androgen activation of AR signaling in PCa cells. We observed that PL inhibits synthetic androgen methyltrienolone (R1881) induced growth of androgen-dependent (AD) LNCaP and AI C4-2 cells. PL treatment was found to inhibit protein levels of AR and AR-target gene PSA and accelerates AR degradation. To analyze the effect of PL on decreased AR transactivation, transient transfections using androgen responsive reporters PSA-luc were carried out. Treatment of LNCaP and C4-2 cells with R1881 led to induction of PSA reporter in these cells. Co-treatment of LNCaP and C4-2 cells with PL led to a dose-dependent decrease in AR transactivation as reflected by a decrease in the PSA reporter activity. These results suggest that PL interferes with androgen-induced transactivation function of AR. It has been reported that IL-6 signaling acts as a ligand for the activation of AR signaling. IL-6 induced phosphorylated Stat3 interacts with the N-terminal domain of AR and activates its target genes. We have previously shown that PL treatment inhibits Stat3 phosphorylation in PCa DU-145 cells xenograft tumor tissues (Cancer Research 2008, PMID: 18974148). Therefore, we hypothesized that PL treatment will inhibit Stat3 and AR N-terminal domain interaction in PCa cells. Reciprocal immunoprecipitation/blotting experiments revealed that PL treatment inhibited IL-6 induced interaction of Stat3 with AR N-terminal domain in both the PCa LNCaP and C4-2 cells. PL treatment also inhibited IL-6 induced Tyr 705 and Ser727 phosphorylation of Stat3. These results establish that PL has the potential to block the androgen and non-androgen activation of AR signaling in PCa and provide further evidence that PL could be used as a chemopreventive natural agent in ongoing anti-hormone therapy of PCa. (Support: NIH grants, CA35368). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3780.

Abstract 1702: Androgen receptor antagonist bicalutamide sensitizes androgen-independent prostate cancer cells to mTOR inhibitor rapamycin by regulating phosphorylation of translation initiation suppressor eIF4E-binding protein 1 (4E-BP1)

Abstract We previously showed that resistance of prostate cancer cells to the mTOR inhibitor rapamycin results from its ability to induce androgen receptor (AR) transcriptional activity, whereas co-treatment with the anti-androgen bicalutamide prevented this effect. Based on our data, collaborators at UC Davis Cancer Center are conducting a clinical trial to test the efficacy of this combination in patients with advanced prostate cancer. However, the mechanism by which the combination of rapamycin and bicalutamide work remains unknown. Investigation of the cause for resistance of prostate cancer cells to rapamycin showed that rapamycin-induced growth inhibition as well as AR transcriptional activity is mediated primarily by p70S6 kinase (S6K1), a downstream target of mTOR, but not by the 4E-BP1/eIF4E/eIF4G pathway, another downstream target of mTOR. Additionally, 4E-BP1 phosphorylation at Ser 65 was dose dependently inhibited by rapamycin in androgen-dependent LNCaP, but not in androgen-independent C4-2 cells, although rapamycin equally affected S6K1phosphorylation in both cells. Comparison of AR expression and 4E-BP1 phosphorylation in various prostate cancer cell lines demonstrated that those expressing high levels of the full-length AR also had increased 4E-BP1 phosphorylation, whereas no such relationship existed with S6K1. Hence, we investigated whether increased AR activity contributed to resistance of C4-2 cells to rapamycin-induced 4E-BP1 dephosphorylation. Bicalutamide treatment as well as culture in medium containing decreased levels of androgens repressed 4E-BP1 phosphorylation, showing that the AR is a positive regulator of 4E-BP1 phosphorylation. The combination of bicalutamide and rapamycin, but not rapamycin alone, inhibited 4E-BP1 phosphorylation in C4-2 cells. Additionally, bicalutamide sensitized PC-3 cells stably transfected with the AR, but not wild-type PC-3 cells that are AR null, to rapamyin-induced 4E-BP1 dephosphorylation. We also compared the effect of bicalutamide in two cell lines derived from CWR22 relapsed tumors: in CWR22R1 cell, which expressed significant levels of the full-length AR, 4E-BP1 phosphorylation was inhibited by bicalutamide, whereas in CWR22Rv1 cells, where the AR lack the ligand-binding domain (LBD), bicalutamide was unable to affect 4E-BP1 phosphorylation. Thus, in cells where bicalutamide is able to bind the AR, this drug sensitizes the cells to the effects of rapamycin. These results were verified in a CWR22 mouse xenograft model of prostate cancer progression. Taken together, our data show that rapamycin treatment increased AR activity by inhibiting S6K1, and that this increase in AR activity in turn induced resistance to rapamycin's ability to repress 4E-BP1 phosphorylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1702.

Abstract 561: Establishment and characterization of matched pairs of human prostate stromal cells to study cancer-stromal interaction

Abstract Prostate stromal cells may play binary roles in the process of prostate cancer development. Being the first to be encountered by infiltrating prostate cancer cells, prostate stromal cells form the first defense line against prostate cancer progression and metastasis. On the other hand, interaction between prostate cancer and stromal cells may facilitate the formation of a tumor microenvironment favoring cancer cell growth and survival. To establish an experimental system for studying the interaction between cancer and stromal cells, we isolated three matched pairs of normal and cancer-associated human prostate stromal cells. The specimens were from three prostate cancer patients who underwent radical prostatectomy in the Department of Urology, Emory University School of Medicine. From each prostate, a cube of prostate tissue was dissected from a histologically normal region distal to the tumor, and another cube was dissected from a cancer-affected zone. Live cells in single cell preparation were cultured in low density for isolation of stromal clones. In contrast to the LNCaP prostate cancer cells, the isolated prostate stromal clones exhibited large fibroblastic morphology with slow growth rate. Growth and survival of the stromal clones were not affected by androgens and were highly resistant to serum starvation. In contrast to the normal counterparts, the cancer-associated stromal clones exhibited differentiated survival ability, as determined by colony formation assay following serum starvation. In direct co-culture, the prostate stromal cells inhibited growth of the LNCaP cells and made the production of Prostate Specific Antigen (PSA) less sensitive to androgen deprivation. Importantly in co-culture experiments, the stromal cells protected some LNCaP prostate cancer cells from death by serum starvation, and cancer-associated stromal clones showed more protection. The surviving LNCaP clones showed features of androgen-independent PSA expression similar to the lineage-related androgen-independent C4-2 cells, suggesting that cancer cells acquired increased malignancy through interaction with the stromal cells. The results from this study indicate that matched stromal cell pairs may serve as models for comparative analysis of molecular changes in the tumor microenvironment. These cells can also be used in co-culture with prostate cancer cells to simulate cancer-stromal interaction in the tumor microenvironment in order to define the role of prostate stromal cells in prostate cancer progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 561.

FKBP51 and Cyp40 are positive regulators of androgen-dependent prostate cancer cell growth and the targets of FK506 and cyclosporin A

Prostate cancer (PCa) growth is dependent on androgens and the androgen receptor (AR), which acts by modulating gene transcription. Tetratricopeptide repeat (TPR) proteins (FKBP52, FKBP51 and Cyp40) interact with AR in PCa cells, suggesting roles in AR-mediated gene transcription and cell growth. We report here that FKBP51 and Cyp40, but not FKBP52, are significantly elevated in PCa tissues and in androgen-dependent (AD) and -independent (AI) cell lines. Overexpression of FKBP51 in AD LNCaP cells increased AR transcriptional activity in the presence and absence of androgen, whereas siRNA knockdown of FKBP51 dramatically decreased AD gene transcription and proliferation. Knockdown of Cyp40 also inhibited androgen-mediated transcription and growth in LNCaP cells. However, disruption of FKBP51 and Cyp40 in the AI C4-2 cells caused only a small reduction in proliferation, indicating that Cyp40 and FKBP51 predominantly regulate AD cell proliferation. Under knock-down conditions, the inhibitory effects of TPR ligands, CsA and FK506, on AR activity were not observed, indicating that Cyp40 and FKBP51 are the targets of CsA and FK506, respectively. Our findings demonstrate that FKBP51 and Cyp40 are positive regulators of AR that can be selectively targeted by CsA and FK506 to achieve inhibition of androgen-induced cell proliferation. These proteins and their cognate ligands thus provide new strategies in the treatment of PCa

Interactions Between Vitamin D and Androgen Receptor Signaling in Prostate Cancer Cells

Prostate cancer is an androgen-dependent disease, and androgen blockade is the primary treatment for metastatic prostate cancer. However, the tumors become resistant within a relatively short time. The recurrent tumors, surprisingly, typically overexpress androgen receptor and a subset of androgen-regulated genes including prostate-specific antigen (PSA). The mechanism for this reactivation is unknown, but alternate therapies that target androgen receptor or other pathways are needed. There is good evidence that vitamin D reduces risk for prostate cancer. Increased exposure to sunlight (our primary source of vitamin D3) is correlated with a decreased incidence of prostate cancer. A more recent study comparing exposure to sunlight and vitamin D receptor (VDR) polymorphisms in advanced prostate cancer patients and controls showed that increased VDR activity decreased risk for prostate cancer. The active form of vitamin D3, 1,25-dihydroxyvitamin D3, abbreviated 1,25(OH)2D, is produced by sequential hydroxylations of the 25 and 1 positions in vitamin D3. In a large study of nearly 20,000 men, the risk of subsequently developing a more aggressive form of prostate cancer was higher in men with low levels of 25(OH)D. Almost all prostate cancer cell lines and primary prostate cancer cells express VDR, and most are inhibited by 1,25(OH)2D. 4-6 1,25(OH)2D-mediated growth inhibition in various types of cancer has been associated with G0/G1 accumulation, 7-11 and, in some cases, with apoptosis. 1,25(OH)2D also reduces invasiveness of DU145 prostate cancer cells, and reduces expression of MMP-9 and cathepsins, proteases that facilitate motility and invasion. Although most prostate cancer cells contain VDR, the extent to which they are growth inhibited varies greatly. The androgen receptor-positive LNCaP cells are much more responsive than the androgen receptor-negative PC-3 or DU145 cells and somewhat more responsive than some other androgen receptor-positive cell lines such as LAPC-4 and 22RV1. A report by Zhao et al. showed that the growth-inhibitory effects of 1,25(OH)2D in the androgen-dependent prostate cancer cell line LNCaP were reduced by treatment with the androgen receptor antagonist Casodex, although Casodex has some growth inhibitory actions of its own. We sought to determine whether this response was a general phenomenon in prostate cancer cell lines, as well as to determine the basis for this differential action. We found that other cells derived from the LNCaP lineage showed the same response, but two other independently derived androgen receptor-containing cell lines (LAPC-4 and 22RV1) did not. Activation of a transiently transfected VDR-responsive reporter was unaffected by treatment with Casodex, so the effect was indirect. Although combined Casodex and 1,25(OH)2D treatment inhibits the growth of LNCaP cells to the same extent as Casodex alone, an examination of some of the effects of 1,25(OH)2D (down-regulation of bcl-2, down-regulation of c-myc, and Go/G1 arrest) revealed that each of these responses was only partially inhibited. The question arises as to whether antagonist-bound androgen receptor has a unique function. In androgenindependent C4-2 cells, a derivative of the androgendependent LNCaP cells, 1,25(OH)2D does inhibit the growth of the cells in medium depleted of androgens. However, this line exhibits some hormone-dependent androgen receptor activity, and PSA is expressed in the absence of androgens. Under these conditions, Casodex partially reverses the growth inhibition caused by 1,25(OH)2D, suggesting a unique function for the antagonist-bound receptor. In an analysis of 1,25(OH)2D action in LNCaP cells, we found that an androgen-induced gene that inhibits cell growth (AS3/APRIN) was also induced by 1,25(OH)2D. 16 Moreover, Casodex reduced induction by 1,25(OH)2D. In contrast, AS3 is not induced by either androgens or 1,25D in 22RV1 cells and there is no Casodex reversal of 1,25(OH)2D-mediated Dr. Weigel is with the Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA. Please address all correspondence to: Dr. Nancy Weigel, Professor, Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Phone: 713-798-6234; Fax: 713790-1275; E-mail: nweigel@bcm.tmc.edu. doi: 10.1301/nr.2007.aug.S116–S117

Suramin-induced decrease in prostate-specific antigen expression with no effect on tumor growth in the LNCaP model of human prostate cancer.

Suramin, a polysulfonated naphthylurea and a recognized antitrypanosomal agent, has shown some promise in phase II clinical trials in the management of hormone-refractory human prostate cancer. Reduction of serum prostate-specific antigen (PSA) levels has been proposed as an end point for evaluating the antitumor efficacy of treatments for hormone-refractory prostate cancer. We examined the antitumor effect of suramin in an in vivo mouse model of hormone-refractory human prostate cancer to determine whether a decrease in PSA levels reflects a reduction in tumor growth (volume). The tumors were induced in castrated, athymic nude mice by use of the androgen-independent, tumorigenic human prostate cancer cell line C4-2, which is a subline of the androgen-dependent, parental nontumorigenic cell line LNCaP. We also evaluated the effects of suramin in vitro on cell growth and the expression of PSA messenger RNA (mRNA) in both LNCaP and C4-2 cells. For the in vivo studies, 24 mice were given a subcutaneous injection of 5 x 10(6) C4-2 cells at each of four sites. Animals (n = 20) with tumor volumes greater than 1 mm3 or less than 5 mm3 were divided equally into two groups. Drug treatment was initiated in one group by administration of 1 mg suramin intraperitoneally, followed by 0.1 mg suramin at 10-day intervals to maintain constant serum levels. Tumor growth and PSA expression levels were monitored. For the in vitro studies, both LNCaP and C4-2 cells were exposed to 100-400 microgram/mL suramin, and cell growth was monitored by a quantitative crystal violet assay. PSA mRNA expression was assessed by northern blot analysis in cells treated with either 250 microgram/mL suramin, 400 ng/mL dihydrotestosterone (DHT) (positive control), or 0.5-75 microgram/mL hydrocortisone (to mimic the clinical use of hydrocortisone during suramin treatment to compensate for the loss of adrenocortical function). In some studies, the combined effect of DHT and suramin on PSA mRNA expression was also evaluated. A two-way analysis of variance was performed to evaluate the treatment differences, and P values were obtained from two-sided tests for statistical significance. In vivo, suramin did not significantly affect the growth of androgen-independent C4-2 tumors (relative to the growth of tumors in 5% glucose-treated control animals; P = .76). However, suramin significantly decreased the ratio of PSA level to tumor volume (ng/mL PSA per mm(3) of tumor) (P<.001). Mice developed bone metastases in both treatment arms. Suramin affected the in vitro growth of LNCaP cells but not of C4-2 cells. Suramin diminished PSA mRNA expression in both LNCaP and C4-2 cells grown in vitro. Hydrocortisone had no effect on PSA mRNA levels. Although suramin inhibited the growth of androgen-dependent LNCaP cells, it did not inhibit the growth of androgen-independent C4-2 cells either in vitro or in vivo. Suramin significantly decreased PSA mRNA expression in both cell lines in vitro and depressed serum PSA levels in mice bearing androgen-independent C4-2 tumors. PSA level should be used with caution as an end point in clinical trials using suramin therapy for hormone-refractory prostate cancer.