SummaryRegulation of prostate epithelial progenitor cells is important in prostate development and prostate diseases. Our previous study demonstrated a function of autocrine cholinergic signaling (ACS) in promoting prostate cancer growth and castration resistance. However, whether or not such ACS also plays a role in prostate development is unknown. Here, we report that ACS promoted the proliferation and inhibited the differentiation of prostate epithelial progenitor cells in organotypic cultures. These results were confirmed by ex vivo lineage tracing assays and in vivo renal capsule recombination assays. Moreover, we found that M3 cholinergic receptor (CHRM3) was upregulated in a large subset of benign prostatic hyperplasia (BPH) tissues compared with normal tissues. Activation of CHRM3 also promoted the proliferation of BPH cells. Together, our findings identify a role of ACS in maintaining prostate epithelial progenitor cells in the proliferating state, and blockade of ACS may have clinical implications for the management of BPH.

SIRT3 is involved in aging-related diseases including cancer, but its role in prostate cancer and detailed regulatory function are not known. We found that SIRT3 was moderately down-regulated in prostate carcinomas. Overexpression of SIRT3 by lentiviral transfection inhibited prostate cancer growth both in vitro and in vivo, whereas knockdown of SIRT3 increased prostate tumor growth. Mechanistically, the tumor suppression effect of SIRT3 was achieved via its inhibition of the PI3K/Akt pathway. Notably, upregulation of SIRT3 suppressed the phosphorylation of Akt, leading to the ubiquitination and degradation of oncoprotein c-MYC; this could be attenuated by constitutive activation of PI3K/Akt signaling. Collectively, our results unveiled SIRT3's tumor suppressive function and the underlying mechanism in prostate cancer, which might provide therapeutic implications for the disease.

Purpose: Although a previous study reported nerve endingderived acetylcholine promoted prostate cancer invasion and metastasis by regulating the microenvironment of cancer cells, the present study aims to determine whether there is autocrine cholinergic signaling in prostate epithelial cells that promotes prostate cancer growth and castration resistance.Experimental design: In this study, IHC was performed to detect protein expression in mouse prostate tissue sections and human prostate cancer tissue sections. Subcutaneously and orthotopically xenografted tumor models were established to evaluate the functions of autocrine cholinergic signaling in regulating prostate cancer growth and castration resistance. Western blotting analysis was performed to assess the autocrine cholinergic signaling-induced signaling pathway.Results: We found the expression of choline acetyltransferase (ChAT), the secretion of acetylcholine and the expression of CHRM3 in prostate epithelial cells, supporting the presence of autocrine cholinergic signaling in the prostate epithelium. In addition, we found that CHRM3 was upregulated in clinical prostate cancer tissues compared with adjacent noncancer tissues. Overexpression of CHRM3 or activation of CHRM3 by carbachol promoted cell proliferation, migration, and castration resistance. On the contrary, blockading CHRM3 by shRNA or treatment with darifenacin inhibited prostate cancer growth and castration resistance both in vitro and in vivo. Furthermore, we found that autocrine cholinergic signaling caused calmodulin/calmodulin-dependent protein kinase kinase (CaM/CaMKK)-mediated phosphorylation of Akt.Conclusions: These findings suggest that blockade of CHRM3 may represent a novel adjuvant therapy for castration-resistant prostate cancer.

Purpose: All uveal melanoma and a fraction of other melanoma subtypes are driven by activation of the G-protein alpha-q (Gαq) pathway. Targeting these melanomas has proven difficult despite advances in the molecular understanding of key driver signaling pathways in the disease pathogenesis. Inhibitors of Gαq have shown promising preclinical results, but their therapeutic activity in distinct Gαq mutational contexts and in vivo have remained elusive. Experimental Design: We used an isogenic melanocytic cellular system to systematically examine hotspot mutations in GNAQ (e.g., G48V, R183Q, Q209L) and CYSLTR2 (L129Q) found in human uveal melanoma. This cellular system and human uveal melanoma cell lines were used in vitro and in in vivo xenograft studies to assess the efficacy of Gαq inhibition as a single agent and in combination with MEK inhibition. Results: We demonstrate that the Gαq inhibitor YM-254890 inhibited downstream signaling and in vitro growth in all mutants. In vivo, YM-254890 slowed tumor growth but did not cause regression in human uveal melanoma xenografts. Through comprehensive transcriptome analysis, we observed that YM-254890 caused inhibition of the MAPK signaling with evidence of rebound by 24 hours and combination treatment of YM-254890 and a MEK inhibitor led to sustained MAPK inhibition. We further demonstrated that the combination caused synergistic growth inhibition in vitro and tumor shrinkage in vivo. Conclusions: These data suggest that the combination of Gαq and MEK inhibition provides a promising therapeutic strategy and improved therapeutic window of broadly targeting Gαq in uveal melanoma. See related commentary by Neelature Sriramareddy and Smalley, p. 1217

Prostate cancer (PCa) is the most frequently diagnosed cancer for men in the developed world. Androgen receptor signaling pathway plays an important role in prostate cancer progression. Recent studies show that microRNA miR-124 exerts a tumor suppressive function in prostate cancer. However, the relationship between AR and miR-124 is unclear. In the present study, we found a negative feedback loop between AR and miR-124 expression. On one hand, miR-124 was a positively regulated target gene of the AR, on the other hand, overexpression of miR-124 inhibited the expression of AR. In addition, we found that miR-124-2 and miR-124-3 promoters were hypermethylated in AR-negative PCa cells. Furthermore, overexpression of miR-124 inhibited proliferation rates and invasiveness capacity of PCa cells in vitro, and suppressed xenograft tumor growth in vivo. Taken together, our results support a negative feedback loop between AR and miR-124 expression. Methylation of miR-124-2 and miR-124-3 may serve as a biomarker for AR-negative PCa cells, and overexpression of miR-124 might be of potential therapeutic value for the treatment of PCa.

Prostate cancer is the most common type of cancer for men in the developed world. Androgen receptor (AR) is very important in prostate cancer progression. TMPRSS2 is an AR signaling downstream gene and closely related to prostate carcinogenesis. DNA methylation is a key mechanism to influence gene expression. Though previous reports have shown that AR signaling plays a critical role in the regulation of TMPRSS2 in prostate cancer, hardly any studies have examined whether the DNA methylation has been involved in the regulation of TMPRSS2. In the present study, we demonstrated that AR-negative prostate cancer (PCa) cells showed low expression levels and hypermethylation of TMPRSS2. In contrast, AR-positive PCa cells displayed high levels and hypomethylation of TMPRSS2. Treatment with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine reversed the low expression levels of TMPRSS2 in the AR-negative PCa cells. Additionally, we found that the level of DNA methyltransferases 1 (DNMT1) was high in AR-negative PCa cells, in which hypermethylation of TMPRSS2 and low expression level of TMPRSS2 were observed. Collectively, these data suggest that the high level of DNMT1 might be the mechanism for the hypermethylation-mediated transcriptional repression of TMPRSS2 in AR-negative PCa cells.

SummaryAlthough STAT3 signaling is demonstrated to regulate sensory cell differentiation and regeneration in the zebrafish, its exact role is still unclear in mammalian cochleae. Here, we report that STAT3 and its activated form are specifically expressed in hair cells during mouse cochlear development. Importantly, conditional cochlear deletion of Stat3 leads to an inhibition on hair cell differentiation in mice in vivo and in vitro. By cell fate analysis, inactivation of STAT3 signaling shifts the cell division modes from asymmetric to symmetric divisions from supporting cells. Moreover, inhibition of Notch signaling stimulates STAT3 phosphorylation, and inactivation of STAT3 signaling attenuates production of supernumerary hair cells induced by a Notch pathway inhibitor. Our findings highlight an important role of the STAT3 signaling during mouse cochlear hair cell differentiation and may have clinical implications for the recovery of hair cell loss-induced hearing impairment.

Polycomb Repressive Complex 2 (PRC2) has oncogenic and tumor suppressor roles in cancer. There is clinical success of targeting this complex in PRC2-dependent cancers, but an unmet therapeutic need exists in PRC2-loss cancer. PRC2 inactivating mutations are a hallmark feature of high-grade malignant peripheral nerve sheath tumor (MPNST), an aggressive sarcoma with poor prognosis and no effective targeted therapy. Through RNAi screening in MPNST, we found that PRC2-inactivation increases sensitivity to genetic or small molecule inhibition of DNA methyltransferase 1 (DNMT1), which results in enhanced cytotoxicity and antitumor response. Mechanistically, PRC2 inactivation amplifies DNMT inhibitor (DNMTi) mediated expression of retrotransposons, subsequent viral mimicry response, and robust cell death in part through PKR-dependent double stranded-RNA (dsRNA) sensor. Collectively, our observations posit DNA methylation as a safeguard against anti-tumorigenic cell fate decisions in PRC2-loss cancer to promote cancer pathogenesis, which can be therapeutically exploited by DNMT1 targeted therapy.