Date of Award

1-1-2022

Embargo Period

4-22-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Drug Discovery and Biomedical Sciences

College

College of Graduate Studies

First Advisor

Eduardo N. Maldonado

Second Advisor

Stephen Duncan

Fourth Advisor

Monika Gooz

Fifth Advisor

Yuri K. Peterson

Sixth Advisor

Danyielle Townsend

Abstract

The contribution of mitochondria to cancer cell metabolism is a continuously growing research field. Enhanced glycolysis and partial suppression of mitochondrial metabolism, reciprocally dependent in many tumors, characterize the pro-survival and proliferative Warburg phenotype. Voltage dependent anion channels (VDAC), in the outer mitochondrial membrane (OMM), regulate the flux of most respiratory substrates entering and exiting mitochondria. Thus, VDAC functions as a switch to turn-on (when open), and off (when closed), mitochondrial metabolism. Previous work from our lab showed that VDAC modulates mitochondrial metabolism in cancer cells, high cytosolic free tubulin closes VDAC, and the synthetic VDAC-binding molecule erastin, antagonize the inhibitory effect of tubulin on the channel. Here, it is demonstrated that mitochondrial dysfunction leading to cell death after the VDAC openers erastin/erastin-like compounds, or the chemotherapeutic agent sorafenib, is induced by a ROS-dependent translocation of activated JNK to mitochondria. Mitochondrial membrane potential (ΔΨm) was used as a global readout of mitochondrial function and observed heterogeneity of ΔΨm, a phenomenon previously described only qualitatively. Using an advanced confocal microscopy approach, ΔΨm was quantified among cells in absolute values and proved that ΔΨm heterogeneity is an intrinsic property of mitochondria. In addition, this quantitative approach to assess ΔΨm by imaging, we developed a novel, sensitive and fast method to assess ΔΨm, NADH generation and electron flow in permeabilized and intact cells using a combination of plate reader and respirometry assays was developed. This method provides fast-tracked medium throughput outcomes in phenotypic assays of mitochondria. Finally, VDAC was used as a pharmacological target to identify in silico small molecules that bind to the NADH-binding pocket of VDAC. NADH binding to the pocket closes the channel so occupying this region with another molecule would prevent VDAC closure by endogenous regulators, maintaining the channel in an open configuration. A lead compound was identified that binds to VDAC, induce mitochondrial dysfunction, and promote cell death. Overall, the work described here contributes to a better understanding of cancer metabolism, provides novel methods to study mitochondrial metabolism, and initiates a potentially highly relevant translational application of VDAC modulation into cancer treatment.

Rights

All rights reserved. Copyright is held by the author.

Download

Share

COinS