Threonine-120 phosphorylation regulated by phosphoinositide-3-kinase/Akt and mammalian target of rapamycin pathway signaling limits the antitumor activity of mammalian sterile 20-like kinase 1.

Mst1/Stk4, a hippo-like serine-threonine kinase, is implicated in many cancers, including prostate cancer. However, the mechanisms regulating Mst1 remain obscure. Here, we characterized the effects of phospho-Thr-120 on Mst1 in prostate cancer cells. We demonstrated that phospho-Thr-120 did not alter the nuclear localization or cleavage of Mst1 in a LNCaP or castration-resistant C4-2 prostate tumor cell model, as revealed by a mutagenesis approach. Phospho-Thr-120 appeared to be specific to cancer cells and predominantly localized in the nucleus. In contrast, phospho-Thr-183, a critical regulator of Mst1 cell death, was exclusively found in the cytoplasm. As assessed by immunohistochemistry, a similar distribution of phospho-Mst1-Thr-120/Thr-183 was also observed in a prostate cancer specimen. In addition, the blockade of PI3K signaling by a small molecule inhibitor, LY294002, increased cytoplasmic phospho-Mst1-Thr-183 without having a significant effect on nuclear phospho-Mst1-Thr-120. However, the attenuation of mammalian target of rapamycin (mTOR) activity by a selective pharmacologic inhibitor, Ku0063794 or CCI-779, caused the up-regulation of nuclear phospho-Mst1-Thr-120 without affecting cytoplasmic phospho-Mst1-Thr-183. This suggests that PI3K and mTOR pathway signaling differentially regulate phospho-Mst1-Thr-120/Thr-183. Moreover, mutagenesis and RNAi data revealed that phospho-Thr-120 resulted in C4-2 cell resistance to mTOR inhibition and reduced the Mst1 suppression of cell growth and androgen receptor-driven gene expression. Collectively, these findings indicate that phospho-Thr-120 leads to the loss of Mst1 functions, supporting cancer cell growth and survival.

Direct regulation of androgen receptor activity by potent CYP17 inhibitors in prostate cancer cells.

TOK-001 and abiraterone are potent 17-heteroarylsteroid (17-HAS) inhibitors of Cyp17, one of the rate-limiting enzymes in the biosynthesis of testosterone from cholesterol in prostate cancer cells. Nevertheless, the molecular mechanism underlying the prevention of prostate cell growth by 17-HASs still remains elusive. Here, we assess the effects of 17-HASs on androgen receptor (AR) activity in LNCaP and LAPC-4 cells. We demonstrate that both TOK-001 and abiraterone reduced AR protein and mRNA expression, and antagonized AR-dependent promoter activation induced by androgen. TOK-001, but not abiraterone, is an effective apparent competitor of the radioligand [(3)H]R1881 for binding to the wild type and various mutant AR (W741C, W741L) proteins. In agreement with these data, TOK-001 is a consistently superior inhibitor than abiraterone of R1881-induced transcriptional activity of both wild type and mutant AR. However, neither agent was able to trans-activate the AR in the absence of R1881. Our data demonstrate that phospho-4EBP1 levels are significantly reduced by TOK-001 and to a lesser extent by abiraterone alcohol, and suggest a mechanism by which cap-dependent translation is suppressed by blocking assembly of the eIF4F and eIF4G complex to the mRNA 5' cap. Thus, the effects of these 17-HASs on AR signaling are complex, ranging from a decrease in testosterone production through the inhibition of Cyp17 as previously described, to directly reducing both AR protein expression and R1881-induced AR trans-activation.

YAP1 Is Involved in Tumorigenic Properties of Prostate Cancer Cells.

The Yes Associated Protein 1 (YAP1) is a transcriptional cofactor negatively regulated by Hippo Pathway. The dysregulation of the pathway has been shown to have a role in tumorigenesis and metastasis in several cancers including prostate cancer (PCa). In this study, YAP1 expression was upregulated in the whole cell lysates and cytoplasmic/nuclear extracts of AR negative (PC3) compared to AR positive (LNCaP) prostate cancer cell lines and primary prostate epithelial cells (PrePEC). pYAP1 expression elevated in LNCaP compared to PC3 and PrePEC in whole cell lysates and cytoplasmic extracts. The treatment of LNCaP and PC3 with YAP1-targeting siRNA oligonucleotides (YAP1 siRNA) significantly reduced their proliferation in vitro. Furthermore, treatment with YAP1 siRNA diminished the clonogenicity, anchorage-independent growth on soft agar, migration and invasion of PC3 cells. Co-IP/WB experiments revealed that YAP1 and AR formed a complex and ChIP/PCR results confirmed that YAP1 was bound to androgen response elements (ARE) core region of the prostate specific antigen (PSA) promoter. The loss of function experiments in LNCaP and PC3 revealed that YAP1 regulates proliferation, colony formation as well as anchorage-independent growth and potentially plays an important role in migration and invasion. Finally, analysis of publicly available data sets indicated that LNCaP had no YAP1 copy number alteration whereas PC3 had gain of YAP1 which was also reflected as an increase in the mRNA level. Moreover, YAP1 copy number gain and elevated YAP1 mRNA expression were detected in clinical samples analyzed in publicly available data sets. Taken together, these results suggested that YAP1 has a role in PCa tumorigenesis.

Increased expression of YAP1 in prostate cancer correlates with extraprostatic extension.

OBJECTIVE: Yes associated protein 1 (YAP1) is a member of the Hippo pathway, acting as a transcriptional coactivator. To elucidate the role of YAP1 and phosphorylated (p)YAP1 in prostate cancer (PCa) tumorigenesis, we investigated their expression in clinical samples of PCa and cell lines. METHODS: Fifty-four tumor, adjacent nontumor, and prostate intraepithelial neoplasia (PIN) tissues from patients with PCa after radical prostatectomy were selected from a retrospective cohort and studied using immunohistochemistry (IHC). Protein and mRNA expression levels of YAP1 were evaluated by Western blot analysis and quantitative real-time reverse transcription PCR, respectively, in cancer cell lines. Publicly available gene expression datasets were downloaded to analyze YAP1 mRNA and protein levels in PCa tissue samples. RESULTS: IHC analysis of PCa tissues revealed that YAP1 staining intensities were moderate to weak in the nucleus and cytoplasm of tumor cells, whereas adjacent normal epithelia showed strong staining. We observed that benign prostates were characterized by higher expression levels of both nuclear (P=0.004) and cytosolic (P=0.005) YAP1. pYAP1 staining was weak in the cytoplasm and absent in the nucleus of all the tissues investigated. YAP1 expression was an indicator of extraprostatic extension (EPE). The level of YAP1 was negatively correlated with the level of the androgen receptor (AR) in The Cancer Genome Atlas dataset and Western blot analysis of cell lines. CONCLUSIONS: Our study suggested that YAP1 expression is heterogeneous in PCa tissue samples; therefore, YAP1 might play different roles in different aspects of PCa progression. This might involve AR-YAP1 interplay in PCa.

MST1 is a multifunctional caspase-independent inhibitor of androgenic signaling.

The MST1 serine-threonine kinase, a component of the RASSF1-LATS tumor suppressor network, is involved in cell proliferation and apoptosis and has been implicated in cancer. However, the physiologic role of MST1 in prostate cancer (PCa) is not well understood. Here, we investigated the possibility of a biochemical and functional link between androgen receptor (AR) and MST1 signaling. We showed that MST1 forms a protein complex with AR and antagonizes AR transcriptional activity as shown by coimmunoprecipitation (co-IP), promoter reporter analysis, and molecular genetic methods. In vitro kinase and site-specific mutagenesis approaches indicate that MST1 is a potent AR kinase; however, the kinase activity of MST1 and its proapoptotic functions were shown not to be involved in inhibition of AR. MST1 was also found in AR-chromatin complexes, and enforced expression of MST1 reduced the binding of AR to a well-characterized, androgen-responsive region within the prostate-specific antigen promoter. MST1 suppressed PCa cell growth in vitro and tumor growth in mice. Because MST1 is also involved in regulating the AKT1 pathway, this kinase may be an important new link between androgenic and growth factor signaling and a novel therapeutic target in PCa.