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1. The Hippo effector YAP1 biochemically and functionally interacts with the nuclear factor-kappa B/RELA transcription factor

   Cinar, B: Al-Mathkour ; Dwead, A ; Boston, A ; Alp, E. ( May 2021 , The FASEB journal)

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   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. 

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2. Abstract P589: Crosstalk Between the Hippo-YAP and Nuclear Factor-Kappa B-RELA Signaling

   Cinar, B ; Said-Bandy, E ; Almathkour, M.M ; Carlos, M. ( December 2019 , ASCB|EMBO 2019 Annual Meeting; December 7-11, 2019; Washington, D.C.)

   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. 

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3. Androgen attenuates the inactivating phospho–Ser-127 modification of yes-associated protein 1 (YAP1) and promotes YAP1 nuclear abundance and activity

   https://doi.org/10.1074/jbc.RA120.013794  

   Cinar, Bekir ; Al-Mathkour, Marwah M. ; Khan, Shafiq A. ; Moreno, Carlos S. ( June 2020 , Journal of Biological Chemistry)

   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. 

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4. Abstract P2386: Androgen Receptor Signaling Promotes YAP1 Nuclear Localization

   Cinar, B: Al-Mathkour ; Khan, S. and ( January 2019 , ASCB-EMBO 2018 Annual Meeting)

   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. 

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5. The Hippo effector YAP1/TEAD1 regulates EPHA3 expression to control cell contact and motility

   https://doi.org/10.1038/s41598-022-07790-4  

   Al-Mathkour, Marwah M. ; Dwead, Abdulrahman M. ; Alp, Esma ; Boston, Ava M. ; Cinar, Bekir ( December 2022 , Scientific Reports)

   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. 

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