Triple Negative Breast Cancer: Notch1-mediated Mechanisms Regulate Tumor-Stroma-Inflammation Networks That Promote Disease Aggressiveness

Introduction: The triple negative subtype of breast cancer (TNBC) is an aggressive disease, in which components of the tumor microenvironment (TME) play key roles in promoting disease progression. Here, based on published studies and on our analyses of patient datasets, we took an integrative approach in which we investigated the pro-metastatic phenotypes of TNBC cells that were exposed to signals delivered by stromal cells, and of the pro-inflammatory TME that resides in TNBC tumors.

Methods and Results: Our findings indicate that TNBC:stroma co-cultures stimulated by the pro-inflammatory cytokine TNFα, expressed exacerbated levels of pro-metastatic chemokines such as CXCL8, largely depending on direct physical contacts between the tumor and the stromal cells. Functionally, TNBC-stroma-inflammation networks have led to increased expression of angiogenic factors, elevated endothelial cell sprouting, tumor cell invasion and migration. Importantly, studies in animal model systems indicated that TNFα stimulation of co-cultures in-vitro has increased the aggressiveness of the TNBC cells in-vivo, leading to higher incidence of mice with lung micro-metastases. Using specific inhibitors, siRNAs, mRNA and patient datasets analyses, we identified complex regulatory roles of the Notch signaling pathway in the cytokine-induced enhanced migratory and invasive properties gained by the tumor cell. Moreover, we identified TNBC-expressed Notch1 as a prime regulator of the contact-dependent CXCL8 induction taking place in TNFα-stimulated TNBC:stroma co-cultures. Using CRISPR/siRNA to p65 in TNBC and/or stromal cells, we found that in TNFα-stimulated TNBC:stroma co-cultures, p65 activation in both TNBC cells and in stromal cells, was required for CXCL8 expression. Furthermore, p65 was also found to be a major regulator of Notch1 activation in TNBC cells, resulting from direct tumor-stromal cells contacts and upon TNFα stimulation.

Conclusions: Overall, our findings identify novel players that regulate processes of tumor-stroma-inflammation interactions that promote the pro-metastatic characteristics of TNBC cells, and pose pro-inflammatory factors and Notch1 as candidate targets whose combined inhibition may prevent metastasis-promoting activities at the TME of the most aggressive subtype of breast cancer.