Date of Award

2019

Embargo Period

6-24-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell and Molecular Pharmacology and Experimental Therapeutics

College

College of Graduate Studies

First Advisor

Elizabeth Yeh

Second Advisor

Robin Muise-Helmericks

Third Advisor

Joe Blumer

Fourth Advisor

Scott Eblen

Fifth Advisor

Besim Ogretmen

Abstract

Despite the availability of targeted therapeutics, the development of resistance to HER2 inhibitors represents a significant barrier to the treatment of HER2+ breast cancers. To enhance the efficacy of these inhibitors, identifying novel molecular targets which facilitate tumorigenesis and resistance is essential. HUNK (Hormonally Upregulated Neu-associated Kinase) is a protein kinase which has been shown to promote HER2+ mammary tumorigenesis in a kinase-dependent fashion. Previous studies also indicate HUNK upregulates autophagy in a HER2+ breast cancer model that is resistant to HER2 inhibitors, though the mechanism for this regulation currently remains unknown. We have recently identified staurosporine (STU) as a HUNK kinase inhibitor, and demonstrate that STU treatment mimics HUNK depletion in a mammary tumor-derived cell model. We found that STU shows synergistic effects with the HER2 inhibitor lapatinib in colony formation, mammosphere formation, and tumor formation in a resistant HER2+ breast cancer cell line. We then used STU as a HUNK inhibitor to identify potential HUNK substrates which regulate autophagy. Because our findings suggested HUNK kinase activity was required for autophagy regulation, we hypothesized that HUNK phosphorylates autophagy-related proteins to upregulate autophagy. We report for the first time that the negative autophagy regulator Rubicon is a novel substrate of HUNK. Using a phospho-deficient Rubicon mutant, we found that HUNK phosphorylates Rubicon on its RUN domain, and that this phosphorylation event is associated with increased autophagic vesicles using confocal microscopy. Collectively, this work has identified the first inhibitor of HUNK enzymatic activity, as well as the first reported HUNK substrate. Because upregulated autophagy is implicated in HER2+ breast cancer resistance as a cytoprotective mechanism, HUNK phosphorylation of Rubicon may promote cytoprotective autophagy to facilitate HER2+ breast tumorigenesis and acquired resistance.

Rights

All rights reserved. Copyright is held by the author.

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