Publication Date

2014-11-03

Availability

Embargoed

Embargo Period

2016-04-26

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Microbiology and Immunology (Medicine)

Date of Defense

2014-10-29

First Committee Member

Samita Andreansky

Second Committee Member

Diana Lopez

Third Committee Member

Eckhard Podack

Fourth Committee Member

Julio Barredo

Fifth Committee Member

Balakrishna Lokeshwar

Sixth Committee Member

Regina Graham

Abstract

Overexpression of the HER-2/neu oncoprotein occurs in approximately 30% of breast cancer cases and is associated with a more aggressive tumor phenotype and poor prognosis for patients. Current treatment strategies are effective in a subset of patients however a significant number of patients relapse due to acquired mechanisms of resistance to current therapy. Alternative approaches, that utilize multiple modes of action, are therefore necessary to combat this severe form of breast cancer. Natural compounds are notorious for their multiple mechanisms of action, which make them ideal therapeutics for complex diseases such as cancer. Withaferin A (WA) is a purified product of Withania somnifera, (commonly known as Ashwaghandha or Indian ginseng) which has been part of Indian ayurvedic medicine for many centuries. Several studies indicate that WA targets several cellular pathways that result in apoptosis of tumor cells by various mechanisms. Using a murine model of HER-2/neu breast cancer and human breast cancer cell lines that express the HER-2 oncoprotein to varying degrees, we investigated the mechanisms by which WA impacts HER-2/neu breast cancer. Our studies indicate that WA exhibits selective potency towards HER-2/neu overexpressing cells. We show that the mechanism of WA induced cell death in HER-2/neu breast cancer is multimodal with WA functioning as both an immunotherapeutic and targeted chemotherapeutic drug. WA induces an immunogenic form of cell death through induction of ER-stress and the unfolded protein response leading to activation of anti-tumor immunity. Systemic administration of WA acts directly at the tumor site and modulates the microenvironment by increasing the numbers of mature dendritic cells, macrophages, CD4 T-cells and B-cells as well as depletes exhausted PD1 expressing CD8 T-cells from the tumor site. In its role as a targeted chemotherapeutic, WA downregulates intracellular and cell surface expression of the HER-2/neu protein. We show that the mechanism of action is related to WA mediated disruption of the HSP90/CDC37 complex by direct targeting of the CDC37 protein, leading to inhibition of HER-2/neu stability and activation. Additionally, WA targets the HER-3 molecule, which also plays a role in maintenance of the tumor phenotype in HER-2/neu breast cancer. Taken together, these studies provide novel mechanisms of action of WA and will serve as a means of developing this natural compound as an adjuvant therapeutic strategy for HER-2/neu cancers not amenable to standard treatments.

Keywords

HER-2/neu breast cancer, Withaferin A, immunogenic cell death, HSP90, CDC37, tumor immunology

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