Tumor-Stroma-Inflammation Networks Promote Aggressiveness in TNBC by Activating the Notch Pathway

Triple-negative breast cancer (TNBC) is an aggressive disease with poor prognosis and high recurrence rate. The lack of known targetable molecules for this specific disease subtype emphasizes the great need for improved understanding of the mechanisms driving TNBC progression. Components of the tumor microenvironment play key roles in promoting disease progression in TNBC; thus, here, we took an integrative approach in which we investigated the combined effects of tumor-stroma-inflammation cross-talks on the pro-metastatic phenotype of TNBC cells. Major focus was put on tumor-promoting functions and molecular events exerted by TNBC cells in the presence of mesenchymal stem cells (MSCs) or patient-derived cancer-associated fibroblasts (CAFs), and their regulation by the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin 1β (IL1β) that are enriched in TNBC tumors.

Our findings indicate that co-cultures of TNBC:stroma cells (MSCs, CAFs) stimulated by TNFα, expressed exacerbated levels of the pro-metastatic chemokines CXCL8, CCL2 and CCL5, largely as a result of direct physical contacts between the tumor and stromal cells; similar findings were obtained in IL-1β-stimulated TNBC:stroma co-cultures. Functionally, TNBC-MSC-TNFα networks increased endothelial cell migration and sprouting, and enhanced invasive and migratory properties of the tumor cells. Furthermore, these elevated pro-metastatic activities were reversible and largely depended on CXCL8-induced signaling, as indicated by using CXCL8 siRNA. Importantly, TNFα stimulation of TNBC:MSC co-cultures has increased the aggressiveness of TNBC cells in vivo, leading to higher incidence of mice with lung metastases .

Mechanistically, we found that activation of the Notch pathway has driven forward the migratory and invasive properties gained by TNBC cells that formed physical contacts with stromal cells in the presence of TNFα. Moreover, by using siRNA/CRISPR approaches, we demonstrated that TNFα has induced p65 activation in TNBC:MSC “Contact” co-cultures, which then has led to Notch1 activation; in turn, Notch1 up-regulated the expression of CXCL8 by the tumor-stroma-inflammation network. Consequently, CXCL8 promoted angiogenesis, as well as TNBC cell migration and invasion.

Overall, our findings identify novel findings on p65-Notch1-mediated tumor-stroma-inflammation interactions that promote pro-metastatic characteristics in TNBC; these findings pose the pro-inflammatory elements CXCL8/p65 and Notch1 as candidate targets whose combined inhibition may prevent metastasis-promoting activities of the most aggressive subtype of breast cancer.