Abstract 2221: Obesity influences the tissue resident breast microbiota impacting complement signaling and tumorigenesis

Abstract Obesity is a risk factor for breast cancer, influencing both the incidence and prognosis of the disease. Obesity alters the breast microbiome in ways that may promote breast carcinogenesis, and microbiome perturbations are evident in breast cancer patients. Yet, it is unclear if these microbial perturbations are drivers of breast cancer. Our group previously showed that diet and anti-cancer therapies modify the breast tissue microbiome. We further showed that certain commensal bacterial species in the mammary gland reduce tumorigenesis in high-fat diet-fed MMTV-PyMT mice, implicating tissue-resident microbiota in modifying breast cancer risk. Thus, we investigated the impact of obesity-induced microbiome alterations on genomic instability in mammary epithelial cells, as a hallmark of breast cancer risk. Our preliminary studies showed that obesity caused elevated systemic and breast tissue levels of lipopolysaccharide (LPS) and flagellin. In vitro and in vivo experiments demonstrated their ability to induce DNA damage in the mammary epithelium. 16S rRNA sequencing on DNA isolated from non-cancerous breast tissue revealed an enrichment of Proteobacteria in obese premenopausal subjects. Bacteria from this phylum harbors highly immunogenic LPS and the majority of them are flagellated. Importantly, Proteobacteria proportional abundance was associated with breast tissue levels of DNA damage. Intriguingly, these associations were absent in postmenopausal women. One key difference between breast tissues of the two menopausal groups was in their complement activity and immune responses to LPS and flagellin. Redox proteomics analysis showed that obesity caused an enrichment in the oxidation of several complement C3 peptides in both menopausal groups. Normally, oxidation of complement C3 causes its activation through the generation of a C3 convertase that cleaves C3 into the active fragments; C3a and C3b. However, this was only observed in premenopausal women where the C3 oxidized peptides showed significant correlations with C3a levels. Complement system dysfunction in postmenopausal women was also manifested by having lower C3a levels, higher B-cell trafficking and lower anti-LPS IgA levels in their breast tissues than premenopausal women. Ongoing experiments are being performed to determine if there are breast tissue differences in complement regulatory proteins such as factor H and CD59. To demonstrate causality, we performed a carcinogen-induced mammary tumorigenesis model in wild-type (WT) and C3 knockout (KO) mice fed a low-fat or high-fat diet. C3 KO mice showed increased tumor-incidence compared to WT in the context of a high-fat diet, but not a low-fat diet, suggesting a role of complement signaling in anti-tumor immunity in the context of obesity. Overall, this work identifies a novel immunity-microbiome signaling axis potentiating obesity-mediated breast cancer risk. Citation Format: Mohamed Gaber, Adam S. Wilson, Adam J. Katz, Karen Haas, Katherine L. Cook. Obesity influences the tissue resident breast microbiota impacting complement signaling and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2221.