Date of Award

2018

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pathology and Laboratory Medicine

College

College of Graduate Studies

First Advisor

Stephen P. Ethier

Second Advisor

Robin C. Muise-Helmericks

Third Advisor

Bartholomeus Smith

Fourth Advisor

Dennis Watson

Fifth Advisor

Steven Rosenzweig

Abstract

A subset of basal breast cancer patients has tumors overexpressing EGFR, loss of PTEN expression and mutated p53; these alterations cooperate and result in a poor prognosis for these patients. PTEN is a direct negative regulator of PI3’Kinase enzymatic activity and loss of PTEN expression is correlated with elevated PI3’Kinase pathway activation. In the presented dissertation, we utilized a panel of PTEN-null basal breast cancer cell lines containing constitutive AKT activation and elevated levels of EGFR to model this subset of breast cancer patients. We first investigated the response of AKT phosphorylation to PTEN re-expression in the cell line panel. We found that PTEN re-expression failed to reverse the constitutive AKT activation present in the HCC-38, SUM-149 and MDA-MB-436 cells; therefore, we used pharmacological inhibitors of PI3’Kinase pathway mediators to inhibit AKT phosphorylation. We found that pan class I PI3’Kinase inhibition (BKM120) and pan mTOR inhibition (KU0063794) failed to ablate AKT phosphorylation in most of the cells within the panel. Furthermore, we found that the SUM-149 and MDA-MB-436 cell lines were resistant to the allosteric AKT inhibitor, MK2206, which led to the observation that these cells harbor elevated levels of AKT3 gene and protein expression. shRNA mediated knockdown of AKT3 revealed that AKT3 was contributing to the resistance of AKT phosphorylation in the SUM-149 cells to inhibition of PI3’Kinase pathway mediators. Immunoprecipitation of AKT1 and AKT3 after treatment of the SUM-149, MDA-MB-436 and HCC-1937 cell lines with inhibitors of PI3’Kinase pathway mediators and PI3’Kianse isoform specific inhibitors, revealed that AKT1 phosphorylation is regulated by canonical PI3’Kinase signaling mechanism and that AKT3 is regulated by non-canonical PI3’Kinase signaling, with emphasis on the class II PI3’Kinases as playing an important role in this regulation. In addition, we demonstrate that PTEN re-expression in PTEN-null basal breast cancer cell lines results in restoration of canonical PI3’Kinase signaling as the primary driver of AKT activation. We conclude that non-canonical PI3’Kinase signaling is activated in PTEN null basal breast cancer cells expressing AKT3, and that this regulation can be reverted to canonical signaling by restoration of PTEN activity.

Rights

All rights reserved. Copyright is held by the author.

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