Abstract 268: Differential regulation of MMP promoters by E2F transcription factors: Potential role of c-MYC and Id1

Abstract

Abstract The retinoblastoma tumor suppressor, Rb, is the major negative regulator of E2F-1 transcription factor, and the Rb-E2F pathway is altered in most cancers. Further, many oncogenic mutations initiate tumors by targeting the Rb-E2F pathway. E2Fs regulate genes involved in a variety of biological processes, including angiogenesis, apoptosis, and differentiation, but it is unclear whether these molecules contribute to cancer cell invasion and metastasis. To investigate the role of E2F in metastasis, we analyzed the promoters of matrix metalloproteinase genes (MMPs), which are major contributors to the invasion and migration programs. We find that many MMP genes had potential E2F binding sites; we focused on MMP2, MMP9, MMP14, and MMP15 promoter, which have multiple E2F binding sites. Chip assays showed Rb, and E2Fs 1-5 could bind to the each MMP promoter. Transient transfection experiments with MMP promoter-luciferase constructs showed thatMMP9, MMP14, and MMP15-luc promoters were induced by E2Fs, whereas the MMP2 promoter was repressed by E2F1-5 in A549, and H1650 NSCLC cell lines. QRT-PCR showed that MMP2 was upregulated in A549 cells transfected with an siRNA targeting E2F1 and E2F3, whereas other MMPs were downregulated. To determine if MMP2 was repressed in an Rb-dependent manner, A549 cells stably expressing shRNA targeting Rb or a non-targeting control were transiently transfected with MMP2-luc construct and E2F1. In both cases, the MMP2-luc promoter was repressed by E2F1, suggesting that the repression is Rb-independent. Further, cells transfected with MMP2-luc promoter and E2F1 had less luciferase activity than cells transfected with MMP2 alone, independent of BRG-1, YY1, HDAC1, prohibitin-1, mSIN3a, or SUV39h1. Further, depletion of these co-repressors did not enhance MMP2 mRNA levels when compared to cells depleted of E2F1 alone. To determine if this repression is through binding site competition, we examined the effect of depleting transcription factors that have potential binding sites in the MMP2 promoter and were in close proximity to the E2F binding sites. We found that depletion of c-Myc and ID1 significantly enhanced MMP2 luciferase activity, and there was less repression from E2F1 in transient transfection experiments. These data suggest that E2F family members can repress the MMP2 promoter through mechanisms that may involve c-MYC and ID1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 268. doi:10.1158/1538-7445.AM2011-268