Yuanhong Chen

KIBRA Regulates Hippo Signaling Activity via Interactions with Large Tumor Suppressor Kinases

The Hippo pathway controls tissue growth and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. Recent genetic studies in Drosophila identified Kibra as a novel regulator of Hippo signaling. Human KIBRA has been associated with memory performance and cell migration. However, it is unclear whether or how KIBRA is connected to the Hippo pathway in mammalian cells. Here, we show that KIBRA associates with and activates Lats (large tumor suppressor) 1 and 2 kinases by stimulating their phosphorylation on the hydrophobic motif. KIBRA overexpression stimulates the phosphorylation of Yes-associated protein (YAP), the Hippo pathway effector. Conversely, depletion of KIBRA by RNA interference reduces YAP phosphorylation. Furthermore, KIBRA stabilizes Lats2 by inhibiting its ubiquitination. We also found that KIBRA mRNA is induced by YAP overexpression in both murine and human cells, suggesting the evolutionary conservation of KIBRA as a transcriptional target of the Hippo signaling pathway. Thus, our study revealed a new connection between KIBRA and mammalian Hippo signaling.

The Hippo Pathway Effector YAP Regulates Motility, Invasion, and Castration-Resistant Growth of Prostate Cancer Cells

ABSTRACT Yes-associated protein (YAP) is an effector of the Hippo tumor suppressor pathway. The functional significance of YAP in prostate cancer has remained elusive. In this study, we first show that enhanced expression of YAP is able to transform immortalized prostate epithelial cells and promote migration and invasion in both immortalized and cancerous prostate cells. We found that YAP mRNA was upregulated in androgen-insensitive prostate cancer cells (LNCaP-C81 and LNCaP-C4-2 cells) compared to the level in androgen-sensitive LNCaP cells. Importantly, ectopic expression of YAP activated androgen receptor signaling and was sufficient to promote LNCaP cells from an androgen-sensitive state to an androgen-insensitive state in vitro, and YAP conferred castration resistance in vivo. Accordingly, YAP knockdown greatly reduced the rates of migration and invasion of LNCaP-C4-2 cells and under androgen deprivation conditions largely blocked cell division in LNCaP-C4-2 cells. Mechanistically, we found that extracellular signal-regulated kinase–ribosomal s6 kinase signaling was downstream of YAP for cell survival, migration, and invasion in androgen-insensitive cells. Finally, immunohistochemistry showed significant upregulation and hyperactivation of YAP in castration-resistant prostate tumors compared to their levels in hormone-responsive prostate tumors. Together, our results identify YAP to be a novel regulator in prostate cancer cell motility, invasion, and castration-resistant growth and as a potential therapeutic target for metastatic castration-resistant prostate cancer (CRPC).

CDK1 Phosphorylation of YAP Promotes Mitotic Defects and Cell Motility and Is Essential for Neoplastic Transformation

The Yes-associated protein, YAP, is a downstream effector of the Hippo pathway of cell-cycle control that plays important roles in tumorigenesis. Hippo-mediated phosphorylation YAP, mainly at S127, inactivates YAP function. In this study, we define a mechanism for positive regulation of YAP activity that is critical for its oncogenic function. Specifically, we found that YAP is phosphorylated in vitro and in vivo by the cell-cycle kinase CDK1 at T119, S289, and S367 during the G2-M phase of the cell cycle. We also found that ectopic expression of a phosphomimetic YAP mutant (YAP3D, harboring T119D/S289D/S367D) was sufficient to induce mitotic defects in immortalized epithelial cells, including centrosome amplification, multipolar spindles, and chromosome missegregation. Finally, we documented that mitotic phosphorylation of YAP was sufficient to promote cell migration and invasion in a manner essential for neoplastic cell transformation. In support of our findings, CDK1 inhibitors largely suppressed cell motility mediated by activated YAP-S127A but not the phosphomimetic mutant YAP3D. Collectively, our results reveal a previously unrecognized mechanism for controlling the activity of YAP that is crucial for its oncogenic function mediated by mitotic dysregulation.

KIBRA Protein Phosphorylation Is Regulated by Mitotic Kinase Aurora and Protein Phosphatase 1

Recent genetic studies in Drosophila identified Kibra as a novel regulator of the Hippo pathway, which controls tissue growth and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. The cellular function and regulation of human KIBRA remain largely unclear. Here, we show that KIBRA is a phosphoprotein and that phosphorylation of KIBRA is regulated in a cell cycle-dependent manner with the highest level of phosphorylated KIBRA detected in mitosis. We further demonstrate that the mitotic kinases Aurora-A and -B phosphorylate KIBRA both in vitro and in vivo. We identified the highly conserved Ser539 as the primary phosphorylation site for Aurora kinases. Moreover, we found that wild-type, but not catalytically inactive, protein phosphatase 1 (PP1) associates with KIBRA. PP1 dephosphorylated Aurora-phosphorylated KIBRA. KIBRA depletion impaired the interaction between Aurora-A and PP1. We also show that KIBRA associates with neurofibromatosis type 2/Merlin in a Ser539 phosphorylation-dependent manner. Phosphorylation of KIBRA on Ser539 plays a role in mitotic progression. Our results suggest that KIBRA is a physiological substrate of Aurora kinases and reveal a new avenue between KIBRA/Hippo signaling and the mitotic machinery.

KIBRA Regulates Aurora Kinase Activity and Is Required for Precise Chromosome Alignment During Mitosis

Background: The memory-associated protein KIBRA regulates cell polarity and migration in non-neuronal cells and a cellular function of KIBRA in mitosis is not defined. Results: KIBRA activates Aurora kinases and is required for precise chromosome alignment and proper spindle organization in mitosis. Conclusion: KIBRA plays a role in mitosis. Significance: The findings mark the importance of KIBRA in association with the mitotic machinery. The Hippo pathway controls organ size and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. KIBRA was recently identified as a novel regulator of the Hippo pathway. Several of the components of the Hippo pathway are important regulators of mitosis-related cell cycle events. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora-A and -B. However, the role KIBRA plays in mitosis has not been established. Here, we show that KIBRA activates the Aurora kinases and is required for full activation of Aurora kinases during mitosis. KIBRA also promotes the phosphorylation of large tumor suppressor 2 (Lats2) on Ser83 by activating Aurora-A, which controls Lats2 centrosome localization. However, Aurora-A is not required for KIBRA to associate with Lats2. We also found that Lats2 inhibits the Aurora-mediated phosphorylation of KIBRA on Ser539, probably via regulating protein phosphatase 1. Consistent with playing a role in mitosis, siRNA-mediated knockdown of KIBRA causes mitotic abnormalities, including defects of spindle and centrosome formation and chromosome misalignment. We propose that the KIBRA-Aurora-Lats2 protein complexes form a novel axis that regulates precise mitosis.

Ajuba Phosphorylation by CDK1 Promotes Cell Proliferation and Tumorigenesis

Recent studies identified the adaptor protein Ajuba as a positive regulator of Yes-associated protein (YAP) oncogenic activity through inhibiting large tumor suppressor (Lats1/2) core kinases of the Hippo pathway, a signaling pathway that plays important roles in cancer. In this study, we define a novel mechanism for phospho-regulation of Ajuba in mitosis and its biological significance in cancer. We found that Ajuba is phosphorylated in vitro and in vivo by cyclin-dependent kinase 1 (CDK1) at Ser119 and Ser175 during the G2/M phase of the cell cycle. Mitotic phosphorylation of Ajuba controls the expression of multiple cell cycle regulators; however, it does not affect Hippo signaling activity, nor does it induce epithelial-mesenchymal transition. We further showed that mitotic phosphorylation of Ajuba is sufficient to promote cell proliferation and anchorage-independent growth in vitro and tumorigenesis in vivo. Collectively, our discoveries reveal a previously unrecognized mechanism for Ajuba regulation in mitosis and its role in tumorigenesis.

KIBRA promotes prostate cancer cell proliferation and motility

KIBRA is a regulator of the Hippo‐yes‐associated protein (YAP) pathway, which plays a critical role in tumorigenesis. In the present study, we show that KIBRA is a positive regulator in prostate cancer cell proliferation and motility. We found that KIBRA is transcriptionally upregulated in androgen‐insensitive LNCaPC4‐2 and LNCaP‐C81 cells compared to parental androgen‐sensitive LNCaP cells. Ectopic expression of KIBRA enhances cell proliferation, migration and invasion in both immortalized and cancerous prostate epithelial cells. Accordingly, knockdown of KIBRA reduces migration, invasion and anchorage‐independent growth in LNCaP‐C4‐2/C81 cells. Moreover, KIBRA expression is induced by androgen signaling and KIBRA is partially required for androgen receptor signaling activation in prostate cancer cells. In line with these findings, we further show that KIBRA is overexpressed in human prostate tumors. Our studies uncover unexpected results and identify KIBRA as a tumor promoter in prostate cancer.

CDK1 phosphorylation of TAZ in mitosis inhibits its oncogenic activity

The transcriptional co-activator with PDZ-binding motif (TAZ) is a downstream effector of the Hippo tumor suppressor pathway, which plays important roles in cancer and stem cell biology. Hippo signaling inactivates TAZ through phosphorylation (mainly at S89). In the current study, we define a new layer of regulation of TAZ activity that is critical for its oncogenic function. We found that TAZ is phosphorylated in vitro and in vivo by the mitotic kinase CDK1 at S90, S105, T326, and T346 during the G2/M phase of the cell cycle. Interestingly, mitotic phosphorylation inactivates TAZ oncogenic activity, as the non-phosphorylatable mutant (TAZ-S89A/S90A/S105A/T326A/T346A, TAZ-5A) possesses higher activity in epithelial-mesenchymal transition, anchorage-independent growth, cell migration, and invasion when compared to the TAZ-S89A mutant. Accordingly, TAZ-5A has higher transcriptional activity compared to the TAZ-S89A mutant. Finally, we show that TAZ-S89A or TAZ-5A (to a greater extent) was sufficient to induce spindle and centrosome defects, and chromosome misalignment/missegregation in immortalized epithelial cells. Together, our results reveal a previously unrecognized connection between TAZ oncogenicity and mitotic phospho-regulation.

Cyclin-dependent kinase 1 (CDK1)-mediated mitotic phosphorylation of the transcriptional co-repressor Vgll4 inhibits its tumor-suppressing activity

The Hippo pathway is an evolutionarily conserved signaling pathway that plays important roles in stem cell biology, tissue homeostasis, and cancer development. Vestigial-like 4 (Vgll4) functions as a transcriptional co-repressor in the Hippo-Yes-associated protein (YAP) pathway. Vgll4 inhibits cell proliferation and tumor growth by competing with YAP for binding to TEA-domain proteins (TEADs). However, the mechanisms by which Vgll4 itself is regulated are unclear. Here we identified a mechanism that regulates Vgll4s tumor-suppressing function. We found that Vgll4 is phosphorylated in vitro and in vivo by cyclin-dependent kinase 1 (CDK1) during antimitotic drug-induced mitotic arrest and also in normal mitosis. We further identified Ser-58, Ser-155, Thr-159, and Ser-280 as the main mitotic phosphorylation sites in Vgll4. We also noted that the nonphosphorylatable mutant Vgll4-4A (S58A/S155A/T159A/S280A) suppressed tumorigenesis in pancreatic cancer cells in vitro and in vivo to a greater extent than did wild-type Vgll4, suggesting that mitotic phosphorylation inhibits Vgll4s tumor-suppressive activity. Consistent with these observations, the Vgll4-4A mutant possessed higher-binding affinity to TEAD1 than wild-type Vgll4. Interestingly, Vgll4 and Vgll4-4A markedly suppressed YAP and β-catenin signaling activity. Together, these findings reveal a previously unrecognized mechanism for Vgll4 regulation in mitosis and its role in tumorigenesis.

CDK1-mediated mitotic phosphorylation of PBK is involved in cytokinesis and inhibits its oncogenic activity

PDZ-binding kinase (PBK) plays a major role in proliferation and in safeguarding mitotic fidelity in cancer cells. Frequently upregulated in many cancers, PBK drives tumorigenesis and metastasis. PBK has been shown to be phosphorylated in mitosis by cyclin-dependent kinase 1 (CDK1)/cyclin B, however, no studies have been done examining PBK mitotic phosphorylation in oncogenesis. Additionally to the previously identified Threonine-9 phosphorylation, we found that Threonine-24, Serine-32, and Serine-59 of PBK are also phosphorylated. PBK is phosphorylated in vitro and in cells by CDK1 during antimitotic drug-induced mitotic arrest and in normal mitosis. We demonstrated that mitotic phosphorylation of Threonine-9 is involved in cytokinesis. The non-phosphorylatable mutant PBK-T9A augments tumorigenesis to a greater extent than wild type PBK in breast cancer cells, suggesting that PBK mitotic phosphorylation inhibits its tumor promoting activity. The PBK-T9A mutant also transforms and increases the proliferation of immortalized breast epithelial cells. Collectively, this study reveals that CDK1-mediated mitotic phosphorylation of PBK is involved in cytokinesis and inhibits its oncogenic activity.