Access Type

Open Access Dissertation

Date of Award

January 2015

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pathology

First Advisor

Q. Ping Dou

Abstract

AMPK activation and Ubiquitin Proteasome System (UPS) inhibition have gained great attention as therapeutic strategies for the treatment of certain types of cancers. While AMPK serves as a master regulator of cellular metabolism, UPS regulates protein homeostasis. Although the crosstalk between them is suggested, the relationship between these two important pathways is not very clear. We observed that proteasome inhibition leads to AMPK activation in human breast cancer cells. We report that a variety of proteasome inhibitors activate AMPK in all of the tested cancer cell lines. Our data using Liver Kinase B1 (LKB1)-deficient cancer cells suggests that proteasome inhibitor-induced AMPK activation is primarily mediated by Calcium/Calmodulin-dependent Kinase Kinase β (CaMKKβ). This hypothesis is supported by pharmacological or genetic inhibition of CaMKKβ that leads to a decrease in proteasome inhibitor-induced AMPK activation. Additionally, the AMPK-activating function of the FDA-approved proteasome inhibitor bortezomib depends on an increase in intracellular calcium levels as calcium chelation abrogates its induced AMPK activation. Finally, bortezomib-mediated upregulation in CaMKKβ levels is due to its enhanced protein synthesis.

It has been shown that proteasome inhibition leads to disturbances in intracellular calcium homeostasis. Hence we hypothesized that calcium channel blockers could be used to sensitize solid tumors to proteasome inhibitors. Although effective for the treatment of hematological malignancies, the FDA approved proteasome inhibitors bortezomib and carfilzomib have limited efficacy in solid tumors including triple negative breast cancer (TNBC). Since absence of specific therapeutic targets, chemotherapy is the only option for treating TNBC. Therefore, development of new TNBC therapeutic strategies has been warranted. When verapamil, a L-type calcium channel blocker with P-glycoprotein (P-gp) and CYP3A4 inhibitory properties, is combined with MG132 or bortezomib or carfilzomib, the cytotoxic effects and apoptosis in TNBC MDA-MB-231 cells were enhanced, compared to each treatment alone. Furthermore, addition of verapamil improved proteasome-inhibitory properties of MG132, bortezomib or carfilzomib in MDA-MB-231 cells, as shown by the increased accumulation of ubiquitinated proteins and proteasome substrates such as IκBα and p27kip1. Additionally, when nicardipine, another P-gp inhibitor, was combined with bortezomib or carfilzomib, enhanced inhibition of MDA-MB-231 cell proliferation was observed. These findings indicate that P-gp inhibitors could sensitize TNBC cells to structurally and functionally diverse proteasome inhibitors and might provide new treatment strategy for TNBC.

Taken together, the studies presented in this dissertation will help to identify additional targets for proteasome inhibitors as well as their crosstalk and might help in designing new combination treatments for difficult to treat solid tumors like TNBC.

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