Doctor of Philosophy (PhD)
Multiple Phos-tag-based screens were conducted in anti-tubulin drug (paclitaxel and nocodazole)-treated cells to explore novel targets implicated in cell cycle regulation, resistance against paclitaxel, and yes-associated protein (YAP) nucleocytoplasmic transport. The Phos-tag-based screen of protein kinases identified that several proteins were degraded or phosphorylated in response to anti-tubulin drug treatment. A tyrosine kinase, fibroblast growth factor receptor 4 (FGFR4), was significantly degraded upon anti-tubulin drug treatment, suggesting its potential role in cell response to paclitaxel. Further functional investigations identified that FGFR4 mediated paclitaxel resistance in ovarian cancer, and specific inhibitors targeting FGFR4 could sensitize ovarian cancer cells to paclitaxel. Mechanistically, FGFR4 modulated paclitaxel resistance by regulating the expression of an anti-apoptotic protein, Bcl-xL. In addition, Protein kinase N1 (PKN1) was noted with prominent phosphorylation during anti-tubulin drug-induced mitotic arrest from the same screening. Subsequent studies validated that PKN1 can be phosphorylated at Ser533/Ser537, Ser562, and Ser916 by cyclin-dependent kinase 1 (CDK1) during mitosis. Of importance, PKN1 mitotic phosphorylation at the above four sites was required for the anchorage-independent growth and migration of ovarian cancer cells. These findings nominated FGFR4 and PKN1 as attractive therapeutic targets for ovarian cancer treatment. Another Phos-tag-based screen in anti-tubulin drug-treated cells was conducted to explore the importin involved in the nucleocytoplasmic transport of YAP, an oncoprotein implicated in pancreatic ductal adenocarcinoma (PDAC) pathogenesis. Importin β1 and importin αs were highly expressed in PDAC and correlated with adverse clinical outcomes. Further functional studies identified the critical roles of importin α1 (KPNA2), importin α5 (KPNA1), and importin α7 (KPNA6) in the maintenance of PDAC cell viability. These importin αs were all phosphorylated upon anti-tubulin drug treatment, and a CDK1-mediated phosphorylation site on KPNA2 was determined. Moreover, double KPNA1 and KPNA2 depletion significantly downregulated YAP expression and increased YAP cytoplasmic sequestration, indicating the regulation of importin αs on YAP activity. This study suggested the promising clinical prospect of targeting the importin α/YAP axis in pancreatic cancer therapy.
Zeng, Renya, "Phos-Tag-Based Screens Identify Novel Therapeutic Targets in Ovarian Cancer and Pancreatic Cancer" (2022). Theses & Dissertations. 650.
Available for download on Sunday, April 28, 2024