Abstract B29: An allelic series of rat mutations reveal a role of TOX3 in mammary gland development, obesity, and breast cancer susceptibility
Authors: Shunkwiler Lauren B. , Pipaliya Royal , Ashy Cody C. , Peel Benjamine Van , Zhao Yang , Guz Jan , Awgulewitsch Alexander , Kern Michael J. , Smits Bart M.G. 
Published: August 2018

Abstract
With the rising incidence of breast cancer cases in the population, it is imperative to understand mechanisms of susceptibility. Genome-wide association studies (GWAS) have identified many variants associated with breast cancer susceptibility. These loci have low penetrance and are common in the population. Some of the alleles are associated with specific subtypes of breast cancer and/or associate specifically within ancestral populations. Variants in the TOX3/LOC643714 locus on human chromosomal band 16q12.1 are strongly associated with risk to estrogen receptor-positive (ER+) breast cancer in cohorts of women of European ancestry. Remarkably, replication of the association of these same variants with breast cancer risk in women of African American ancestry failed, but other variants in a more distal area of the locus were found to be associated with risk. The correlated polymorphisms comprising these variants exist in noncoding regions, providing the possibility that these polymorphisms alter gene expression. The most likely causal gene, TOX3, encodes a transcription factor that has previously been found to regulate estrogen receptor alpha (ERα)-responsive genes in breast cancer cells. We hypothesize that noncoding breast cancer-associated polymorphisms on 16q12.1 regulate TOX3, subsequently modifying risk of developing ER+ breast cancer.
To genetically dissect this locus, we implemented the CRISPR-Cas9 genetic engineering system in the rat model organism. The rat is the preferred rodent model for ER+ breast cancer. We engineered an allelic series of mutations generating rats with deletions in the portion of the Tox3 locus orthologous to the human risk-associated regions. We obtained viable mutants across all genotypes, despite partial embryonic lethality in homozygous Tox3 knockouts. The mutants in the allelic series display variable levels of Tox3 downregulation in the mammary gland, suggesting the presence of multiple Tox3-regulatory elements. Mutants showing altered Tox3 expression also show significant effects on mammary gland development, implicating a role for Tox3 in mammary stem/progenitor cell biology potentially through ERα gene regulation. In accordance with the association of the human risk-increasing allele with lower TOX3 transcript level, preliminary data from mammary carcinogenesis experiments in our rat model indicate that lower Tox3 levels result in increased mammary carcinoma multiplicity. This result suggests that Tox3 is a breast cancer susceptibility gene. Interestingly, Tox3 knockout rats present an obesity phenotype, male and female sterility, and a behavioral phenotype (increased anxiety), indicating pleiotropic effects of TOX3 loss-of-function mutations.
Ongoing studies are focused on Tox3 in rat mammary gland biology and carcinogenesis to elucidate the mechanism of Tox3 regulation in susceptibility to ER+ breast cancer. Understanding mechanisms of susceptibility genes will ultimately lead to innovative strategies aimed at preventing breast cancer.
Citation Format: Lauren B. Shunkwiler, Royal Pipaliya, Cody C. Ashy, Benjamine Van Peel, Yang Zhao, Jan Guz, Alexander Awgulewitsch, Michael J. Kern, Bart M.G. Smits. An allelic series of rat mutations reveal a role of TOX3 in mammary gland development, obesity, and breast cancer susceptibility [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B29.