Abstract 2177: A novel protein interaction between Smad4 and NRF1 establishes a molecular switch on p15Ink4b expression

Abstract

Abstract We have been investigating TGFβ signaling particularly concentrating on Smad4 as a key element in this biologically important pathway in cancer. In order to demonstrate the novel molecular ways in regulation of Smad4 in tumorigenesis, we previously have performed a baculovirus protoarray and identified nuclear respiratory factor 1 (NRF1) as one of the putative interacting protein partners of Smad4. NRF1 is the first mammalian transcriptional factor reported to act on nuclear respiratory genes. Other than mitochondrial respiratory function and biogenesis, NRF1 has also been associated with a subset of E2F-responsive genes, suggesting a role in DNA replication, mitosis and cytokinesis. In this study, we firstly demonstrated the novel interaction between NRF1 and Smad4 via using bimolecular fluorescence complementation (BiFC) assay. NRF1 amino-terminal domain, carrying a nuclear localization signal, was found to interact with Smad4 and direct the heterodimer into nucleus. Subsequently, to investigate further the role of NRF1 on Smad4 expression, a manual search of probable NRF1 binding sites on Smad4 promoter was carried out and putative sequences were identified. Upon ectopic expression of NRF1, luciferase reporter assays as well as western blot analysis resulted in decrements of both Smad4 transcriptional activity and protein expression. The core binding region of NRF1 was discovered within -186/-175 nucleotide region of Smad4 promoter via utilizing a site-directed mutagenesis approach. Thirdly, to further substantiate the role of Smad4 and NRF1's interaction in tumorigenesis, p15INK4b, a cell cycle regulator, was identified as a putative common target of both NRF1 and Smad4 through a comparison among previously reported subsets of target promoters. Indeed, the induction of p15INK4b promoter activity by Smad4 was found to be diminished to a basal level by overexpression of NRF1. Furthermore, the nature of this inhibition was shown to be both dose and time dependent in the presence of NRF1. Under cell synchronized conditions, both transcriptional factors were shown to bind p15INK4b promoter region in close proximity, whereas NRF1 was found to slightly inhibit Smad4's binding to its regulatory region when cells proceed in cell cycle. Finally, a computational analysis of gastric and breast cancer microarray expression public data on other putative common targets of NRF1 and Smad4 revealed a negative correlation between the effects of these two transcriptional factors on gene expressions. Thus, NRF1 appears to negatively interact with Smad4 and inhibit p15INK4b expression, through which it suggests a possible role in tumorigenesis. We will be representing our current findings in this research. 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 2177. doi:10.1158/1538-7445.AM2011-2177