Progression Of Androgen-independent Prostate Cancer Research Articles

MicroRNA-147b induces neuroendocrine differentiation of prostate cancer cells by targeting ribosomal protein RPS15A.

Prostate cancer (PCa) is the leading cause of cancer related deaths in men, often androgen deprivation therapy (ADT) leads to the progression of androgen independent PCa (AIPC) which further leads to Neuroendocrine PCa (NEPC). Identifying the molecular mechanisms which navigate the neuroendocrine differentiation(NED) of PCa cells is clinically relevant. It has been suggested that the micro RNAs (miRNAs) play an important role in the regulation of intrinsic mechanisms relevant to tumor progression, resistance as a result leads to poor prognosis. miR-147b has been transpiring as one of the deregulated miRNAs associated with the occurrence of multiple cancers. The present study has studied the role of miRNA-147b in inducing NEPC. To investigate the functional role of miR-147b in NEPC, we have expressed miRNA mimics or inhibitors in PCa cells and monitored the progression of NEPC along with PCa cell proliferation and survival. The molecular mechanism miRNA-147b follows was studied using western blot and reverse transcriptionpolymerase chain analysis. miRNA target prediction using bioinformatics tools followed by target validation using luciferase reporter assays was performed. In the present study, we found that miR-147b is highly expressed in AIPC cell lines in particular neuroendocrine cells NCI-H660 and NE-LNCaP derived from LNCaP. Mechanistic studies revealed that overexpression of miR-147b or miRNA mimics induced NED in LNCaP cells in in-vitro while its inhibitor reversed the NE features (increased NE markers and reduced prostate specific antigen) of PC3, NCI-H660 and NE-LNCaP cells. In addition, miR-147b reduced the proliferation rate of LNCaP cells via elevated p27kip1 and lowered cyclin D1 for promoting differentiation. In reporter assays, we have identified ribosomal protein S15A (RPS15A) is a direct target of miRNA-147b and RPS15A expression was negatively regulated by miR-147b in PCa cells. Furthermore, we also report that RPS15A is downregulated in NEPC cells and its expression is inversely correlated with NE markers. Targeting the miR-147b - RPS15A axis may overcome the progression of NEPC and serve as a novel therapeutic target to attenuate NED progression of PCa.

Qi Ling Inhibits Progression of Androgen-Independent Prostate Cancer via Negative Regulation of TRIM66/HP1γ/AR Axis

Aim: This study aimed to understand the molecular mechanism underlying the therapeutic effect of Qi Ling (QL) against androgen-independent prostate cancer. Methods: The relative expression of TRIM66 in prostate tumor was interrogated by microarray. Real-time polymerase chain reaction and Western blotting were performed to determine the transcript abundances and protein expressions of TRIM66, HP1γ, AR, c-Myc, and GAPDH. Cell proliferation and apoptosis were analyzed by cell counting kit-8 method and flow cytometry. The regulatory action of c-Myc on TRIM66 was interrogated with luciferase reporter plasmid and the direct binding was demonstrated by chromatin immunoprecipitation. The secretory prostate-specific antigen was quantified by enzyme-linked immunosorbent assay. Results: TRIM66 was aberrantly overexpressed in prostate cancer and associated with unfavorable prognosis. TRIM66/HP1γ/AR was upregulated during the androgen-independent transition in hormone-deprived medium. The TRIM66 level positively linked to cell proliferation and negatively linked to cell apoptosis in androgen-independent prostate cancer cells. QL treatment specifically inhibited c-Myc and therefore directly downregulated TRIM66 via binding to its promoter. Ectopic introduction of TRIM66 significantly reversed the anti-tumor effects of QL against androgen-independent prostate cancer. Conclusion: Our study uncovered the importance of downregulated TRIM66/HP1γ/AR signaling in mediating the anti-tumor properties of QL.

LncRNA LEF1-AS1 promotes metastasis of prostatic carcinoma via the Wnt/\u03b2-catenin pathway

BackgroundLong noncoding RNAs (lncRNAs) are important functional regulators of many biological processes of cancers. However, the mechanisms by which lncRNAs modulate androgen-independent prostate cancer (AIPC) development remain largely unknown.MethodsNext-generation sequencing technology and RT-qPCR were used to assess LEF1-AS1 expression level in AIPC tissues and adjacent normal tissues. Functional in vitro experiments, including colony formation, EDU and transwell assays were performed to assess the role of LEF1-AS1 in AIPC. Xenograft assays were conducted to assess the effect of LEF1-AS1 on cell proliferation in vivo. Chromatin immunoprecipitation (ChIP) and RNA binding protein immunoprecipitation (RIP) assays were performed to elucidate the regulatory network of LEF1-AS1.ResultsThe next-generation sequencing results showed that LEF1-AS1 is significantly overexpressed in AIPC. Furthermore, our RT-qPCR assay data showed that LEF1-AS1 is overexpressed in AIPC tissues. Functional experiments showed that LEF1-AS1 promotes the proliferation, migration, invasion and angiogenic ability of AIPC cells in vitro and tumour growth in vivo by recruiting the transcription factor C-myb to the promoter of FZD2, inducing its transcription. Furthermore, LEF1-AS1 was shown to function as a competing endogenous RNA (ceRNA) that sponges miR-328 to activate CD44.ConclusionIn summary, the results of our present study revealed that LEF1-AS1 acts as a tumour promoter in the progression of AIPC. Furthermore, the results revealed that LEF1-AS1 functions as a ceRNA and regulates Wnt/β-catenin pathway activity via FZD2 and CD44. Our results provide new insights into the mechanism that links the function of LEF1-AS1 with AIPC and suggests that LEF1-AS1 may serve as a novel potential target for the improvement of AIPC therapy.

Stabilization of ADAM9 by N-\u03b1-acetyltransferase 10 protein contributes to promoting progression of androgen-independent prostate cancer

N-α-Acetyltransferase 10 protein (Naa10p) was reported to be an oncoprotein in androgen-dependent prostate cancer (PCa; ADPC) through binding and increasing transcriptional activity of the androgen receptor (AR). PCa usually progresses from an androgen-dependent to an androgen-independent stage, leading to an increase in the metastatic potential and an incurable malignancy. At present, the role of Naa10p in androgen-independent prostate cancer (AIPC) remains unclear. In this study, in silico and immunohistochemistry analyses showed that Naa10 transcripts or the Naa10p protein were more highly expressed in primary and metastatic PCa cancer tissues compared to adjacent normal tissues and non-metastatic cancer tissues, respectively. Knockdown and overexpression of Naa10p in AIPC cells (DU145 and PC-3M), respectively, led to decreased and increased cell clonogenic and invasive abilities in vitro as well as tumor growth and metastasis in AIPC xenografts. From the protease array screening, we identified a disintegrin and metalloprotease 9 (ADAM9) as a potential target of Naa10p, which was responsible for the Naa10p-induced invasion of AIPC cells. Naa10p can form a complex with ADAM9 to maintain ADAM9 protein stability and promote AIPC’s invasive ability which were independent of its acetyltransferase activity. In contrast to the Naa10p-ADAM9 axis, ADAM9 exerted positive feedback regulation on Naa10p to modulate progression of AIPC in vitro and in vivo. Taken together, for the first time, our results reveal a novel cross-talk between Naa10p and ADAM9 in regulating the progression of AIPC. Disruption of Naa10p–ADAM9 interactions may be a potential intervention for AIPC therapy.

2012P - Heat shock protein 90 chaperones and protein kinase D3 regulates androgen-independent prostate cancer development

BackgroundProstate cancer is the second leading cause of cancer related deaths in men worldwide. Heat Shock Protein 90 (Hsp90) is expressed in tumour cells at high levels – 3-5% of total proteins – and regulates the function of oncogenes and other tumour related proteins. Protein kinase D3 (PKD3) has a proven role in the progression of androgen-independent prostate cancer. In the present study we set out to explore the impact of Hsp90 and PKD3, respectively, on prostate cancer growth and their potential interaction. MethodsWe employed the DU145 and PC3 well-characterized androgen-independent prostate cancer cell lines. Cell viability was determined by Trypan Blue exclusion cell counting. Apoptosis analysis was performed by flow cytometry after AnnexinV and propidium-iodide co-staining. Protein levels were detected by western blot and protein-protein interactions were investigated by co-immunoprecipitation. ResultsWe found that the clinically used Hsp90 inhibitor ganetespib induced apoptosis and significantly reduced the viability of the androgen-independent DU145 and PC3 cell lines. The pan-PKD inhibitor CRT0066101 also decreased cell viability of the prostate cancer cells in a dose-dependent manner. Further, we demonstrated that ganetespib reduced PKD3 protein level in a concentration-dependent manner and induced its proteasomal degradation. Finally, a co-immunoprecipitation study revealed a physical connection between PKD3 and Hsp90. ConclusionsWe identified and confirmed an Hsp90-PKD3 chaperone client interaction, which may be important in prostate cancer cell survival. Further studies are under way to characterize the biological significance of our findings. Our results contribute to better understand the pathological signalling of androgen-independent prostate cancer cells and to find novel treatment strategies. Legal entity responsible for the studyMTA-SE Pathobiochemistry Research Group. FundingNational Research, Development and Innovation Office - Hungary. DisclosureAll authors have declared no conflicts of interest.

Abstract 3061: Role of ID4 (inhibitor of DNA binding protein 4) in FKBP52-AR pathway

Abstract Introduction: Inhibitor of DNA binding/differentiation protein 4 (ID4), acts as a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR through selective interaction with FKBP52. Pharmacological inhibition of FKBP52, by MJC13, attenuated the tumor growth, weight, and volume of LNCaP cells lacking ID4(L(-)ID4) xenografts. The L(-)ID4 cells also gains CRPC phenotype through FKBP52-mediated AR signaling. In this study we investigated the role of ID4 in pharmacological inhibition of FKBP52 with GMC1 (Initial hit molecule by in-silico screening with the most potent inhibition of FKBP52) in regulating the AR pathway. Experimental procedures: GMC1 drug treatments in LNCaP(-)ID4 and CW22RV1 generated xenograft tumors were further analyzed for tumor volume and tumor weight. We also performed immunohistochemistry on GMC1 treated xenografts to determine AR, PSA, FKBP51 and FKBP52 protein expression. Our in vivo studies with GMC1 drug treatments in castrated male SCID mice injected with LNCaP-ID4 and CW22RV1 cells showed decreased tumor size and volume when compared to untreated mice tumors. Results: IHC results on GMC1 treated LNCaP(-)ID4 and CW22RV1 generated xenograft tumors showed no change in ID4 expression with decrease expression of AR, PSA, FKBP51, FKBP52 and KI67 when compared to untreated xenograft tumors. These results suggest that inhibition of FKBP52-regulated AR activity (via GMC1) demonstrated a significant reduction in the tumor volume of subcutaneous xenografts in vivo. Overall, ID4 appears as a potential tumor suppressor gene that selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52-mediated AR signaling. Conclusions: In conclusion, we demonstrate that loss of ID4 in LNCaP cells elevates FKBP52 levels, transcriptionally potentiates AR activity which in turn then leads to progression of androgen-independent prostate cancer. GMC1 is a novel inhibitor that inhibits the binding of FKBP52 to AR thereby prevents the AR transcriptional hyperactivity in PCa Acknowledgement: These studies were supported by DOD, Grant # W81XWH-17-1-0437 Citation Format: Shravan Kumar Komaragiri. Role of ID4 (inhibitor of DNA binding protein 4) in FKBP52-AR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3061.

Oncogenic and osteolytic functions of histone demethylase NO66 in castration-resistant prostate cancer.

Epigenetic changes that cause dysregulated gene expression during progression of androgen-independent prostate cancer (PCa) and metastatic skeletal lesions remain elusive. Here, we explored the role of histone demethylase NO66 in the pathogenesis of PCa and bone metastasis-related skeletal lesions. Tissue and cDNA microarrays of PCa were analyzed for NO66 mRNA and protein levels. We examined the effects of gain and loss of NO66 function on cell viability, colony formation, migration, invasion, and tumor-induced skeletal lesions in femoral bone. RNAseq and ChIPseq were performed to elucidate NO66-target genes in PCa. We report that NO66 levels were upregulated in advanced primary prostate tumors compared to normal tissue or tumors with low Gleason scores. Forced expression of NO66 promoted cell survival and invasion of PCa cells; whereas, knockdown of NO66 resulted in decreased cell survival and increased sensitivity to docetaxel. NO66-overexpressing PC3 cells implanted into the femoral bone of male SCID mice caused massive bone loss and stimulation of mouse osteoclast-promoting genes, including Dickkopf1, Cathepsin K, Nf-kβ,; and Calcr, suggesting a role for NO66 in tumor growth in bone and osteoclast activity. Combined RNAseq and ChIP-seq revealed that NO66 activates the survival gene MCL1, the invasion-associated genes IGFBP5 and MMP3, the pro-oncogenic genes CTNNB1 and CCND1, and the epigenetic modifier gene KMT2A in androgen-independent PCa. Our findings uncover the role of NO66 as a key oncogenic driver in PCa, causing osteolytic lesions through upstream epigenetic regulation of key genes for survival, invasion and metastasis, and pro-osteoclastic factors.

Mutagenesis Screens for Prostate Cancer Using Replication-Incompetent Lentiviral Vectors.

Prostate cancer (PC) is the second leading cause of cancer-related deaths in US men, and progression to androgen-independent PC (AIPC) typically results in metastasis and is lethal. However, the mechanisms whereby PC progresses from androgen dependence to androgen independence are not completely understood. Mutagenesis screens to identify novel genes involved in the progression to AIPC have been performed using replication-incompetent lentiviral vectors (LVs). In this approach the LV acts both as a mutagen and as molecular tag to identify nearby genes that may have been dysregulated by the vector provirus, and are candidate AIPC genes. Here we describe protocols for generation of replication-incompetent LV preparations and performing a mutagenesis screen to identify AIPC genes in vitro.

Abstract 5468: Neratinib significantly inhibits responses to androgen in human prostate cancer cells

Abstract Background: Prostate cancer (PCa) is the most commonly diagnosed male cancer in the Western world. Tumor growth is initially androgen dependent - and driven by the androgen receptor (AR). The mainstays of prostate cancer treatment are androgen ablation and antiandrogen treatment, which block AR signalling. However, PCa often relapses to an androgen- independent disease. Androgens can transactivate genes directly via the AR-mediated transcription factor and indirectly via less well understood signal transduction pathways. These signal transduction pathways become increasingly relevant as prostate cancer cells progress to anti-androgen resistance, with HER2 being associated with higher relapse rates. Neratinib is an orally available tyrosine kinase inhibitor that irreversibly binds and inhibits EGFR, HER2 and HER4 receptor tyrosine kinases. This study aimed to examine the effect of neratinib on androgen signalling and on the expression of androgen-regulated genes in prostate cancer cells. Methods: Changes in protein phosphorylation after androgen treatment of hormonally starved prostate cancer cells (LNCaP) was assessed using protein microarrays (Kinexus, Canada). Changes in gene expression after neratinib or androgen treatment were ascertained using AmpliSseq® technology or standard qPCR, and analysed via IPA Ingenuity software. Results: Androgen treatment of hormonally starved prostate cancer cells (LNCaP) caused phosphorylation of several members of the signal transduction cascade including HER2 and Src within 2 hours, indicating a role for HER2 in rapid androgen signalling. Additionally, from over 1000 genes upregulated by androgen treatment (>2fold within 2 hours), 87% showed downregulation with neratinib treatment. Ingenuity pathway analysis indicated that STAT3, ETS-family and NF-κB transcription factors may be responsible for the rapid androgen-induced gene upregulation observed, and that these pathways were inhibited by neratinib treatment. Q-Quantitative PCR analysis of PSA expression in LNCaP cells stimulated with androgen in the presence of increasing concentrations of neratinib resulted in a dose-dependent inhibition of androgen activity. Conclusions: These results show that neratinib is able to inhibit the responses of prostate cancer cells to androgens, and that a strong potential signaling cross-talk exists between the androgen receptor and the certain signal transduction pathways - pathways known to be involved in the progression of androgen-independent prostate cancer. Citation Format: Dafydd A. Dart, Alshad S. Lalani, Francesca Avogadri-Connors, Richard Bryce, Wen G. Jiang. Neratinib significantly inhibits responses to androgen in human prostate cancer cells [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 5468.

Replication-incompetent gammaretroviral and lentiviral vector-based insertional mutagenesis screens identify prostate cancer progression genes.

Replication-incompetent gammaretroviral (γRV) and lentiviral (LV) vectors have both been used in insertional mutagenesis screens to identify cancer drivers. In this approach the vectors stably integrate in the host cell genome and induce cancers by dysregulating nearby genes. The cells that contain a retroviral vector provirus in or near a proto-oncogene or tumor suppressor are preferentially enriched in a tumor. γRV and LV vectors have different integration profiles and genotoxic potential, making them potentially complementary tools for insertional mutagenesis screens. We performed screens using both γRV and LV vectors to identify driver genes that mediate progression of androgen-independent prostate cancer (AIPC) using a xenotransplant mouse model. Vector transduced LNCaP cells were injected orthotopically into the prostate gland of immunodeficient mice. Mice that developed tumors were castrated to create an androgen-deficient environment and metastatic tumors that developed were analyzed. A high-throughput modified genomic sequencing PCR (MGS-PCR) approach identified the positions of vector integrations in these metastatic tumors. OR2A14, FER1L6, TAOK3, MAN1A2, MBNL2, SERBP1, PLEKHA2, SPTAN1, ADAMTS1, SLC30A5, ABCC1, SLC7A1 and SLC25A24 were identified as candidate prostate cancer (PC) progression genes. TAOK3 and ABCC1 expression in PC patients predicted the risk of recurrence after androgen deprivation therapy. Our data shows that γRV and LV vectors are complementary approaches to identify cancer driver genes which may be promising potential biomarkers and therapeutic targets.

Lentiviral vector-mediated insertional mutagenesis screen identifies genes that influence androgen independent prostate cancer progression and predict clinical outcome.

Prostate cancer (PC) is the second leading cause of cancer related deaths in US men. Androgen deprivation therapy (ADT) improves clinical outcome, but tumors often recur and progress to androgen independent prostate cancer (AIPC) which no longer responds to ADT. The progression to AIPC is due to genetic alterations that allow PC cancer cells to grow in the absence of androgen. Here we performed an insertional mutagenesis screen using a replication-incompetent lentiviral vector (LV) to identify the genes that promote AIPC in an orthotopic mouse model. Androgen sensitive PC cells, LNCaP, were mutagenized with LV and injected into the prostate of male mice. After tumor development, mice were castrated to select for cells that proliferate in the absence of androgen. Proviral integration sites and nearby dysregulated genes were identified in tumors developed in an androgen deficient environment. Using publically available datasets, the expression of these candidate androgen independence genes in human PC tissues were analyzed. A total of 11 promising candidate AIPC genes were identified: GLYATL1, FLNA, OBSCN, STRA13, WHSC1, ARFGAP3, KDM2A, FAM83H, CLDN7, CNOT6, and B3GNT9. Seven out the 11 candidate genes; GLYATL1, OBSCN, STRA13, KDM2A, FAM83H, CNOT6, and B3GNT6, have not been previously implicated in PC. An in vitro clonogenic assay showed that knockdown of KDM2A, FAM83H, and GLYATL1 genes significantly inhibited the colony forming ability of LNCaP cells. Additionally, we showed that a combination of four genes, OBSCN, FAM83H, CLDN7, and ARFGAP3 could significantly predicted the recurrence risk in PC patients after prostatectomy (P = 5.3 × 10-5 ). © 2015 Wiley Periodicals, Inc.

Abstract 3253: Id4 and FKBP52 interaction regulates androgen receptor activity in normal prostate and prostate cancer

Abstract The androgen receptor (AR) is a ligand activated transcription factor and important member of the steroid hormone nuclear receptors. Androgen signaling through AR plays a critical role in prostate tumorigenesis. AR transcriptional enhancement normally requires FKBP52 mediated peptidyl-prolyl cis-trans isomerase (PPIase) activity as well as HSP90-binding ability. FK506 binding protein 4 (FKBP52) is an AR folding factor that has critically important physiological roles in male reproductive tissues. Despite several experimental evidences, the molecular mechanism by which FKBP52 promotes AR signaling in prostate cancer still remains to be investigated. Our previous lab studies have demonstrated that Id4 is regulated by androgens in normal prostate epithelial and LNCaP cells. However, the interaction of Id4 and with the androgen receptor pathway in regulating the development and function of the normal prostate still remains elusive. Here we demonstrate that Id4 (inhibitor of differentiation-4) a dominant negative regulator of bHLH transcription factors can physically interact with AR, FKBP52 and HSP90 in early stage prostate cancer LNCaP cells and thereby selectively regulating AR transcriptional activity. Id4 is highly expressed in the normal prostate and decreased in prostate cancer due to promoter hypermethylation. Loss of Id4 in LNCaP cells resulted in ligand independent AR activation characterized by increased AR nuclear translocation and expression of PSA, FKBP51, FKBP52 and ARD1, the transcriptional targets of AR. Differential protein expression between LNCaP and LNCaP-Id4 demonstrated that loss of Id4 cells significantly elevated the levels of FBKP52 and Hsp27, the two known transcriptional regulators and nuclear transporters of AR. More importantly, LNCaP-Id4 cells were able to synthesize significantly higher levels of testosterone as compared to the androgen-dependent LNCaP cells. In conclusion, we demonstrate that loss of Id4 in LNCaP cells elevates FKBP52 levels, transcriptionally potentiates AR activity which in turn then leads to progression of androgen-independent prostate cancer. NIH grant #R01CA128914 Citation Format: Jugal B. Joshi. Id4 and FKBP52 interaction regulates androgen receptor activity in normal prostate and 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 3253. doi:10.1158/1538-7445.AM2014-3253

Paired box 2 upregulates androgen receptor gene expression in androgen-independent prostate cancer.

Androgen-independent prostate cancer is known as a hormone-refractory disease. Although the androgen receptor (AR) is considered to be a key regulator of androgen-independent prostate cancer progression, the mechanism through which AR gene expression is regulated is not well understood. In the present study, we showed that the AR gene was upregulated by paired box 2 (PAX2) in androgen-independent prostate cancer. When PAX2 upregulated AR gene expression, a decrease in DNA methylation of the AR gene locus was also observed. PAX2 was highly expressed and promoted cell growth in an androgen-independent prostate cancer cell line (22Rv1). The cell growth inhibition by PAX2 knockdown was rescued by AR overexpression in 22Rv1 cells. In a mouse xenograft model of androgen-independent prostate cancer, PAX2 knockdown inhibited tumor growth and AR gene expression and also increased DNA methylation of the AR gene. Consistent with this, AR and PAX2 expression levels were positively correlated in prostate cancer patients. These findings suggested that PAX2 promoted cancer cell growth in androgen-independent prostate cancer by regulating AR gene expression through an epigenetic mechanism.

Abstract 2126: Inhibition of hedgehog and androgen receptor signaling pathways produced synergistic suppression of androgen independent prostate cancer progression.

Abstract Introduction: Prostate cancer (PCa) is the second most common cause of death from cancer and the leading cancer diagnosed in the US men. Approximately 1 in 6 American men will be diagnosed with prostate cancer during his lifetime. Estimated new cases and deaths from prostate cancer in the United States for 2012 include: 241,740 new cases and 28,710 deaths. Initial treatment for metastatic PCa involves androgen ablation therapy, which causes regression of androgen-dependent tumors. However they all eventually relapse resulting in recurrent androgen independent prostate cancer (AIPC). Methods: This study aims at assessing the role of Hedgehog (Hh) and Androgen receptor (AR) signaling in the development of AIPC. Quantitative RT-PCR analysis was performed for AIPC cells in vitro and for AIPC xenograft after castration, to assess the levels of Hh and AR signaling components. Hh inhibitors, LDE225 (Novartis) and Cyclopamine, and Androgen receptor inhibitor Pyrvinium pamoate were tested individually and in combination to assess the suppression of AIPC progression both in vitro and in vivo. AR-responsive and Gli-responsive promoter-luciferase assay and MTT assay were used for in vitro studies. Intraperitoneal administration of drugs and bioluminescence imaging for monitoring orthotopic tumors in mice were used to study the effect of drugs on the growth of AIPC in vivo. Results: We demonstrate that androgen deprivation leads to increase in Hedgehog (Hh) signaling components in the AIPC cells in vitro and in vivo. AIPC cells also show increased sensitivity to low concentrations of androgen, suggesting increased androgen receptor expression. Together, these results suggest that increased Hh signaling and androgen receptor expression in the absence of androgen agonist plays an important role in the development and progression of AIPC. Our results show that inhibiting either Hh or androgen receptor signaling pathway individually was not effective in preventing AIPC cell proliferation in vitro as well as in inhibiting xenograft tumor growth in mice. In contrast, combination of an Hh pathway inhibitor with an androgen receptor inhibitor synergistically suppressed the growth of AIPC cells in vitro as assessed by MTT assay and reduced tumor burden in a mouse xenograft model of AIPC as compared to individual treatments.Conclusion: Upregulated Hh signaling and AR expression mediates development and progression of AIPC. Combined inhibition of Hh and androgen receptor signaling pathways may be a novel effective therapeutic strategy for the treatment of androgen independent prostate cancer. Citation Format: pramod gowda, Jianhong Deng, Sweta Mishra, LuZhe Sun. Inhibition of hedgehog and androgen receptor signaling pathways produced synergistic suppression of androgen independent prostate cancer progression. [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 2126. doi:10.1158/1538-7445.AM2013-2126

Contrasted effects of the multitarget TKi vandetanib on docetaxel-sensitive and docetaxel-resistant prostate cancer cell lines

ObjectivesOverexpression of epidermal growth factor receptor (EGFR) and angiogenic factors is associated with the progression of androgen-independent prostate cancer (AIPC). We examined the effects of vandetanib, an inhibitor of vascular endothelial growth factor (VEGFR), EGFR, and rearranged during transfection (RET) tyrosine-kinase activities, alone or combined with docetaxel, on PC3 docetaxel-sensitive (PC3wt) or docetaxel-resistant (PC3R) AIPC cell growth in vivo and in vitro. MethodsMice bearing PC3wt or PC3R tumors were treated for 3 weeks with vandetanib (25 or 50 mg/kg/d p.o., 5 d/wk), docetaxel (10 or 30 mg/kg i.p., 1 d/wk), or their combination (low or high doses). Xenograft tumors were analyzed for expression of Ki-67, EGFR, VEGFR2, and production of VEGFA. ResultsOn PC3wt, vandetanib at both doses stimulated tumor growth, whereas docetaxel at both doses exerted strong growth-inhibiting effects. The low-dose vandetanib-docetaxel combination resulted in tumor growth similar to that of control, whereas the high-dose combination induced a significant antiproliferative effect. In contrast, on PC3R, the low-dose of vandetanib had no effect on tumor growth, whereas the high-dose of vandetanib significantly inhibited tumor growth. Docetaxel at both doses exerted moderate and transient antitumor effects. The combination of high-dose vandetanib with high-dose docetaxel resulted in antiproliferative effects, which were lower than expected from the sum of individual drug effects. Importantly, tumor analyses revealed overexpression of the EGFR/VEGFR pathways in PC3R relative to PC3wt. ConclusionPresent results suggest that vandetanib should not be associated with docetaxel in treatment-naive or docetaxel-resistant prostate cancer (CaP). The use of high-dose vandetanib alone may warrant further investigation in patients with docetaxel-resistant AIPC overexpressing VEGFR/EGFR pathways.

Abstract 3927: Loss of bHLH transcription factor Id4 modulates androgen receptor activity in prostate cancer progression

Abstract Inhibitor of differentiation 4 (Id4), a crucial developmental gene also expressed in the normal prostate, has been shown to act as a tumor suppressor in prostate cancer. Promoter hypermethylation of Id4 is observed in several advanced stage cancers including prostate cancer. The loss of Id4 expression correlates with androgen independent prostate cancer progression. Our studies suggest that Id4 plays a crucial role in mediating the transition from androgen dependent to androgen independent prostate cancer by mediating the AR axis potential and reprogramming several cellular mechanisms including migration, senescence and proliferation. Stable transfection of a full length Id4 plasmid into the androgen-independent prostate cancer cell line DU145 resulted in re-expression of AR and downstream AR response gene PSA. Id4 transfected cells also observed an androgen regulated rate of proliferation in responsive to treatment with AR antagonist, Casodex. AR antagonism also significantly reduced the rate of migration as observed by a scratch wound assay. Under closer observation, AR antagonist treatment with Casodex also inhibited translocation of AR to the nucleus of DU145 cells as observed by immunofluorescence. Previous studies in our laboratory have demonstrated that DU145+Id4 transfected cells undergo a significant level of senescence (∼ 32%) compared to normal DU145 cells (∼ 4%). Treatment of DU145+Id4 cells with the AR antagonist Casodex demonstrate that the rate of senescence in DU145+Id4 transfected cells is closely related to inhibition of AR activity. Our results suggest that Id4 may play a crucial role in the loss of AR activity in advanced stage prostate cancer progression. These results also suggest that the antagonism of AR nuclear translocation post Id4 induction closely regulates the rate of proliferation and the induction of a senescent phenotype. These results provide a significant correlation between Id4 expression and AR activity during prostate cancer progression. Loss of AR activity through Id4 promoter hypermethylation may play a crucial role in understanding castration resistant prostate cancer. Therefore, induction of Id4 may provide a novel therapeutic target for advanced stage prostate cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3927. doi:1538-7445.AM2012-3927

Abstract B109: Aldo-keto reductase(AKR) 1C3 expression is frequently observed in human prostate cancer cases after androgen therapy and promotes prostate cancer cell growth in castrated mice model.

Abstract Introduction: Prostate cancer (PCa) is the most common malignancies and the second leading cause of death of men in US. Because PCa require androgen for its growth, anti-androgen therapy is favored for treating advanced PCa. Although anti-androgen therapy is initially effective for most PCa cases, virtually all of the cases will relapse after certain period of treatment. Recently several studies have revealed that androgen level was elevated in prostate tumor tissue even after anti-androgen therapy, while serum androgen level was reduced. In human male, the major sauce of androgen production is the testis. In addition adrenal glands also produce androgen in lesser extent. However adrenal androgens, dehydroepiandrosterone (DHEA), have weaker activity compared with testicular androgen, testosterone. DHEA could be converted to testosterone by only two enzymes, AKR1CE and HSD3B2, normally expressed in the testis. Recently it has been reported that AKR1C3 and HSD3B2 were over-expressed in prostate cancer tissue after anti-androgen therapy. However the precise origin of their production and biological relevance of these enzymes for androgen-independent PCa progression have yet to be clarified. Method: Seventy cases who received prostatectomy for PCa in our hospital were selected. Among the cases examined, 19 cases were treated with anti-androgen therapy prior to the prostatectomy. The expression of AKR1C3 and HSD3B in PCa tissues were examined immunohistochemically. The expression of AKR1C3 were also examined by Western blot in three androgen dependent PCa cell lines, LNCap, VCap and MDA-PCa2B, and one independent PCa cell line, PC3. For castrated mice model, one million (1×10∘6) of LNCap cells transduced with Mock or AKR1C3 (LNCap-mock or AKR1C3) by lenti-virus system were transplanted in mammary fat pad of intact or castrated nude mice. The tumor sizes were measured weekly, and developed tumors were histologically examined 5 weeks after implantation. Results: In human PCa cases, AKR1C3 expression was observed in 18 of 70 (25.7%) cases. The frequency of AKR1C3 expression was higher (p<0.01) in the cases that received anti-androgen therapy prior to prostatectomy (11/19; 57.9%) compared with the cases without prior therapy (7/58; 12.1%). No significant difference in terms of frequency of expression was observed in HSD3B2 (15/19; 78.9% vs. 45/58; 77.6%). In normal growth condition, AKR1C3 expression was observed in VCap and PC3, but not in LNCap or MDA-PCa2b. The AKR1C3 expression was enhanced or induced in VCap and LNCap in hormone depleted condition. Adione, which can be converted to testosterone by AKR1C3, stimulated LNCap-AKR1C3 growth but not LNCap-Mock. In castrated mice model, all the cases (6/6) of LNCap-AKR1C3 developed tumor, while only one out of five (1/5) case of LNCap-Mock did after 5 weeks of transplantation. Conclusion: AKR1C expression might be induced by anti-androgen therapy and could facilitate tumor cell proliferation in androgen depleted condition. Thus the molecular targeting therapy against AKR1C3, combination with ordinary anti-androgen therapy, will be promising for treating PCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B109.

Overexpression of Fn14 promotes androgen-independent prostate cancer progression through MMP-9 and correlates with poor treatment outcome

Fibroblast growth factor-inducible 14 (Fn14), a transmembrane receptor binding to the multifunctional cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is known to modulate many cellular activities including cancer progression. Here, we demonstrated the significant role of Fn14 in invasion, migration and proliferation of androgen-independent prostate cancer (AIPC) cells. Fn14 and its ligand TWEAK were highly expressed in two AIPC cell lines, DU 145 and PC-3, whereas expression was weak in androgen-sensitive LNCaP cells. Fn14 knockdown using small-interfering RNAs attenuated migration, invasion and proliferation and enhanced apoptosis in the AIPC cell lines. Both forced overexpression of Fn14 by stable Fn14 complementary DNA transfection to PC-3 cells (PC-3/Fn14) and ligand activation by recombinant TWEAK in PC-3 cells enhanced invasion. Fn14 was shown to modulate expression of matrix metalloproteinase (MMP)-9, and MMP-9 mediated the invasive potential influenced by Fn14 in PC-3 cells. In vivo, subcutaneous xenografts of PC-3/Fn14 grew significantly faster than xenograft of PC-3/Mock, and the invasive capacity in PC-3/Fn14 was found to be higher than that of PC-3/Mock as evaluated in an invasion model of the diaphragm. Furthermore, the messenger RNA expressions of MMP-9 in PC-3/Fn14 xenografts were significantly higher than those in PC-3/Mock xenografts. Clinically, high expression of Fn14 was significantly associated with higher prostate-specific antigen recurrence rate in patients who underwent radical prostatectomy. In conclusion, the overexpression of Fn14 may contribute to multiple malignant cellular phenotypes associated with prostate cancer (PCa) progression, in part via MMP-9. TWEAK-Fn14 signaling may be a novel therapeutic target of PCa.

Abstract 249: Inhibition of protein phosphatase 2A supports androgen-independent growth of prostate cancer cells

Abstract Clinical progression of prostate cancer (PCa) is characterized by a transition from androgen-dependent (AD) to androgen-independent (AI) stage. Once the PCa has recurred in AI form, it progresses to a highly aggressive disease and poses an increased risk of morbidity and death. Therefore, understanding the mechanisms involved in AI progression of PCa is a significant area of research. Earlier, we identified PPP2CA, which encodes for alpha-isoform of the protein phosphatase 2A catalytic subunit (PP2Acα), as one of the downregulated genes in AI PCa cells. PP2A is a ser/thr phosphatase and a potent tumor suppressor involved in broad cellular functions; however, its role in PCa has not yet been determined. Here, we have investigated the effect of PP2A downregulation on the growth of AD PCa (LNCaP) cells under steroid-deprived condition. Furthermore, we have examined the effect of PP2A inhibition on the signaling pathways and delineated their role in AI growth of LNCaP cells. Our data show that the downregulation of PP2A activity by pharmacological inhibition or siRNA-mediated PPP2CA silencing sustains the growth of AD PCa cells under androgen-deprived condition by relieving the androgen-deprivation-induced cell cycle arrest and preventing apoptosis. Immunoblot analysis revealed enhanced phosphorylation of Akt, ERK, BAD, increased expression of cyclins (cyclin A1 and cyclin D1) and decreased expression of cyclin inhibitor (p27) upon PP2A downregulation. Furthermore, our data show that PP2A inhibition partially maintains AR signaling through its increased expression and ligand-independent phosphorylation, which is also supported by AR transcriptional activity assay and its target gene, KLK3, expression. Pharmacological inhibition of Akt, ERK and AR confirmed a role of these signaling pathways in facilitating the AI growth of LNCaP cells. Altogether, our findings suggest that modulation of PP2A activity may represent an alternative therapeutic approach for the treatment of advanced androgen-independent prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 249. doi:10.1158/1538-7445.AM2011-249

Abstract 2156: c-Myb promotes growth, invasion and androgen-independence of prostate cancer cells

Abstract Most prostate cancer (PCa) patients treated with first line androgen-deprivation therapy (ADT) initially exhibit a dramatic regression of the androgen-dependent (AD) cancer cells; however, the tumors eventually progress to an androgen-independent (AI) stage. The resulting hormone-refractory tumors are highly aggressive and resistant to conventional therapies. The causal mechanisms for such a transition remain largely unknown; however, a number of genetic and epigenetic alterations are believed to be involved in the process of AI progression of PCa. In an earlier study, c-Myb was identified among the genes that are amplified at higher frequency in hormone-refractory PCa. The c-Myb proto-oncogene is a cellular progenitor of v-Myb oncogenes, which encodes for a transcription factor and confers its oncogenic activity by regulating the expression of several target genes. In the present study, we have investigated the functional role of c-Myb in prostate cancer cells. Our data showed that c-Myb was overexpressed in AI (C4-2) PCa cells as compared to AD (LNCaP) PCa cells of the same genotypic lineage. Furthermore, we found that c-Myb was regulated by androgens in a biphasic manner and its expression correlated with the effect of androgens on prostate cancer cell growth. Short-hairpin RNA (shRNA)-mediated silencing of c-Myb in C4-2 cells reduced their growth, clonogenicity, motility and invasion, whereas its ectopic expression in LNCaP cells had opposite effects. Moreover, we observed that the overexpression of c-Myb in LNCaP and C4-2 cells was associated with their growth in androgen-depleted media, suggesting its role in androgen-independence. Immunoblot analysis demonstrated loss of mesenchymal markers N-cadherin, vimentin, Slug, Snail, and Twist, and gain of epithelial marker, E-cadherin in c-Myb-silenced C4-2 cells. In contrast, c-Myb overexpressing LNCaP cells exhibited a loss of epithelial and gain of mesenchymal markers, thus indicating a role of c-Myb in epithelial to mesenchymal transition (EMT) of PCa cells. Altogether, our studies provide first experimental evidence for a functional role of c-Myb in altered growth, androgen-independence and malignant behavior of the prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2156. doi:10.1158/1538-7445.AM2011-2156