Androgen-independent Proliferation Research Articles

TPM2 attenuates progression of prostate cancer by blocking PDLIM7-mediated nuclear translocation of YAP1

BackgroundProstate cancer (PCa) is a common malignant tumor of the genitourinary system. Clinical intervention in advanced PCa remains challenging. Tropomyosins 2 (TPM2) are actin-binding proteins and have been found as a biomarker candidate for certain cancers. However, no studies have explored the role of TPM2 in PCa and its regulatory mechanism.MethodsTPM2 expression was assessed in Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) PCa patient dataset. The effect of TPM2 on PCa progression was assessed in vitro and in vivo by quantifying proliferation, migration, invasion and tumor growth assays, and the mechanism of TPM2 in PCa progression was gradually revealed by Western blotting, immunoprecipitation, and immunofluorescence staining arrays.ResultsTPM2 was found to be severely downregulated in tumor tissues of PCa patients compared with tumor-adjacent normal tissues. In vitro experiments revealed that TPM2 overexpression inhibited PCa cell proliferation, invasion and androgen-independent proliferation. Moreover, TPM2 overexpression inhibited the growth of subcutaneous xenograft tumors in vivo. Mechanistically, this effect was noted to be dependent on PDZ-binding motif of TPM2. TPM2 competed with YAP1 for binding to PDLIM7 through the PDZ-binding motif. The binding of TPM2 to PDLIM7 subsequently inhibited the nuclear transport function of PDLIM7 for YAP1. YAP1 sequestered in the cytoplasm phosphorylated at S127, resulting in its inactivation or degradation which in turn inhibited the expression of YAP1 downstream target genes.ConclusionsThis study investigated the role of TPM2, PDLIM7, and YAP1 in PCa progression and castration resistance. TPM2 attenuates progression of PCa by blocking PDLIM7-mediated nuclear translocation of YAP1. Accordingly, targeting the expression or functional modulation of TPM2, PDLIM7, or YAP1 has the potential to be an effective therapeutic approach to reduce PCa proliferation and prevent the progression of castration-resistant prostate cancer (CRPC).

Abstract 725: LIFR promotes SUCLG2-dependent castration resistance and neuroendocrine differentiation of prostate cancer

Abstract Neuroendocrine (NE) differentiation is a well-recognized phenotypic change of prostate cancer after androgen-deprivation therapy (ADT) that ultimately develops an aggressive subtype. An increasing number of patients with NE histologic type prostate cancer present with metabolic genes disorder. However, the metabolic pathways that influence the malignant transformation and the NE differentiation of prostate cancer remain unclear. In this study, we found that ADT induced the upregulation of leukemia inhibitory factor receptor (LIFR), which contributes to the expression of succinate-CoA ligase GDP-forming beta subunit (SUCLG2), thus activating metabolic reprogramming to enable NE differentiation and increase aggressiveness. Activated LIF-LIFR-STAT3 axis stimulates SUCLG2 expression, subsequently upregulating glucose metabolism and the NE differentiation of prostate cancer. SUCLG2 knockdown markedly reduced mitochondrial DNA content and inhibited the enzymatic activity of succinyl-CoA synthetase and nucleoside diphosphate kinase (NDPK) in prostate cancer cells. SUCLG2 overexpression promoted androgen-independent proliferation; however, SUCLG2 knockdown suppressed NE differentiation and the ADT resistance of prostate cancer cells. Analysis of prostate tissue samples showed an increased intensity of LIFR associated with SUCLG2 in castration-resistant prostate cancer tumors. Our study identifies SUCLG2 as a potential prognostic marker of ADT-resistant prostate cancer and suggests it could be a target for prostate cancer treatment. Citation Format: Hsiu-Lien Yeh, Shian-Ren Lin, Kuo-Ching Jiang, Wei-Hao Chen, Yen-Nien Liu. LIFR promotes SUCLG2-dependent castration resistance and neuroendocrine differentiation of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 725.

Role of Cav3.2 T-type Ca2+ channels in prostate cancer cells

Among voltage-gated Ca2+ channels, T-type Ca2+ channels, which are activated by low voltages, regulate neuronal excitability, spontaneous neurotransmitter release, hormone secretion, etc. and also participate in proliferation of distinct cancer cells. Among three isoforms of T-type Ca2+ channels, Cav3.2 is detectable in 100% of biopsy samples from prostate cancer patients. In general, prostate cancer cells are highly sensitive to androgen deprivation therapy, but often acquire hormone-therapy resistance. The androgen deprivation may trigger neuroendocrine (NE)-like differentiation of some prostate cancer cells. We have analyzed the expression and function of Cav3.2 in human prostate cancer LNCaP cells during NE-like differentiation. NE-like LNCaP cells overexpress Cav3.2 through the CREB/Egr-1 pathway and also cystathionine-γ-lyase (CSE), which generates H2S that enhances the channel activity of Cav3.2. H2S generated by upregulated CSE appears to enhance the activity of upregulated Cav3.2 after the differentiation. The enhanced Cav3.2 activity in NE-like cells may contribute to increased secretion of mitogenic factors essential for androgen-independent proliferation of surrounding prostate cancer cells. It is known that increased extracellular glucose levels enhance Cav3.2 activity through asparagine (N)-linked glycosylation of Cav3.2, which might contribute to diabetic neuropathy. We then found that high glucose accelerates the enhanced channel function and overexpression of Cav3.2 in NE-like LNCaP cells, which might be associated with clinical evidence for diabetes-related poor prognosis of prostate cancer and development of hormone therapy resistance. Thus, Cav3.2 is considered to play a role in the pathophysiology of prostate cancer, and may serve as a therapeutic target.

High OGT activity is essential for MYC-driven proliferation of prostate cancer cells.

O-GlcNAc transferase (OGT) is overexpressed in aggressive prostate cancer. OGT modifies intra-cellular proteins via single sugar conjugation (O-GlcNAcylation) to alter their activity. We recently discovered the first fast-acting OGT inhibitor OSMI-2. Here, we probe the stability and function of the chromatin O-GlcNAc and identify transcription factors that coordinate with OGT to promote proliferation of prostate cancer cells.Methods: Chromatin immunoprecipitation (ChIP) coupled to sequencing (seq), formaldehyde-assisted isolation of regulatory elements, RNA-seq and reverse-phase protein arrays (RPPA) were used to study the importance of OGT for chromatin structure and transcription. Mass spectrometry, western blot, RT-qPCR, cell cycle analysis and viability assays were used to establish the role of OGT for MYC-related processes. Prostate cancer patient data profiled for both mRNA and protein levels were used to validate findings.Results: We show for the first time that OGT inhibition leads to a rapid loss of O-GlcNAc chromatin mark. O-GlcNAc ChIP-seq regions overlap with super-enhancers (SE) and MYC binding sites. OGT inhibition leads to down-regulation of SE-dependent genes. We establish the first O-GlcNAc chromatin consensus motif, which we use as a bait for mass spectrometry. By combining the proteomic data from oligonucleotide enrichment with O-GlcNAc and MYC ChIP-mass spectrometry, we identify host cell factor 1 (HCF-1) as an interaction partner of MYC. Inhibition of OGT disrupts this interaction and compromises MYC's ability to confer androgen-independent proliferation to prostate cancer cells. We show that OGT is required for MYC-mediated stabilization of mitotic proteins, including Cyclin B1, and/or the increased translation of their coding transcripts. This implies that increased expression of mRNA is not always required to achieve increased protein expression and confer aggressive phenotype. Indeed, high expression of Cyclin B1 protein has strong predictive value in prostate cancer patients (p=0.000014) while mRNA does not.Conclusions: OGT promotes SE-dependent gene expression. OGT activity is required for the interaction between MYC and HCF-1 and expression of MYC-regulated mitotic proteins. These features render OGT essential for the androgen-independent, MYC-driven proliferation of prostate cancer cells. Androgen-independency is the major mechanism of prostate cancer progression, and our study identifies OGT as an essential mediator in this process.

Inhibition of the androgen receptor induces a novel tumor promoter, ZBTB46, for prostate cancer metastasis.

Current therapeutic regimens for prostate cancer focus on targeting androgen receptor (AR) signaling. However, the AR is a key factor in luminal epithelium differentiation and was shown to have a role as a tumor suppressor. Thus, its inhibition may activate oncogenic pathways that contribute to metastatic castration-resistant prostate cancer (CRPC). Herein, we report a novel tumor promoter, ZBTB46, which is negatively regulated by AR signaling via microRNA (miR)-1-mediated downregulation. ZBTB46 is associated with malignant prostate cancer and is essential for metastasis. Its overexpression can overcome the antitumor effects of miR-1 and promote androgen-independent proliferation. We demonstrated that ZBTB46 can transcriptionally regulate SNAI1, a key epithelial-to-mesenchymal transition (EMT) driver, which could contribute to induction of the EMT after androgen-deprivation therapy and metastasis. Our findings are supportive of the model that disruption of AR's function may predispose prostate cancer to progress to metastatic CRPC.

Abstract 1583: Inhibition of proline isomerase Pin1 interrupts the function of the androgen receptor N-terminal domain and suppresses androgen-independent growth of prostate cancer cells

Abstract Patients with advanced prostate cancer continue to develop lethal castration-resistant prostate cancer (CRPC) despite hormone therapy and maintaining castrate levels of serum androgen. Most CRPC appears to be dependent on the androgen receptor (AR), but instead of the C-terminal ligand-binding domain, it is the N-terminal domain (NTD) harboring a powerful transactivation domain that drives AR transcriptional activity. This was supported by the discovery of AR splice variants (ARv567es and V7) which are constitutively active, do not encode a functional ligand-binding domain, and correlate with poor patient outcome. The AR NTD is intrinsically disordered, but it contains several putative binding sites for Pin1, a proline isomerase specific for phosphorylated-Ser/Thr-Pro motifs. Since the innate ability of the AR NTD to adopt multiple transient structures is important for transactivation of AR, we aimed to determine whether Pin1 regulates motifs within the AR NTD. We tested several known inhibitors of Pin1 in cell-based assays that measure proliferation or transcription mediated by AR. Our results demonstrated that inhibition of Pin1 interrupted the function of the AR NTD. The Pin1 inhibitor juglone effectively and specifically blocked transcription mediated by AR induced by androgen, as well as transactivation of the AR NTD in the presence of IL-6. We found that Pin1 predominantly interacted with a specific region of the AR NTD containing two Pin1 binding sites, and by inhibiting Pin1 the interactions between endogenous AR and STAT3 became attenuated. Furthermore, Pin1 inhibitors were more effective than second-generation anti-androgens in blocking androgen-independent proliferation of LNCaP95 cells driven by AR variants. Here we describe that Pin1 is a critical factor for transcription mediated by AR, regardless of ligand, by regulating the AR NTD. Understanding the molecular mechanisms that may promote AR signaling in the absence of androgen will aid the development of more effective therapies for CRPC. Citation Format: Jacky K. Leung, Yusuke Imamura, Minoru Kato, Nasrin R. Mawji, Marianne D. Sadar. Inhibition of proline isomerase Pin1 interrupts the function of the androgen receptor N-terminal domain and suppresses androgen-independent growth of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1583. doi:10.1158/1538-7445.AM2017-1583

TCF7 is suppressed by the androgen receptor via microRNA-1-mediated downregulation and is involved in the development of resistance to androgen deprivation in prostate cancer.

Resistance to androgen deprivation therapy (ADT) represents a key step in the malignant progression of prostate cancer, and mutation to androgen receptor (AR) is one major driver to an androgen-independent phenotype. However, alternative oncogenic pathways that bypass AR signaling have emerged as an important mechanism promoting resistance to ADT. It is known that AR activation can prevent the interaction between β-catenin and T cell factor/lymphoid enhancer-binding factor (TCF/LEF) family, inhibiting the Wnt signaling pathway. The aim of this study was to determine the role of transcription factor 7 (TCF7), a transcription factor best known as a Wnt effector that forms a complex with β-catenin, in the development of advanced prostate cancer. We further investigated the molecular mechanisms by which TCF7 is induced when AR signaling is inactivated. A novel AR signaling pathway that induces microRNA-1 (miR-1) to suppress metastatic prostate cancer was recently demonstrated (AR-miR-1 signaling axis), and its regulation of Wnt signaling was explored in the current study. Clinical data sets were analyzed for potential targets of AR-miR-1 signaling in the TCF/LEF family, and tissue samples were utilized to validate the relationship. The molecular mechanism and biological functions were demonstrated in prostate cancer cell lines and a mouse xenograft model. We demonstrated a molecular mechanism of AR signaling suppressing TCF7 partly through miR-1-mediated downregulation. TCF7 exhibited oncogenic properties and compromised the tumor-suppressive effects of miR-1. Our results also showed that overexpression of TCF7 or disruption of miR-1 function promoted androgen-independent proliferation. We demonstrated that the AR-miR-1 axis negatively regulates the novel oncogenic factor, TCF7. Dysregulation of TCF7 promoted a survival advantage and resistance to androgen deprivation, suggesting its therapeutic potential for castration-resistant prostate cancer.

Src promotes castration-recurrent prostate cancer through androgen receptor-dependent canonical and non-canonical transcriptional signatures.

Progression of prostate cancer (PC) to castration-recurrent growth (CRPC) remains dependent on sustained expression and transcriptional activity of the androgen receptor (AR). A major mechanism contributing to CRPC progression is through the direct phosphorylation and activation of AR by Src-family (SFK) and ACK1 tyrosine kinases. However, the AR-dependent transcriptional networks activated by Src during CRPC progression have not been elucidated. Here, we show that activated Src (Src527F) induces androgen-independent growth in human LNCaP cells, concomitant with its ability to induce proliferation/survival genes normally induced by dihydrotestosterone (DHT) in androgen-dependent LNCaP and VCaP cells. Src induces additional gene signatures unique to CRPC cell lines, LNCaP-C4-2 and CWR22Rv1, and to CRPC LuCaP35.1 xenografts. By comparing the Src-induced AR-cistrome and/or transcriptome in LNCaP to those in CRPC and LuCaP35.1 tumors, we identified an 11-gene Src-regulated CRPC signature consisting of AR-dependent, AR binding site (ARBS)-associated genes whose expression is altered by DHT in LNCaP[Src527F] but not in LNCaP cells. The differential expression of a subset (DPP4, BCAT1, CNTNAP4, CDH3) correlates with earlier PC metastasis onset and poorer survival, with the expression of BCAT1 required for Src-induced androgen-independent proliferation. Lastly, Src enhances AR binding to non-canonical ARBS enriched for FOXO1, TOP2B and ZNF217 binding motifs; cooperative AR/TOP2B binding to a non-canonical ARBS was both Src- and DHT-sensitive and correlated with increased levels of Src-induced phosphotyrosyl-TOP2B. These data suggest that CRPC progression is facilitated via Src-induced sensitization of AR to intracrine androgen levels, resulting in the engagement of canonical and non-canonical ARBS-dependent gene signatures.

Nimbolide inhibits androgen independent prostate cancer cells survival and proliferation by modulating multiple pro-survival signaling pathways.

Prostate cancer is the most prominent cancer in men, experiencing a relapse in disease often express high serum TNF-α levels. It has been correlated with increased cell survival and proliferation of prostate cancer cells. Previous studies reported that nimbolide, a terpenoid derived from the leaves and flowers of neem tree inhibits cancer growth through selective modulation of cell signaling pathways linked to inflammation, survival, proliferation, angiogenesis and metastasis. The present study aimed to examine the effect of nimbolide at 1 and 2μM concentrations on TNF-α/TNFR1 mediated signaling molecules involved in cell survival and proliferation in PC-3 cell line via NF-κB and MAPK pathways by real time PCR and western blot. Protein and compound interaction were performed by Molecular docking analysis. Our results indicate that nimbolide treatment suppressed expression of TNF-α, SODD, Grb2, SOS mRNA and modulated TNF-α/TNFR1 regulated NF-κB and MAPK signaling molecules in PC-3 cells. Additional molecular dynamics simulation studies confirmed the stability of nimbolide and signaling molecules binding interactions. Binding pose analysis revealed the significance of hydrogen bond interactions for effective stabilization of virtual ligand protein complexes. Nimbolide inhibited prostate cancer cell survival and proliferation via NF-κB and MAPK pathways.

MP90-03 REACTIVE OXYGEN SPECIES INDUCTION BY CABAZITAXEL VIA INHIBITION OF THE SESTRIN FAMILY IN CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP90-03 REACTIVE OXYGEN SPECIES INDUCTION BY CABAZITAXEL VIA INHIBITION OF THE SESTRIN FAMILY IN CASTRATION-RESISTANT PROSTATE CANCER Takeo Kosaka, Hiroshi Hongo, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, and Mototsugu Oya Takeo KosakaTakeo Kosaka More articles by this author , Hiroshi HongoHiroshi Hongo More articles by this author , Yasumasa MiyazakiYasumasa Miyazaki More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Akira MiyajimaAkira Miyajima More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2547AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cabazitaxel (CBZ) has become the most effective cytotoxic agent to demonstrate an improvement in survival in men with docetaxel-refractory CRPC. Accumulating evidence has suggested reactive oxygen species (ROS) production induced by taxanes in cancer cells may influence taxane-induced cell death or drug resistance. However, ROS production by CBZ or its mechanistic action remains to be characterized in CRPC. In this study, we investigated PCa cell sensitivity to docetaxel (DTX) or CBZ by measurement of ROS production. METHODS Three cell lines were used: LNCaP, a human PCa cell line that exhibits androgen-dependent proliferation; C4-2, a human PCa cell line that exhibits androgen-independent proliferation; and C4-2AT6, a CRPC cell line derived from C4-2. These cells were treated with increasing concentrations of docetaxel or CBZ in vitro. Gene expression profiles of CBZ- or DTX-treated cells were analyzed using a whole human genome microarray. FACS analysis was performed using a fluorogenic marker of ROS. Furthermore, we investigated the changes in expression of antioxidant enzymes induced by DTX and CBZ. RESULTS Cabazitaxel showed significantly higher cytotoxicity than docetaxel in human CRPC cells. FACS analysis revealed that treatment with cabazitaxel significantly increased ROS generation than docetaxel in C4-2AT6 cells, accompanied by higher expression of phospho-p38. Microarray analysis revealed significant changes in ROS regulation-related genes by CBZ. qPCR analysis revealed CBZ induced C4-2AT6 cell death, accompanied by significantly decreased expression of Sestrin-3 (SESN3). To investigate whether CBZ-mediated cell death was caused by induction of ROS generation, C4-2AT6 cells were incubated with CBZ in the presence of the antioxidant N-acetylcysteine (NAC), which was observed to reduce cabazitaxel-induced cytotoxicity. Microarray analysis revealed dynamic changes in ROS regulation-related genes by CBZ, and qPCR analysis demonstrated CBZ reduced mRNA expression of SESN3. We observed that ROS reduction by SESN3 was significantly inhibited by cabazitaxel in a dose-dependent manner, but not by docetaxel. When C4-2AT6 cells were treated with cabazitaxel in the presence of siRNAs against SESN3, significant cytotoxicity was observed, accompanied by enhanced ROS production. CONCLUSIONS Our results indicated that the higher sensitivity of human CRPC to CBZ, compared with DTX, involves ROS production via inhibition of SESN3 expression, an antioxidant enzyme. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1145 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Takeo Kosaka More articles by this author Hiroshi Hongo More articles by this author Yasumasa Miyazaki More articles by this author Eiji Kikuchi More articles by this author Akira Miyajima More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Functional upregulation of the H2S/Cav3.2 channel pathway accelerates secretory function in neuroendocrine-differentiated human prostate cancer cells

Neuroendocrine-differentiated prostate cancer cells may contribute to androgen-independent proliferation of surrounding cells through Ca2+-dependent secretion of mitogenic factors. Human prostate cancer LNCaP cells, when neuroendocrine-differentiated, overexpress Cav3.2 T-type Ca2+ channels that contribute to Ca2+-dependent secretion. Given evidence for the acceleration of Cav3.2 activity by hydrogen sulfide (H2S), we examined the roles of the H2S/Cav3.2 pathway and then analyzed the molecular mechanisms of the Cav3.2 overexpression in neuroendocrine-differentiated LNCaP cells. LNCaP cells were differentiated by dibutyryl cyclic AMP. Protein levels and T-type Ca2+ channel-dependent currents (T-currents) were measured by immunoblotting and whole-cell pacth-clamp technique, respectively. Spontaneous release of prostatic acid phosphatase (PAP) was monitored to evaluate secretory function. The differentiated LNCaP cells exhibited neurite outgrowth, androgen-independent proliferation and upregulation of mitogenic factors, and also showed elevation of Cav3.2 expression or T-currents. Expression of cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS), H2S-forming enzymes, and spontaneous secretion of PAP increased following the differentiation. The augmented T-currents were enhanced by H2S donors and suppressed by inhibitors of CSE, but not CBS. The PAP secretion was reduced by inhibition of CSE or T-type Ca2+ channels. During differentiation, Egr-1 and REST, positive and negative transcriptional regulators for Cav3.2, were upregulated and downregulated, respectively, and Egr-1 knockdown prevented the Cav3.2 overexpression. Our data suggest that, in neuroendocrine-differentiated LNCaP cells, H2S formed by the upregulated CSE promotes the activity of the upregulated Cav3.2, leading to the elevated secretory functions. The overexpression of Cav3.2 appears to involve upregulation of Egr-1 and downregulation of REST.

Identification of novel genes that regulate androgen receptor signaling and growth of androgen-deprived prostate cancer cells.

Prostate cancer progression to castration refractory disease is associated with anomalous transcriptional activity of the androgen receptor (AR) in an androgen-depleted milieu. To identify novel gene products whose downregulation transactivates AR in prostate cancer cells, we performed a screen of enzymatically-generated shRNA lenti-libraries selecting for transduced LNCaP cells with elevated expression of a fluorescent reporter gene under the control of an AR-responsive promoter. The shRNAs present in selected populations were analyzed using high-throughput sequencing to identify target genes. Highly enriched gene targets were then validated with siRNAs against selected genes, testing first for increased expression of luciferase from an AR-responsive promoter and then for altered expression of endogenous androgen-regulated genes in LNCaP cells. We identified 20 human genes whose silencing affected the expression of exogenous and endogenous androgen-responsive genes in prostate cancer cells grown in androgen-depleted medium. Knockdown of four of these genes upregulated the expression of endogenous AR targets and siRNAs targeting two of these genes (IGSF8 and RTN1) enabled androgen-independent proliferation of androgen-dependent cells. The effects of IGSF8 appear to be mediated through its interaction with a tetraspanin protein, CD9, previously implicated in prostate cancer progression. Remarkably, homozygous deletions of IGSF8 are found almost exclusively in prostate cancers but not in other cancer types. Our study shows that androgen independence can be achieved through the inhibition of specific genes and reveals a novel set of genes that regulate AR signaling in prostate cancers.

S3-2 Roles of the hydrogen sulfide-CAV3.2 T-type calcium channel pathway in neuronal and neuroendocrine differentiation

H2S, a gasotransmitter, is formed by multiple enzymes including cystathionine-gamma-lyase (CSE). H2S enhances function of Cav3.2 T-type calcium channels in NG108-15 cells, isolated dorsal root ganglion neurons and Cav3.2-transfected HEK293 cells, and the H2S/Cav3.2 pathway plays a role in somatic and visceral pain signaling. H2S also causes Cav3.2-dependent neuronal differentiation characterized by neuritogenesis in NG108-15 cells. Interestingly, androgen-sensitive human prostate cancer LNCaP cells exhibit neuroendocrine-like differentiation following elevation of intracellular cyclic AMP levels or androgen deprivation, and the differentiated cells abundantly express Cav3.2 responsible for calcium-dependent secretion of proliferation factors. We have found that neuroendocrine-like differentiation causes upregulation of CSE as well as Cav3.2, and endogenous H2S formed by CSE facilitates Cav3.2 function and then secretory responses in LNCaP cells. Expression of Egr1 at an early stage appears to be involved in transcriptional upregulation of Cav3.2. Our study thus suggest that H2S formed by upregulated CSE regulates secretory function via activation of upregulated Cav3.2 in neuroendocrine-like differentiated LNCaP cells, contributing to androgen-independent proliferation of surrounding cells, and the CSE/H2S/Cav3.2 pathway might serve as targets for pharmacological interventions for preventing acquisition of hormone therapy resistance of prostate cancer.

Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV.

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

Abstract 2152: Paroxetine, a selective serotonin re-uptake inhibitor, induces growth inhibition and apoptosis in prostate cancer cells

Abstract Introduction and Objective: Recently, neuroendocrine cells is suggested to be involved in prostate cancer (PC) progression through the secretion of a variety of neuroendocrine products, such as serotonin, which have mitogenic effects on adjacent cancer cells and thus can contribute to the androgen independent proliferation of PC cells. With having experience that a patient with hormone refractory prostate cancer responded to paroxetine, a selective serotonin re-uptake inhibitor (SSRI), in serum prostate specific antigen (PSA) level, we investigated the effect of paroxetine on the growth and apoptosis induction in PC cell lines. Methods: Paroxetine was administered to four patients with hormone refractory, metastatic PC and their serum PSA levels were determined. The effects of paroxetine on the growth and apoptosis induction in PC cell lines (PC3, DU145, 22RV1 and LNCaP) were confirmed. Cell growth inhibition was assessed in vitro by cell number count. The induction of apoptosis was examined by flow cytometry analysis and caspase activations were confirmed by western blot analysis. Results: Serum PSA levels decreased soon after the administration of paroxetine but increased again in all four patients but one in whom it decreased from 0.485 ng/ml to undetectable level (less than 0.008 ng/ml) for over three years. In vitro study, paroxetine inhibited cell growth in a dose and time dependent manner. The 50% inhibitory concentration (IC50 values) of paroxetine in PC3, 22RV1, DU145 and LNCaP cells were 38.8, 30.1, 35.9 and 21µM, respectively. Apoptosis induction following the treatment of paroxetine was revealed in prostate cancer cells by the increase of sub-G1 population in flow cytometry analysis and a cleavage of procaspase-3 in western blot analysis. Conclusions: A potent SSRI, paroxetine, induces growth inhibition and apoptosis in PC cells. These results suggest that SSRIs have the potential of anti-tumor effect against PC. 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 2152. doi:10.1158/1538-7445.AM2011-2152

103 HIGH DENSITY LIPOPROTEIN INDUCES PROLIFERATION AND MIGRATION OF HUMAN PROSTATE ANDROGEN INDEPENDENT CANCER CELLS VIA ATP-BINDING CASSETTE TRANSPORTER A1 BY A CHOLESTEROL-INDEPENDENT MECHANISM

You have accessJournal of UrologyProstate Cancer: Basic Research I1 Apr 2010103 HIGH DENSITY LIPOPROTEIN INDUCES PROLIFERATION AND MIGRATION OF HUMAN PROSTATE ANDROGEN INDEPENDENT CANCER CELLS VIA ATP-BINDING CASSETTE TRANSPORTER A1 BY A CHOLESTEROL-INDEPENDENT MECHANISM Yoshitaka Sekine, Yosuke Furuya, Hidekazu Koike, Kazuhiro Suzuki, and Alan Remaley Yoshitaka SekineYoshitaka Sekine Bethesda, MD More articles by this author , Yosuke FuruyaYosuke Furuya Maebashi, Japan More articles by this author , Hidekazu KoikeHidekazu Koike Maebashi, Japan More articles by this author , Kazuhiro SuzukiKazuhiro Suzuki Maebashi, Japan More articles by this author , and Alan RemaleyAlan Remaley Bethesda, MD More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.152AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgen deprivation therapy in men with prostate cancer (PC) leads to a significant increase of HDL. The increase is generally viewed as beneficial, particularly for cardiovascular disease, but the effects of HDL on prostate cancer are unknown. ATP-binding Cassette Transporter A1 (ABCA1), ATP-binding Cassette Transporter G1 and scavenger receptor class B member 1 are receptors for HDL and have important roles in HDL-induced signal transductions. In this study, we investigated the effect of HDL on prostate cancer cell proliferation, migration, cholesterol levels, and signal transductions, and if they are mediated by one of the receptors for HDL. METHODS HDL was isolated from health volunteer men. mRNA expressions of PC cells and human prostate biopsy samples were evaluated by quantitative real-time PCR. Cell viability was determined by MTS assay. Cell migration was shown by would healing assay. The activation of MAPK and Akt were detected by Western blot analysis. The functional role of ABCA1 in any of the observed phenomena was examined after transfection with a siRNA against ABCA1. RESULTS HDL could induce PC-3 and DU145 cell proliferations, migrations and both MAPK and Akt signal transductions, but not in LNCaP cells. Treatment with HDL did not significantly alter cellular cholesterol levels in PC cells. In cells grown in 10% FCS, the expression of ABCA1 was much lower in LNCaP than PC-3 and DU145. After culturing in androgen-free condition, ABCA1 expression increased 10 times in LNCaP cells. In human prostate biopsy samples, ABCA1 expression was significantly higher in androgen deprivation therapy group than BPH and pre-treatment prostate cancer group. Knockdown of ABCA1 by siRNA inhibited HDL-induced cell proliferation, migration and both MAPK and Akt signal transductions in PC-3. Moreover, after treatment of androgen free condition for 96 hour, HDL could induce MAPK activation, and knockdown of ABCA1 expression by siRNA inhibited HDL-induced MAPK activation in LNCaP cells. Simvastatin, which inhibited ABCA1 expression in PC-3 and DU145 cells, inhibited HDL-induced PC-3 and DU145 cell proliferation, migration and both MAPK and Akt signal transduction. CONCLUSIONS HDL can induce androgen independent prostate cancer cell proliferation and migration via ABCA1, which can be reversed by simvastatin. These results suggest that therapies that modulate HDL and lipid metabolism can potentially affect prostate cancer growth. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e42-e43 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yoshitaka Sekine Bethesda, MD More articles by this author Yosuke Furuya Maebashi, Japan More articles by this author Hidekazu Koike Maebashi, Japan More articles by this author Kazuhiro Suzuki Maebashi, Japan More articles by this author Alan Remaley Bethesda, MD More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

677 IDENTIFICATION OF EP4 AS A POTENTIAL TARGET FOR THE TREATMENT OF CASTRATION-RESISTANT PROSTATE CANCER USING A NOVEL XENOGRAFT MODEL

More effective therapeutic approaches for castration-resistant prostate cancer (CRPC) are urgently needed, thus reinforcing the need to understand how prostate tumors progress to castration resistance. We have established a novel mouse xenograft model of prostate cancer, KUCaP-2, which expresses the wild-type an- drogen receptor (AR) and which produces the prostate-specific antigen (PSA). In this model, tumors regress soon after castration, but then reproducibly restore their ability to proliferate after 1 to 2 months without AR mutation, mimicking the clinical behavior of CRPC. In the present study, we used this model to identify novel therapeutic targets for CRPC. Evaluating tumor tissues at various stages by gene expression profiling, we discovered that the prostaglandin E receptor EP4 subtype (EP4) was significantly upregulated during progression to castration resistance. Immunohistochemical results of human prostate cancer tissues con- firmed that EP4 expression was higher in CRPC compared with hormone-naive prostate cancer. Ectopic overexpression of EP4 in LNCaP cells (LNCaP-EP4 cells) drove proliferation and PSA production in the ab- sence of androgen supplementation in vitro and in vivo. Androgen-independent proliferation of LNCaP-EP4 cells was suppressed when AR expression was attenuated by RNA interference. Treatment of LNCaP-EP4 cells with a specific EP4 antagonist, ONO-AE3-208, decreased intracellular cyclic AMP levels, suppressed PSA production in vitro, and inhibited castration-resistant growth of LNCaP-EP4 or KUCaP-2 tumors in vivo. Our findings reveal that EP4 overexpression, via AR activation, supports an important mechanism for castration-resistant progression of prostate cancer. Furthermore, they prompt further evaluation of EP4 antagonists as a novel therapeutic modality to treat CRPC. Cancer Res; 70(4); 1606-15. ©2010 AACR.

Identification of EP4 as a Potential Target for the Treatment of Castration-Resistant Prostate Cancer Using a Novel Xenograft Model

More effective therapeutic approaches for castration-resistant prostate cancer (CRPC) are urgently needed, thus reinforcing the need to understand how prostate tumors progress to castration resistance. We have established a novel mouse xenograft model of prostate cancer, KUCaP-2, which expresses the wild-type androgen receptor (AR) and which produces the prostate-specific antigen (PSA). In this model, tumors regress soon after castration, but then reproducibly restore their ability to proliferate after 1 to 2 months without AR mutation, mimicking the clinical behavior of CRPC. In the present study, we used this model to identify novel therapeutic targets for CRPC. Evaluating tumor tissues at various stages by gene expression profiling, we discovered that the prostaglandin E receptor EP4 subtype (EP4) was significantly upregulated during progression to castration resistance. Immunohistochemical results of human prostate cancer tissues confirmed that EP4 expression was higher in CRPC compared with hormone-naïve prostate cancer. Ectopic overexpression of EP4 in LNCaP cells (LNCaP-EP4 cells) drove proliferation and PSA production in the absence of androgen supplementation in vitro and in vivo. Androgen-independent proliferation of LNCaP-EP4 cells was suppressed when AR expression was attenuated by RNA interference. Treatment of LNCaP-EP4 cells with a specific EP4 antagonist, ONO-AE3-208, decreased intracellular cyclic AMP levels, suppressed PSA production in vitro, and inhibited castration-resistant growth of LNCaP-EP4 or KUCaP-2 tumors in vivo. Our findings reveal that EP4 overexpression, via AR activation, supports an important mechanism for castration-resistant progression of prostate cancer. Furthermore, they prompt further evaluation of EP4 antagonists as a novel therapeutic modality to treat CRPC.

Wnt-11 promotes neuroendocrine-like differentiation, survival and migration of prostate cancer cells

BackgroundWnt-11 is a secreted protein that modulates cell growth, differentiation and morphogenesis during development. We previously reported that Wnt-11 expression is elevated in hormone-independent prostate cancer and that the progression of prostate cancer from androgen-dependent to androgen-independent proliferation correlates with a loss of mutual inhibition between Wnt-11- and androgen receptor-dependent signals. However, the prevalence of increased expression of Wnt-11 in patient tumours and the functions of Wnt-11 in prostate cancer cells were not known.ResultsWnt-11 protein levels in prostate tumours were determined by immunohistochemical analysis of prostate tumour tissue arrays. Wnt-11 protein was elevated in 77/117 of tumours when compared with 27 benign prostatic hypertrophy specimens and was present in 4/4 bone metastases. In addition, there was a positive correlation between Wnt-11 expression and PSA levels above 10 ng/ml. Androgen-depleted LNCaP prostate cancer cells form neurites and express genes associated with neuroendocrine-like differentiation (NED), a feature of prostate tumours that have a poor prognosis. Since androgen-depletion increases expression of Wnt-11, we examined the role of Wnt-11 in NED. Ectopic expression of Wnt-11 induced expression of NSE and ASCL1, which are markers of NED, and this was prevented by inhibitors of cyclic AMP-dependent protein kinase, consistent with the known role of this kinase in NED. In contrast, Wnt-11 did not induce NSE expression in RWPE-1 cells, which are derived from benign prostate, suggesting that the role of Wnt-11 in NED is specific to prostate cancer. In addition, silencing of Wnt-11 expression in androgen-depleted LNCaP cells prevented NED and resulted in apoptosis. Silencing of Wnt-11 gene expression in androgen-independent PC3 cells also reduced expression of NSE and increased apoptosis. Finally, silencing of Wnt-11 reduced PC3 cell migration and ectopic expression of Wnt-11 promoted LNCaP cell invasion.ConclusionsThese observations suggest that the increased level of Wnt-11 found in prostate cancer contributes to tumour progression by promoting NED, tumour cell survival and cell migration/invasion, and may provide an opportunity for novel therapy in prostate cancer.