Chromatin , Gene , and RNA Regulation MnSOD Promotes Tumor Invasion via Upregulation of FoxM 1 – MMP 2 Axis and Related with Poor Survival and Relapse in Lung Adenocarcinomas

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme responsible for the elimination of superoxide radical. The role of MnSOD in tumor progression in different human cancers is still controversial. In the present study, MnSOD expression in lung cancer cells was explored by knockdown or overexpression using transfection of a short hairpin RNA (shRNA) or an expression vector, respectively, to determine whether MnSOD expression mediates lung cancer cell migration, invasion, and oncogenic potential by increasing FoxM1 and MMP2 expression. Western blotting showed that FoxM1 and MMP2 expression was dependent on MnSOD expression, suggesting that FoxM1 could be upregulated by MnSOD. Three FoxM1 promoters were constructed to verify this activation of FoxM1 by MnSOD and to determine the transcription factors responsible. Luciferase reporter and chromatin immunoprecipitation assays indicated that MnSOD overexpression in lung cancer cells promoted binding of E2F1 and Sp1 to their putative FoxM1 promoter-binding sites and activated FoxM1 reporter activity. MnSOD also enhanced the potential for cell migration, invasion, and anchorage-independent colony growth on soft-agar plates, again via upregulation of FoxM1 and MMP2 expression. In patients with lung cancer, evaluation of MnSOD expression in lung tumors by immunohistochemistry indicated a positive correlation between FoxM1 and MMP2 mRNA expressions. Kaplan–Meier and Cox regression analysis revealed a poorer overall survival (OS) and relapse-free survival (RFS) in patients with MnSOD-positive tumors than with MnSOD-negative tumors. We conclude that MnSOD may promote tumor aggressiveness via upregulation of the FoxM1–MMP2 axis, and that MnSOD expression can independently predict survival and relapse in patients with resected lung adenocarcinoma. Mol Cancer Res; 11(3); 261–71. 2012 AACR. Introduction The manganese superoxide dismutase (MnSOD) is an important antioxidant enzyme that eliminates superoxide radical (O2 ) by converting it into H2O2 (1). MnSODmay also function as a tumor suppressor; for example, it may reduce growth of tumors associated with melanoma (2), pancreatic adenocarcinoma (3, 4), colorectal cancer (5), ovarian cancer (6), breast cancer (7), and multiple myeloma (8). Conversely, MnSOD may promote tumor progression in cancers, such as fibrosarcoma (9), gastric cancer (10, 11), glioblastoma (12), T-cell lymphoma (13), breast cancer (14, 15), cervical cancer (16), prostate cancer (17), and lung cancer (18). Therefore, the precise role of MnSOD in tumorigenesis remains elusive. MnSOD-dependent H2O2 production is involved in the regulation of expression of matrix metalloproteinases (MMP; refs. 19, 20), which are major contributors to the stromal degradation associated with tumor invasion. Zhang and colleagues showed activation of MMP2 by MnSOD in human breast cancer MCF-7 cells via the regulation of intracellular reactive oxygen species (ROS; ref. 21). A study on immunodeficient mice showed that MnSOD promoted the metastastic potential of fibrosarcoma cells via increased levels of MMP1 mRNA (22). However, no connection has yet been established between MnSOD upregulation of MMP1 or MMP2 and the Authors' Affiliations: Institute of Medical and Molecular Toxicology; Institute of Medicine, Chung Shan Medical University; Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC; Department of Surgery, China Medical University; Department of Thoracic Surgery, Taichung Veteran General Hospital, Taichung; Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Miaoli; Graduate Institute of Cancer Biology and Drug Discovery, Taipei Medical University, Taipei, Taiwan, ROC; Department of Health Services Management, China Medical University and Hospital, Taichung, Taiwan, ROC. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/) Corresponding Author: Huei Lee, Graduate Institute of Cancer Biology and Drug Discovery, Taipei Medical University, Room 5, 12F, No. 3, Park Street, Nankang District, Taipei 115, Taiwan, ROC. Phone: 886-2-27361661; E-mail: hl@tmu.edu. tw doi: 10.1158/1541-7786.MCR-12-0527 2012 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals. org 261 on June 16, 2017. © 2013 American Association for Cancer Research. mcr.aacrjournals. org Downloaded from Published OnlineFirst December 27, 2012; DOI: 10.1158/1541-7786.MCR-12-0527