YAP-mediated GPER signaling impedes proliferation and survival of prostate epithelium in benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) occurs when there is an imbalance between the proliferation and death of prostate cells, which is regulated tightly by estrogen signaling. However, the role of G protein-coupled estrogen receptor (GPER) in prostate cell survival remains ambiguous. In this study, we observed that prostates with epithelial hyperplasia showed increased yes-associated protein 1 (YAP) expression and decreased levels of estrogen and GPER. Blocking YAP through genetic or drug interventions led to reduced proliferation and increased apoptosis in the prostate epithelial cells. Interestingly, GPER agonists produced similar effects. GPER activation enhanced the phosphorylation and degradation of YAP, which was crucial for suppressing cell proliferation and survival. The Gαs/cAMP/PKA/LATS pathway, downstream of GPER, transmitted signals that facilitated YAP inhibition. This study investigated the interaction between GPER and YAP in the prostate epithelial cells and its contribution to BPH development. It lays the groundwork for future research on developing BPH treatments.

Potent antitumour of the mTORC1/2 dual inhibitor AZD2014 in docetaxel-sensitive and docetaxel-resistant castration-resistant prostate cancer cells.

Recent studies indicate mammalian target of rapamycin (mTOR) may play an important role in PCa progression and drug resistance. Here, we investigated the effects of a novel mTORC1/C2 dual inhibitor, AZD2014, on naive and docetaxel (Doc)-pre-treated castration-resistant PCa (CRPC) cells and explored its therapeutic potential in CRPCs. In the current study, AZD2014 has a greater inhibitory effect against 4EBP1 and AKT phosphorylation than rapamycin in CRPC cells and prevented the feedback activation of AKT signalling. Importantly, AZD2014 suppressed CRPC cell growth in vitro by suppressing proliferation, apoptosis, cell cycle arrest at G1 phase and autophagy to a greater extent than rapamycin. Moreover, AZD2014 was more efficacious than rapamycin in inhibiting migration, invasion and EMT progression in Doc-sensitive and Doc-resistant CRPC cells. Overall, AZD2014 showed significant antitumour effects. Thereby, the current study highlights a reliable theoretical basis for the clinical application of AZD2014 in both Doc-sensitive and Doc-resistant CRPCs.

M2 macrophage-mediated interleukin-4 signalling induces myofibroblast phenotype during the progression of benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is a progressive disease in elderly men, but potential factors accelerating its progression remain largely unknown. The aim of this study was to elucidate the factors affecting BPH progression by understanding the complex mechanisms causing early- progressed BPH, which progresses rapidly and requires surgical intervention before the age of 50. Three groups of human prostate tissue samples, from patients with early-progressed BPH, age-matched prostate and elderly BPH tissues, were collected (n = 25 each). We compared these tissues to determine the histologic features and molecular mechanisms underlying BPH progression. We found that early-progressed BPH samples were characterised by aberrant stromal hyper-proliferation, collagen deposition and increased M2 macrophage infiltration, compared to those from age-matched prostate and elderly BPH tissues. The M2 macrophage-fibroblast co-culture system demonstrated that the myofibroblast phenotypes were strongly induced only in fibroblasts from the early-progressed BPH samples, while the co-cultured M2 macrophages expressed high levels of pro-fibrotic cytokines, such as IL4 and TGFß1. M2 macrophage-derived IL4, but not TGFß1, selectively induced the myofibroblast phenotype through the JAK/STAT6, PI3K/AKT and MAPK/ERK signalling pathways in the early-progressed BPH prostate fibroblasts. Taken together, our results indicate that induction of the myofibroblast phenotype may lead to BPH progression through M2 macrophage-mediated IL4 signalling, and that IL4 may represent a potential therapeutic target, allowing the prevention of M2 macrophage activation and fibroblast-to-myofibroblast differentiation.

CD8+ T cells promote proliferation of benign prostatic hyperplasia epithelial cells under low androgen level via modulation of CCL5/STAT5/CCND1 signaling pathway.

Previous studies by our group have shown that low intra-prostatic dihydrotestosterone (DHT) induced BPH epithelial cells (BECs) to recruit CD8+ T cells. However, the influence of the recruited CD8+ T cells on BECs under a low androgen level is still unknown. Here, we found CD8+ T cells have the capacity to promote proliferation of BECs in low androgen condition. Mechanism dissection revealed that interaction between CD8+ T cells and BECs through secretion of CCL5 might promote the phosphorylation of STAT5 and a higher expression of CCND1 in BECs. Suppressed CCL5/STAT5 signals via CCL5 neutralizing antibody or STAT5 inhibitor Pimozide led to reverse CD8+ T cell-enhanced BECs proliferation. IHC analysis from Finasteride treated patients showed PCNA expression in BECs was highly correlated to the level of CD8+ T cell infiltration and the expression of CCL5. Consequently, our data indicated infiltrating CD8+ T cells could promote the proliferation of BECs in low androgen condition via modulation of CCL5/STAT5/CCND1 signaling. The increased secretion of CCL5 from the CD8+ T cells/BECs interaction might help BECs survive in a low DHT environment. Targeting these signals may provide a new potential therapeutic approach to better treat BPH patients who failed the therapy of 5α-reductase inhibitors.

DLEC1, a 3p tumor suppressor, represses NF-κB signaling and is methylated in prostate cancer.

UNLABELLED: Deleted in lung and esophageal cancer 1 (DLEC1), located at 3p22-p21.3, is involved in the carcinogenesis of multiple cancers, but its role in prostate cancer (PrCa) remains unclear. Here, we studied the epigenetic alteration of DLEC1 and its functions in prostate cancer. We found that DLEC1 was highly expressed in normal prostate tissues, normal prostatic epithelium cell line (RWPE-1), and benign prostatic hyperplasia cell line (BPH-1), but frequently downregulated by promoter methylation in PrCa cell lines. Pharmacologic demethylation could restore DLEC1 expression. DLEC1 was downregulated in prostate tumor tissues compared with their adjacent non-malignant tissues. DLEC1 was methylated in 76/110 primary tumors, but rarely in benign prostatic hyperplasia tissues. DLEC1 methylation was associated with higher PSA levels (p = 0.016), higher Gleason scores (p = 0.015), and more advanced tumor stages (p = 0.003). Furthermore, DLEC1 methylation was detected in 11/30 urine sediment samples from PrCa patients, but seldom in ones from BPH patients. Ectopic expression of DLEC1 inhibited the colony formation of PrCa cells, through inducing cell apoptosis. DLEC1 also suppressed PrCa cell migration. Moreover, DLEC1 inhibited NF-κB transcription activity in PrCa and HEK293 cells. Taken together, our data demonstrate that DLEC1 functions as a tumor suppressor but is frequently methylated in prostate cancer. DLEC1 methylation is associated with prostate cancer progression, which could be a non-invasive epigenetic biomarker for PrCa diagnosis. KEY MESSAGES: • Promoter methylation of DLEC1 is a potential prognostic biomarker for PrCa. • DLEC1, a functional tumor suppressor, is frequently methylated in PrCa. • DLEC1 suppresses prostate cancer growth and metastatic behavior. • DLEC1 mediates tumor-suppressive activities through NF-κB signaling.

Low intraprostatic DHT promotes the infiltration of CD8+ T cells in BPH tissues via modulation of CCL5 secretion.

Clinical studies suggested thatandrogen might be associated with infiltrating T cells in prostate of benign prostatic hyperplasia (BPH) patients, but detail of T-cell subset and mechanism still remained unclear. The present study tested the hypothesis that intraprostatic 5 α -dihydrotestosterone (DHT) exerts effects on T cells recruitment by BPH epithelial cells. Prostate tissues from 64 cases of BPH patients after transurethral resection of prostate (TURP) were divided into 2 groups: (1) no medication history; (2) administration of 5 α -reductase type II inhibitor-finasteride 5 mg daily for at least 6 months before surgery. Group 2 presented significantly higher CD8+ T cells infiltration than group 1, but no changes in CD4+ T cells (immunohistochemistry and flow cytometry). In vitro study more CD8+ T cell migrated to the prostate tissue lysates from group 2 and BPH-1 cells in low DHT condition. Transcription of chemokine (C-C motif) Ligand 5 (CCL5) mRNA in BPH-1 cells and chemokine (C-C motif) receptor 5 (CCR5) mRNA in CD8+ T cells were upregulated in low DHT condition (q-PCR). CCL5 expression was also identified to be higher in group 2 prostate tissues by IHC. This study suggested that intraprostatic DHT may participate in regulating inflammatory response which was induced by human prostatic epithelial cell, via modulating CCL5 secretion.

Evidence of TGF-ß1 mediated epithelial-mesenchymal transition in immortalized benign prostatic hyperplasia cells.

Expression of epithelial-mesenchymal transition (EMT) markers has been detected clinically in benign prostatic hyperplasia (BPH) tissues. To understand the molecular basis, we investigated the role of stromal microenvironment in the progression of EMT in BPH cells. First, we used cell culture supernatant from normal prostate stromal WPMY-1 cells to provide supernatant-conditioned medium (WSCM) to culture the BPH-1 cell line. Then, the morphological changes and migratory capacity were detected in BPH-1 cells. The expression of EMT markers was examined in BPH-1 cells by Western blot and immunofluorescent analysis. Finally, to investigate the role of transforming growth factor beta 1 (TGF-ß1) in this process, the WSCM-cultured cells were treated with monoclonal antibody against TGF-ß1 to study its effect on EMT. We found that the morphology of BPH-1 cells changed to a spindle-like shape after cultured in WSCM, and the levels of E-cadherin and cytokeratin 5/8 (CK5/8) were significantly lower than the cells cultured in ordinary medium. These BPH-1 cells were also tested positive for mesenchymal markers vimentin and a-smooth muscle actin (SMA) as well as Snail. We also found WSCM can increase the migratory capacity of BPH-1 cells. In addition, when they were treated with anti-TGF-ß1, upregulation of E-cadherin and CK5/8 levels was observed but no expression of vimentin, alpha-SMA or Snail was detected. Furthermore, phosphorylated-Smad3 expression in WSCM-cultured BPH-1 cells was also suppressed by anti-TGF-ß1 treatment. Our results demonstrated that stromal cell supernatant was able to induce EMT in BPH-1 cells, possibly through secreting TGF-ß1 to activate Smad signaling. Our results suggest novel molecular targets for clinical treatment of BPH by modification of stromal microenvironment through inhibiting TGF-ß1/Smad expression.

RNA interference targeting human integrin α6 suppresses the metastasis potential of hepatocellular carcinoma cells.

BACKGROUND: Increased metastasis has been proved to be associated with a poor prognosis for hepatocellular carcinoma (HCC). There are higher-level expressions of integrin α6 in the tissues of HCC patients with a higher fatality rate. The aim of this study is to investigate the effect of short hairpin RNA (shRNA) silencing integrin α6 expression on the proliferation and metastasis in HCC cell lines. METHODS: Two human HCC cell lines, HepG2 and Bel-7402 were transfected with shRNA targeting human integrin α6. Protein and mRNA expression level were determined by western blot and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to detect the transfected efficacy. The metastasis potential of HCC cells was evaluated by their proliferation, adhesion and invasion abilities. Cell proliferation was measured by MTT assay. Adhesion ability was measured by adhesion and spreading assays. The expression of matrix metalloproteinases (MMPs) was measured by qRT-PCR. The potential of invasion was measured by qRT-PCR and Transwell chamber assay. PI3K inhibitor LY294002 was used to explore the signal pathways of integrin α6 in HCC cells. RESULTS: Western blot and qRT-PCR detection showed that over 75% of integrin α6 expression in HCC cells was through knockdown by shRNA. Proliferation, adhesion, spreading and invasion of HepG2 and Bel-7402 cells were dramatically decreased in cells transfected with shRNA compared to the control cells. P-ERK and p-AKT were reduced by shRNA targeting integrin α6 and PI3K inhibitor LY294002. CONCLUSION: Knockdown integrin α6 can inhibit the proliferation and metastasis of HCC cells through PI3K/ARK and MAPK/ERK signal pathways by shRNA in vitro. Integrin α6 can mediate the metastasis potential, and can be used as a candidate target for therapy in HCC resulting in improved patients' survival.

[Effects of clinically effective dose of lovastatin on prostate cancer PC3 cells].

OBJECTIVE: To investigate the effects of clinically achievable dose of lovastatin on prostate cancer PC3 cells. METHODS: PC3 prostate cancer cells were treated with dimethyl sulfoxide(DMSO),or lovastain only,or lovastatin with mevalonic acid for 24, 48 and 72 hours respectively. MTT assay was used to detect the cell viability. By means of [3H] thymidine incorporation tests, the effects of lovastatin on cell proliferation were analyzed. Western blot was used to detect activated casepase3, caspase7, and cleaved PARP (cPARP), the important molecules on the apoptosis pathway. RESULTS: Cell proliferation of PC3 was significantly inhibited by 39.29%[(63.69%+/-3.69%) vs (102.98%+/-6.84%), P=0.000] after 48 h treatment with lovastatin at its clinically achievable dose of 2 micromol/L. After 72 hours the cell proliferation was inhibited by 44.24% [(52.79%+/-9.88% ) vs (97.03%+/-0.87%), P=0.048]. The cell number was also markedly decreased (4.86x10(5)+/-0.10x10(5)) vs (9.66x10(5)+/-0.10x10(5)), P=0.000] after 72 h treatment at this low concentration of 2 micromol/L. The viability of PC3 cells was significantly decreased 50.12% (56.52%+/-6.40%) vs (106.64%+/-5.27%), P=0.000] and 60.05% (41.99%+/-11.64%) vs (102.94%+/-8.49%), P=0.000] after 48 h and 72 h treatment, respectively. In addition, 2 micromol/L lovastatin induced activation of casepase7 and led the death substrate PARP to cleavage. CONCLUSION: Clinically achievable dose of lovastatin inhibits prostate cancer PC3 cell proliferation and induces PC3 cell apoptosis.

[Toxin-neutralizing monoclonal antibodies to the different domains of anthrax protective antigen].

Anthrax toxin from Bacillus anthracis is a three-component toxin consisting of lethal factor (LF), edema factor (EF), and protective antigen (PA). PA binds to target cells and transports LF or EF into the cell cytosol where they carry out their enzymatic functions. PA can induce protective immunity to the infection of the bacterium and is the major component in the only anthrax vaccine approved by FDA of USA. Mouse hybridoma clones specifically secreting anti-PA monoclonal antibodies (MAbs) were generated by cell fusion technique and their ability to neutralize anthrax lethal toxin activities was screened in vitro on a toxin-sensitive cell line. Nine toxin-neutralizing MAbs obtained were then characterized for the domains of PA they recognize, and the epitope regions they bind were analyzed by competitive binding ELISA. It was found that these MAbs bind four potential neutralizing epitope regions in three different domains of PA. Four MAbs bind to two non-overlapping epitope regions in domain 4 of PA and may prevent the binding of PA to its cell receptor. Four MAbs bind to domain 2, a domain involved in membrane insertion. One MAb binds to domain 3, a region involved in the oligomerization of PA. The results provided supporting evidence that PA has several neutralizing epitopes, and offered potential immunotherapeutic agents for the treatment of anthrax.