Dissecting Signaling Pathways Regulating Tumor Angiogenesis And Metastasis in the Breast Carcinoma Microenvironment
Limoge, Michelle Kay
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Breast cancer is the most commonly diagnosed cancer and second leading cause of cancer-related death in women in the United States. Limited treatment options are available for patients with triple-negative breast cancers, recurrent and metastatic disease. Accumulating evidence implicates the tumor microenvironment (TME) in breast cancer progression and response to therapy. However, an increased understanding of the interactions between the cellular components of the TME and what mediates those interactions is required. Once identified, these mediators may be viable as therapeutic targets. The objective of this work was to explore the interaction of tumor cells and stromal fibroblasts, the main cellular component of the TME, in breast carcinoma growth using in vivo and in vitro co-culture models. Cancer-associated fibroblasts (CAFs) have been linked to the progression of breast cancer and tumor growth. CAFs have been shown to secrete numerous cytokines including TGFβ, TNFα and IL1β. MMP9, an important factor in angiogenesis, can be regulated by these cytokines. We investigated the contribution of fibroblasts to tumor growth and asked if the mechanism involved crosstalk of cytokines. In a mouse xenograft model, co-injection of tumor cells and fibroblasts increased tumor growth and increased the diameter of the tumor vasculature compared to tumor cells alone. Tumoral MMP9 was found to be a key player in this response. Investigation into the mechanism in vitro, in a direct co-culture model, revealed that tumor-fibroblast co-cultures increase expression of cytokines which can act together upon tumor cells to induce expression of MMP9. This cooperation does not occur through cross-activation of canonical signaling, but through co-stimulation of MAPK-AP1 signaling which together with TNFα-induced TAK1-IKK-NFκB were found to be essential components for MMP9 regulation. Elevated levels of p38 signaling are found in breast carcinomas and this has been correlated with tumor progression. p38 signaling has been linked to breast cancer invasion, EMT and metastasis and may be involved in MMP9 regulation. Interestingly, p38 can also act as a tumor suppressor. A further understanding of the role of p38 in tumor growth and regulation of the TME is required. We have demonstrated that p38 signaling within tumor cells is critical for breast carcinoma growth, vascularization and invasive and metastatic capacities. Overexpression of a dominant-negative form of p38 (dnp38) in tumor cells showed reduced growth and metastasis and decreased tumor blood vessels. Interestingly, the increase observed in tumor xenograft growth and vascularization seen upon co-injection of fibroblasts with tumor cells were negated by expression of dnp38 in the tumor cells. We found that p38 signaling contributes to the expression of pro-angiogenic extracellular factors such as fibronectin and VEGFA, IL8 and HBEGF cytokines. Co-culturing tumor cells with fibroblasts resulted in an increase in these factors, which was negated by expression of dnp38 in tumor cells. Given the importance of the TME to breast cancer pathogenesis, we sought to further our understanding of the interaction of tumor cells and fibroblasts in the TME. We identified novel ways that fibroblasts can contribute to tumor growth and vascularization by stimulating cytokine signaling crosstalk and p38 activity in tumor cells. Several important mediators have been identified as potential targets of breast cancer therapeutics.