Graduation Date

Spring 5-8-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pathology & Microbiology

First Advisor

Jixin Dong

Abstract

The Hippo pathway is an evolutionarily conserved signaling pathway that plays important roles in stem cell biology, tissue homeostasis, and cancer development. The Hippo core signaling pathway features a kinase cascade consisting of mammalian sterile-20 like protein 1/2 (Mst1/2) and large tumor suppressor 1/2 (Lats1/2). Inactivation of the Hippo pathway is correlated with the promotion of proliferation and anti-apoptosis through activation of the transcriptional co-activator Yes-associated protein (YAP). YAP functions through binding with TEA-domain transcription factors (TEAD1–4) to activate target genes.

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 non-phosphorylatable 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 a higher-binding affinity to TEAD1 than wild-type Vgll4. Interestingly, Vgll4 and Vgll4-4A markedly suppressed YAP and -catenin signaling activity. Together, these findings reveal a previously unrecognized mechanism for Vgll4 regulation in mitosis and its role in tumorigenesis.

Paclitaxel is one of the anti-tubulin drugs and has been widely used in ovarian, breast, non-small cell lung cancers, and recently pancreatic cancer. Despite their wide use in cancer treatment, the patient response rate is still low, and drug resistance is a major clinical obstacle. Through a Phos-tag-based kinome-wide screen, we identified microtubule affinity-regulating kinase 2 (MARK2) as a critical regulator for Paclitaxel chemosensitivity in pancreatic ductal adenocarcinoma (PDAC). We show that MARK2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) in response to anti-tubulin chemotherapeutics and in unperturbed mitosis in a kinase activity-independent manner. Phosphorylation is essential for MARK2 in regulating mitotic progression and Paclitaxel cytotoxicity in PDAC cells. Mechanistically, our findings also suggest that MARK2 controls Paclitaxel chemosensitivity by regulating class IIa histone deacetylase (HDACs). MARK2 directly phosphorylates HDAC4 specifically during anti-tubulin treatment. Phosphorylated HDAC4 promotes YAP activation and controls the expression of YAP target genes induced by Paclitaxel. Importantly, a combination of HDAC inhibition (by knockdown or Vorinostat) and Paclitaxel overcomes chemoresistance in preclinical PDAC animal models. Furthermore, the expression levels of MARK2, class IIa HDACs, and YAP are upregulated and positively correlated in PDAC patients. Inhibition of MARK2 or class IIa HDACs potentiates Paclitaxel cytotoxicity by inducing mitotic abnormalities in PDAC cells. Together, our findings identify the MARK2-HDAC axis as a druggable target for overcoming chemoresistance in PDAC.

Download

Share

COinS