Metastatic cancers remain an important problem for cancer medicine. Transforming growth factor beta 1 (TGF-b1) is a potent tumor suppressor, but, paradoxically, TGF-b1 promotes tumor spreading in advanced cancers. Given the dual function of TGF-b1, it is important to develop therapies that specifically suppress oncogenic function of TGF-b1. The current study identified specific components of the TGF-b1 pathway contributing to the oncogenic function of TGF-b1 in cancer cells. We show that TGF-β signaling in breast cancer MDA-MB-231 cells is critical for tumor angiogenesis and invasion. Studies in orthotopic and subcutaneous xenograft models showed that disruption of autocrine TGF-b1 signaling in tumor cells by dominant-negative TGF-b type I receptor ALK5-K232R (DN-ALK5) reduces metastasis and, importantly, the micro-vascular density. In contrast, active ALK5 enhances tumor angiogenesis and reduces tumor cell apoptosis. Our study identified matrix metalloproteinase MMP-9/gelatinase-B as a critical TGF-b1 target in tumor angiogenesis. Suppression of MMP-9 by RNA interference (RNAi) in tumor cells delayed tumor growth and reduced tumor neovasculature and metastasis. The analysis of the signaling pathways revealed that MEK-ERK is required for up-regulation of MMP-9 by TGF-β-ALK5 signaling, whereas JNK, p38 MAPK, and Smad4 are dispensable. Adenoviral transduction of dominant-negative MEK1 blocked and constitutively active MEK1 enhanced expression of MMP-9. In contrast, inhibition of p38MAPK by DN-p38alpha or RNAi-p38alpha, JNK by kinase inhibitors, or Smad4 by RNAi had no effect. Given that TGF-β-Smad4 signaling functions as tumor suppressor, our studies open the possibility for selective suppression of TGF-β-mediated tumor angiogenesis and metastasis by targeting MAPK and MMP-9. Our studies also suggest that the ALK5-MEK-MMP-9 pathway enhances recruitment of vascular cells (endothelial cells, pericytes, myofibroblasts) from stromal compartments. To test whether stromal cells can rescue the defect in tumor angiogenesis of DN-ALK5 xenografts, tumor cells were placed subcutaneously alone or in combination with non-tumorigenic stromal cells. The studies showed that stromal cells restored tumor growth and angiogenesis in the DN-ALK5 xenografts to the control levels. MRI analysis of tumor xenografts confirmed the increase in blood flow in tumors containing stromal cells. Thus, the TGF-b1-ALK5-MEK-MMP-9 pathway in cancer cells promotes tumor growth and metastasis by enhancing tumor angiogenesis via recruitment of vasculature-forming cells from stromal compartments. Novel approaches for suppression of TGF-b-mediated tumor angiogenesis and metastasis will be discussed.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA