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Abstract The EPHA3 protein tyrosine kinase, a member of the ephrin receptor family, regulates cell fate, cell motility, and cell–cell interaction. These cellular events are critical for tissue development, immunological responses, and the processes of tumorigenesis. Earlier studies revealed that signaling via the STK4 -encoded MST1 serine-threonine protein kinase, a core component of the Hippo pathway, attenuated EPHA3 expression. Here, we investigated the mechanism by which MST1 regulates EPHA3. Our findings have revealed that the transcriptional regulators YAP1 and TEAD1 are crucial activators of EPHA3 transcription. Silencing YAP1 and TEAD1 suppressed the EPHA3 protein and mRNA levels. In addition, we identified putative TEAD enhancers in the distal EPHA3 promoter, where YAP1 and TEAD1 bind and promote EPHA3 expression. Furthermore, EPHA3 knockout by CRISPR/Cas9 technology reduced cell–cell interaction and cell motility. These findings demonstrate that EPHA3 is transcriptionally regulated by YAP1/TEAD1 of the Hippo pathway, suggesting that it is sensitive to cell contact-dependent interactions. 

RNA binding proteins (RBPs) regulate all aspects of RNA biogenesis from transcription, splicing, and translation to degradation, and they have a critical role in cellular homeostasis and functional diversity. Recent studies have indicated that altered expressions of RBPs are associated with many human diseases ranging from neurologic disorders to cancer. The transcriptional coregulator yes-associated protein 1 (YAP1), a critical nuclear effector of the mammalian Hippo pathway, regulates cell fate, cell contact, metabolism, and developmental processes. This study demonstrates a link between YAP1 and nucleophosmin1 (NPM1) protein. NPM1 is an RNA-binding protein that regulates many cellular activities, including ribosome biogenesis, RNA processing, chromatin remodeling, DNA repair, and genomic stability. We identified NPM1 from YAP1 protein complexes of androgen-responsive human cancer cells using proteomics approaches. Our proximity ligation assay demonstrated that YAP1 and NPM1 physically interacted with each other. The interaction between YAP1 and NPM1 occurred in cell nuclei and was regulated by androgen hormone signaling. In addition, our GST-pulldown assay demonstrated that NPM1 formed a protein complex with the proline-rich domain of YAP1. Furthermore, our enhanced RNA interactome capture (eRIC) assay showed that androgen also regulated the interaction of RBPs to polyA+ mRNA within the cell. Consistent with this observation, our eRIC assay combined with the mass spectrometry method enabled us to identify distinct RBP patterns in human cancer cells that are genetically related but phenotypically different. These observations indicate that global alterations of RBPs under changing environmental conditions may have essential roles in cellular physiology and disease biology. 

Sex comb on midleg-like-2 (SCML2), a conserved polycomb group protein, functions as a transcriptional repressor. SCML2 binds monomethylated lysine residues on histones and regulates homeotic gene expression during development in mammals and the fly. Using proteomic approaches, we have identified SCML2 as a binding partner of the YAP1 protein complexes isolated from nuclei of prostate cancer cell lines. Both SCML2 and YAP1 are known to regulate basic cellular biology, including stem cell maintenance and carcinogenesis. Our western blot analysis showed that, unlike androgen receptor (AR)-negative cancerous and non-cancerous prostate epithelium, AR-positive cell lines express the high levels of SCML2, suggesting a possible link between androgen hormonal signaling and SCML2. In addition, our immunofluorescence imaging revealed that androgen hormone signaling promoted the subcellular localization of SCML2 and YAP1 proteins compared with mock control. Enzalutamide, a potent pharmacological inhibitor of AR, significantly prevented the subcellular distribution ofYAP1 and SCML2. Consistent with this observation, our proximity ligation assay demonstrated that androgen also regulated the physical interaction between SCML2 and YAP1proteins that occurred primarily in cell nuclei. Enzalutamide also prevented protein-protein interaction between YAP and SCML2. Besides, our GST-pulldown assay revealed that SCML2 and proteins physically interact with each other in the test tube. Furthermore, our promoter-reporter assay showed that transfection of two different SCML2 siRNA enhanced the activation of the YAP-responsive promoter-reporter gene four-fold compared to mock siRNA control. These observations suggest that the interaction between SCML2 and YAP1 is biologically functional and crucial in human physiology and disease. 

Cell-cell interaction is critical for tissue development and repair, immunological responses, and cancer cell metastasis. The tyrosine kinase EPHA3 (erythropoietin‑producing hepatocellular carcinoma cell surface type-A receptor 3) regulates cell-cell interaction, cell differentiation, and cancer cell survival. Previously, our published study indicated that the theSTK4-encoded MST1 signaling, a core kinase component of the Hippo pathway, suppressedEPHA3 expression in the prostate cancer cell models. However, the mechanism is unknown. Here, we have demonstrated that the YAP1 and TEAD1 proteins, critical nuclear effectors of the Hippo pathway, mediate EPHA3 expression. First, we showed that AR-positive cell lines express the highest levels of EPHA3 and its ligand, ephrin-A5, transcripts compared with other EPH family members. Second, we demonstrated the induction of MST1/STK4attenuated the EPHA3 protein and transcripts, consistent with our initial observation. Next, we demonstrated that the knockdown of YAP1 by siRNA suppressed EPHA3 protein and mRNA expression. Similarly, the silencing of the TEAD1-4 proteins, critical mediators of YAP1-dependent gene transcription, revealed that the TEAD1 is a crucial inducer of EPHA3expression. Moreover, bioinformatics tools allowed the identification of three putative TEAD binding sites (p<0.001) in the promoter region of the EPHA3 gene. Furthermore, CRISPR/Cas9-aided EPHA3 knockout significantly (p<0.01), decreased cell growth in monolayer and sphere formation in 3D cultures, and caused androgen-independent cells to become sensitive to enzalutamide, a potent direct inhibitor of AR activity. These observations suggest that the YAP/TEAD1 transcriptionally regulates EPHA3 and its cellular biology. 

The transcriptional co-activator YAP1 (yes-associated protein 1) is a critical nuclear effector of the Hippo pathway. The Hippo pathway regulates cell growth, cell motility, cell migration, and carcinogenesis, but poorly defined mechanism. We investigated biochemical and functional interactions between YAP1 and the nuclear factor (NF)-kappa B/RELA subunit in prostate cancer cell models. We demonstrated that endogenous YAP1 and RELA form protein complexes in the cell, as revealed by co-immunoprecipitation and western blotting. Compared with control, we found that combined treatment of cells with androgen and SDF-1a (stromal cell-derived factor-1 alpha) or RANKL (receptor activator of NF-kappa B ligand) enhanced the protein-protein interaction between YAP1 and RELA, as showed by proximity ligation assay. Our confocal microscopy experiment further showed that combined SDF-1aand androgen treatment promoted the YAP1 and RELA colocalization instead of single-agent treatment. Moreover, our promoter-reporter and RNAi experiments showed that knockdown of YAP1 or TEAD, a key mediator of the YAP transcription, significantly reduced the NF-Kappa B promoter-reporter gene activity. Also, disruption of YAP1 activity attenuated the TEAD-RELA interaction. Furthermore, the controlled expression of MST1/STK4, a potent inhibitor of YAP1, attenuated the NF-Kappa B-promoter reporter activity. Additionally, our unbiased bioinformatics analysis of the exiting ChIP-seq (chromatin immunoprecipitation-sequencing) data sets identified several genes that are likely co-regulated by the YAP1/TEAD and NF-Kappa B/RELA transcription factors. These findings suggest that cooperative androgen and cytokine signaling regulates Hippo/YAP and NF-Kappa B interaction. Thus, the YAP1/TEAD and NF-Kappa B/RELA interaction may have critical roles in cellular biology and human diseases. 

The transcriptional coactivator YAP1 (yes-associated protein 1) regulates cell proliferation, cell–cell interactions, organ size, and tumorigenesis. Post-transcriptional modifications and nuclear translocation of YAP1 are crucial for its nuclear activity. The objective of this study was to elucidate the mechanism by which the steroid hormone androgen regulates YAP1 nuclear entry and functions in several human prostate cancer cell lines. We demonstrate that androgen exposure suppresses the inactivating post-translational modification phospho–Ser-127 in YAP1, coinciding with increased YAP1 nuclear accumulation and activity. Pharmacological and genetic experiments revealed that intact androgen receptor signaling is necessary for androgen's inactivating effect on phospho–Ser-127 levels and increased YAP1 nuclear entry. We also found that androgen exposure antagonizes Ser/Thr kinase 4 (STK4/MST1) signaling, stimulates the activity of protein phosphatase 2A, and thereby attenuates the phospho–Ser-127 modification and promotes YAP1 nuclear localization. Results from quantitative RT-PCR and CRISPR/Cas9–aided gene knockout experiments indicated that androgen differentially regulates YAP1-dependent gene expression. Furthermore, an unbiased computational analysis of the prostate cancer data from The Cancer Genome Atlas revealed that YAP1 and androgen receptor transcript levels correlate with each other in prostate cancer tissues. These findings indicate that androgen regulates YAP1 nuclear localization and its transcriptional activity through the androgen receptor–STK4/MST1–protein phosphatase 2A axis, which may have important implications for human diseases such as prostate cancer. 

The transcriptional co-activator YAP1 (yes-associated protein 1), a crucial effector of the Hippo pathway in mammals, regulates cell growth, cell motility, cell migration, and carcinogenesis. The STK4/Hippo kinase phosphorylates Ser127 and inactivate YAP1 activity in mammalian cells. Cytokines such as receptor activator of nuclear factor Kappa B (RANKL) regulate the immune system and bone remodeling. Similarly, stroma-cell derived factor 1 alpha (SDF1α) produced by the bone marrow stromal cell, is directly linked to cell migration and metastasis. We hypothesize that RANKL/SDF1α attenuates phospho-Ser127 and enhances YAP nuclear localization. We conducted immunological assays to evaluate the effects of SDF1α or RANKL on YAP1 in the LNCaP prostate cancer cell line. We showed that SDF1α and RANKL modulate phospho-Ser127 and total YAP1 protein in a time-dependent manner, as demonstrated by western blotting. We also showed that SDF1α exposure promoted YAP1 nuclear localization, as revealed by immunofluorescence imaging with confocal microscopy. These findings suggest that cytokines positively regulate YAP1 activity, possibly by counteracting with the STK4/Hippo signaling. The results of this study imply that cytokines secreted by the tumor cell environment promote an invasive cancer cell phenotype by modulating the Hippo-YAP1 pathway. 

The Hippo pathway controls cell-cell interaction and organ size by regulating cell proliferation. The study aimed to determine whether Hippo/YAP and NF-kappa B/RELA signaling interact. Here, we have demonstrated that native YAP1/TEAD and RELA proteins biochemically and functionally interact with each other in human LNCaP and C4-2 cell lines. Our co-immunoprecipitation (co-IP), western blot (WB), and proximity ligation assay (PLA) showed that endogenous YAP/TEAD and RELA physically interact within the cell. Our immunofluorescence assays revealed that the expression of YAP1 and RELA proteins overlapped in the cytoplasm and the nucleus. Combined treatment of cells with RANKL (receptor activator of nuclear factor-kappa Β ligand) and androgen hormone enhanced YAP1 and RELA colocalization and interaction, as demonstrated by co-IP/WB experiments. Moreover, our PLA confirmed that co-treatment of cells with androgen and SDF1a (stromal cell-derived factor 1 alpha) or RANKL increased YAP1 and RELA interaction cytoplasm and nucleus compared with controls. Our promoter-reporter assays showed that the knockdown of YAP1 by siRNA significantly reduced the activity of an NF-Kappa B responsive promoter-reporter gene. We also showed that controlled expression of MST1/STK4, a potent inhibitor of YAP1, attenuated the NF-Kappa B promoter reporter activity. Our unbiased bioinformatics analysis of the chromatin immunoprecipitation data has revealed that YAP/TEAD and NF-kappa B signaling regulates several genes. These findings suggest that interaction between the Hippo/YAP and NF-Kappa B/RELA plays a critical role in broad cellular biology. 

Dysregulation of the receptor tyrosine kinases (RTKs) by means of mutation, amplification or overexpression plays a crucial role in cell growth, cell survival, cell motility during cancer progression and metastasis. EPHA3 (erythropoietin-producing hepatocellular carcinoma cell surface type A receptor 3) is a member of the RTKs. Evidence indicates that the upregulation of the EPHA3 activity is implicated in the pathobiology of various cancers, including prostate cancer, and thus, it is a prime therapeutic target in cancer. However, the role of EPHA3 signaling in prostate cancer progression remains obscure. Currently, the development of castration-resistant prostate cancer (CRPC) poses a clinical challenge because it is lethal. The molecular mechanisms that contribute to lethal prostate cancer are largely unknown. The objective of this study is to investigate whether EPHA3 signaling plays a critical role in prostate cancer progression and therapeutic relapse. Our analysis of the prostate cancer public datasets revealed that the EPHA3 gene was amplified up to 19% of metastatic CRPC cases with the neuroendocrine phenotype. Our immunological assay confirmed the positive staining of EPHA3 protein in human prostate cancer specimens. Our semi-quantitative and quantitative PCR assays demonstrated that the levels of EPHA3 vary among established prostate cancer cell lines. Nevertheless, we consistently found that the levels of EPHA3 mRNA in CRPC cell line, C4-2, were 3-fold higher than its castration-sensitive parental LNCaP cells. Furthermore, we demonstrated that an increase in expression of EPHA3 mRNA in C4-2 compared with LNCaP cells coincided with the upregulation of the EPHA3 protein, as independently confirmed by western blotting and immunofluorescence imaging. These findings indicate that EPHA3 may confer an aggressive prostate cancer cell phenotype. Because androgen receptor (AR) signaling is a potent mediator of CRPC cell growth and survival, the targeting of EPHA3 signaling alone or together with AR may improve the efficacy of current therapies for patients with advanced prostate cancer. 

The transcriptional coactivator YAP1 (yes-associated protein 1) is a critical nuclear effector of the Hippo pathway. The serine/threonine protein kinases STK3/4 and LATS1/2, core components of the Hippo pathway, phosphorylate and inhibit YAP1 nuclear localization. Previously, we reported that the interaction of nuclear YAP1 with androgen receptor (AR) might play a critical role in prostate cancer progression and therapeutic relapse (Kuser-Abali et al., Nat. Commun. 2015). Here, we investigated the regulation of YAP1 by androgens in isogenic, androgen-responsive LNCaP and androgen non-responsive C4-2 prostate cancer cell models. We demonstrated that androgen suppressed the inhibitory phospho-Ser127 site on YAP1 in LNCaP cells, but the effects of androgen on phospho-Ser127 was modest in C4-2 cells. In agreement with this observation, androgen increased the presence of nuclear YAP1 in LNCaP cells, whereas regardless of androgen exposure the YAP1 protein was primarily expressed in C4-2 cell nuclei. We also demonstrated that androgen exposure suppressed the levels of phospho-Ser127 induced by okadaic acid, which is a potent inhibitor of the Ser/Thr phosphatases PP1 and PP2A. Moreover, the pharmacological inhibition of androgen receptor (AR) signaling by enzalutamide reversed the inhibitory effects of androgen on phospho-Ser127, which coincided with the inhibition of YAP1 nuclear localization. Similarly, the genetic inhibition of AR signaling by small interfering RNA (siRNA) reduced phospho-Ser127 levels. Additionally, the silencing of the STK3/4 and LATS1/2 signaling by siRNA resulted in increases in YAP1 protein levels. Furthermore, our analysis of the TCGA (The Cancer Genome Atlas) prostate adenocarcinoma data set indicates that the levels of YAP1 and AR mRNA expression were positively correlated in prostate cancer clinical samples. These observations suggest that AR signaling promotes YAP1 nuclear localization by suppressing phospho-Ser127, possibly through the protein phosphatases PP1 and PP2A, and supporting a new mechanism of YAP1 regulation and YAP1-mediated cancer cell growth and survival.