Mutations in the SOD2 Promoter Reveal a Molecular Basis for An

Mutations in the SOD2 Promoter Reveal a Molecular Basis for an Activating Protein 2-Dependent Dysregulation of Manganese Superoxide Dismutase Expression in Cancer Cells Yong Xu,1 Fang Fang,2 Sanjit K. Dhar,1 Antonio Bosch,3 William H. St. Clair,2 Edward J. Kasarskis,1,4 and Daret K. St. Clair1 1Graduate Center for Toxicology and Departments of 2Radiation Oncology, 3Internal Medicine, and 4Neurology, University of Kentucky, Lexington, Kentucky Abstract metabolizing enzyme systems (1).Oxidative stress is thought to A primary antioxidant enzyme in mitochondria, be one of the important pathogenic factors of cancer manganese superoxide dismutase (MnSOD), plays a development and abnormal levels of MnSOD in cancer have critical role in the survival of aerobic life. It is well been documented.MnSOD expresses at a lower level in many documented that, compared with normal cell types of transformed and neoplastic cells, suggesting that loss counterparts, MnSOD level is decreased in neoplastic of MnSOD activity may be a general characteristic of transformed cells but is increased in aggressive tumorigenesis (reviewed in ref.2).Consistent with this cancers. However, the underlying mechanism for the possibility, overexpression of MnSOD has been shown to observed dysregulation of MnSOD in cancer is suppress tumor growth in nude mice and to inhibit the unknown. We have identified previously a unique set of metastasis of transplanted tumors (3).MnSOD mimetic mutations located in the promoter region of the SOD2 pretreatment also significantly reduces 7,12-dimethylben- gene in several types of cancer cells. We found that a z[a]anthracene/12-O-tetradecanoylphorbol-13-acetate-induced C-to-T transition at -102 and an insertion of A at -93 skin tumor incidence (4). down-regulate MnSOD transcription by interrupting the In contrast to the association between reduction of MnSOD formation of a single-stranded loop that is essential for a and tumorigenesis, evidence that a high level of MnSOD is n n high level of promoter activity. Here, we show that the correlated with nuclear factor- B (NF- B) activation is found n additional downstream mutation, C-to-G transversion at in various aggressive tumors.Activation of the NF- B pathway, -38, creates a binding site for the transcription factors leading to the induction of prosurvival proteins including specificity protein 1 (Sp1) and activating protein 2 (AP-2). MnSOD, has been implicated in the resistance of cancer cells to The promoter function is regulated by the relative chemotherapy and radiotherapy (reviewed in ref.5).Conse- n levels of Sp1 and AP-2. In cytokine-induced quently, suppressing MnSOD by inhibiting the NF- B pathway expression of the SOD2 gene, Sp1 cooperates with a results in enhanced radiosensitivity of prostate cancer cells transcriptional complex containing nuclear factor-KB (6, 7).Although some studies have implicated MnSOD in the and nucleophosmin. The presence of AP-2 attenuates differential regulation of cancer cells, the mechanistic link this induction. Our results suggest that the high level of between MnSOD reduction and induction at different stages of MnSOD observed in aggressive cancer cells may be cancer remains unknown. Human MnSOD is an 88.6-kDa tetrameric protein contain- due, in part, to the absence of AP-2 transcriptional 2+ repression. (Mol Cancer Res 2008;6(12):1881–93) ing a Mn -associated with each subunit.It is encoded by the human SOD2 gene located at 6q25 and is highly conserved in mammals (8).The human SOD2 gene is activated by a Introduction proximal promoter region that is characterized by the absence Manganese superoxide dismutase (MnSOD), a nuclear- of TATA or CAAT box and the presence of multiple CpG encoded mitochondrial primary antioxidant enzyme, catalyzes islands (9).Transcription factors specificity protein 1 (Sp1) and superoxide radicals into molecular oxygen and hydrogen activating protein 2 (AP-2) can directly bind to the CpG islands peroxide, which are further reduced into water by peroxide- to differently regulate promoter activity (10).Sp1 plays a central role in transcriptional activation of the SOD2 gene, which is consistent with observations from studies of many other human genes that contain TATA- or CAAT-less Received 6/2/08; revised 9/10/08; accepted 9/11/08. Grant support: NIH grants CA 49797 and CA 73599 (D.K. St. Clair). promoters.In contrast, AP-2 plays a negative role in The costs of publication of this article were defrayed in part by the payment of transcriptional regulation of the SOD2 gene by attenuating advertisement page charges.This article must therefore be hereby marked in Sp1 function (11). accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Daret K.St.Clair, Graduate Center for Toxicology, MnSOD expression is rapidly up-regulated in response to University of Kentucky, Lexington, KY 40536.Phone: 859-257-3956; Fax: 859- oxidative stress.Numerous studies have shown that MnSOD is 323-1059.E-mail: [email protected] Copyright D 2008 American Association for Cancer Research. induced in various types of cells and tissues by toxic stimuli doi:10.1158/1541-7786.MCR-08-0253 and treatments, such as tumor necrosis factor-a, interleukin-1h, Mol Cancer Res 2008;6(12). December 2008 1881 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2008 American Association for Cancer Research. 1882 Xu et al. and 12-O-tetradecanoylphorbol-13-acetate (TIT; 12), dinitro- Mutation at -38 Creates Binding Motifs for Sp1and AP-2 phenol (13), paraquat (14), UV (15), and ionizing radiation (6). To determine how mutations affect transcriptional regulation A second intron 2 element (I2E) containing a NF-nB-binding by Sp1 and AP-2, Sp1 and AP-2a expression constructs were site has been identified in both human and mouse sod2 genes cotransfected with promoter-reporter constructs into HepG and is responsible for cytokine-mediated induction of MnSOD cells.Increasing Sp1 level in HepG2 cells markedly elevates (16, 17).Interaction between the Sp1-based promoter and the promoter activity.In contrast, expression of AP-2 a in HepG2 NF-nB-based enhancer is thought to be essential for transcrip- cells significantly suppresses both constitutive and Sp1- tional induction.A recent insight from our investigations activated promoter activities (Fig.2A).The presence of showed that nucleophosmin (NPM) functions from a distance M1-M2 reduces promoter activity.In contrast, the presence of as a coordinator between the proximal promoter and the NF-nB M3 leads to an enhanced effect by Sp1 and a repressed effect by enhancer (12, 18). AP-2a.The repressive effect of AP-2 on the wild-type or the In an effort to determine the cause for the deregulation of mutant promoter was further analyzed by transfecting various MnSOD expression in cancer, we have identified several concentrations of the AP-2a expression construct into the mutations located in the promoter region of the SOD2 gene of HepG2 cells (Fig.2B).The promoter activity is reduced in a several cancer cell lines (19).One of these mutations was dose-dependent manner when the cells express AP-2a. confirmed in molecular epidemiologic studies (20).In the Importantly, AP-2a has a greater negative effect on the mutant present study, we conducted cell-based transcriptional analysis promoter than on the wild-type promoter. using both cancer and transformed cell lines to delineate the To verify the effect of M3, electrophoretic mobility shift transcriptional mechanism altered by these mutations.The assay was done to determine whether Sp1 and AP-2a bind to results suggest that the mutations in the SOD2 promoter the promoter region containing M3.A promoter fragment was facilitate an AP-2-dependent modification of MnSOD expres- radiolabeled to generate both wild-type and M3 probes.AP-2 a sion in cancer cells. protein was ectopically expressed in HepG2 cells.Nuclear extracts from the transfected cells were probed and specific bindings of Sp1 and AP-2 were detected by the respective Results antibodies (Fig.2C).As shown in Fig.2C, in comparison with Mutations Identified In Cancer Cells Alter SOD2 Promoter the wild-type probe without detectable binding activity, the M3 Activity probe shows both Sp1 and AP-2 binding activities with HepG2 We identified previously three mutations in the proximal nuclear extracts.Importantly, the AP-2 binding activity is dose- promoter region of the SOD2 gene from human cancer cell dependently increased as the concentration of AP-2a is lines (19).As illustrated in Fig.1A, these mutations contain increased.Thus, the results of electrophoretic mobility shift C-to-T transition at -102 (M1), A insertion at -93 (M2), and assay are consistent with the reporter assay, suggesting that the C-to-G transversion at -38.Transcription factor database effect of M3 on transcription is dependent on the relative level searching indicates that the M3 creates a binding site for both of AP-2 and Sp1. Sp1 and AP-2.In addition, although M1 and M2 are unlikely to change the binding motif for known transcription factors, the Promoter Mutations Affect Transcriptional Induction sequence change apparently interrupts the formation of a single- To detect whether the mutant promoter also affects stranded loop, which has been shown to be an important transcriptional induction of the SOD2 gene, enhancer- structure for the promoter function (12).To verify that Sp1 and promoter-reporter plasmids containing mutations in the AP-2 regulate MnSOD expression, the levels of the three promoter region were

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