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Significance Although germline heterozygous BRCA1 mutations predispose human carriers to cancer, heterozygous mouse BRCA1 mutations do not. We find that exposure to a source of upper gastrointestinal replication stress (RS) elicited a marked cancer incidence in Brca1+/−;Trp53+/− heterozygous mice but not in wild-type mice (Brca1+/+;Trp53+/−). Oral delivery of 4 nitroquinoline-1-oxide induced esophageal epithelial RS, an increased esophageal mutation rate, loss of esophageal Brca1 heterozygosity (LOH), and accelerated esophageal tumorigenesis. These data underscore the necessity of combining otherwise nontumorigenic BRCA1 heterozygosity with simultaneous induction of RS for the generation of not only BRCA1 mutant esophageal cancer, but also a dramatically accelerated form thereof. These results strongly imply that RS is both a major contributor to BRCA1 cancer development and a marked accelerant thereof., BRCA1 germline mutations are associated with an increased risk of breast and ovarian cancer. Recent findings of others suggest that BRCA1 mutation carriers also bear an increased risk of esophageal and gastric cancer. Here, we employ a Brca1/Trp53 mouse model to show that unresolved replication stress (RS) in BRCA1 heterozygous cells drives esophageal tumorigenesis in a model of the human equivalent. This model employs 4-nitroquinoline-1-oxide (4NQO) as an RS-inducing agent. Upon drinking 4NQO-containing water, Brca1 heterozygous mice formed squamous cell carcinomas of the distal esophagus and forestomach at a much higher frequency and speed (∼90 to 120 d) than did wild-type (WT) mice, which remained largely tumor free. Their esophageal tissue, but not that of WT control mice, revealed evidence of overt RS as reflected by intracellular CHK1 phosphorylation and 53BP1 staining. These Brca1 mutant tumors also revealed higher genome mutation rates than those of control animals; the mutational signature SBS4, which is associated with tobacco-induced tumorigenesis; and a loss of Brca1 heterozygosity (LOH). This uniquely accelerated Brca1 tumor model is also relevant to human esophageal squamous cell carcinoma, an often lethal tumor.

Women with mutations in breast cancer (BrCa) gene BRCA1 are highly predisposed to BrCa. In these families, cancer develops almost exclusively in females and often at very young ages. The molecular pathogenic steps, especially the earliest ones that drive the transition of normal mammary epithelial cells (BRCA1mut/+) to tumor cells, are largely unknown. Mouse model-based studies have not successfully addressed this question, in part because, unlike women with BRCA1 (B1) mutations, B1 heterozygous mice do not form mammary tumors any faster than the wild-type (wt) mice. A mouse model that can biologically mimic what is happening in the mammary tissue of B1 mutation carriers would be an invaluable tool to study B1 mutant BrCa. Furthermore, most of the efforts to study breast tumor-associated gene changes have been based on GWAS studies or RNA-seq based analysis of the breast tumor samples. Such studies have shown that the transcriptional network is altered in the breast tumors compared to wild-type tissue. However, whether those changes are tumor initiating, i.e., “driver” changes, or are “passenger” mutations/changes is still not clear. Given the role of B1 in suppressing replication stress (RS), a tumor-promoting event, and the increased RS observed in B1 heterozygous cells, we have established an RS-driven B1 BrCa mouse model to study the early genetic changes that drive B1 mutant cancer. We find that intraductal injections of 4-nitroquinoline-1-oxide (4NQO1, a replication stress-inducing agent) leads to breast adenocarcinoma in B1flx/wt;Trp53flx/flx,K14Cre mice on average 84 days post injections unlike B1 wt mice that do not make tumors. This is the first RS-induced BrCa model that can successfully recapitulate B1 mutant BrCa. The fast timeline of tumor formation provides significant advantage for studying tumor formation, progression, and response to therapy. Furthermore, single-cell RNA sequencing analysis of the breast adenocarcinoma and tissue collected early during injections in these mice has revealed presence of a highly proliferative “transdifferentiated population” that shares properties of both luminal and basal cells (e.g., K8+K18+) and also exists in the hyperplastic tissue. No such population was found in control PBS injected mammary fat pad of B1flx/wt;Trp53flx/flx,K14Cre mice, indicating that this population might be dependent on RS. We also find that this population is enriched very early during mammary tumorigenesis and some of the top hits include cancer stem cell markers. We have also confirmed the presence of this population by immune histochemistry-based experiments. Finally, our study provides evidence for a BrCa mouse model where basal-like tumor formation is driven by RS. We also found evidence for an early precancerous transdifferentiated population that is enriched upon RS and have identified early markers that can potentially drive breast tumorigenesis in B1 mutant BrCa. Identification of such candidate genes is critical in the design of future preventive and/or therapeutic strategies. Citation Format: Joshua Rivera, Delan Khalid, Stevenson Tran, Kemmie Kibaja, Shailja Pathania. Tracking the path of breast tumorigenesis in BRCA1 mutant breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B36.