Tumor-Suppressive Activity of the Cell Death Activator GRIM-19 on a Constitutively Active Signal Transducer and Activator of Transcription 3 Sudhakar Kalakonda, 1 Shreeram C. Nallar, 1 Daniel J. Lindner, 3 Jiadi Hu, 1 Sekhar P. Reddy, 2 and Dhananjaya V. Kalvakolanu 1 1 Department of Microbiology and Immunology, Greenebaum Cancer Center, University of Maryland School of Medicine; 2 Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland; and 3 Taussig Cancer Center, The Cleveland Clinic Foundation, Cleveland, Ohio Abstract Signal transducers and activators of transcription 3 (STAT3) was originally identified as a transcription factor that mediates cytokine-induced responses. In these pathways, Janus-activated kinase (JAK)–induced transient tyrosine phos- phorylation of STAT3 promotes gene expression in response to a number of cytokines, which is inhibited by feedback mechanisms. A number of studies have shown that STAT3 is constitutively activated in human cancer cells, leading to cell proliferation. It is unclear, apart from a chronic tyrosyl phosphorylation of STAT3, what mechanisms contribute to the STAT3 deregulation in tumors. Earlier, we have isolated a novel growth inhibitory gene product, gene associated with retinoid-IFN–induced mortality 19 (GRIM-19), using a genetic approach. GRIM-19 is an IFN/retinoic acid–regulated growth suppressor. Subsequent analyses have shown that GRIM-19 binds to STAT3 and prevents interleukin-6–induced transcrip- tion of cellular genes. However, its effects on a constitutively active STAT3 and cellular transformation are unknown. In this study, we show that GRIM-19 suppresses constitutive STAT3- induced cellular transformation in vitro and in vivo by down- regulating the expression of a number of cellular genes involved in cell proliferation and apoptosis. [Cancer Res 2007;67(13):6212–20] Introduction Oncogenic proteins alter gene expression patterns during cellular transformation. DNA binding oncoproteins like c-MYC and FRA-1 have been shown to promote cell growth by activating specific sets of genes (1, 2). Antioncogenic proteins reverse these processes to restore normal cell growth. We have been studying the mechanisms of antitumor action of IFN-h and retinoic acid (IFN/ RA) in human tumor cells. IFN/RA exhibited strong antitumor actions in a number of animal models and in clinical studies (3, 4). Using a genetic technique, we have isolated a novel gene product, gene associated with retinoid-IFN–induced mortality 19 (GRIM-19), which promotes IFN/RA–induced cell death (5). More recently, we have shown that a loss of GRIM-19 expression in human renal cell carcinomas (6) and an inhibition of its proapoptotic activity by viral oncoproteins occur, indicating a potential tumor suppressor- like function for this protein. Overexpression of GRIM-19 in a number of human tumor cell lines induces apoptosis (5–7). Some esophageal tumors seem to harbor inhibitors of GRIM-19 (8). A portion of the total GRIM-19 in cells is also present in the mitochondrion (9–11). Very recently, GRIM-19 has been coupled to reactive oxygen species generation in an IFN/RA–induced pathway in HeLa and MCF-7 cells (12). We and others have shown that GRIM-19 binds to signal transducer and activator of transcription 3 (STAT3) transcription factor and inhibits STAT3-dependent cytokine-induced gene transcription (13, 14). However, its role in regulating oncogenic cell proliferation and, in particular, its effects on a ligand- independent, constitutively active STAT3 and cellular transforma- tion are unclear. To address these issues, we have used a cellular system in which a constitutively active STAT3 was sufficient to induce oncogenic transformation (15). We show here that GRIM-19 overrides cellular transformation caused by constitutive STAT3 and suppresses the expression of endogenous genes involved in cell proliferation. GRIM-19 also inhibits injury-induced cell migration and formation of tumors in vivo . These data for the first time show a direct tumor-suppressive effect of GRIM-19 and establish an ‘‘oncogene-tumor suppressor’’ like relationship between STAT3 and GRIM-19. Materials and Methods Plasmids and antibodies. The pRC/CMV flag-STAT3C (A 661 C and N 663 C) that expresses a constitutively dimerizing version of STAT3 (15) was a gift from Jackie Bromberg (Sloan-Kettering Cancer Center, New York, NY). Mammalian expression vector pCXN2-myc and its derivative pCXN2-GRIM- 19-myc were described elsewhere. TKS3-Luc, cyclinB1-Luc, and cdc2-Luc were described in our previous publication (14). APRE-Luc contains three copies of acute phase response element (APRE) upstream of the SV40 minimal promoter. c-fos-Luc (16) and Bcl-X L -Luc (17) were described earlier. Lentiviral expression vectors carrying GRIM-19 and a scrambled short hairpin RNA (shRNA) were purchased from Open Biosystems, Inc., and virus stocks were prepared as recommended by the supplier. Antibodies specific for STAT3, phospho-STAT3-Y705, and phospho-STAT3-S727 (Cell Signaling Technology); actin, myc-epitope, and Flag-epitope (Sigma- Aldrich); and Ki-67 (Oncogene Science) were used in these studies. Establishment of stable cells. Rat fibroblast 3Y1 cells were grown in DMEM supplemented with 10% heat fetal bovine serum (FBS), 100 units/mL penicillin, and 100 Ag/mL streptomycin. Cells were electroporated either individually or in combination with pCXN2-myc, STAT3C, or pCXN2-GRIM 19 (1 Ag of each plasmid) using the Nucleofector technology (Amaxa Biosystems). Transfection efficiency was f45% with this method. Twenty- four hours posttransfection, cells were selected with G418 (750 Ag/mL) for Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). S. Kalakonda and S.C. Nallar contributed equally to this study. Requests for reprints: Dhananjaya V. Kalvakolanu, University of Maryland Cancer Center, Howard Hall, Room 324, 660 W. Redwood Street, Baltimore, MD 21201. Phone: 410-328-1396; Fax: 410-328-6559; E-mail: dkalvako@umaryland.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-0031 Cancer Res 2007; 67: (13). July 1, 2007 6212 www.aacrjournals.org Research Article Research. on January 28, 2016. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from