Abstract 2964: Transcriptional regulation of manganese superoxide dismutase gene reveals a novel mechanism by which FUS/TLS functions as a tumor suppressor
Authors: Sanjit K. Dhar, Edward Kasarskis, Haining Zhu, Bert C. Lynn, Daret K. St. Clair
Journal: Cancer Research (2012)

Abstract Fused in Sarcoma/Translocation in Lipo-Sarcoma (FUS/TLS), also known as hetero-nuclear ribonucleo-protein, is an evolutionarily conserved nuclear protein that possesses RNA/DNA binding capability and has diverse functions, including gene transcription. Although FUS/TLS as a FUS-CHOP fusion protein is implicated as playing a role in Sarcoma, recent studies demonstrate that FUS may also play an important role in prostate cancer malignancy, leading to the suggestion that FUS can function as a new type of tumor suppressor gene. Previously, we demonstrated that nucleophosmin (NPM), a RNA binding protein, plays a very important role in coactivating Sp1- and NF-κB- mediated mitochondria-localized manganese superoxide dismutase (MnSOD) gene transcription. In this study, we have identified FUS as an additional RNA binding protein that physically interacts with NPM by the use of coimmunoprecipitation followed by proteomic analysis. To elucidate the role of FUS on MnSOD gene transcription, prostate cancer cells (PC3) were transfected with FUS expression vector along with a full-length MnSOD promoter construct, and MnSOD reporter gene activity, mRNA and protein levels were assessed. The results demonstrate that overexpression of FUS increases MnSOD gene transcription in a dose-dependent manner. Consistent with the role of FUS as a transcription coregulator of MnSOD expression, knock-down of FUS by FUS siRNA leads to inhibition of MnSOD gene transcription. To further elucidate whether FUS is directly associated with MnSOD promoter or enhancer, we generated a series of MnSOD deletion constructs linked to the luciferase reporter gene and the effects of FUS overexpression on the reporter gene activity of those deletion constructs were investigated. The results demonstrate that the far upstream region of the MnSOD promoter is important for FUS -mediated gene transcription. Chromatin Immunoprecipitation (ChIP) assay and affinity chromatography show that FUS binds to the MnSOD promoter and interacts with Sp1. The binding of FUS to MnSOD promoter is further confirmed by Surface Plasmon Resonance (SPR) analysis. SPR data demonstrate that FUS binds with the MnSOD promoter in a sequence specific manner with a Kd of 1.08 x 10−7 M, whereas mutations in the promoter sequences drastically reduce the binding affinity (Kd of 3.37 x 10−4 M). Mutation in FUS abrogates promoter binding activity, leading to significant loss of MnSOD transcription. These results identify FUS as a key regulator of MnSOD and suggest that disruption of FUS may lead to the loss of normal cellular antioxidant capacity that is associated with the development of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2964. doi:1538-7445.AM2012-2964