LncRNA PCBP1-AS1-mediated AR/AR-V7 deubiquitination enhances prostate cancer enzalutamide resistance

The refractory of castration-resistant prostate cancer (CRPC) is mainly reflected in drug resistance. The current research on the resistance mechanism of CRPC is still in its infancy. In this study, we revealed for the first time the key role of LncRNA PCBP1-AS1 in CRPC drug resistance. Through detailed in vivo and in vitro studies, we found that PCBP1-AS1 may enhance the deubiquitination of AR/AR-V7 by stabilizing the USP22-AR/AR-V7 complex, thereby preventing AR/AR-V7 from being degraded through the ubiquitin–proteasome pathway. Targeting PCBP1-AS1 can significantly restore the drug sensitivity of enzalutamide-resistant tumors in vivo and in vitro. Our research further expands the function of LncRNA in castration-resistant prostate cancer, which may provide new potential for clinical diagnosis and targeted therapy.Subject terms: Prostate cancer, Ubiquitylation, Long non-coding RNAs     

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     

Beyond T and DHT - Novel Steroid Derivatives Capable of Wild Type Androgen Receptor Activation

While androgen deprivation therapy (ADT) remains the primary treatment for metastatic prostate cancer (PCa), castration does not eliminate androgens from the prostate tumor microenvironment, and residual intratumoral androgens are implicated in nearly every mechanism by which androgen receptor (AR)-mediated signaling promotes castration-resistant disease. The uptake and intratumoral (intracrine) conversion of circulating adrenal androgens such as dehydroepiandrosterone sulfate (DHEA-S) to steroids capable of activating the wild type AR is a recognized driver of castration resistant prostate cancer (CRPC). However, less well-characterized adrenal steroids, including 11-deoxcorticosterone (DOC) and 11beta-hydroxyandrostenedione (11OH-AED) may also play a previously unrecognized role in promoting AR activation. In particular, recent data demonstrate that the 5α-reduced metabolites of DOC and 11OH-AED are activators of the wild type AR. Given the well-recognized presence of SRD5A activity in CRPC tissue, these observations suggest that in the low androgen environment of CRPC, alternative sources of 5α-reduced ligands may supplement AR activation normally mediated by the canonical 5α-reduced agonist, 5α-DHT. Herein we review the emerging data that suggests a role for these alternative steroids of adrenal origin in activating the AR, and discuss the enzymatic pathways and novel downstream metabolites mediating these effects. We conclude by discussing the potential implications of these findings for CRPC progression, particularly in context of new agents such as abiraterone and enzalutamide which target the AR-axis for prostate cancer therapy.     

Clinicopathological characteristics of androgen receptor splicing variant 7 (AR-V7) expression in patients with castration resistant prostate cancer: A systematic review and meta‐analysis

Background Studies have shown that AR-V7 may be correlated with the poor prognosis of castration resistant prostate cancer (CRPC), however, clinicopathological characteristics of AR-V7 have not been fully elucidated.ObjectiveThis study aimed at evaluating the clinicopathological features of AR-V7 in CRPC patients.Materials and methodsTo evaluate the clinicopathological features of AR-V7 in CRPC patients. A search of PubMed, Embase, and Web of Science was performed using the keywords prostate cancer, prostate tumor, prostate neoplasm, prostate carcinoma, AR-V7, AR3, androgen receptor splicing variant-7, or androgen receptor-3. Twenty-four trials published by February 2020 were included in this study.ResultsThe proportion of Gleason score ≥ 8 was found to be significantly higher in AR-V7-positive CRPC (69.5%) than negative (54.9%) (OR 1.68, 95% CI 1.25–2.25, p < 0.001), while the rates of T3/T4 stage (OR 1.16, 95% CI 0.60–2.24, p = 0.65) and N1 stage (OR 0.99, 95% CI 0.65–1.51, p = 0.96) were not statistically correlated with AR-V7 status. The AR-V7-positive patients exhibited a significantly higher proportion of any site metastasis (61.3% versus 35.0%; OR 2.19, 95% CI 1.57–3.05, p < 0.001) and bone metastasis (81.7% versus 69.0%; OR 1.97, 95% CI 1.44–2.69, p < 0.001), and a trend close to significance was expected in visceral metastasis (28.8% versus 22.1%; OR 1.29, 95% CI 0.96–1.74, p = 0.09). Incidences of pain in AR-V7-positive CRPC (54.6%) were significantly higher than in negative CRPC (28.1%; OR 4.23, 95% CI 2.52–7.10, p < 0.001), line with worse ECOG performance status (56.7% versus 35.0%, OR 2.18, 95% CI 1.51–3.16, P < 0.001). Limitations of the study include differences in sample sizes and designs, AR-V7 detection assays, as well as disease characteristics of the included studies.ConclusionsAR-V7 positivity is associated with a higher Gleason score, bone or any site metastasis, pain and worse ECOG performance scores in CRPC. However, it is not correlated with tumor stage or lymph node metastasis. More studies are needed to confirm these findings.     

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.     

Similarities and Distinctions in Actions of Surface-Directed and Classic Androgen Receptor Antagonists

The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells. These effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide. Microarray analysis and confirmatory qRT-PCR reveals that MJC13 and flutamide inhibit dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells. Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists (MDV3100, ARN-509) at selected genes. MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects castrate resistant prostate cancer (CRPC) reveals that it inhibits flutamide activation of an AR mutant (ART877A) that emerges during flutamide withdrawal syndrome, but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. Importantly, MJC13 inhibits AR interactions with SRC2 and β-catenin in the nucleus and, unlike flutamide, strongly inhibits amplification of AR activity obtained with transfected SRC2 and β-catenin. MJC13 also inhibits DHT and β-catenin-enhanced cell division in LNCaP cells. Thus, a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in particular forms of CRPC.     

Genomic Deregulation of the E2F/Rb Pathway Leads to Activation of the Oncogene EZH2 in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is a highly aggressive lung neoplasm with extremely poor clinical outcomes and no approved targeted treatments. To elucidate the mechanisms responsible for driving the SCLC phenotype in hopes of revealing novel therapeutic targets, we studied copy number and methylation profiles of SCLC. We found disruption of the E2F/Rb pathway was a prominent feature deregulated in 96% of the SCLC samples investigated and was strongly associated with increased expression of EZH2, an oncogene and core member of the polycomb repressive complex 2 (PRC2). Through its catalytic role in the PRC2 complex, EZH2 normally functions to epigenetically silence genes during development, however, it aberrantly silences genes in human cancers. We provide evidence to support that EZH2 is functionally active in SCLC tumours, exerts pro-tumourigenic functions in vitro, and is associated with aberrant methylation profiles of PRC2 target genes indicative of a “stem-cell like” hypermethylator profile in SCLC tumours. Furthermore, lentiviral-mediated knockdown of EZH2 demonstrated a significant reduction in the growth of SCLC cell lines, suggesting EZH2 has a key role in driving SCLC biology. In conclusion, our data confirm the role of EZH2 as a critical oncogene in SCLC, and lend support to the prioritization of EZH2 as a potential therapeutic target in clinical disease.     

Androgen Receptor Splice Variant AR3 Promotes Prostate Cancer via Modulating Expression of Autocrine/Paracrine Factors

Deregulation of androgen receptor (AR) splice variants has been implicated to play a role in prostate cancer development and progression. To understand their functions in prostate, we established a transgenic mouse model (AR3Tg) with targeted expression of the constitutively active and androgen-independent AR splice variant AR3 (a.k.a. AR-V7) in prostate epithelium. We found that overexpression of AR3 modulates expression of a number of tumor-promoting autocrine/paracrine growth factors (including Tgfβ2 and Igf1) and expands prostatic progenitor cell population, leading to development of prostatic intraepithelial neoplasia. In addition, we showed that some epithelial-mesenchymal transition-associated genes are up-regulated in AR3Tg prostates, suggesting that AR3 may antagonize AR activity and halt the differentiation process driven by AR and androgen. This notion is supported by our observations that the number of Ck5⁺/Ck8⁺ intermediate cells is increased in AR3Tg prostates after castration, and expression of AR3 transgene in these intermediate cells compromises prostate epithelium regeneration upon androgen replacement. Our results demonstrate that AR3 is a driver of prostate cancer, at least in part, through modulating multiple tumor-promoting autocrine/paracrine factors.     

PRPF6 promotes androgen receptor/androgen receptor-variant 7 actions in castration-resistant prostate cancer cells

Androgen receptor (AR) and its variants play vital roles in development and progression of prostate cancer. To clarify the mechanisms involved in the enhancement of their actions would be crucial for understanding the process in prostate cancer and castration-resistant prostate cancer transformation. Here, we provided the evidence to show that pre-mRNA processing factor 6 (PRPF6) acts as a key regulator for action of both AR full length (AR-FL) and AR variant 7 (AR-V7), thereby participating in the enhancement of AR-FL and AR-V7-induced transactivation in prostate cancer. In addition, PRPF6 is recruited to cis-regulatory elements in AR target genes and associates with JMJD1A to enhance AR-induced transactivation. PRPF6 also promotes expression of AR-FL and AR-V7. Moreover, PRPF6 depletion reduces tumor growth in prostate cancer-derived cell lines and results in significant suppression of xenograft tumors even under castration condition in mouse model. Furthermore, PRPF6 is obviously highly expressed in human prostate cancer samples. Collectively, our results suggest PRPF6 is involved in enhancement of oncogenic AR signaling, which support a previously unknown role of PRPF6 during progression of prostate cancer and castration-resistant prostate cancers.     

Divergent Androgen Receptor and Beta-Catenin Signaling in Prostate Cancer Cells

Despite decades of effort to develop effective therapy and to identify promising new drugs, prostate cancer is lethal once it progresses to castration-resistant disease. Studies show mis-regulation of multiple pathways in castration-resistant prostate cancer (CRPC), reflecting the heterogeneity of the tumors and also hinting that targeting androgen receptor (AR) pathway alone might not be sufficient to treat CRPC. In this study, we present evidence that the Wnt/β-catenin pathway might be activated in prostate cancer cells after androgen-deprivation to promote androgen-independent growth, partly through enhanced interaction of β-catenin with TCF4. Androgen-independent prostate cancer cells were more prone to activate a Wnt-reporter, and inhibition of the Wnt/β-catenin pathway increased sensitivity of these cells to the second-generation antiandrogen, enzalutamide. Combined treatment of enzalutamide and Wnt/β-catenin inhibitor showed increased growth repression in both androgen-dependent and -independent prostate cancer cells, suggesting therapeutic potential for this approach.     

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.     

EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer

Recent experiments have demonstrated that the Polycomb group (PcG) gene EZH2 is highly expressed in metastatic prostate cancer and in lymphomas. EZH2 is a component of the PRC2 histone methyltransferase complex, which also contains EED and SUZ12 and is required for the silencing of HOX gene expression during embryonic development. Here we demonstrate that both EZH2 and EED are essential for the proliferation of both transformed and non-transformed human cells. In addition, the pRB-E2F pathway tightly regulates their expression and, consistent with this, we find that EZH2 is highly expressed in a large set of human tumors. These results raise the question whether EZH2 is a marker of proliferation or if it is actually contributing to tumor formation. Significantly, we propose that EZH2 is a bona fide oncogene, since we find that ectopic expression of EZH2 is capable of providing a proliferative advantage to primary cells and, in addition, its gene locus is specifically amplified in several primary tumors.