TLR9 Downregulation in Breast Cancer: Its Role in Tumor Immunity, Inflammatory Response, and Cellular Senescence

Introduction: Toll-like receptor 9 (TLR9) is primarily expressed in human dendritic and B cells and recognizes double-stranded DNA motifs from pathogens to initiate an inflammatory response. Recent studies have revealed TLR9s' involvement beyond its conventional role in the immune response, notably during the tumorigenesis of various cancers such as head and neck, cervical, and ovarian cancers. Methods: In this study patient biopsies of breast cancer tumors and normal breast epithelium were analyzed by immunohistochemistry to examine TLR9 expression. The study also investigated downregulation in transformed breast cancer cell lines compared to untransformed breast epithelial cells by analyzing gene or protein expression, including TLR9, IL-6, CCL2, CXCL1, and GM-CSF. MDA-MB-361 cells were engineered to express exogenous TLR9, and the effects on colony growth and senescence were assessed using colony formation assays, senescence staining, cytokine analysis, and flow cytometry. Results: TLR9 levels in breast cancer tumors were significantly reduced compared to normal breast tissue epithelium. This downregulation was also observed in several transformed breast cancer cell lines compared to untransformed breast epithelial cell lines. Furthermore, MDA-MB-361 breast cancer cells expressing exogenous TLR9 exhibited reduced colony growth and an increase in the senescence marker IL-6, pro-inflammatory cytokine CCL2, CXCL1 chemokine; and growth factor GM-CSF. Conclusion: These findings support TLR9’s regulatory role in mitigating breast cancer and highlight its critical connection between the innate immunity and tumor cell growth.