CRISPRa screen identifies a role for c-KIT signaling in tamoxifen resistance, potentially through upregulation of ABC transporters

Abstract

Resistance to endocrine therapy is a common problem in patients with estrogen receptor alpha (ER) positive breast cancer. In this study, we took a non-biased genome-wide approach to identify novel mechanisms of endocrine resistance using a clustered regularly interspaced short palindromic repeats (CRISPR) activating (CRISPRa) screen. Results from the screen identified 109 candidate resistance-associated genes, with several of these genes, such as EGFR and SRC, having been previously associated with endocrine resistance. One candidate gene that has not been previously associated with endocrine resistance is the tyrosine kinase receptor, c-KIT. We further tested for associations between c-KIT and endocrine resistance and found that c-KIT overexpressing cells proliferate more rapidly in the presence of tamoxifen compared to control cell lines. To gain deeper insight into the potential role of c-KIT signaling in tamoxifen resistance, we next performed precision nuclear run-on and sequencing (PRO-seq) analysis of c-KIT overexpressing cells to identify downstream factors that may mediate the c-KIT response. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the overexpressed genes found that the only class of factors that was significantly induced by c-KIT was the ATP-binding cassette (ABC) transporters; specifically, ABCA1, ABCA4, and ABCG1. Interestingly, overexpression of two of these ABC transporters, ABCA1 and ABCG1, significantly correlated with worse prognosis in ER+ breast cancer patients following endocrine therapy. We then tested for potential therapeutic effects of c-KIT inhibition on endocrine resistance and found that the c-KIT inhibitor Gleevec appears to synergize with tamoxifen to suppress MCF-7-S cell growth. Together, our findings support the hypothesis that c-KIT signaling promotes endocrine resistance via the induction of ABC transporter activity. Additionally, our studies suggest that inhibition of c-KIT signaling may represent a novel strategy for preventing or overcoming endocrine resistance in ER+ patients.