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theses

TP53 is the most commonly mutated gene in cancer; therefore, small molecule restoration of wild type structure and function to mutant p53 (hereafter referred to as “reactivation”) is a highly sought-after goal in cancer therapeutics. Mutations that impair the p53 protein’s ability to bind zinc (zinc deficient mutants) are exceedingly prevalent in cancer. Zinc metallochaperones (ZMCs) are a class of small molecules that reactivate zinc deficient p53 mutants by restoring zinc binding. ZMCs raise intracellular zinc levels by functioning as zinc ionophores, causing mutant p53 to refold properly, which we have termed the “ON” switch. Here we have investigated the cancer cell’s response to this zinc surge and demonstrate that within hours, cellular zinc homeostatic mechanisms normalize zinc levels leading to mutant p53 misfolding and inactivation of the drug, which we have termed the “OFF” switch. This ON/OFF switch imparts a pulsatile nature to the mechanism of ZMCs which indicates that only a brief exposure of a ZMC is necessary for its p53-mediated cell kill. Cell growth inhibition experiments in which we varied the time of exposure to ZMC1 demonstrate an on-target effect in as little as 15 minutes. This switch mechanism is unique in cancer therapeutics and imparts an advantage over other targeted therapeutics in that efficacy can be achieved with minimal exposure which minimizes toxicity and maximizes the therapeutic window. We have extended our understanding of the role of cellular zinc homeostatic mechanisms in ZMC function to serving as a source of innate resistance. HCC1395 is a human triple-negative breast cancer cell line that expresses a zinc-deficient mutant p53 (p53-R175H) but is resistant to ZMC1 withno evidence of refolding of the p53-R175H. We observe that the ZMC1-induced zinc kinetics are markedly reduced when compared to a ZMC1-sensitive cell line. Expression of several cytosolic zinc binding proteins are higher in the resistant cell line, and knockdown of their expression markedly increases the zinc levels in response to ZMC1. Supplementation of ZnCl2 results in a large zinc influx which causes refolding of p53-R175H and sensitizes the cells to ZMC1.Together these data support the hypothesis that cellular zinc homeostatic mechanisms function as an “OFF” switch to regulate the mechanism of ZMCs and also contribute to innate resistance to ZMCs. The latter indicates that high metallothionein levels may serve as a biomarker of resistance to ZMCs.

ETD_9040

Ph.D.

Includes bibliographical references

by Samuel Kogan

doi:10.7282/t3-t89p-7725

ETD doctoral

The author owns the copyright to this work.