Selective degradation of AR-V7 to overcome castration resistance of prostate cancer

Androgen receptor splice variant 7 (AR-V7), a form of ligand-independent and constitutively activating variant of androgen receptor (AR), is considered as the key driver to initiate castration-resistant prostate cancer (CRPC). Because AR-V7 lacks ligand-binding domain, the AR-targeted therapies that aim to inactivate AR signaling through disrupting the interaction between AR and androgen are limited in CRPC. Thus, the emergence of AR-V7 has become the greatest challenge for treating CRPC. Targeting protein degradation is a recently proposed novel avenue for cancer treatment. Our previous studies have been shown that the oncoprotein AR-V7 is a substrate of the proteasome. Identifying novel drugs that can trigger the degradation of AR-V7 is therefore critical to cure CRPC. Here we show that nobiletin, a polymethoxylated flavonoid derived from the peel of Citrus fruits, exerts a potent anticancer activity via inducing G0/G1 phase arrest and enhancing the sensitivity of cells to enzalutamide in AR-V7 positive PC cells. Mechanically, we unravel that nobiletin selectively induces proteasomal degradation of AR-V7 (but not AR). This effect relies on its selective inhibition of the interactions between AR-V7 and two deubiquitinases USP14 and USP22. These findings not only enrich our understanding on the mechanism of AR-V7 degradation, but also provide an efficient and druggable target for overcoming CRPC through interfering the stability of AR-V7 mediated by the interaction between AR-V7 and deubiquitinase.Subject terms: Drug development, Translational research     

PLCε knockdown overcomes drug resistance to androgen receptor antagonist in castration-resistant prostate cancer by suppressing the wnt3a/β-catenin pathway

Most prostate cancers (Pcas) develop into castration-resistant prostate cancer (CRPC) after receiving androgen deprivation therapy (ADT). The expression levels of PLCε and wnt3a are increased in Pca and regulate androgen receptor (AR) activity. However, the biological function and mechanisms of PLCε and wnt3a in CRPC remain unknown. In this study, we found that the expression levels of PLCε, wnt3a, and AR were significantly increased in CRPC tissues as well as bicalutamide-resistant-LNCaP and enzalutamide-resistant-LNCaP cells. In addition, PLCε knockdown partly restored the sensitivity of drug-resistant cells to bicalutamide and enzalutamide by inhibiting the activity of the wnt3a/β-catenin/AR signaling axis. Interestingly, the resistance of LNCaP cells docetaxel is related to PLCε but not the wnt3a/β-catenin pathway. We also found that the combination of PLCε knockdown and enzalutamide treatment synergistically suppressed cell proliferation, tumor growth, and bone metastasis using in vitro and in vivo experiments. Our study revealed that PLCε is involved in the progression of drug-resistance in CRPC and could be a new target for the treatment of CRPC.     

Androgen Suppresses the Proliferation of Androgen Receptor-Positive Castration-Resistant Prostate Cancer Cells via Inhibition of Cdk2, CyclinA,and Skp2

The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3^AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3^AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27^Kip1; and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3^AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3^AR cells partially blocked accumulation of p27^Kip1 and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.     

Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas

Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor–derived xenografts (PDX) revealed that AR–negative SCPC (AR^?SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR–positive castration-resistant adenocarcinomas (AR⁺ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR^? and AR⁺ PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR^?SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR^?SCPC cell lines. We conclude that the epigenome of AR^? is distinct from that of AR⁺ castration-resistant prostate carcinomas, and that the AR^? phenotype can be reversed with epigenetic drugs.     

The androgen receptor antagonist enzalutamide induces apoptosis,dysregulates the heat shock protein system,and diminishes the androgen receptor and estrogen receptor β1 expression in prostate cancer cells

Enzalutamide's accepted mode of action is by targeting the androgen receptor's (AR) activity. In clinical practice, enzalutamide demonstrates a good benefit-risk profile for the treatment of advanced prostate cancer (PC), even after poor response to standard antihormonal treatment. However, since both, well-established antiandrogens and enzalutamide, target AR functionality, we hypothesized that additional unknown mechanisms might be responsible for enzalutamide's superior anticancer activity. In the current study, PC cells were incubated with enzalutamide and enzalutamide-dependent modulation of apoptotic mechanisms were assessed via Western blot analysis, TDT-mediated dUTP-biotin nick end-labeling assay, and nuclear morphology assay. Alterations of heat shock protein (HSP), AR, and estrogen receptor (ER) expression were examined by Western blot analysis. Enzalutamide attenuated the proliferation of PC cells in a time- and dose-dependent manner. In the presence of enzalutamide, apoptosis occurred which was shown by increased BAX expression, decreased Bcl-2 expression, nuclear pyknosis, and genomic DNA fragmentation. Moreover, enzalutamide inhibited the expression of HSPs primarily involved in steroid receptor stabilization and suppressed AR and ERβ1 expression. This study demonstrates for the first time that enzalutamide treatment of PC cells triggers varying molecular mechanisms resulting in antiproliferative effects of the drug. In addition to the well-characterized antagonistic inhibition of AR functionality, we have shown that enzalutamide also affects the intracellular synthesis of steroid receptor-associated HSPs, thereby diminishing the expression of AR and ERβ1 proteins and inducing apoptotic pathways. According to an indirect attenuation of HSP-associated factors such as steroid receptors, endometrial carcinoma, uterine leiomyosarcoma, and mamma carcinoma cells also demonstrated inhibited cell growth in the presence of enzalutamide. Our data, therefore, suggest that enzalutamide's high efficacy is at least partially independent of AR and p53 protein expression, which are frequently lost in advanced PC.     

Construction of enzalutamide-resistant cell model of prostate cancer and preliminary screening of potential drug-resistant genes

Among many factors of causing castration-resistant prostate cancer (CRPC) progression, a growing number of evidences have shown androgen receptors play a critical role. Therefore, blocking androgen receptor remains a therapeutic goal of CRPC. However, resistance to androgen receptor inhibitors, for example, enzalutamide, limits therapeutic efficacy for many patients. In this study, to develop an enzalutamide-resistant cell model for molecular mechanism investigation of enzalutamide-resistance, we continuously treated C4-2B cells with multiplied concentrations of enzalutamide. The IC50 of resistant cells was identified as 14.7705 µM, and the resistance index was calculated as 12.4. In addition, we verified the resistance of resistant cells through experiments in vivo and found the genes in androgen receptor signaling pathway (androgen receptor, Jagged1, Notch1) and those in androgen receptor alternative signaling pathways behaved the opposite. For some of the former, their mRNA and protein expression reduced markedly while for the latter, for example, CXCR7, AKT, STAT3, FOXP3, they rose dramatically in the expression level of protein and mRNA. More importantly, the tumor volume, tumor wet weight, PSA and VEGF secretion level, positive staining rate of Ki67 nuclei in resistant strain heterogeneous tumor treated with enzalutamide were significantly higher than those of maternal cell heterogeneous tumor treated with enzalutamide, whereas no obvious difference was detected between resistant strain heterogeneous tumor treated with enzalutamide and those of the resistant strain treated with reference drug. Finally, we identified 654 differentially expression genes and 2 compounds (atracurium besilate, methotrexates) associated with the amelioration of enzalutamide-resistance. Overall, we successfully established an enzalutamide-resistance cell model and screened out some resistance genes and candidate small molecule drugs.     

Differential gene-expression patterns in genital fibroblasts of normal males and 46,XY females with androgen insensitivity syndrome: evidence for early programming involving the androgen receptor

Background  

Androgen insensitivity syndrome (AIS) comprises a range of phenotypes from male infertility to complete feminization. Most individuals with AIS carry germline mutations of the androgen receptor (AR) that interfere with or ablate its function. As genital fibroblasts retain expression of the AR in vitro, we used genital skin fibroblasts from normal males and 46,XY females with complete AIS due to known AR mutations to gain insights into the role of the AR in human genital differentiation.     

Effects of orlistat combined with enzalutamide and castration through inhibition of fatty acid synthase in a PC3 tumor-bearing mouse model

Androgen deprivation therapy (ADT) is one of the typical treatments used for patients with prostate cancer (PCa). ADT, however, may fail when PCa develops castration-resistance. Fatty acid synthase (FASN), a critical enzyme involved in fatty acid synthesis, is found to be up-regulated in PCa. Since enzalutamide and ADT are frequently used for the treatment of PCa, the present study aimed to unravel the underlying mechanism of combination of orlistat, an FASN inhibitor, and enzalutamide using PC3 cell line; and orlistat and castration in PC3 tumor-bearing animal model. Cytotoxicity was determined by AlamarBlue assay. Drug effects on the cell cycle and protein expressions were assayed by the flow cytometry and Western blot. Electromobility shift assay was used to evaluate the NF-κB activity. The tumor growth delay, expressions of the signaling-related proteins, and histopathology post treatments of orlistat and castration were evaluated in PC3 tumor-bearing mouse model. The results showed that orlistat arrested the PC3 cells at the G₁ phase of the cell cycle and enhanced the cytotoxic effects of enzalutamide synergistically. Pretreatment with orlistat combined with castration inhibited the tumor growth significantly compared with those of castration and orlistat treatments alone in PC3 tumor-bearing mice. Combination treatment reduced both FASN and NF-κB activities and their downstream effector proteins. The present study demonstrated the synergistic effects of orlistat combined with enzalutamide in vitro and castration in vivo on human PCa.     

Blocking GRP/GRP-R signaling decreases expression of androgen receptor splice variants and inhibits tumor growth in castration-resistant prostate cancer

Clinical management of castration-resistant prostate cancer (CRPC) resulting from androgen deprivation therapy (ADT) remains challenging. Many studies indicate that androgen receptor splice variants (ARVs) play a critical role in the development of CRPC, including resistance to the new generation of inhibitors of androgen receptor (AR) action. ARVs are constitutively active and lack the ligand-binding domain (LBD), thereby allowing prostate cancer (PC) to maintain AR activity despite therapies that target the AR (full-length AR; AR-FL). Previously, we have reported that long-term ADT increases the neuroendocrine (NE) hormone – Gastrin Releasing Peptide (GRP) and its receptor (GRP-R) expression in PC cells. Further, we demonstrated that activation of GRP/GRP-R signaling increases ARVs expression by activating NF-κB signaling, thereby promoting cancer progression to CRPC. Most importantly, as a cell surface protein, GRP-R is easily targeted by drugs to block GRP/GRP-R signaling. In this study, we tested if blocking GRP/GRP-R signaling by targeting GRP-R using GRP-R antagonist is sufficient to control CRPC progression. Our studies show that blocking GRP/GRP-R signaling by targeting GRP-R using RC-3095, a selective GRP-R antagonist, efficiently inhibits NF-κB activity and ARVs (AR-V7) expression in CRPC and therapy-induced NEPC (tNEPC) cells. In addition, blocking of GRP/GRP-R signaling by targeting GRP-R can sensitize CRPC cells to anti-androgen treatment (such as MDV3100). Further, preclinical animal studies indicate combination of GRP-R antagonist (targeting ARVs) with anti-androgen (targeting AR-FL) is sufficient to inhibit CRPC and tNEPC tumor growth.     

Ack1-mediated androgen receptor phosphorylation modulates radiation resistance in castration-resistant prostate cancer

Androgen deprivation therapy has been the standard of care in prostate cancer due to its effectiveness in initial stages. However, the disease recurs, and this recurrent cancer is referred to as castration-resistant prostate cancer (CRPC). Radiotherapy is the treatment of choice; however, in addition to androgen independence, CRPC is often resistant to radiotherapy, making radioresistant CRPC an incurable disease. The molecular mechanisms by which CRPC cells acquire radioresistance are unclear. Androgen receptor (AR)-tyrosine 267 phosphorylation by Ack1 tyrosine kinase (also known as TNK2) has emerged as an important mechanism of CRPC growth. Here, we demonstrate that pTyr(267)-AR is recruited to the ATM (ataxia telangiectasia mutated) enhancer in an Ack1-dependent manner to up-regulate ATM expression. Mice engineered to express activated Ack1 exhibited a significant increase in pTyr(267)-AR and ATM levels. Furthermore, primary human CRPCs with up-regulated activated Ack1 and pTyr(267)-AR also exhibited significant increase in ATM expression. The Ack1 inhibitor AIM-100 not only inhibited Ack1 activity but also was able to suppress AR Tyr(267) phosphorylation and its recruitment to the ATM enhancer. Notably, AIM-100 suppressed Ack1 mediated ATM expression and mitigated the growth of radioresistant CRPC tumors. Thus, our study uncovers a previously unknown mechanism of radioresistance in CRPC, which can be therapeutically reversed by a new synergistic approach that includes radiotherapy along with the suppression of Ack1/AR/ATM signaling by the Ack1 inhibitor, AIM-100.