The protein homeostasis network in breast cancer therapeutic response

The protein homeostasis network restores homeostasis and enhances cell survival upon exposure to diverse proteotoxic stresses and is co-opted by tumor cells to survive the myriad stresses associated with cancer. Indeed, the heat-shock response, unfolded protein response, oxidative stress response, and autophagy are activated in breast cancers and are strongly associated with poor clinical outcomes. Since this network is poised to both sense and shape the cellular environment in which small molecule drug targets operate, activation of this network may influence the breast cancer therapeutic response landscape. Our preliminary data indicates that loss of heat-shock factor 1 (HSF1) alters the drug response of transformed cells. Here, we will systematically assess the effect of silencing the major regulators of the protein homeostasis network in breast cancer cells and examine how these perturbations affect therapeutic response. To avoid limiting our study to a single cell line, we will develop an innovative strategy that employs next-generation sequencing to monitor the viability of barcoded mixtures of cell lines exposed to a library of cancer drugs. By co-culturing the cell lines in pools, we observed that MDA-MB-231 cells outcompete ZR-75-1, SKBR3, HCC-38, and BT-20 cells over four weeks. We used the relative cell abundances over time to mathematically model and experimentally validate the optimal initial seeding densities so that each cell line will be similarly represented in the drug screen. We are currently introducing doxycycline-inducible Cas9 into the cell lines, testing for functional Cas9 activity, screening multiple guide RNAs for each of the ten genes we plan to target, and testing whether the guide RNAs remain functional when barcoded. Overall, this study aims to improve our understanding of the protein homeostasis network in cancer biology. It will also reveal novel pharmacogenetic interactions in breast cancer that can be utilized for diagnostic and therapeutic benefit.