RESUMEN
BACKGROUND: In the present study we determined the relative contribution of two processes to breast cancer progression: (1) Intrinsic events, such as activation of the Ras pathway and down-regulation of p53; (2) The inflammatory cytokines TNFα and IL-1ß, shown in our published studies to be highly expressed in tumors of >80% of breast cancer patients with recurrent disease. METHODS: Using MCF-7 human breast tumor cells originally expressing WT-Ras and WT-p53, we determined the impact of the above-mentioned elements and cooperativity between them on the expression of CXCL8 (ELISA, qRT-PCR), a member of a "cancer-related chemokine cluster" that we have previously identified. Then, we determined the mechanisms involved (Ras-binding-domain assays, Western blot, luciferase), and tested the impact of Ras + TNFα on angiogenicity (chorioallantoic membrane assays) and on tumor growth at the mammary fat pad of mice and on metastasis, in vivo. RESULTS: Using RasG12V that recapitulates multiple stimulations induced by receptor tyrosine kinases, we found that RasG12V alone induced CXCL8 expression at the mRNA and protein levels, whereas down-regulation of p53 did not. TNFα and IL-1ß potently induced CXCL8 expression and synergized with RasG12V, together leading to amplified CXCL8 expression. Testing the impact of WT-Ras, which is the common form in breast cancer patients, we found that WT-Ras was not active in promoting CXCL8; however, TNFα has induced the activation of WT-Ras: joining these two elements has led to cooperative induction of CXCL8 expression, via the activation of MEK, NF-κB and AP-1. Importantly, TNFα has led to increased expression of WT-Ras in an active GTP-bound form, with properties similar to those of RasG12V. Jointly, TNFα + Ras activities have given rise to increased angiogenesis and to elevated tumor cell dissemination to lymph nodes. CONCLUSIONS: TNFα cooperates with Ras in promoting the metastatic phenotype of MCF-7 breast tumor cells, and turns WT-Ras into a tumor-supporting entity. Thus, in breast cancer patients the cytokine may rescue the pro-cancerous potential of WT-Ras, and together these two elements may lead to a more aggressive disease. These findings have clinical relevance, suggesting that we need to consider new therapeutic regimens that inhibit Ras and TNFα, in breast cancer patients.