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AR-mTOR-SRF Axis Regulates HMMR Expression in Human Prostate Cancer Cells.

ABSTRACT: The elevated expression of the hyaluronan-mediated motility receptor (HMMR) is known to be highly associated with tumor progression in prostate cancer, but the molecular mechanisms underlying the regulation of HMMR expression remain unclear. Here, we report that mammalian target of rapamycin (mTOR) is a key regulator of HMMR expression, for which its kinase activity is required. Pharmacological inhibitors of mTOR, such as rapamycin and Torin2, markedly suppressed the mRNA level as well as the protein level of HMMR in LNCaP and PC-3 cells. Our data demonstrate that such regulation occurs at the transcription level. HMMR promoter reporter assays revealed that the transcription factor SRF is responsible for the mTOR-mediated transcriptional regulation of HMMR gene. Consistently, the suppression of HMMR expression by Torin2 was noticeably reversed by the overexpression of SRF. Moreover, our findings suggest that the SRF binding sites responsible for the transcriptional regulation of HMMR through the mTOR-SRF axis are located in HMMR promoter sequences carrying the first intron, downstream of the translational start site. Furthermore, the upregulation of HMMR by DHT was abolished by stimulation with rapamycin, prior to DHT treatment, suggesting that mTOR activity is required for the induction of HMMR expression by androgen. Collectively, our study provides new mechanistic insights into the role of mTOR/SRF/AR signaling in HMMR regulation in prostate cancer cells.

SUBMITTER: Sun Y

PROVIDER: S-EPMC8551732 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Tumor suppressive miR-99b-5p as an epigenomic regulator mediating mTOR/AR/SMARCD1 signaling axis in aggressive prostate cancer.

Project description:IntroductionAfrican American (AA) men exhibited 2.3-fold higher PCa incidence and 1.7-fold higher PCa mortality rates when compared to the European American (EA) men. Besides the socioeconomic factors, emerging evidence has highlighted that biological risk factors may play critical roles in the AA PCa disparities. Previously, we have shown that downregulated miR-99b-5p and upregulated mTOR cooperatively promotes the AA PCa aggressiveness and drug resistance.MethodsIn this study, we aimed to explore the miR-99b-5p/mTOR/AR/SMARCD1 signaling axis in AA PCa aggressiveness. The analyses used in the study included immunofluorescence, western blot, in-vitro functional assays (TUNEL, colony forming, and MTT), and chromatin immunoprecipitation (ChIP)-qPCR assays in 2D and/or 3D culture model of EA PCa and AA PCa cell lines.ResultsSpecifically, the immunofluorescence staining, and western blot analysis has revealed that nuclear mTOR, AR, and SMARCD1 were highly expressed in AA PCa (MDA PCa 2b) compared to EA PCa (LNCaP) cell line. Western blot analysis further revealed that miR-99b-5p inhibited protein levels of mTOR, AR/AR-V7 and SMARCD1 in cytoplasm and nuclei of EA and AA PCa. The in-vitro functional (MTT, TUNEL, and clonogenic) assays have demonstrated that miR-99b-5p effectively inhibited cell proliferation/survival and induced cell apoptosis in EA and AA PCa cells. Moreover, combination of miR-99b-5p and enzalutamide (Enz) synergistically enhances the cytotoxicity against aggressive AA PCa and castration-resistant prostate cancer (CRPC). mTOR ChIP-qPCR assays further demonstrated that miR-99b-5p or miR-99b-5p/Enz significantly reduces the recruitment of mTOR to the genes involved in the metabolic reprogramming in CRPC.DiscussionTaken together, miR-99b-5p may function as an epigenomic driver to modulate the mTOR/AR/SMARCD1 signaling axis in AA PCa and resistant CRPC.

| S-EPMC10661933 | biostudies-literature

AFF4 regulates osteogenic potential of human periodontal ligament stem cells via mTOR-ULK1-autophagy axis.

Project description:Scaffold protein AF4/FMR2 family member 4 (AFF4) has been found to play a role in osteogenic commitment of stem cells. However, function of AFF4 in human periodontal ligament stem cells (hPDLSCs) has not been studied yet. This present study aims to investigate the biological effect of AFF4 on osteogenic differentiation of hPDLSCs and potential mechanistic pathway. First, AFF4 expression profile was evaluated in conditions of periodontitis and osteogenic differentiation of hPDLSCs by immunohistochemical staining, western blot and qRT-PCR. Next, si-RNA mediated knockdown and lentiviral transduction mediated overexpression of AFF4 were adopted to explore impact of AFF4 on osteogenic capacity of hPDLSCs. Then, possible mechanistic pathway was identified. At last, pharmacological agonist of autophagy, rapamycin, was utilized to affirm the role of autophagy in AFF4-regulated osteogenesis of hPDLSCs. First, AFF4 expressions were significantly lower in inflamed periodontal tissues and lipopolysaccharides-treated hPDLSCs than controls, and were up-regulated during osteogenic differentiation of hPDLSCs. Next, osteogenic potential of hPDLSCs was impaired by AFF4 knockdown and potentiated by AFF4 overexpression. Moreover, AFF4 was found to positively regulate autophagic activity in hPDLSCs. At last, rapamycin treatment was shown to be able to partly restore AFF4 knockdown-suppressed osteogenic differentiation. Our study demonstrates that AFF4 regulates osteogenic potential of hPDLSCs via targeting autophagic activity. The involvement of AFF4 in periodontal homeostasis was identified for the first time.

| S-EPMC10849782 | biostudies-literature

The HIF/PHF8/AR axis promotes prostate cancer progression.

Project description:Recent studies provide strong evidence that the androgen receptor (AR) signaling pathway remains active in castration-resistant prostate cancer (CRPC). However, the underlying mechanisms are not well understood. In this study, we demonstrate that plant homeo domain finger protein 8 (PHF8 )interacts with and functions as an essential histone demethylase activity-dependent AR coactivator. Furthermore, we demonstrate that the expression of PHF8 is induced by hypoxia in various prostate cancer cell lines. Knockdown of either hypoxia-inducible factor HIF2? or HIF1? almost completely abolished hypoxia-induced PHF8 expression. Importantly, we observed that PHF8 is highly expressed in clinical androgen deprived prostate cancer samples and expression of PHF8 correlates with increased levels of HIF1? and HIF2?. Moreover, elevated PHF8 is associated with higher grade prostate cancers and unfavorable outcomes. Our findings support a working model in which hypoxia in castrated prostate cancer activates HIF transcription factors which then induces PHF8 expression. The elevated PHF8 in turn promotes the AR signaling pathway and prostate cancer progression. Therefore, the HIF/PHF8/AR axis could serve as a potential biomarker for CRPC and is also a promising therapeutic target in combating CRPC.

| S-EPMC5177772 | biostudies-literature

JMJD6 Is a Druggable Oxygenase That Regulates AR-V7 Expression in Prostate Cancer.

Project description:Endocrine resistance (EnR) in advanced prostate cancer is fatal. EnR can be mediated by androgen receptor (AR) splice variants, with AR splice variant 7 (AR-V7) arguably the most clinically important variant. In this study, we determined proteins key to generating AR-V7, validated our findings using clinical samples, and studied splicing regulatory mechanisms in prostate cancer models. Triangulation studies identified JMJD6 as a key regulator of AR-V7, as evidenced by its upregulation with in vitro EnR, its downregulation alongside AR-V7 by bromodomain inhibition, and its identification as a top hit of a targeted siRNA screen of spliceosome-related genes. JMJD6 protein levels increased (P < 0.001) with castration resistance and were associated with higher AR-V7 levels and shorter survival (P = 0.048). JMJD6 knockdown reduced prostate cancer cell growth, AR-V7 levels, and recruitment of U2AF65 to AR pre-mRNA. Mutagenesis studies suggested that JMJD6 activity is key to the generation of AR-V7, with the catalytic machinery residing within a druggable pocket. Taken together, these data highlight the relationship between JMJD6 and AR-V7 in advanced prostate cancer and support further evaluation of JMJD6 as a therapeutic target in this disease. SIGNIFICANCE: This study identifies JMJD6 as being critical for the generation of AR-V7 in prostate cancer, where it may serve as a tractable target for therapeutic intervention.

| S-EPMC8025710 | biostudies-literature

ELF5-Mediated AR Activation Regulates Prostate Cancer Progression.

Project description:The transcription factor E74-like factor 5 (ELF5) is a potent antioncogene that can prevent epithelial-mesenchymal transition (EMT) and metastasis in prostate cancer (PCa). However, little is known how it suppress the tumor growth and if it can interact with androgen receptor (AR). In this study, we find that the ELF5 is frequently expressed in AR activated PCa cells, where it binds to AR acting as a physiological partner and negatively regulates its transcriptional activity. In addition, the interaction between ELF5 and AR is androgen-dependent. Downregulation of ELF5 by shRNA increases the expression of AR-response genes and the progression of PCa. Moreover, ELF5 is a AR-dependent gene that its expression can be induced by androgen and suppressed by antiandrogen treatment. Notably, forced reduction of ELF5 in LNCaP cells facilitates the binding of AR to ARE in ELF5 gene and enabling its transcription, so that low level ELF5 can turn up its own expression by the negative feedback loop.

| S-EPMC5347131 | biostudies-literature

A COP1-GATA2 axis suppresses AR signaling and prostate cancer.

Project description:Androgen receptor (AR) signaling is crucial for driving prostate cancer (PCa), the most diagnosed and the second leading cause of death in male patients with cancer in the United States. Androgen deprivation therapy is initially effective in most instances of AR-positive advanced or metastatic PCa. However, patients inevitably develop lethal castration-resistant PCa (CRPC), which is also resistant to the next-generation AR signaling inhibitors. Most CRPCs maintain AR expression, and blocking AR signaling remains a main therapeutic approach. GATA2 is a pioneer transcription factor emerging as a key therapeutic target for PCa because it promotes AR expression and activation. While directly inhibiting GATA2 transcriptional activity remains challenging, enhancing GATA2 degradation is a plausible therapeutic strategy. How GATA2 protein stability is regulated in PCa remains unknown. Here, we show that constitutive photomorphogenesis protein 1 (COP1), an E3 ubiquitin ligase, drives GATA2 ubiquitination at K419/K424 for degradation. GATA2 lacks a conserved [D/E](x)xxVP[D/E] degron but uses alternate BR1/BR2 motifs to bind COP1. By promoting GATA2 degradation, COP1 inhibits AR expression and activation and represses PCa cell and xenograft growth and castration resistance. Accordingly, GATA2 overexpression or COP1 mutations that disrupt COP1-GATA2 binding block COP1 tumor-suppressing activities. We conclude that GATA2 is a major COP1 substrate in PCa and that COP1 promotion of GATA2 degradation is a direct mechanism for regulating AR expression and activation, PCa growth, and castration resistance.

| S-EPMC9618149 | biostudies-literature

Aurora A regulates expression of AR-V7 in models of castrate resistant prostate cancer.

Project description:Androgen receptor variants (AR-Vs) provide a mechanism of therapy evasion in castrate-resistant prostate cancer (CRPC), yet mechanisms of regulation remain largely unknown. Here we investigate the role of Aurora A kinase on AR-Vs in models of CRPC and show depletion of Aurora A reduces AR-V target gene expression. Importantly, knockdown of Aurora A reconfigures splicing of AR pre-mRNA to discriminately down-regulate synthesis of AR-V transcripts, including AR-V7, without effecting full-length AR mRNA; and as a consequence, AR-V-driven proliferation and survival of CRPC cells is markedly reduced. Critically, these effects are reproduced by Aurora A inhibition. We show that Aurora A levels increase in advanced disease and AURKA is an AR-V target gene demonstrating a positive feedback mechanism of androgenic signalling in CRPC. In all, our data suggests that Aurora A plays a pivotal role in regulation of AR-V7 expression and represents a new therapeutic target in CRPC.

| S-EPMC5311967 | biostudies-literature

The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells.

Project description:CD4(+)Foxp3(+) regulatory T (T reg) cells play an essential role in maintaining immunological tolerance via their suppressive function on conventional CD4(+) T (Tconv) cells. Repertoire studies suggest that distinct T cell receptor signaling pathways lead to T reg differentiation, but the signals that regulate T reg specification are largely unknown. We identify AKT as a strong repressor of entry into the T reg phenotype in vitro and in vivo. A constitutively active allele of AKT substantially diminished TGF-beta-induced Foxp3 expression in a kinase-dependent manner and via a rapamycin-sensitive pathway, implicating the AKT-mammalian target of rapamycin axis. The observed impairment in Foxp3 induction was part of a broad dampening of the typical T reg transcriptional signature. Expression of active AKT at a stage before Foxp3 turn on during normal T reg differentiation in the thymus selectively impaired differentiation of CD4(+)Foxp3(+) cells without any alteration in the positive selection of Tconv. Activated AKT, in contrast, did not affect established Foxp3 expression in T reg cells. These results place AKT at a nexus of signaling pathways whose proper activation has a strong and broad impact on the onset of T reg specification.

| S-EPMC2275380 | biostudies-literature

Analytic Validation of RNA In Situ Hybridization (RISH) for AR and AR-V7 Expression in Human Prostate Cancer.

Project description:PurposeRNA expression of androgen receptor splice variants may be a biomarker of resistance to novel androgen deprivation therapies in castrate-resistant prostate cancer (CRPC). We analytically validated an RNA in situ hybridization (RISH) assay for total AR and AR-V7 for use in formalin-fixed paraffin-embedded (FFPE) prostate tumors.Experimental designWe used prostate cell lines and xenografts to validate chromogenic RISH to detect RNA containing AR exon 1 (AR-E1, surrogate for total AR RNA species) and cryptic exon 3 (AR-CE3, surrogate for AR-V7 expression). RISH signals were quantified in FFPE primary tumors and CRPC specimens, comparing to known AR and AR-V7 status by IHC and RT-PCR.ResultsThe quantified RISH results correlated significantly with total AR and AR-V7 levels by RT-PCR in cell lines, xenografts, and autopsy metastases. Both AR-E1 and AR-CE3 RISH signals were localized in nuclear punctae in addition to the expected cytoplasmic speckles. Compared with admixed benign glands, AR-E1 expression was significantly higher in primary tumor cells with a median fold increase of 3.0 and 1.4 in two independent cohorts (P < 0.0001 and P = 0.04, respectively). While AR-CE3 expression was detectable in primary prostatic tumors, levels were substantially higher in a subset of CRPC metastases and cell lines, and were correlated with AR-E1 expression.ConclusionsRISH for AR-E1 and AR-CE3 is an analytically valid method to examine total AR and AR-V7 RNA levels in FFPE tissues. Future clinical validation studies are required to determine whether AR RISH is a prognostic or predictive biomarker in specific clinical contexts. Clin Cancer Res; 22(18); 4651-63. ©2016 AACR.

| S-EPMC5026571 | biostudies-literature

ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells.

Project description:Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. 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. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, we examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function.

| S-EPMC4991035 | biostudies-literature

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