Bekir Cinar

The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1

Akt kinases mediate cell growth and survival. Here, we report that a pro‐apoptotic kinase, Mst1/STK4, is a physiological Akt1 interaction partner. Mst1 was identified as a component of an Akt1 multiprotein complex isolated from lipid raft‐enriched fractions of LNCaP human prostate cancer cells. Endogenous Mst1, along with its paralog, Mst2, acted as inhibitors of endogenous Akt1. Surprisingly, mature Mst1 as well as both of its caspase cleavage products, which localize to distinct subcellular compartments and are not structurally homologous, complexed with and inhibited Akt1. cRNAs encoding full‐length Mst1, and N‐ and C‐terminal caspase Mst1 cleavage products, reverted an early lethal phenotype in zebrafish development induced by expression of membrane‐targeted Akt1. Mst1 and Akt1 localized to identical subcellular sites in human prostate tumors. Mst1 levels declined with progression from clinically localized to hormone refractory disease, coinciding with an increase in Akt activation with transition from hormone naïve to hormone‐resistant metastases. These results position Mst1/2 within a novel branch of the phosphoinositide 3‐kinase/Akt pathway and suggest an important role in cancer progression.

Membrane rafts as potential sites of nongenomic hormonal signaling in prostate cancer

Recent evidence indicates that nuclear receptors for steroid hormones can signal by nongenomic mechanisms that operate independently of their transcription function. These signal-transduction processes occur within seconds to minutes after initiation with agonist and involve interactions between nuclear receptors and other signaling proteins in the cytoplasm and at membrane surfaces. This review provides an overview of published information on possible nongenomic activities of the androgen receptor (AR) and other nuclear receptors, focusing on the potential involvement of these processes in prostate cancer. We discuss the hypothesis that the cholesterol-rich lipid-raft compartment(s) of cancer-cell membranes might provide privileged sites for nongenomic signals mediated by the AR.

Bruton tyrosine kinase is commonly overexpressed in mantle cell lymphoma and its attenuation by Ibrutinib induces apoptosis.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that characteristically shows overexpression of cyclin-D1 due to an alteration in the t(11;14)(q13;q32) chromosomal region. Although there are some promising treatment modalities, great majority of patients with this disease remain incurable. The B-cell antigen receptor (BCR) signaling plays a crucial role in B-cell biology and lymphomagenesis. Bruton tyrosine kinase (BTK) has been identified as a key component of the BCR signaling pathway. Evidence suggests that the blockade of BTK activity by potent pharmacologic inhibitors attenuates BCR signaling and induces cell death. Notably, the expression levels and the role of BTK in MCL survival are still elusive. Here, we demonstrated a moderate to strong BTK expression in all MCL cases (n=19) compared to benign lymphoid tissues. Treatment of MCL cell lines (Mino or Jeko-1) with a potent BTK pharmacologic inhibitor, Ibrutinib, decreased phospho-BTK-Tyr(223) expression. Consistent with this observation, Ibrutinib inhibited the viability of both Mino and JeKo-1 cells in concentration- and time-dependent manners. Ibrutinib also induced a concentration-dependent apoptosis in both cell lines. Consistently, Ibrutinib treatment decreased the levels of anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 protein. These findings suggest that BTK signaling plays a critical role in MCL cell survival, and the targeting of BTK could represent a promising therapeutic modality for aggressive lymphoma.

Post-transcriptional regulation of the androgen receptor by mammalian target of rapamycin

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), an ErbB1 ligand and prostate stromal growth factor, is an antagonist of androgen receptor (AR) function. In the LNCaP prostate cancer model, HB-EGF reduced AR protein levels and AR transactivation without affecting AR mRNA level or protein turnover. The signal to attenuate AR was mediated by the mammalian target of rapamycin, as shown by genetic and pharmacologic methods, and was independent of ErbB2/HER-2, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase pathways. Additional evidence suggests that AR protein levels are highly sensitive to regulation by cap-dependent mRNA translation. These findings reveal a novel mechanism for regulation of AR by a classic growth factor system and indicate that a rapamycin-sensitive post-transcriptional pathway can attenuate or possibly bypass AR-mediated signaling.

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

Background: Abiraterone and TOK-001 are inhibitors of CYP17 activity, a crucial enzyme for the synthesis of testosterone in prostate cancer cells. Results: These 17-heteroarylsteroids also directly down-regulate the expression and activation of the androgen receptor. Conclusion: TOK-001 and abiraterone, down-regulate androgen receptor signaling via multiple mechanisms. Significance: Anti-CYP17 17-heteroarylsteroids may have multiple mechanisms of action 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 [3H]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.

Identification of a negative regulatory cis-element in the enhancer core region of the prostate-specific antigen promoter: implications for intersection of androgen receptor and nuclear factor-kappaB signalling in prostate cancer cells

The NF-kappaB (nuclear factor-kappaB) transcription factors mediate activation of a large number of gene promoters containing diverse kappaB-site sequences. Here, PSA (prostate-specific antigen) was used as an AR (androgen receptor)-responsive gene to examine the underlying mechanism by which the NF-kappaB p65 transcription factor down-regulates the transcriptional activity of AR in cells. We observed that activation of NF-kappaB by TNFalpha (tumour necrosis factor alpha) inhibited both basal and androgen-stimulated PSA expression, and that this down-regulation occurred at the promoter level, as confirmed by the super-repressor IkappaBalpha (S32A/S36A), a dominant negative inhibitor of NF-kappaB. Using a linker-scanning mutagenesis approach, we identified a cis -element, designated XBE (X-factor-binding element), in the AREc (androgen response element enhancer core) of the PSA promoter, which negatively regulated several AR-responsive promoters, including that of PSA. When three copies of XBE in tandem were juxtaposed to GRE4 (glucocorticoid response element 4), a 4-6-fold reduction of inducible GRE4 activity was detected in three different cell lines, LNCaP, ARCaP-AR and PC3-AR. Bioinformatics and molecular biochemical studies indicated that XBE is a kappaB-like element that binds specifically to the NF-kappaB p65 subunit; consistent with these observations, only NF-kappaB p65, but not the NF-kappaB p50 subunit, was capable of inhibiting AR-mediated PSA promoter transactivation in LNCaP cells. In addition, our data also showed that AR binds to XBE, as well as to the kappaB consensus site, and that the transfection of AR inhibits the kappaB-responsive promoter in transient co-transfection assays. Collectively, these data indicate that cross-modulation between AR and NF-kappaB p65 transcription factors may occur by a novel mechanism involving binding to a common cis -DNA element.

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

Background: The molecular mechanisms that regulate the hippo-like Mst1 protein kinase remain elusive. Results: PI3-kinase and mTOR signaling regulate Mst1 phosphorylation in a discrete cell location. Conclusion: Phospho-Thr-120 restricts Mst1 functions, leading cancer cell growth and survival. Significance: Mst/Hippo signaling is a promising drug target in human cancers. 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.

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.

Overexpression of MYC and EZH2 cooperates to epigenetically silence MST1 expression

Hippo-like MST1 protein kinase regulates cell growth, organ size, and carcinogenesis. Reduction or loss of MST1 expression is implicated in poor cancer prognosis. However, the mechanism leading to MST1 silencing remains elusive. Here, we report that both MYC and EZH2 function as potent suppressors of MST1 expression in human prostate cancer cells. We demonstrated that concurrent overexpression of MYC and EZH2 correlated with the reduction or loss of MST1 expression, as shown by RT-qPCR and immunoblotting. Methylation sensitive PCR and bisulfite genomic DNA sequencing showed that DNA methylation caused MST1 silencing. Pharmacologic and RNAi experiments revealed that MYC and EZH2 silenced MST1 expression by inhibiting its promoter activity, and that EZH2 was a mediator of the MYC-induced silencing of MST1. In addition, MYC contributed to MST1 silencing by partly inhibiting the expression of microRNA-26a/b, a negative regulator of EZH2. As shown by ChIP assays, EZH2-induced DNA methylation and H3K27me3 modification, which was accompanied by a reduced H3K4me3 mark and RNA polymerase II occupancy on the MST1 promoter CpG region, were the underlying cause of MST1 silencing. Moreover, potent pharmacologic inhibitors of MYC or EZH2 suppressed prostate cancer cell growth in vitro, and the knockdown of MST1 caused cells’ resistance to MYC and EZH2 inhibitor-induced growth retardation. These findings indicate that MYC, in concert with EZH2, epigenetically attenuates MST1 expression and suggest that the loss of MST1/Hippo functions is critical for the MYC or EZH2 mediation of cancer cell survival.

Concurrent inhibition of MYC and BCL2 is a potentially effective treatment strategy for double hit and triple hit B-cell lymphomas

Double hit lymphoma or triple hit lymphoma (DHL/THL) is a rare form of aggressive B-Cell Lymphoma. Overexpression of MYC, BCL2 or/and BCL6 due to genomic rearrangements are the key molecular features of DHL/THL. Patients with DHL/THL show very aggressive disease course and poor survival due to the lack of effective treatment modalities. Here, we established new THL cell model and assessed its in vitro growth characteristics along with the DHL cell line in response to potent MYC inhibitors, 10058-F4 and JQ-1, and a BCL2 inhibitor, ABT-199, with or without chemotherapeutic agent vincristine or doxorubicin. We found that 10058-F4, JQ-1 or ABT-199 exposure as a single agent inhibited the growth of DHL/THL cells in a dose-dependent manner. Combined exposure of 10058-F4 or JQ-1 and ABT-199 as well as vincristine or doxorubicin markedly suppressed the growth of DHL/THL cells compared with the single treatment. As assessed by multiple approaches, apoptosis induced by ABT-199, 10058-F4 or JQ-1 was underlying cause of the observed growth suppression. These findings suggest that co-inhibition of MYC and BCL2 signaling is a promising therapeutic strategy for patients with DHL/THL lymphomas.