Cyclin-dependent kinase 1-mediated AMPK phosphorylation regulates chromosome alignment and mitotic progression.

AMP-activated protein kinase (AMPK), a heterotrimeric serine/threonine kinase and cellular metabolic sensor, has been found to regulate cell cycle checkpoints in cancer cells in response to energetic stress, to harmonize proliferation with energy availability. Despite AMPK's emergent association with the cell cycle, it still has not been fully delineated how AMPK is regulated by upstream signaling pathways during mitosis. We report, for the first time, direct CDK1 phosphorylation of both the catalytic α1 and α2 subunits, as well as the ß1 regulatory subunit, of AMPK in mitosis. We found that AMPK-knockout U2OS osteosarcoma cells have reduced mitotic indexes and that CDK1 phosphorylation-null AMPK is unable to rescue the phenotype, demonstrating a role for CDK1 regulation of mitotic entry through AMPK. Our results also denote a vital role for AMPK in promoting proper chromosomal alignment, as loss of AMPK activity leads to misaligned chromosomes and concomitant metaphase delay. Importantly, AMPK expression and activity was found to be critical for paclitaxel chemosensitivity in breast cancer cells and positively correlated with relapse-free survival in systemically treated breast cancer patients.

Zyxin promotes colon cancer tumorigenesis in a mitotic phosphorylation-dependent manner and through CDK8-mediated YAP activation.

Zyxin is a member of the focal adhesion complex and plays a critical role in actin filament polymerization and cell motility. Several recent studies showed that Zyxin is a positive regulator of Yki/YAP (Yes-associated protein) signaling. However, little is known about the mechanisms by which Zyxin itself is regulated and how Zyxin affects Hippo-YAP activity. We first showed that Zyxin is phosphorylated by CDK1 during mitosis. Depletion of Zyxin resulted in significantly impaired colon cancer cell proliferation, migration, anchorage-independent growth, and tumor formation in xenograft animal models. Mitotic phosphorylation is required for Zyxin activity in promoting growth. Zyxin regulates YAP activity through the colon cancer oncogene CDK8. CDK8 knockout phenocopied Zyxin knockdown in colon cancer cells, while ectopic expression of CDK8 substantially restored the tumorigenic defects of Zyxin-depletion cells. Mechanistically, we showed that CDK8 directly phosphorylated YAP and promoted its activation. Fully activated YAP is required to support the growth in CDK8-knockout colon cancer cells in vitro and in vivo. Together, these observations suggest that Zyxin promotes colon cancer tumorigenesis in a mitotic-phosphorylation-dependent manner and through CDK8-mediated YAP activation.

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 Vgll4's 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 Vgll4's 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 ß-catenin signaling activity. Together, these findings reveal a previously unrecognized mechanism for Vgll4 regulation in mitosis and its role in tumorigenesis.

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 Ser(119) and Ser(175) 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.

A Second Drug Binding Site in P2X3.

Purinergic P2X3 receptors form trimeric cation-gated channels, which are activated by extracellular ATP. P2X3 plays a crucial role in chronic cough and affects over 10% of the population. Despite considerable efforts to develop drugs targeting P2X3, the highly conserved structure within the P2X receptor family presents obstacles for achieving selectivity. Camlipixant, a potent and selective P2X3 antagonist, is currently in phase III clinical trials. However, the mechanisms underlying receptor desensitization, ion permeation, principles governing antagonism, and the structure of P2X3 when bound to camlipixant remain elusive. In this study, we established a stable cell line expressing homotrimeric P2X3 and utilized a peptide scaffold to purify the complex and determine its structure using cryo-electron microscopy (cryo-EM). P2X3 binds to camlipixant at a previously unidentified drug-binding site and functions as an allosteric inhibitor. Structure-activity studies combined with modeling and simulations have shed light on the mechanisms underlying the selective targeting and inhibition of P2X3 by camlipixant, distinguishing it from other members of the P2X receptor family.

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.

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.

The hippo pathway effector YAP regulates motility, invasion, and castration-resistant growth of prostate cancer cells.

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).