Abstract P5-01-02: Single cell RNA transcriptomics reveals tumor promoting mammary cell subpopulation upon replication stress in BRCA1 mutant breast cancer mouse model
Authors: Pathania Shailja , Rivera Joshua , Khalid Delan , Manne Monica , Tran Stevenson , Kibaja Kemmie , Li Carman MC , Brugge Joan 
Published: March 2022

Abstract
Women with BRCA1 (B1) mutation have an exceptionally high risk of developing breast cancer. The only effective preventive strategy currently offered to these women is the life altering prophylactic mastectomy. In light of limited treatment options available, it is critical that new therapeutic and preventive strategies be identified. Design of such strategies requires an understanding of early events in the breast cells that drive tumorigenesis. B1 heterozygous mouse models can help us identify these early changes in mammary tissue as the cells become tumor cells. However, despite the well-established association between B1 heterozygosity and cancer predisposition in humans, there are currently no such B1 mouse models that faithfully recapitulate this high risk of tumor formation. B1 heterozygous mice are not tumor-prone. This makes it difficult to use these models to study the role of B1 heterozygosity and to identify early tumor promoting changes in the breast tissue. We have now established a mouse model that induces mammary tumors in B1 heterozygous (Brca1wt/flx,Trp53flx/flx,K14cre) mice upon replication stress (RS), thus giving us a tool to study early tumor promoting changes in B1 heterozygous breast tissue. Our approach is based on our published work that reveals haploinsufficiency for RS suppression in B1 heterozygous cells. Given the importance of RS development in tumorigenesis, this effect would be a logical contributor to B1 mutant cancer development. Indeed, increasing RS in B1 heterozygous mice resulted in accelerated mammary tumorigenesis. RS in this mouse model was delivered by injecting DNA-adduct forming 4-nitroquinoline-1-oxide via mammary intraductal injection over a course of 7 weeks. RS served as an efficient and abnormally rapid driver of tumor formation (30 days post completion of injection regimen) in B1 heterozygous, but not B1 wild type mice. B1 heterozygous mice formed mammary adenocarcinoma and ductal carcinoma in-situ. Immunofluorescence based tissue section analysis and transcriptomic analysis reveals that adenocarcinomas formed in B1 heterozygous mice carry a basal epithelial phenotype like those found in human breast cancer. Such an accelerated tumor model system could prove to be invaluable in understanding the earliest events in B1 mutant breast cancer.Furthermore, our scRNAseq-based analysis has revealed early changes that occur in the breast tissue as different cell types (luminal and basal) respond to RS, and have identified new cell populations that emerge exclusively in B1 heterozygous mammary tissue undergoing RS. For this analysis, cells were collected from naïve mammary tissue, tissue collected midway through injections, and post-tumor tissue. This analysis identified a unique population of trans-differentiated cells expressing prognostic markers that have correlation to poor outcome in human breast cancer. This RS-induced mammary cell population in B1 heterozygous tissue also expresses both luminal progenitor and basal epithelial markers. Interestingly, this population was enriched for proliferation markers like Top2a, Ube2c, mKi67, and Ccnb2. Given that such proliferation markers are a hallmark of cancer stem cells, we suspect that this transdifferentiated population, which is primarily enriched in B1 heterozygous mammary tissue undergoing RS, marks some of the early cancer promoting changes in the breast tissue.Altogether, our integrative approach reveals that B1 heterozygosity in combination with RS leads to accumulation and proliferation of a specific mammary cell population that contributes to breast tumorigenesis. Identification of such early drivers is critical for the design of effective preventive and therapeutic strategies for women with B1 mutation.
Citation Format: Shailja Pathania, Joshua Rivera, Delan Khalid, Monica Manne, Stevenson Tran, Kemmie Kibaja, Carman MC Li, Joan Brugge. Single cell RNA transcriptomics reveals tumor promoting mammary cell subpopulation upon replication stress in BRCA1 mutant breast cancer mouse model [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-02.