Genes In Prostate Cancer Cells Research Articles

Amplification and protein expression of androgen receptor gene in prostate cancer cells: Fluorescence in situ hybridization analysis.

Prostate cancer (PCa) is the most frequently diagnosed non-skin cancer and the second highest cause of cancer-related mortality in adult males worldwide. PCa is highly dependent upon androgen receptor (AR) signaling for cell proliferation and survival. The AR therefore plays a vital role in the development and function of normal and malignant prostate cells or PCa recurrence. The present study aimed to examine the ubiquity of AR amplification in PCa recurrence, even in the absence of androgen. For this purpose, specimens were collected from 37 patients. The amplification of AR and the number of X chromosomes were determined by two-colored fluorescence in situ hybridization analysis. The automated image analysis was used to determine the protein expression of AR. Clinical characteristics and survival in patients whose tumors showed or did not show AR amplification and in X-chromosome polysomy with PCa recurrence has also been compared. The results showed that >35% of patients (13 specimens) exhibited AR amplification. It was also observed that AR was immunostained more intensely in the tumors with amplified AR compared with those tumors with non-amplified AR. This study demonstrated an influential role of AR in tumor growth and progression even after the deprivation of androgen, as well as showing the potential contribution of AR amplification to AR activation even in the relative absence of androgen.

MP46-17 TARGETING MULTIPLE FUNCTIONS OF E3 UBIQUITIN LIGASE-1 (WWP1) SIGNALING BY MIR-452 INHIBITS CANCER CELL MIGRATION AND INVASION IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2015MP46-17 TARGETING MULTIPLE FUNCTIONS OF E3 UBIQUITIN LIGASE-1 (WWP1) SIGNALING BY MIR-452 INHIBITS CANCER CELL MIGRATION AND INVASION IN PROSTATE CANCER Yusuke Goto, Akira Kurozumi, Rika Nishikawa, Mayuko Kato, Shinichi Sakamoto, Satoko Kojima, Yukio Naya, Hideki Enokida, Masayuki Nakagawa, Tomohiko Ichikawa, and Naohiko Seki Yusuke GotoYusuke Goto More articles by this author , Akira KurozumiAkira Kurozumi More articles by this author , Rika NishikawaRika Nishikawa More articles by this author , Mayuko KatoMayuko Kato More articles by this author , Shinichi SakamotoShinichi Sakamoto More articles by this author , Satoko KojimaSatoko Kojima More articles by this author , Yukio NayaYukio Naya More articles by this author , Hideki EnokidaHideki Enokida More articles by this author , Masayuki NakagawaMasayuki Nakagawa More articles by this author , Tomohiko IchikawaTomohiko Ichikawa More articles by this author , and Naohiko SekiNaohiko Seki More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1580AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES MicroRNAs (miRNAs), a class of small noncoding RNAs, regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. A growing body of evidence suggests that miRNAs contribute to prostate cancer (PCa) progression, development, and metastasis. Our recent study revealed that miR-224 functions as a tumor suppressor, especially contributes to cancer cell metastasis directly targeting miR-224-TPD52 signaling (FEBS Letters, 588:1973-82, 2014). In human genome, miR-452 is located nearby tumor-suppressive miR-224, forming clustered miRNAs on chromosome Xq28 region. The aim of the study was to investigate the functional significance of miR-452 and to identify novel miR-452-mediated cancer pathways and responsible genes in PCa cells. METHODS Expression levels of miR-452 and its candidate target genes were evaluated in PCa cell lines (PC3 and DU145) and PCa clinical specimens (29 prostate tissues and 54 PCa tissues) by PCR methods. Gain-of-function studies using mature miR-452 was performed to investigate cell proliferation, migration and invasion in PCa cell lines (PC3 and DU145). To identify the molecular pathways potentially regulated by miR-452, we applied gene expression data and in silico analysis using TargetScan database [http://www.targetscan.org/] and publicly available gene expression data set in GEO. Loss-of-function assays were performed to investigate the functional significance of miR-452 target genes. RESULTS The expression level of miR-452 was significantly lower in PCa tissues and cell lines compared to normal tissues (P < 0.0001). Low expression of miR-452 was associated with shorter progression-free interval (P = 0.0414). Restoration of miR-452 in PCa cell lines revealed significant inhibition of cancer cell migration and invasion. In silico analysis and oligomicroarray analysis showed that WW domain-containing E3 ubiquitin ligase-1 (WWP1) was candidate target of miR-452 regulation. Silencing of WWP1 significantly inhibited cancer cell proliferation, migration and invasion. Up-regulation of WWP1 was detected in PCa specimens. CONCLUSIONS Down-regulation of miR-452 activates WWP1 signaling in PCa cells and these events contribute to PCa oncogenesis and metastasis. WWP1 signaling plays a variety of cellular biological processes and important roles in cancer cells. Therefore, recognition of tumor suppressive miRNA-mediated PCa pathways provides new therapeutic strategies for the disease. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e550 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yusuke Goto More articles by this author Akira Kurozumi More articles by this author Rika Nishikawa More articles by this author Mayuko Kato More articles by this author Shinichi Sakamoto More articles by this author Satoko Kojima More articles by this author Yukio Naya More articles by this author Hideki Enokida More articles by this author Masayuki Nakagawa More articles by this author Tomohiko Ichikawa More articles by this author Naohiko Seki More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Upregulation of CRMP4, a new prostate cancer metastasis suppressor gene, inhibits tumor growth in a nude mouse intratibial injection model

Prostate cancer, the most commonly diagnosed male cancer in North America, has a high incidence of bone metastasis. Our previous study showed collapsin response mediator protein 4 (CRMP4) gene inhibited prostate cancer migration and invasion. In this study, we investigated whether overexpression of CRMP4 gene in prostate cancer cells inhibit tumor bone metastasis. The stable prostate cancer cells overexpressing the CRMP4 gene were constructed using lentivirus infection. Prostate cancer bone metastasis nude mouse model was built though orthotopic prostate implantation, intracardiac injection and intratibial injection with CRMP4 overexpress and control cancer cells. Small animal PET/CT scanning results showed no difference of bone metastatic capacity in orthotopic and intracardiac injection models between CRMP4 overexpression and control group, while CRMP4 overexpression inhibited tumor growth in the intratibial injection model. Moreover, our invitro study showed CRMP4 overexpression downregulates the Neuropilin1 (NRP1) expression and upregulate the Noggin expression. Immunohistochemical staining of the hind limbs of intratibial injection model was confirmed with cytological experiments. Taken together, our research indicated CRMP4 inhibits prostate cancer cells growth in the nude mouse bone microenvironment and this effect may relate with regulation of NRP1 and Noggin expression.

The pluripotency factor Nanog is directly upregulated by the androgen receptor in prostate cancer cells.

The Androgen Receptor (AR) is a nuclear hormone receptor that functions as a critical oncogene in all stages of prostate cancer progression, including progression to castration-resistance following androgen-deprivation therapy. Thus, identifying and targeting critical AR-regulated genes is one potential method to block castration-resistant cancer proliferation. Of particular importance are transcription factors that regulate stem cell pluripotency; many of these genes are emerging as critical oncogenes in numerous tumor cell types. Of these, Nanog has been previously shown to increase the self-renewal and stem-like properties of prostate cancer cells. Thus, we hypothesized that Nanog is a candidate AR target gene that may impart castration-resistance. We modulated AR signaling in LNCaP prostate cancer cells and assayed for Nanog expression. Direct AR binding to the NANOG promoter was tested using AR Chromatin Immunoprecipation (ChIP) and analyses of publically available AR ChIP-sequencing data-sets. Nanog over-expressing cells were analyzed for cell growth and cytotoxicity in response to the AR antagonist enzalutamide and the microtubule stabilizing agent docetaxel. AR signaling upregulates Nanog mRNA and protein. AR binds directly to the NANOG promoter, and was not identified within 75 kb of the NANOGP8 pseudogene, suggesting the NANOG gene locus was preferentially activated. Nanog overexpression in LNCaP cells increases overall growth, but does not increase resistance to enzalutamide or docetaxel. Nanog is a novel oncogenic AR target gene in prostate cancer cells, and stable expression of Nanog increases proliferation and growth of prostate cancer cells, but not resistance to enzalutamide or docetaxel.

Dietary lutein modulates growth and survival genes in prostate cancer cells.

Lutein is a carotenoid pigment present in fruits and vegetables that has anti-inflammatory and antitumor properties. In this study, we examined the effect of lutein on proliferation and survival-associated genes in prostate cancer (PC-3) cells. We found that in vitro culture of PC-3 cells with lutein induced mild decrease in proliferation that improved in combination treatment with peroxisome proliferator-activated receptor gamma (PPARγ) agonists and other chemotherapeutic agents. Flow cytometry analyses showed that lutein improved drug-induced cell cycle arrest and apoptosis in prostate cancer. Gene array and quantitative reverse transcription-polymerase chain reaction analyses showed that lutein altered the expression of growth and apoptosis-associated biomarker genes in PC-3 cells. These findings highlight that lutein modulates the expression of growth and survival-associated genes in prostate cancer cells.

Lycopene induce apoptosis in human prostate cells and alters the expression of Bax and Bcl-2 genes

Prostate cancer is the most common malignancy in men and the second leading cause of cancer-related mortality in men of the Western world. Lycopene has received attention due to its potential to prevent cancer. Alterations of the balance between proliferation and apoptosis are associated with cancer. In the present study, we evaluated the influence of lycopene on cell viability and apoptosis of human prostate cancer (PCa) cells and benign prostate hyperplasia (BPH) cells. We have studied prostate cells treated with lycopene for 96 h. Using MTT assay, we observed a decrease of viable PCa cells after treatment with lycopene. Flow cytometer analysis revealed that lycopene promoted up to two-fold increase of apoptotic cells in PCa cells when compared to the control group. Using real time PCR assay, we found that lycopene promoted an up-regulation of Bax gene and a downregulation of Bcl-2 gene in PCa cells. Less expressive effects were observed in BPH cells. Taken together, the present study supports the proposal that lycopene may have a protective effect on prostate cancer.

Genistein downregulates onco-miR-1260b and upregulates sFRP1 and Smad4 via demethylation and histone modification in prostate cancer cells.

Background:Recently several microRNAs (miRNAs) have been found to be regulated by genistein in cancer cells. In this study, we focused on the gene regulatory effect of genistein on microRNA and its target genes in prostate cancer (PC).Methods:Initially, we investigated the effect of genistein on prostate cancer cells and identified that the expression of miRNA-1260b was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA-1260b expression and prostate cancer patient outcomes. Two target genes (sFRP1 and Smad4) of miR-1260b were identified based on computer algorithm and 3′UTR luciferase assay was carried out to determine direct miRNA regulation of the genes.Results:Genistein promoted apoptosis while inhibiting prostate cancer cell proliferation, invasion and TCF reporter activity in PC cells. MiR-1260b was highly expressed in prostate cancer tissues and significantly downregulated by genistein in PC cells. After knocking down miR-1260b, cell proliferation, invasion, migration and TCF reporter activity were decreased in PC cells. Western analysis and 3′UTR luciferase assay showed that the two target genes (sFRP1 and Smad4) were directly regulated by miR-1260b. The expression of sFRP1 and Smad4 was significantly decreased in prostate cancer tissues. Genistein also increased expression of these two genes via DNA demethylation and histone modifications.Conclusions:Our data suggest that genistein exerts its anti-tumour effect via downregulation of miR-1260b that targeted sRRP1 and Smad4 genes in prostate cancer cells. The expression of sFRP1 and Smad4 was also modulated by genistein via DNA methylation or histone modifications in PC cell lines.

Triptolide Inhibits Histone Methyltransferase EZH2 and Modulates the Expression of Its Target Genes in Prostate Cancer Cells

The histone methyltransferase EZH2 (enhancer of zeste homolog 2) plays critical roles in prostate cancer (PCa) development and is a potential target for PCa treatment. Triptolide possesses anti-tumor activity, but it is unknown whether its therapeutic effect relates with EZH2 in PCa. Here we described EZH2 as a target for Triptolide in PCa cells. Our data showed that Triptolide suppressed PCa cell growth and reduced the expression of EZH2. Overexpression of EZH2 attenuated the Triptolide induced cell growth inhibition. Moreover, Triptolide treatment of PC-3 cells resulted in elevated mRNA levels of target genes (ADRB2, CDH1, CDKN2A and DAB2IP) negatively regulated by EZH2 as well as reduced mRNA levelsan of EZH2 positively regulated gene (cyclin D1). Our findings suggest the PCa cell growth inhibition mediated by Triptolide might be associated with downregulation of EZH2 expression and the subsequent modulation of target genes.

Characterisation of the androgen regulation of glycine N-methyltransferase in prostate cancer cells

The development and growth of prostate cancer is dependent on androgens; thus, the identification of androgen-regulated genes in prostate cancer cells is vital for defining the mechanisms of prostate cancer development and progression and developing new markers and targets for prostate cancer treatment. Glycine N-methyltransferase (GNMT) is a S-adenosylmethionine-dependent methyltransferase that has been recently identified as a novel androgen-regulated gene in prostate cancer cells. Although the importance of this protein in prostate cancer progression has been extensively addressed, little is known about the mechanism of its androgen regulation. Here, we show that GNMT expression is stimulated by androgen in androgen receptor (AR) expressing cells and that the stimulation occurs at the mRNA and protein levels. We have identified an androgen response element within the first exon of the GNMT gene and demonstrated that AR binds to this element in vitro and in vivo. Together, these studies identify GNMT as a direct transcriptional target of the AR. As this is an evolutionarily conserved regulatory element, this highlights androgen regulation as an important feature of GNMT regulation.

Abstract 1206: Evaluation of androgen receptor function index (ARFI) in prostate cancer.

Abstract Introduction: Initially prostate cancer is driven by the male hormone androgen through the androgen receptor (AR). However, in some cases androgen receptor is functionally altered at late stages of tumorigenesis. Early knowledge of androgen receptor dysfunctions may assist in patient stratification for emerging therapeutic strategies. Although AR function can be altered by numerous mechanisms, the net effect of these changes is reflected in defective transcription factor functions of the AR. To develop readouts for AR function in prostate cancer we have evaluated the expression of a functionally relevant panel of AR-regulated genes in a prostate cancer cell culture model and in whole-mounted prostate tumor tissues. Methods: In the VCaP cell culture model of prostate cancer that endogenously express, AR, TMPRSS2-ERG fusion (ERG), KLK3 (PSA), NKX3.1, PMEPA1, ODC1 and AMD1 genes we have evaluated the dose and time kinetics of these AR regulated genes in response to androgen (R1881) treatment at protein and mRNA levels. Radical prostatectomy specimens representing favorable or poor prognostic cases were selected for the study. Each prostate was fixed, sectioned and immunohistochemical (IHC) staining on adjacent four-micron sections of the whole-mounted blocks were performed by using mouse monoclonal anti-AR, anti-ERG antibody (clone 9FY), rabbit polyclonal anti-PSA and anti-NKX3.1 antibodies. IHC intensities were scored and the final score was determined after multiplying the intensity score and percentage of positively stained area in the respective lesions. Results: Towards defining the expression levels of AR regulated genes in prostate cancer cells we have evaluated and confirmed the androgen dose and time kinetic response of endogenously expressed KLK3(PSA), PMEPA1, NKX3.1, ODC1, AMD1 and TMPRSS2-ERG (ERG) genes in VCaP cell culture model. Assessment of AR dysfunction by the immunohistochemical evaluation of AR regulated genes in whole-mounted prostate cancer specimens showed reduced expression of AR regulated genes in a sub-set of prostate cancer cases with notable inter- and intratumoral heterogeneity. Conclusions: Androgen regulation of examined genes was confirmed in VCaP prostate cancer cell culture model. Assessment of AR, ERG, NKX3.1 and PSA protein levels by IHC indicated reduced expression of AR-regulated genes in a subset of the cases. The analysis revealed inter- and intratumoral heterogeneity, as well as, challenges in the quantitative assessment of IHC. The observed readouts of AR dysfunction may provide a new tool for improved prognostic accuracy and patient stratification at early stages of prostate cancer treatment. Citation Format: Albert Dobi, Denise Young, Wei Huang, Lakshmi Ravindranth, Shashwat Sharad, Hua Li, Gyorgy Petrovics, David G. McLeod, Isabell Sesterhenn, Shiv Srivastava. Evaluation of androgen receptor function index (ARFI) in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1206. doi:10.1158/1538-7445.AM2013-1206

Abstract 1324: PMEPA1 enhances the prostate cancer cell growth inhibitory effect of MDV3100 and bicalutamide.

Abstract Introduction and Objective: PMEPA1 is a highly androgen-inducible gene in prostate cancer cells, recruits the NEDD4-1, an E3 ubiquitin ligase, to degrade AR. We hypothesize that the frequently (65%) observed decrease or loss of PMPEA1 expression in human prostate tumors may lead to enhanced AR functions and accelerated prostate tumorigenesis. Therefore, we investigated the growth and survival of prostate cancer cells in the presence of AR inhibitors in response to PMEPA1 knockdown. Methods: PMEPA1 was silenced by PMEPA1 Mission shRNA lentivirus infection and puromycin selection in LNCaP and VCaP cells respectively. The PMEPA1 deficient LNCaP and VCaP cells were treated with the AR inhibitors (MDV3100 and bicalutamide), as well as the artificial synthesized androgen R1881. Ectopic NEDD4-1 was expressed by pCMV-XL4-NEDD4-1 eukaryotic expression vector. Cell growth was monitored by cell counting assay, BrdU incorporation assay and colony plating efficiency assay. Cell cycle was analyzed with propidium iodide staining based flowcytometry assay. Protein levels of AR, PMEPA1, PSA and NEDD4-1 were assessed by immunoblot assays. Results: PMEPA1 silenced LNCaP and VCaP cells had higher AR, PSA protein levels, suggesting activated AR signaling. Consistent with this observation, inhibition of PMEPA1-1 by shRNA resulted in increased cell growth, stronger BrdU incorporation ability and higher colony plating efficiency in both LNCaP and VCaP cells. Cell cycle assay showed more S-phase cells in PMEPA1 deficient LNCaP and VCaP cells in regular medium or R1881 supplemented charcoal-striped medium. Ectopic expression of NEDD4-1 could not inhibit cell growth in PMEPA1 silenced LNCaP cells. More interestingly, the PMEPA1 deficient CaP cells (both LNCaP and VCaP cells) demonstrated more resistance to the growth inhibitory effects of MDV3100 and bicalutamide. Conclusions: The frequently observed decreased expression of PMEPA1 results in elevated AR protein levels and activated AR signaling, which in turn promotes cell growth in androgen responsive prostate cancer cells. The PMEPA1 deficient cells develop more resistance to AR inhibitors. Our findings further suggest the important role of decreased PMEPA1 in AR related prostate tumorigenesis. These data highlight that PMEPA1 deficiency may confer resistance to AR inhibitors during CaP progression. This study is supported by the NIH grant (R01CA106653) to SS. Citation Format: Hua E. Li, Daniel Heidenberg, Albert Dobi, Shiv Srivastava. PMEPA1 enhances the prostate cancer cell growth inhibitory effect of MDV3100 and bicalutamide. [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 1324. doi:10.1158/1538-7445.AM2013-1324

Abstract 5340: MicroRNA-720 promotes cell proliferation and invasion in prostate cancer cells.

Abstract MicroRNAs (miRNAs) are highly conserved, single stranded, non-coding RNAs of approximately 21 - 24 nucleotides that regulate gene expression by posttranscriptional silencing of specific target RNAs, by repressing translation or cleaving RNA transcripts by binding to the 3’ untranslated region (3’UTR) of the target messenger RNAs. miRNAs regulate diverse cellular processes such as cell-cycle progression, proliferation, apoptosis and development. miRNAs also have been shown to function as oncogenes or tumor suppressor genes. MicroRNA profiling has shown that miR-720 was up-regulated in cancer tissues, however the function of miR-720 in cancer has not been studied. Here we studied the functional effects of miR-720 on prostate cancer cells. We found that miR-720 was up-regulated in human prostate cancer tissues compared to adjacent normal tissues. We also found that miR-720 was up-regulated in prostate PC-3 cancer cell line compared with normal prostate cell line, RWPE1 cells. Transfection of miR-720 into prostate cancer cells, PC-3 and DU145 cells, decreased apoptosis, promoted cell proliferation and cell invasion. Transfection of anti-miR-720 into PC-3 cells increased apoptosis, inhibited cell proliferation and cell invasion. These results indicated that miR-720 has oncogenic functions in prostate cancer cells. In-silico analysis suggests that miR-720 targets genes of DR1, a down-regulator of global transdcription, and SWI5, a component of a complex required for double-strand break repair, which have tumor suppressive functions. Luciferase assays indicated that miR-720 binds to the 3’ UTR of these genes in prostate cancer cells, suggesting that these genes are targets of miR-720. Our results suggest that miR-720 promotes malignancy by targeting DR1 and SWI5 in prostate cancer cells. Funding: National Institutes of Health through Grant Number RO1CA138642, RO1CA130860 and RO1CA160079 and VA Merit Review and VA Program Project. Citation Format: Soichiro Yamamura, Takeshi Chiyomaru, Shinichiro Fukuhara, Sharanjot Saini, Shahana Majid, Guoren Deng, Vary Shahryary, Sumit Arora, Inik Chang, Yuichiro Tanaka, Peter Carroll, Rajvir Dahiya. MicroRNA-720 promotes cell proliferation and invasion in prostate cancer cells. [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 5340. doi:10.1158/1538-7445.AM2013-5340

Genistein Up-Regulates Tumor Suppressor MicroRNA-574-3p in Prostate Cancer

Genistein has been shown to inhibit cancers both in vitro and in vivo, by altering the expression of several microRNAs (miRNAs). In this study, we focused on tumor suppressor miRNAs regulated by genistein and investigated their function in prostate cancer (PCa) and target pathways. Using miRNA microarray analysis and real-time RT-PCR we observed that miR-574-3p was significantly up-regulated in PCa cells treated with genistein compared with vehicle control. The expression of miR-574-3p was significantly lower in PCa cell lines and clinical PCa tissues compared with normal prostate cells (RWPE-1) and adjacent normal tissues. Low expression level of miR-574-3p was correlated with advanced tumor stage and higher Gleason score in PCa specimens. Re-expression of miR-574-3p in PCa cells significantly inhibited cell proliferation, migration and invasion in vitro and in vivo. miR-574-3p restoration induced apoptosis through reducing Bcl-xL and activating caspase-9 and caspase-3. Using GeneCodis software analysis, several pathways affected by miR-574-3p were identified, such as ‘Pathways in cancer’, ‘Jak-STAT signaling pathway’, and ‘Wnt signaling pathway’. Luciferase reporter assays demonstrated that miR-574-3p directly binds to the 3′ UTR of several target genes (such as RAC1, EGFR and EP300) that are components of ‘Pathways in cancer’. Quantitative real-time PCR and Western analysis showed that the mRNA and protein expression levels of the three target genes in PCa cells were markedly down-regulated with miR-574-3p. Loss-of-function studies demonstrated that the three target genes significantly affect cell proliferation, migration and invasion in PCa cell lines. Our results show that genistein up-regulates tumor suppressor miR-574-3p expression targeting several cell signaling pathways. These findings enhance understanding of how genistein regulates with miRNA in PCa.

The Stress Response Mediator ATF3 Represses Androgen Signaling by Binding the Androgen Receptor

Activating transcription factor 3 (ATF3) is a common mediator of cellular stress response signaling and is often aberrantly expressed in prostate cancer. We report here that ATF3 can directly bind the androgen receptor (AR) and consequently repress AR-mediated gene expression. The ATF3-AR interaction requires the leucine zipper domain of ATF3 that independently binds the DNA-binding and ligand-binding domains of AR, and the interaction prevents AR from binding to cis-acting elements required for expression of androgen-dependent genes while inhibiting the AR N- and C-terminal interaction. The functional consequences of the loss of ATF3 expression include increased transcription of androgen-dependent genes in prostate cancer cells that correlates with increased ability to grow in low-androgen-containing medium and increased proliferative activity of the prostate epithelium in ATF3 knockout mice that is associated with prostatic hyperplasia. Our results thus demonstrate that ATF3 is a novel repressor of androgen signaling that can inhibit AR functions, allowing prostate cells to restore homeostasis and maintain integrity in the face of a broad spectrum of intrinsic and environmental insults.

Slug, a Unique Androgen-Regulated Transcription Factor, Coordinates Androgen Receptor to Facilitate Castration Resistance in Prostate Cancer

Prostate cancer (PCa) becomes lethal when cancer cells develop into castration-resistant PCa (CRPC). Androgen receptor (AR) gene mutation, altered AR regulation, or overexpression of AR often found in CRPC is believed to become one of the key factors to the lethal phenotype. Here we identify Slug, a member of the Snail family of zinc-finger transcription factors associated with cancer metastasis, as a unique androgen-responsive gene in PCa cells. In addition, the presence of constitutively active AR can induce Slug expression in a ligand-independent manner. Slug overexpression will increase AR protein expression and form a complex with AR. In addition, Slug appears to be a novel coactivator to enhance AR transcriptional activities and AR-mediated cell growth with or without androgen. In vivo, elevated Slug expression provides a growth advantage for PCa cells in androgen-deprived conditions. Most importantly, these observations were validated by several data sets from tissue microarrays. Overall, there is a reciprocal regulation between Slug and AR not only in transcriptional regulation but also in protein bioactivity, and Slug-AR complex plays an important role in accelerating the androgen-independent outgrowth of CRPC.

Activation of silenced tumor suppressor genes in prostate cancer cells by a novel energy restriction‐mimetic agent

Targeting tumor metabolism by energy restriction-mimetic agents (ERMAs) has emerged as a strategy for cancer therapy/prevention. Evidence suggests a mechanistic link between ERMA-mediated antitumor effects and epigenetic gene regulation. Microarray analysis showed that a novel thiazolidinedione-derived ERMA, CG-12, and glucose deprivation could suppress DNA methyltransferase (DNMT)1 expression and reactivate DNA methylation-silenced tumor suppressor genes in LNCaP prostate cancer cells. Thus, we investigated the effects of a potent CG-12 derivative, CG-5, vis-à-vis 2-deoxyglucose, glucose deprivation and/or 5-aza-deoxycytidine, on DNMT isoform expression (Western blotting, RT-PCR), DNMT1 transcriptional activation (luciferase reporter assay), and expression of genes frequently hypermethylated in prostate cancer (quantitative real-time PCR). Promoter methylation was assessed by pyrosequencing analysis. SiRNA-mediated knockdown and ectopic expression of DNMT1 were used to validate DNMT1 as a target of CG-5. CG-5 and glucose deprivation upregulated the expression of DNA methylation-silenced tumor suppressor genes, including GADD45a, GADD45b, IGFBP3, LAMB3, BASP1, GPX3, and GSTP1, but also downregulated methylated tumor/invasion-promoting genes, including CD44, S100A4, and TACSTD2. In contrast, 5-aza-deoxycytidine induced global reactivation of these genes. CG-5 mediated these epigenetic effects by transcriptional repression of DNMT1, which was associated with reduced expression of Sp1 and E2F1. SiRNA-mediated knockdown and ectopic expression of DNMT1 corroborated DNMT1's role in the modulation of gene expression by CG-5. Pyrosequencing revealed differential effects of CG-5 versus 5-aza-deoxycytidine on promoter methylation in these genes. These findings reveal a previously uncharacterized epigenetic effect of ERMAs on DNA methylation-silenced tumor suppressor genes, which may foster novel strategies for prostate cancer therapy.

Abstract 992: Credentialing UHRF1 as a target for prostate cancer therapy

Abstract Alterations in DNA methylation are a hallmark of prostate carcinogenesis. UHRF1 is an essential component in the machinery required for the maintenance of DNA methylation in actively dividing cells. In S-phase, UHRF1 binds nascent hemimethylated DNA and recruits DNA methyltransferases to copy the methylation pattern to the daughter DNA strand in a manner analogous to semi-conservative replication. Additionally, it has been shown that UHRF1 interacts with methylated histones and histone methyltransferases, making it an important epigenetic regulator in proliferating cells. Because of its ties to epigenetic gene silencing and cell proliferation, we hypothesized that UHRF1 could serve as a target for prostate cancer treatment. In this study, we have taken steps to credential UHRF1 as a therapeutic target in prostate cancer and have developed a high-throughput UHRF1-DNA binding assay to screen for UHRF1 inhibitors. We show that UHRF1 is overexpressed in a panel of prostate cancer cell lines and it appears to track with proliferation in prostate cancer tissue. Through loss-of-function studies in prostate cancer cell lines PC3 and DU145, we show that long-term UHRF1 depletion leads to a decrease in clonogenic survival and an overall reduction in global levels of DNA methylation. Further, we find that UHRF1 is required to maintain DNA methylation and epigenetic silencing of hypermethylated genes in prostate cancer cells. Additionally, ChIP experiments revealed that UHRF1 can be bound at the promoter CpG islands of epigenetically silenced genes in prostate cancer cells. With the goal of ultimately developing UHRF1 inhibitors, we have developed a time-resolved fluorescence resonance energy transfer (TR-FRET)-based assay, compatible with high-throughput screening, that can detect the binding of the SRA domain of UHRF1 to hemimethylated DNA. In summary, we have shown that UHRF1 plays a critical role in maintaining the DNA methylation of epigenetically silenced genes in prostate cancer and have developed an assay for high-throughput screening of UHRF1 inhibitors. 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 992. doi:1538-7445.AM2012-992

Abstract 2989: Identification of a potential tumor suppressor function for SNX9 by mining public genomic data

Abstract Members of the sorting nexin (SNX) family of proteins are involved in intracellular trafficking and endocytosis processes, with functions essential to regulation of cell surface receptor levels and subsequent signaling by receptor-mediated endocytosis. Detrimental mutations and altered expression of SNX genes have been found in several cancers and tumor cell lines. A recent publication (Mao et al. AJCR 2011) identified a chromosomal translocation that created a SNX9:UNC5C fusion gene in prostate cancer cells. UNC5C is a known tumor suppressor gene. Although SNX9 is known to regulate degradation of EGFR, a well-established prognostic marker for lung cancer, no tumorigenic role has been identified for SNX9. We investigated associations of SNX9 to cancer phenotypes by mining curated public genomic data available in NextBio. Integration of diverse datatypes such as gene/miRNA expression, methylation, and copy-number variation in NextBio allowed us to uncover several lines of evidence which indicated that SNX9 activity is altered in many types of cancer. i) Loss of the SNX9 gene was observed in many different cancers including multiple myeloma, leukemia, lymphoma, melanoma, glioblastoma, renal clear cell carcinoma, and breast cancer. ii) Hypermethylation of SNX9, indicative of overall gene silencing, occurred in colorectal cancer, breast cancer and glioblastoma. iii) The miRNA predicted to target SNX9, miR-493-5p, was up-regulated in prostate, testicular, liver, and lung cancers. iv) SNX9 gene expression was down regulated relative to normal tissues in multiple myeloma, leukemia, lymphoma, cancers of the skin, breast, prostate and more. Correlations within the data suggest an association between the loss of SNX9 and more aggressive or recurrent disease; SNX9 is reduced in higher grade, metastatic clear cell carcinoma relative to lower grade, non-metastatic clear cell carcinoma. In myeloma and leukemia patients, lower SNX9 expression in primary tumors correlated with earlier relapsing disease. SNX9 expression was also lower when comparing recurring with primary acute myeloid leukemia. Querying our PharmacoAtlas application revealed that SNX9 gene expression was altered by a number of anti-neoplastic therapies. For example, paclitaxel, bortezomib, and letrozole treatments were each found to increase SNX9 gene expression in breast cancer cells whereas doxorubicin treatment resulted in decreased SNX9 expression. The integrative analysis of curated public genomic data drawn from thousands of studies strongly supports a hypothesis that SNX9 is a putative tumor suppressor and biomarker for more aggressive forms of cancer. As genomic technologies are applied to patient evaluation and therapeutic guidance, integrating multiple lines of evidence from research and clinical data to identify biomarkers such as SNX9 will increasingly inform clinical decision making. 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 2989. doi:1538-7445.AM2012-2989

Abstract 4205: Identification of glycine N-methyltransferase-regulated genes in prostate cancer cells

Abstract Glycine N-methyltransferase (GNMT) is a S-adenosylmethionine (SAM)-dependent methyltransferase which catalyses the transfer of the methyl group on SAM to the amino acid glycine to generate N-methyl glycine (sarcosine), a compound with poorly defined biological functions. In prostate cancer GNMT may act as a tumour-susceptible gene. Its expression has been shown to be abundant in normal prostate and benign prostatic hyperplasia tissues and diminished in prostate cancer. It is therefore important to determine the significance of GNMT in the development and progression of prostate cancer and investigate the gene expression changes upon the loss of its expression in prostate cancer cells. Using gene expression microarray analysis of the androgen-responsive LNCaP cell line, we previously identified genes that were differentially expressed upon treatment with the synthetic androgen R1881 (Ngan et al 2009 Oncogene 128: 2051-2063). GNMT was the third most significant up-regulated gene, showing up-regulation by 19-fold after 24 hours of R1881 treatment. Using a panel of prostate cancer cell lines, GNMT expression was seen to be highest in androgen receptor (AR) positive cells, where expression was increased after treatment with R1881 and reduced following addition of the anti-androgen bicalutamide. Together, these findings highlight the importance of GNMT in the androgen-mediated regulation of prostate cancer. In the present study, we aimed to gain new insight into the specific function of GNMT in prostate cancer. We used siRNAs to investigate the effects of GNMT knockdown on global gene expression. The expression profiling was carried out in the LNCaP cell line using the Illumina HT-12 v4 array platform. To minimize off-target effects, LNCaP cells were treated with two different siRNAs targeting the GNMT transcript and a non-targeting siRNA was used as control. Differentially expressed genes that were statistically significant in both siRNA-mediated GNMT knockdown were validated by TaqMan Real-Time RT-PCR and considered for further analysis. In addition, to provide a detailed and comprehensive assessment of the androgen response in LNCaP cells, the LNCaP transcriptome was also analysed after AR knockdown compared to non-targeting siRNA control and R1881 treatment compared to no ligand control. To our knowledge, this is the first study that reveals potential GNMT downstream targets in prostate cancer cells. Further analysis will better characterize the molecular pathways triggered by the enzyme in the progression of the cancer. 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 4205. doi:1538-7445.AM2012-4205

Abstract A8: Androgen regulation of SRF target genes in prostate cancer cells is mediated in part by the small GTPase RhoA

Abstract Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen-responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate and correlate with aggressive disease. Moreover, the SRF-AR expression signature is associated more strongly with biochemical recurrence than similarly sized sets of AREdriven AR target genes. Thus, understanding the mechanism(s) by which SRF conveys androgen effects to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we explore the role of the RhoA signaling axis, a well-known activating pathway upstream of SRF, in androgen activation of SRF action. PCa cells were treated with inhibitors of RhoA signaling or transfected with siRNAs targeting RhoA, the critical SRF cofactor MAL or control siRNAs in the presence or absence of the synthetic androgen R1881. Real-time RT-PCR and western blotting was done to assess effects on expression of SRF target genes. Cell fractionation experiments evaluated the cellular localization of AR, MAL and SRF. ChIP was performed to assess recruitment of MAL to SRF target genes. Wild-type, dominant-active and dominant-negative versions of RhoA were overexpressed to determine effects on androgen regulation of SRF target genes. Immunohistochemistry was done to evaluate RhoA expression in clinical PCa samples and a tissue microarray derived from a CWR22 PCa xenograft model that mimics progression from androgen-stimulated (AS) to castration-recurrent (CR) disease. Effects on AS and CR PCa cell biology were assessed using proliferation and migration assays. Here, we show that the small GTPase RhoA mediates androgen responsiveness of more than half of SRF target genes tested. Interference with expression of RhoA, activity of the RhoA effector RhoA-associated kinase (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor MAL, all prevented full androgen-regulation of SRF target genes. Androgen treatment increased the nuclear content of MAL and led to MAL recruitment to the promoter of the SRF target gene FHL2. Moreover, in clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival following radical prostatectomy. Overexpression of RhoA markedly increased the androgen responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. Cell lines and a xenograft model that mimics the transition from AS to CR PCa indicate that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Silencing of RhoA, use of ROCK inhibitors or an inhibitor of SRF-MAL interaction blunted (androgen-regulated) cell viability and attenuated AS and CR PCa cell migration. Taken together, our work demonstrates that the RhoA signaling axis mediates clinically relevant AR action in PCa. In addition, these studies are the first to report on overexpression of RhoA in PCa and its association with aggressive disease and poor outcome. Note: This abstract was not presented at the conference because the presenter was unable to attend. Citation Format: Lucy Schmidt, Thomas Sebo, Donald Tindall, Hannelore Heemers, Kelly Duncan, Neelu Yadav, Kevin Regan, Christine Lohse, Elena Pop, Kristopher Attwood, Gregory Wilding, James Mohler. Androgen regulation of SRF target genes in prostate cancer cells is mediated in part by the small GTPase RhoA [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 A8.