Notch-Mediated Tumor-Stroma-Inflammation Networks Promote Invasive Properties and CXCL8 Expression in Triple-Negative Breast Cancer

Stromal cells and pro-inflammatory cytokines play key roles in promoting the aggressiveness of triple-negative breast cancers (TNBC; Basal/Basal-like). In our previous study we demonstrated that stimulation of TNBC and mesenchymal stem cells (MSCs) co-cultures by the pro-inflammatory cytokine tumor necrosis factor alpha (TNF alpha) has led to increased metastasis-related properties in vitro and in vivo. In this context, elevated release of the pro-metastatic chemokines CXCL8 (IL-8) and CCL5 (RANTES) was noted in TNF alpha- and interleukin-1 beta (IL-1 beta)-stimulated TNBC:MSC co-cultures; the process was partly (CXCL8) and entirely (CCL5) dependent on physical contacts between the two cell types. Here, we demonstrate that DAPT, inhibitor of gamma-secretase that participates in activation of Notch receptors, inhibited the migration and invasion of TNBC cells that were grown in Contact co-cultures with MSCs or with patient-derived cancer-associated fibroblasts (CAFs), in the presence of TNF alpha. DAPT also inhibited the contact-dependent induction of CXCL8, but not of CCL5, in TNF alpha- and IL-1 beta-stimulated TNBC:MSC/CAF co-cultures; some level of heterogeneity between the responses of different TNBC cell lines was noted, with MDA-MB-231:MSC/CAF co-cultures being the most sensitive to DAPT. Patient dataset studies comparing basal tumors to luminal-A tumors, and mRNA analyses of Notch receptors in TNBC and luminal-A cells pointed at Notch1 as possible mediator of CXCL8 increase in TNF alpha-stimulated TNBC:stroma Contact co-cultures. Accordingly, down-regulation of Notch1 in TNBC cells by siRNA has substantially reduced the contact-dependent elevation in CXCL8 in TNF alpha- and also in IL-1 beta-stimulated TNBC:MSC Contact co-cultures. Then, studies in which CXCL8 or p65 (NF-kappa B pathway) were down-regulated (siRNAs; CRISPR/Cas9) in TNBC cells and/or MSCs, indicated that upon TNF alpha stimulation of Contact co-cultures, p65 was activated and led to CXCL8 production mainly in TNBC cells. Moreover, our findings indicated that when tumor cells interacted with stromal cells in the presence of pro-inflammatory stimuli, TNF alpha-induced p65 activation has led to elevated Notch1 expression and activation, which then gave rise to elevated production of CXCL8. Overall, tumor:stroma interactions set the stage for Notch1 activation by pro-inflammatory signals, leading to CXCL8 induction and consequently to pro-metastatic activities. These observations may have important clinical implications in designing novel therapy combinations in TNBC.