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Angiogenesis plays a significant role in cancer by providing increased blood supply to the affected tissues and thus bringing in growth factors, cytokines, and various nutrients for tumor growth. VEGF is the most prominent angiogenic agent that is markedly induced in cancer. Induction of VEGF has been widely studied but as cancer cells are quite adept at acquiring new alternative processes to circumvent surrounding environmental pressures, our understanding of the molecular mechanisms regulating VEGF expression in cancer, especially in triple-negative breast cancer cells, remains incomplete. Here, we present evidence of a novel mode of VEGF induction in triple-negative MDA-MB-231 breast cancer cells that is regulated by serum amyloid A activating factor 1 (SAF-1) transcription factor. Inhibition of SAF-1 by antisense short hairpin RNA profoundly reduces VEGF expression along with reduction in endothelial cell proliferation and migration. By both in vitro and in vivo molecular studies, we show that the effect of SAF-1 is mediated through its direct interaction with the VEGF promoter. In correlation, DNA-binding activity of SAF-1 is found to be significantly higher in MDA-MB-231 breast cancer cells. Examination of several breast cancer samples further revealed that SAF-1 is overexpressed in clinical breast cancer tissues. Taken together, these findings reveal that SAF-1 is a hitherto unrecognized participant in inducing VEGF expression in triple-negative breast cancer cells, an aggressive form of breast cancer that currently lacks effective treatment options. Suppression of SAF-1 activity in these cells can inhibit VEGF expression, providing a possible new method to control angiogenesis. Mol Cancer Res; 9(8); 1030–41. ©2011 AACR.

A disintegrin and metalloproteinase-12 (ADAM-12), a member of multifunctional family of proteins, is upregulated in many cancers, including breast, lung, liver, prostate, gastric, and bladder. The multidomain structure, composed of a prodomain, a metalloproteinase, disintegrin-like, epidermal growth factor–like, cysteine-rich and transmembrane domains, and a cytoplasmic tail, allows ADAM-12 to promote matrix degradation, cell-cell adhesion, and intracellular signaling capacities and thereby to play a critical role in cancer growth and metastasis. Despite ample evidence linking increased ADAM-12 expression with cancer, the mechanisms controlling its upregulation are still unknown. In the present study, transforming growth factor-β1 (TGF-β1) is shown to increase ADAM-12 mRNA expression in MDA-MB-231 breast carcinoma cells. We have identified a promoter element responsible for TGF-β1–mediated ADAM-12 induction. We show interaction of NF-κB with ADAM-12 promoter and that high level of NF-κB activity in breast carcinoma cells results in the upregulation of ADAM-12 expression. Site-directed mutagenesis of the NF-κB element in ADAM-12 promoter and inhibition of NF-κB activity by Bay-11-7085 and MG-132 significantly reduced TGF-β1–mediated increase of ADAM-12 promoter-driven gene expression. Transfection of cells with a dominant-negative mutant form of IκBα (IκBαΔN), which inhibits activation of NF-κB, significantly reduced transcription from ADAM-12 promoter-reporter in TGF-β1–stimulated MDA-MB-231 cancer cells. In correlation, overexpression of NF-κB induced ADAM-12 expression in a dose-dependent manner. DNA-binding and ChIP assays indicated that p65 subunit of NF-κB binds to ADAM-12 promoter. Together, our study identified a cellular mechanism for induction of ADAM-12, which involves NF-κB and its activation by TGF-β1. Mol Cancer Res; 8(9); 1261–70. ©2010 AACR.