Androgen Receptor Negative Cells Research Articles

L1CAM mediates neuroendocrine phenotype acquisition in prostate cancer cells.

A specific type of prostate cancer (PC) that exhibits neuroendocrine (NE) differentiation is known as NEPC. NEPC has little to no response to androgen deprivation therapy and is associated with the development of metastatic castration-resistant PC (CRPC), which has an extremely poor prognosis. Our understanding of genetic drivers and activated pathways in NEPC is limited, which hinders precision medicine approaches. L1 cell adhesion molecule (L1CAM) is known to play an oncogenic role in metastatic cancers, including CRPC. However, the impact of L1CAM on NEPC progression remains elusive. L1CAM expression level was investigated using public gene expression databases of PC cohorts and patient-derived xenograft models. L1CAM knockdown was performed in different PC cells to study in vitro cell functions. A subline of CRPC cell line CWR22Rv1 was established after long-term exposure to abiraterone to induce NE differentiation. The androgen receptor-negative cell line PC3 was cultured under the tumor sphere-forming condition to enrich cancer stemness features. Several oxidative stress inducers were tested on PC cells to observe L1CAM-mediated gene expression and cell death. L1CAM expression was remarkably high in NEPC compared to CRPC or adenocarcinoma tumors. L1CAM was also correlated with NE marker expressions and associated with the adenocarcinoma-to-NEPC progression in gene expression databases and CRPC cells with NE differentiation. L1CAM also promoted cancer stemness and NE phenotypes in PC3 cells under cancer stemness enrichment. L1CAM was also identified as a reactive oxygen species-induced gene, by which L1CAM counteracted CRPC cell death triggered by ionizing radiation. Our results unveiled a new role of L1CAM in the acquisition of the NE phenotype in PC, contributing to the NE differentiation-related therapeutic resistance of CRPC.

Abstract 3294: The GSPT1 molecular glue degrader MRT-2359 is active against prostate cancer

Abstract We have previously described our GSPT1 molecular glue degrader MRT-2359, which was optimized to achieve preferential antiproliferative activity in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) expressing high N-MYC or L-MYC mRNA. A clinical trial with this new drug candidate is ongoing (NCT05546268). Here, we present pre-clinical profiling of MRT-2359 across hundreds of cancer cell lines representing multiple cancer types, aiming at identifying other cancers where preferential sensitivity is associated with high expression of a MYC family member. These studies showed that prostate cancer cell lines clearly segregate into groups that are either sensitive or insensitive. Sensitive prostate cancer cell lines could be further divided into two subgroups: (1) androgen receptor positive cell lines displaying higher c-MYC mRNA expression levels than the other prostate cancer cell lines, (2) neuroendocrine prostate cancer cell lines with or without high N-MYC. The latter is in line with our previous observation that the neuroendocrine phenotype is generally associated with heightened sensitivity to MRT-2359 independently of MYC status. The insensitive cell lines were androgen receptor and neuroendocrine negative and displayed lower c-MYC mRNA levels. GSPT1 degradation in androgen receptor-positive cell lines led to rapid and deep loss of c-MYC protein as well as reduction of androgen receptor, including the splice variant AR-V7 that is associated with resistance to agents targeting androgen signaling. In androgen receptor-negative cell lines, the levels of c-MYC protein decreased only modestly upon GSPT1 degradation. Finally, in immunocompromised mice, xenografts of the AR-V7-positive cell line 22RV1 and the neuroendocrine prostate cell line NCI-H660, both of which are sensitive to MRT-2359 in vitro, were treated with several MRT-2359 dose regimens including continuous as well as intermittent (5 days on/9 days off) dosing. Most regimens led to marked tumor regression. Tumors of both models fully regressed after a 4-week course of 10 mg/kg MRT-2359 once daily, and no tumor regrowth could be detected after cessation of treatment. No significant in vivo response was observed for xenografts of the insensitive cell line PC-3. These data support the clinical investigation of MRT-2359 in prostate cancer. Citation Format: Ralph Tiedt, Martin Schillo, Arnaud Osmont, Débora Bonenfant, Rajiv Narayan, Owen Wallace, Markus Warmuth. The GSPT1 molecular glue degrader MRT-2359 is active against prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3294.

Abstract 652: An androgen receptor-associated transcription factor regulates fatty acid metabolism driving tumor growth in androgen receptor-positive breast cancer

Abstract Despite advances in the treatment of breast cancer, it is still the second leading cause of cancer-related death in women worldwide. A large number of patients develop recurrence and die of advanced metastatic disease. More than 70% of metastatic breast cancers (mBC) express androgen receptor (AR) representing a potential target for anti-hormone therapies. AR is suggested to directly interact with an associated transcription factor (ARaTF) in breast cancer, however the functional role of ARaTF and its interaction with AR remains to be elucidated. We find that AR expression highly correlates with ARaTF in patients. To study its functional role driving tumorigenesis and metastatic outgrowth, we utilize patient-derived xenograft cell lines derived from liquid biopsies of mBC patients which reflect the highly heterogenous disease. Using genetically manipulated PDX cell models, we demonstrate that repression of ARaTF significantly reduces tumor growth of AR positive (AR+) BC cells in vivo. Notably, we observe the opposite phenotype in an AR negative cell model suggesting a tumor suppressive function when AR is not present. Mechanistically, we find that ARaTF regulates key metabolic processes: (1) Pharmacological inhibition of AR or silencing of ARaTF restricts mitochondrial respiration activity resulting in decreased energy production. (2) We also find that ARaTF positively regulates genes encoding enzymes involved in de novo fatty acid biosynthesis in AR+ BC. We further observe upregulation of the CD36 fatty acid transporter facilitating fatty acid uptake as compensatory pathway upon downregulation of ARaTF. Finally, abolishing CD36 expression in AR+ BC cells significantly reduces cell viability upon ARaTF downregulation. These findings suggest that lipid and energy metabolism are transcriptionally regulated by ARaTF facilitating cell survival in nutrient-depleted environments and thus facilitating metastatic outgrowth. These findings provide knowledge about the functional role of ARaTF regulating the lipogenic metabolic profile of AR+ breast cancer, and might help to identify patients who benefit from an anti-androgen treatment. Citation Format: Sarah-Jane Neuberth, Corinna Schumacher, Marteinn Thor Snaebjörnsson, Roberto Würth, Franziska Maria Zickgraf, Ornella Kossi, Felix Geist, Andrea Geist, Vera Thiel, Jonas Schwickert, Mattia Falcone, Massimo Saini, Almut Schulze, Andreas Trumpp, Martin R. Sprick. An androgen receptor-associated transcription factor regulates fatty acid metabolism driving tumor growth in androgen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 652.

Ablation of LGR4 signaling enhances radiation sensitivity of prostate cancer cells

AimOur previous study has shown that leucine-rich repeat containing GPCR-4 (LGR4, or GPR48) LGR4 plays a role in cell migration, invasion, proliferation and apoptosis of prostate cancer (PCa). In this study, we aimed to explore whether LGR4 would affect radiation response in PCa. Materials and methodsLGR4 expression was silenced by shRNA transfection. qRT-PCR was employed to determine mRNA expression of LGR4 and DNA damage repair genes. Western blot was used to evaluate protein expression of LGR4, RSPO1-4, androgen receptor (AR), cyclic AMP response-element binding protein (CREB1), γH2A.X, and H2A.X. Cell proliferation was detected by CCK-8 assay and apoptosis was assayed by flow cytometry. Additionally, a xenograft model was also established to validate the role of LGR4 in PCa cells after radiation. Key findingsLGR4 expression was enhanced in PCa cells by radiation treatment in dose- and time-dependent means. RSPO1-4 were also upregulated post-radiation. Furthermore, LGR4 knockdown exacerbated apoptosis, reduced cell viabilities and strengthened nuclear γH2A.X staining in AR positive PCa cells but not in AR negative cells in the presence of radiation. Likewise, LGR4 ablation diminished AR and CREB1 expression induced by radiation. In contrast, RSPO1 stimulation augmented cell viabilities, promoted AR and CREB1 expression, and upregulated DNA repair gene expression, which could be reversed by enzalutamide, except for AR expression. Additionally, LGR4 knockdown further suppressed tumor growth and AR/CREB1 expression but enhanced γH2A.X expression in xenografts. SignificanceIn all, our study suggested that LGR4 might serve as an important regulator of radiation sensitivity in PCa.

Abstract 2959: Glycogen synthase kinase 3-β expression in prostate cancer (PCa) correlates with aggressive pathological features and its blockade with 9-ING-41 inhibits viability of PCa cell lines

Abstract Castration-resistant prostate cancer (CRPC) represents a lethal stage of disease with limited treatment options beyond androgen receptor (AR) inhibitors and chemotherapy. GSK-3β is a serine/threonine kinase established as a therapeutic target in several solid tumors. GSK-3β inhibitors reduce prostate cancer cell growth and inhibit AR-V7 transcriptional activity in vitro (Rinnab L et al 2008; Schütz SV et al 2011; Nakata et al 2017). This study aimed to characterize the GSK-3β expression in molecular subtypes of PCa and the antitumor activity of 9-ING-41, a selective small molecule GSK-3β inhibitor currently in phase 1/2 clinical studies (NCT03678883). We hypothesized that GSK3-β expression may correlate with sensitivity to GSK3-β inhibition as well as suppression of anti-apoptotic pathways. We evaluated the expression of GSK-3β in a tissue microarray of 134 specimens of PCa tumors from radical prostatectomies (median age 69, serum PSA 10.5 ± 7.6 ng/ml; grade groups (GG): 18 - GG 1 (13.4%), 67 - GG 2 (48.5%), 29 - GG 3 (21.4%), 7 - GG 4 (5.2%), 13 - GG 5 (9.7%); 72 patients (54.9%) had pT2 tumors, and 52 (39,1%) were pT3. Seven patients (5.7%) had positive lymph node (pN1 disease). ERG expression and PTEN loss were observed in 52% (71/134) and 42%, respectively. The GSK-3β histologic score (% of positive tumor cells multiplied by intensity 0-3) correlated with higher Gleason grade (p<0.05), extraprostatic extension (pT3a, p<0.05), but not with serum PSA, tumor volume, margin status or size of index nodule. Cases with predominant nuclear localization of GSK-3β (5%; N=7) had higher Gleason score, pathologic stage, and all but one had PTEN loss. The antiproliferative effect of 9-ING-41 in four PCa human cell lines (PC3, DU145, LNCAP and 22rV1) was investigated using Cell-Titer-Glo (CTG) viability assay. 9-ING-41 demonstrated a dose-dependent decrease in proliferation of AR positive (IC50s 0.3 μM LNCAP; 0.8 μM 22rV1) and AR negative cell lines (IC50 0.6 μM PC3, 0.2 μM DU145). 9-ING-41 induced robust apoptosis (cleaved PARP) in LNCAP and PC3 cells, but not in DU145. All four cell lines expressed GSK-3β, the target of 9-ING-41 and its level were not altered by treatment. 9-ING-41 decreased the expression of phosphorylated NF-kβ (Ser536), anti-apoptotic proteins MCL-1 and BCL-2 by western immunoblotting. Interestingly, the most sensitive cell line, DU145, had lower levels of NF-kβ and suppressed both MCL-1 and BCL-2 after exposure to 9-ING-41. Our current work is evaluating the extent of apoptosis versus growth arrest, especially in the DU145 cell line, where PARP cleavage was not observed. We are also evaluating the effects of the 9-ING-41 on cellular targets of GSK-3β as potential markers of drug efficacy. 9-ING-41 has potent anti-proliferative activity against PCa cell lines. These data support the inclusion of patients with CRPC in clinical studies of 9-ING-41 and its further investigation for the treatment of CRPC. Citation Format: Fabio Tavora, Tamara Lotan, Marclesson Alves, Lanlan Zhou, Ali Amin, Navaraj Arunasalam, Andre De Souza, Anthony Mega, Dragan Golijanin, Frank Giles, Wafik El-Deiry, Benedito Carneiro. Glycogen synthase kinase 3-β expression in prostate cancer (PCa) correlates with aggressive pathological features and its blockade with 9-ING-41 inhibits viability of PCa cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2959.

Abstract 5904: Post-transcriptional regulation of androgen receptor by DDX3 in prostate cancer progression

Abstract Purpose Prostate cancer (CaP) driven by androgen receptor (AR) can be targeted therapeutically by androgen deprivation therapy (ADT); however, in 10-20% of cases, ADT fails, allowing disease recurrence. Mechanisms for recurrence include dysregulation of AR at the gene, RNA, and protein level. Recently, AR negative cell growth has been implicated as a mechanism of resistance. This study aims to identify mechanisms of drug resistance in castration-resistant prostate cancer (CRPC) that can be exploited therapeutically to reduce disease recurrence. Experimental Procedures Using the BCaP model, a novel series of cell lines derived from a single human prostate epithelial cell, we can study the progression of CaP from benign to metastasis. Additionally, using the LNCaP-C4 series we can study progression to CRPC. To determine the RNA binding capacity of DDX3, we utilized 1) RIP-qPRC for identification of mRNA targets and 2) RNAscope for visual colocalization. Changes in RNA and protein expression were determined using qPCR, Western blot, and IF. Results DDX3 is an ATP-dependent RNA helicase that can aid translation of target mRNAs, or in stress conditions, form ribonucleoprotein granules that prevent translation. While DDX3 is implicated in several cancers, its role as a translational regulator in CaP remains unstudied. In BCaP and LNCaP-C4, DDX3 protein expression increases through progression, concurrent with localization to cytoplasmic puncta. RIP-qPCR identified AR as an mRNA target of DDX3 in the metastatic and CRPC cell lines. Because of the dual role of DDX3 in translational control, AR expression in these models was investigated; while AR mRNA expression increased through progression, AR protein expression decreased. Treatments in vitro with RK33, a commercially available inhibitor of DDX3, restored the protein expression of AR and downstream AR signaling (PSA, Nkx3.1). These data suggest DDX3 acts as a translational repressor of AR in metastasis and CRPC. To confirm that increased DDX3 expression is sufficient to reduce AR protein expression, we overexpressed DDX3 using two models: induced cellular stress and a DDX3 expression vector. Here, we saw DDX3 localized to puncta, and subsequent reduction in AR and PSA protein. Because DDX3 is sufficient to reduce AR protein expression in metastases and CRPC, co-treatment of DDX3 inhibitors with anti-androgen therapy may prevent AR negative cell growth underlying recurrence. in vitro co-treatments with RK33 and bicalutamide, an AR antagonist, show decreased proliferation compared to either treatment alone, suggesting increased efficacy of bicalutamide with DDX3 inhibition. Conclusions DDX3 as a repressor of AR translation could have clinical implications as a mechanism of resistance to ADT. Based on preliminary data, DDX3 could be contributing to the regulation of AR post-transcriptionally, and targeting DDX3 could reduce resistance and disease recurrence. Citation Format: Jordan E. Vellky, William Ricke. Post-transcriptional regulation of androgen receptor by DDX3 in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5904.

Evaluation of SHetA2 Analogs as Anti‐Cancer Agents on Human Prostate Cancer Cells

BackgroundSHetA2 is a flexible heteroarotinoid compound that has shown potential anticancer effects on many cancer cell lines as well as animal models of cancer. However, due to its high lipophilicity, poor target specificity and low bioavailability, it may not be an ideal drug candidate. To overcome these limitations, over twenty thiochroman analogs of SHetA2 were synthesized and screened for their growth inhibitory effects in three different human prostate cancer cells. The screening yielded three analogs, SL‐01‐18, SL‐01‐30 and SL‐01‐39 that showed growth inhibitory effects comparable to SHetA2. All three analogs were more potent in inhibiting the growth of androgen receptor (AR) positive cell line, LNCaP, than the AR negative cell lines, DU145 and PC3. Here, we further evaluated the mechanism of action of these three analogs in LNCaP cell line.ObjectiveThe aim of the study was to evaluate the mechanism of action of the three SHetA2 analogs and compare their effects to SHetA2 in androgen receptor positive cell line, LNCaP.MethodsApoTox‐Glo assay was performed on LNCaP cells after 24 hours exposure to SHetA2 and its three analogs to determine the cell viability, cytotoxicity and caspase 3/7 activity. Bromodeoxyuridine (BrdU) incorporation assay was performed to measure the proportion of LNCaP cells in S‐phase of the cell cycle after drug treatments. Western blot analysis was performed on LNCaP cells treated with the SHetA2 analogs to analyze the effect on the expression of androgen receptor (AR) protein. Finally, prostate specific antigen (PSA)‐ELISA assay was performed to determine PSA concentration on LNCaP supernatants upon treatment with SHetA2 and its analogs.ResultsApoTox‐Glo assay results showed decreased cell viability, increased caspase activity and very little cytotoxicity in LNCaP cells treated with SHetA2 and its analogs. BrdU incorporation assay in LNCaP cells revealed a significant reduction in the proportion of cells in the S‐phase of the cell cycle with all four drug treatments, Finally, Western blot analysis revealed time and concentration‐dependent decrease in AR expression in treated cells while PSA‐ELISA assay indicated significant decrease in PSA secretion from LNCaP cells treated with SHetA2 and its analogs for 72 hours.ConclusionSHetA2 and its thiochroman analogs, SL‐01‐18, SL‐01‐30 and SL‐01‐39 inhibited cell growth of AR positive LNCaP cells while not being cytotoxic to these cells. In addition to stalling the cells from entering the S‐phase of the cell cycle, these agents also raised the likelihood of the cells to undergo apoptosis by activating the executioner caspase 3/7. A decrease in secreted PSA levels as well as concentration‐dependent decrease in AR protein expression suggests that the SHetA2 analogs may elicit their action by interfering with androgen receptor signaling. However, understanding how exactly are the analogs affecting androgen receptor signaling, inducing cell cycle arrest and/or causing programmed cell death requires further investigation.Support or Funding InformationTouro CA COP‐MS Program for funding this research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Downregulation of miR-17-92a cluster promotes autophagy induction in response to celastrol treatment in prostate cancer cells

Celastrol has potential application for the treatment of prostate cancer. However it causes autophagy as a protective response in prostate and other types of cancers, thus unveiling the underlying mechanisms may benefit its future application. In the present study, we demonstrate that the miR-17-92a cluster plays a negative role in celastrol induced-autophagy. Dissection of miR-17-92a cluster revealed the role of miR-17 seed family (miR-20a and miR-17) in autophagy inhibition in the context of prostate cancer cells. Autophagy-related gene ATG7 was validated as a target of miR-17 seed family by dual-luciferase assay and qPCR. Celastrol induced autophagy was inhibited by miR-20a or miR-17, while the inhibitory effects were rescued in the presence of pcDNA-ATG7 lacking 3′ UTR, demonstrating that these two members target ATG7 to inhibit celastrol-induced autophagy. As celastrol degrades androgen receptor (AR), a key transcription factor in prostate cancer cells, we further investigated whether AR affected miR-17-92a expression in prostate cancer cells. AR binding sites were found in the promoter and two introns of miR-17-92a. In addition, higher expression levels of miR-17-92a were observed in AR positive cells compared with AR negative cells. Ectopic expression of AR could enhance the expression of miR-17-92a cluster in AR-negative prostate cancer cells while knockdown of AR decreased miR-17-92a expression in AR-positive cells, demonstrating the regulation of AR on miR-17-92a transcription. In summary, our results demonstrate that celastrol downregulates AR and its target miR-17-92a, leading to autophagy induction in prostate cancer cells.

The E2F1/DNMT1 axis is associated with the development of AR negative castration resistant prostate cancer

Research on castration resistant prostate cancer (CRPC) has focused primarily on functional alterations of the androgen receptor (AR). However, little is known about the loss of AR gene expression itself and the possible contribution of AR negative cells to CRPC. Human and murine prostate cancer tissue microarrays (TMAs) were evaluated with antibodies specific for E2F1, DNA methyltransferase 1 or AR. The human prostate cancer TMA consisted of clinical samples ranging from normal tissue to samples of metastatic disease. The murine TMA was comprised of benign, localized or metastatic prostate cancer acquired from TRAMP mice treated with castration and/or 5'-Aza-2'-deoxycytidine (5Aza). Immunohistochemical analysis revealed increased nuclear DNMT1 staining in localized PCa (P < 0.0001) and metastatic PCa (P < 0.0001) compared to normal tissue. Examination of specific diagnoses revealed that Gleason seven tumors exhibited greater nuclear DNMT1 staining than Gleason six tumors (P < 0.05) and that metastatic tissue exhibited greater levels of nuclear DNMT1 than Gleason seven tumors (P < 0.01). Evaluation of the murine tissue cores revealed that 8.2% and 8.1% of benign tissue cores stained positive for E2F1 and DNMT1 respectively, while 97.0% were AR positive. Conversely, 81% and 100% of tumors were positive for E2F1 and DNMT1 respectively. This was in stark contrast to only 18% of tumors positive for AR. Treatment of mice with 5Aza reduced DNMT1 staining by 30%, while AR increased by 27%. These findings demonstrate that the E2F1/DNMT1 inhibitory axis of AR transcription is activated during the emergence of CRPC.

Suppressors of cytokine signalling-3 and -1 in human carcinogenesis

The role of suppressors of cytokine signaling (SOCS)-3 and -1 has been investigated in various cancers. These proteins have been identified as endogenous controllers of activation of Janus kinase/signal transducers and activators of transcription factors pathway factors under physiological conditions and in disease. SOCS-3 expression is lost in several cancers due to epigenetic mechanisms, mostly promoter methylation. In liver, lung, and squamous head and neck cancer, and several hematological malignancies SOCS-3 acts as a classic tumor suppressor. In prostate cancer, SOCS-3 effects are cell type-dependent. It prevents apoptosis in androgen receptor-negative cells. However, in androgen-sensitive cells, it could act as a negative feedback factor for androgenic regulation. Melanoma cells which overexpress SOCS-3 confer a growth advantage. SOCS-1 is in most cancers a tumor suppressor which may inhibit expression of cyclin-dependent kinases and cyclins. In general, the mechanisms responsible for the different effects of SOCS in cancer cell lines have to be further investigated. The results discussed in the present review may have an impact on personalized approaches in cancer medicine.

227 CALCIUM CHANNEL BLOCKER MODULATES ANDROGEN RECEPTOR-MEDIATED GENE EXPRESSION AND INDUCES CYTOTOXICITY IN PROSTATE CANCER CELLS

INTRODUCTION AND OBJECTIVES: Calcium (Ca ), a ubiquitous secondary messenger, is involved in several cellular processes such as apoptosis, proliferation and carcinogenesis. It has also been shown that Ca signaling is involved in androgen-induced PSA secretion. Recently published epidemiologic data demonstrated that patients on calcium channel blockers had significantly lower incidences of prostate cancer. Therefore, we examined the role of various voltagegated calcium channels in androgen signaling and prostate cancer development. METHODS: Multiple prostate cancer cell lines including LNCaP, LAPC-4, and C4-2 (all androgen receptor positive) were treated with calcium channel blockers nifedipine, mibefradil, and tetrandine. The medications were added to the cell culture media at a dose range of 5-50 micromolar for various time points between 2 and 24 hours. Cell proliferaion was measured using an MTS assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine expression of several Ca channel genes in different cell lines. Knockdown of Ca channel expression using an siRNA was also utilized to determine effects on cell lines. RESULTS: Blocking L-type channels with nifedipine and tetrandrine significantly suppressed cell proliferation and androgen receptormediated gene expression in prostate cancer cells. Higher doses of tetrandrine also induced a dramatic cytotoxic effect. In contrast, a T-type specific channel blocker mibefradil induced androgen receptor transactivation without androgen addition and also drastically enhanced androgen receptor transactivation in a reporter gene assay. RT-PCR demonstrated that only L-type CACNA1D (cav1.3) and T-type CACNA1H (cav3.2) alpha-subunit genes are highly expressed in androgen receptor-positive prostate cancer cell lines, while L-type CACNA1F (cav1.4) is only weakly expressed in androgen receptorpositive cell lines but highly expressed in androgen receptor-negative cell lines. Other subunits are not expressed in any prostate cancer cells. Furthermore, consistently, knocking down CACNA1D (cav1.3) expression by the siRNA approach abrogated AR transactivation while knocking down CACNA1H (cav3.2) expression enhanced AR transactivation. CONCLUSIONS: These results suggest that the L-type calcium channel subunit (cav1.3) has the potential as a therapeutic target for prostate cancer intervention, although further clinical testing is warranted.

Abstract B5: Epigenetic modification of AR in PC3 cells negatively regulates mTORC1/2 activity

Abstract Wild type androgen receptor (AR) acts as a tumor suppressor and mediates the differentiation of non-malignant prostate epithelial cells. However, in the development of prostate cancer AR activity becomes critical for the development and progression of disease. Previous studies demonstrate that re-expressing wild type AR within an AR negative cell line (PC3) possesses tumor suppressor quality by reducing cell proliferation and tumorgenicity in mice. Further, it is documented that acetylation of AR in non-malignant cells is a post-translational modification associated with increased transcriptional activity of AR, whereas acetylation of AR in tumor cells results in negative regulation of AR transcriptional activity. We therefor are seeking to investigate the regulation of re-expressed AR within PC3 cells by induced acetylation post HDAC inhibition. Our preliminary data reveals that sensitivity to HDAC inhibition is increased with the re-expression of AR in PC3 cells. Further, epigenetic modification of AR in this model negatively regulates mTORC1 and mTORC2 expression and activity, independent of AR transcriptional activity. Ongoing experiments are being conducted to investigate further the role of epigenetic post-translational modification of AR and its negative regulation of mTORC1/2 signaling. Citation Format: Leigh Ellis, Remi Adelaiye, Shengyu Ku, Alejandro Godoy, Roberto Pili. Epigenetic modification of AR in PC3 cells negatively regulates mTORC1/2 activity [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B5.

Statins reduce the androgen sensitivity and cell proliferation by decreasing the androgen receptor protein in prostate cancer cells

Statins (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors) are cholesterol-lowering drugs that are widely used to prevent and treat atherosclerotic cardiovascular disease. Recent epidemiological studies suggest that statins reduce serum prostate-specific antigen (PSA) levels and decrease the risk of prostate cancer. In the present study, we determined the molecular mechanisms related to the regulation of PSA, androgen receptor (AR) and cell proliferation in prostate cancer cell lines by statins. Western blotting, quantitative real-time polymerase chain reaction, cytotoxicity analysis and a cell proliferation assay were used to resolve the regulatory role of statins (mevastatin and simvastatin) in three prostate cancer cell lines, RWPE-1, 22Rv1, and LNCaP. Western blotting revealed that both mevastatin and simvastatin downregulated AR and PSA protein. However, these statins did not downregulate AR mRNA expression, while they decreased PSA mRNA. The protease inhibitor MG132 inhibited the downregulation of AR protein which suggested that statins decreased AR protein levels by increasing AR proteolysis. Furthermore, statins reduced cell proliferation in AR positive cells but not in AR negative cells, suggesting that statins regulate cell proliferation via AR expression. In addition, cell proliferation assay at various concentrations of dihydrotestosterone (DHT) showed that statins decreased androgen sensitivity in LNCaP cells. Statins decreased AR protein by proteolysis but not mRNA transcription. The drop in AR levels resulted in a reduction in androgen sensitivity and a decrease in cell proliferation in AR positive prostate cancer cells.

Abstract 602: Silencing of the androgen receptor (AR) with a novel mRNA antisense oligonucleotide causes antitumor effects in xenograft models of prostate cancer

Abstract Background: The AR is a ligand-activated transcriptional factor that plays an important role in prostate cancer. While androgen-deprivation therapies typically inhibit tumor growth, tumor recurrence due to the castration resistant form of the disease frequently occurs and leads to mortality. Nevertheless, castration-resistant tumors are still dependent on a functional (AR). Therefore, inhibition of AR expression, rather than systemic androgen deprivation, may provide a novel strategy for treatment of advanced prostate cancer. We have developed a novel locked nucleic acid (LNA)-based antisense oligonucleotide (ON), EZN-4176, which down-modulated AR and inhibited tumor growth. Methods: The target mRNA down-modulation, growth inhibition, luciferase activity, protein inhibition, and reduction of prostate specific antigen (PSA; an AR downstream target and biomarker of prostate cancer) were evaluated by qRT-PCR, MTS, SteadyGlo, western blot analysis, and ELISA assay, respectively, in LNCaP cancer cell lines. A mismatched LNA antisense oligo (EZN-4176MM) and an AR negative cell line (PC-3 prostate cancer) served as controls. Additionally, the effect of EZN-4176 on DHT-induced AR transcriptional activity was evaluated in LNCaP cells containing an AR-responsive luciferase reporter. In vivo, therapeutic efficacy of EZN-4176 was evaluated in the AR-positive CWR22 (androgen dependent). Results: In vitro, in the absence of any transfection reagent, EZN-4176 but not the mismatched control (EZN-4176MM) inhibited the growth of DHT-induced LNCaP cells. The growth reduction correlated with AR down-modulation and its down-stream target gene expression and AR-responsive luciferase activity. In vivo, EZN-4176, prepared in the absence of a delivery vehicle, demonstrated dose-dependent inhibition of mRNA of AR, PSA, and TMPRSS2 as well as protein level of AR in androgen-sensitive CWR22 tumor xenografts, resulting in significant tumor growth inhibition (TGI) (∼55%) (similar to high-dose Casodex® [bicalutamide] treatment). Cy5.5-labeled EZN-4176 was shown to localize in subcutaneous CWR22 or LNCaP tumor xenografts using xenogen imaging system. In castration-resistant tumor cells, EZN-4176 potently and specifically inhibited the growth of C4-2b cells as well as down-regulated AR-luciferase reporter systems. More importantly, AR and PSA mRNA were inhibited in both castration-resistant C4-2b and 22RV1 tumor xenografts. Conclusions: Our data suggest that AR mRNA down modulation by EZN-4176 inhibits prostate tumor growth and provides a novel strategy to treat advanced prostate cancer. 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 602.

99 UTILIZING METFORMIN TO ENHANCE THE EFFICACY OF ANDROGEN DEPRIVATION THERAPY IN THE TREATMENT OF PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Prostate cancer (PCa) incidence varies dramatically by geographic location, with developed countries exhibiting significantly higher levels of disease. Some attribute this to the ‘Westernized lifestyle’ of high energy diets and limited physical activity with consequent obesity. Obesity and obesity-related diseases like diabetes, cause hyperinsulinemia, which upregulates pro-survival insulin/insulin-like growth factor signalling. Our previous work shows diet-induced hyperinsulinemia to enhance PCa tumor growth in vivo. Metformin, a treatment for diabetes, reduces hyperinsulinemia, and has recently been shown to exhibit anti-neoplastic properties. We assessed the potential additive benefit of combining a standard PCa treatment (androgen ablation therapy with bicalutamide) with metformin in vitro and in vivo. METHODS: Using clonogenic assays we assessed the effect of bicalutamide and/or metformin on colony formation rates in LNCaP, PC3, DU145 and PC3AR2 PCa cell lines. Western blot and cell cycle analyses were used to elucidate any mechanism of interaction between the two drugs in androgen receptor (AR) positive (LNCaP) and AR negative (PC3) cell lines. The combination treatment regimen was then assessed in vivo using a LNCaP murine xenograft model. RESULTS: Micromolar bicalutamide or millimolar metformin caused significant dose-dependent reduction in colony formation rates (p 0.001). Combination treatment further significantly reduced colony formation rates (p 0.005). The effect was more pronounced in AR positive cells. Western blot and cell cycle analyses suggested differing mechanisms of interaction in AR positive and negative cell lines. Following combination treatment LNCaP cells exhibited altered cell proliferation (decreased PCNA) and perturbed cell cycle kinetics (G1/S arrest). Conversely, PC3 cells showed evidence of enhanced apoptosis (increased BAX, decreased caspase 3 and phospho-Akt). Preliminary in vivo results show significantly diminished tumor growth in response to the combination treatment regimen (p 0.0001). CONCLUSIONS: Combining bicalutamide and metformin significantly reduces PCa cell colony formation rates further than either monotherapy. In AR positive cells this effect is mediated by reducing cellular proliferation rates, whereas in AR negative cells the combination treatment regimen promotes apoptosis. This combination drug regimen may potentially improve prostate-cancer specific survival via the direct anti-neoplastic properties outlined.

Regulation of IκB Kinase ε Expression by the Androgen Receptor and the Nuclear Factor-κB Transcription Factor in Prostate Cancer

Although several genes have been associated with prostate cancer progression, it is clear that we are far from understanding all the molecular events implicated in the initiation and progression of the disease to a hormone-refractory state. The androgen receptor is a central player in the initiation and proliferation of prostate cancer and its response to hormone therapy. Nuclear factor-kappaB has important proliferative and antiapoptotic activities that could contribute to the development and progression of cancer cells as well as resistance to therapy. In this study, we report that IkappaB kinase epsilon (IKKepsilon), which is controlled by nuclear factor-kappaB in human chondrocytes, is expressed in human prostate cancer cells. We show that IKKepsilon gene expression is stimulated by tumor necrosis factor-alpha treatment in LNCaP cells and is inhibited by transfection of a dominant-negative form of IkappaBalpha, which prevents the nuclear translocation of p65. Furthermore, we found that tumor necrosis factor-alpha-induced IKKepsilon expression is inhibited by an androgen analogue (R1881) in androgen-sensitive prostate cancer cells and that this inhibition correlates with the modulation of IkappaBalpha expression by R1881. We also noted constitutive IKKepsilon expression in androgen-independent PC-3 and DU145 cells. To our knowledge, this is the first report of an IkappaB kinase family member whose expression is modulated by androgen and deregulated in androgen receptor-negative cells.

Characterization of prostate cancer DU145 cells expressing the recombinant androgen receptor.

Prostate cancer (PC) develops as a consequence of abnormal androgenic stimulation. Unfortunately, most of the PC cell lines are androgen independent (like DU145), or express mutated forms of androgen receptor (AR). We have produced and characterized a new stably transfected PC line expressing the AR (DU145-AR). Untreated DU145-AR cells showed a lower proliferation rate than mock transfected cells, but responded to testosterone treatment. PSA mRNA, undetectable in mock DU145 cells, was present and upregulated by testosterone in DU145-AR. About 5% of DU 145-AR cells showed modification of morphology and enriched of f-actin after testosterone treatment. Moreover, in DU145-AR plasminogen activator (PA) activity and secreted urokinase type plasminogen activator (uPA) protein were lower than in AR negative cells; again testosterone induced PA activity and uPA protein only in DU145-AR. These results indicate that, in general, the effects of unactivated AR is to suppress function(s) in DU145 cells and the addition of testosterone restores the normal properties associated with the untransfected cells. Some of the effects described may thus be mediated by a ligand-independent activation of AR in DU145 cells.

Identification of the Phosphatidic Acid Phosphatase Type 2a Isozyme as an Androgen-regulated Gene in the Human Prostatic Adenocarcinoma Cell Line LNCaP

Differential display was used to identify novel androgen-regulated genes in the human prostatic adenocarcinoma cell line LNCaP. A 322-base pair cDNA fragment that was consistently induced by the synthetic androgen R1881 revealed 100% homology with the human phosphatidic acid phosphatase type 2a isozyme very recently reported by Kai et al. (PAP-2a; Kai., M., Wada, I., Imai, S.-i., Sakane, F., and Kanoh, H. (1997) J. Biol. Chem. 272, 24572-24578). The fragment was used to clone the corresponding cDNA from a human prostate library. The deduced amino acid sequence confirmed the identity with human PAP-2a. The inducibility of PAP-2a mRNA by androgens was confirmed by Northern blot hybridization. The effect was time- and dose-dependent with a maximal stimulation (4-fold) after 24 h of treatment with 10(-9) M R1881. The steroid specificity of PAP-2a mRNA regulation was found to be in agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, supporting the involvement of the androgen receptor in the induction process. Furthermore, low basal levels of PAP-2a mRNA and absence of androgen inducibility were observed in the poorly differentiated and androgen receptor-negative cell lines PC-3 and DU-145. Induction of PAP-2a mRNA was not affected by the protein synthesis inhibitor cycloheximide and was accompanied by a marked increase in PAP-2 activity as measured by the conversion of phosphatidic acid into diacylglycerol in membrane fractions of LNCaP. Comparison of the expression of PAP-2a mRNA in 50 different human tissues revealed ubiquitous expression. The highest levels, however, were observed in the prostate. Since PAP-2 plays a pivotal role in the control of signal transduction by lipid mediators such as phosphatidate, lysophosphatidate, and ceramide-1-phosphate, the ability of androgens to stimulate the expression and activity of this enzyme in prostatic cells may provide an important opportunity for cross-talk between signaling pathways involving lipid mediators and androgens.

Regulatory Mechanism of Human Factor IX Gene: Protein Binding at the Leyden-Specific Region

Hemophilia B-Leyden is characterized by the gradual amelioration of bleeding after the onset of puberty. All Leyden phenotype mutations found to date lie within the Leyden-specific region, which spans roughly nt-40 to +20 in the 5' end of the human factor IX gene. With HepG2 cell nuclear extracts, the Leyden-specific region and its immediate neighboring region of the normal factor IX gene showed five DNase I footprints: FP-I (nt +4 to +19), FP-II (nt -16 to -3), FP-III (nt -27 to -19), FP-IV (nt -67 to -49), and FP-V (nt -99 to -77). Protein binding affinities of short oligonucleotides containing sequences of FP-I, FP-II, or FP-III were substantially reduced in the presence of Leyden phenotype mutations in these areas, correlating well with the negative effects of these mutations on factor IX gene expression. A Leyden phenotype mutation at nt -20 (T to A) caused a loss of both footprints FP-III and FP-II but generated a new footprint, FP-III' (nt -34 to -23), partially overlapping with FP-III, indicating mutation-dependent competitive protein binding at these sites. Although the FP-III' area contains an androgen responsive element-like sequence, the nuclear protein that binds at FP-III' is not androgen receptor. The protein was not recognized by anti-androgen receptor antibody and, furthermore, was present not only in liver but also in both androgen receptor-positive and androgen receptor-negative cells in electrophoretic mobility shift assays. The nuclear concentration of this protein increased significantly upon treatment of the HepG2 cells with testosterone. Its binding affinity to an oligonucleotide (-32sub) containing the FP-III' sequence was greatly reduced in the presence of exogenous androgen receptor, suggesting a possible interaction of this protein with androgen receptor. The affinities of both this protein and a protein which binds to FP-III (presumably HNF-4) to -32sub with a mutation at nt -26 were grossly lowered. These findings suggest that the amelioration of hemophilia B-Leyden with a mutation at nt -20 after puberty involves binding of a specific non-androgen receptor nuclear protein at FP-III' and it is able to substitute for the function of a protein bound at FP-III in the normal gene optimally through its elevated interaction with androgen receptor upon a surge of testosterone.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphoinositide metabolism in human prostate cancer cells in vitro

To understand better the mechanism by which 5-alpha-dihydrotestosterone (5-alpha-DHT) influences prostate epithelial cell function, we examined the effects of 5-alpha-DHT on phosphoinositide metabolism in human prostate cancer cell lines. Androgen receptor-positive LN-CaP cells showed dose-responsive, steady-state elevations in phosphoinositide metabolism when treated with 5-alpha-DHT. The intracellular pool of 3H-myoinositol decreased and the incorporation of 3H-myoinositol into cellular lipids increased with increasing concentrations of 5-alpha-DHT. 5-alpha-DHT increased the release of 3H-inositol phosphates into the media. The inactive stereoisomer, 5-beta-DHT, did not increase phosphoinositide metabolism. In androgen receptor-negative cells, phosphoinositide metabolism was not altered by 5-alpha-DHT. The slow induction of phosphoinositide metabolism by 5-alpha-DHT suggests that the effects may be mediated through other factors that serve as intermediates in 5-alpha-DHT modulation of intracellular signalling. We conclude that this modulation involves increased turnover of phosphatidylinositol, incorporation of myoinositol into cellular lipids, and alterations in the aqueous intracellular myoinositol pool size, possibly as a result of altered transport mechanisms.