[CANCER RESEARCH 61, 3276 –3280, April 15, 2001] Advances in Brief Overexpression of a Dominant-Negative Signal Transducer and Activator of Transcription 3 Variant in Tumor Cells Leads to Production of Soluble Factors That Induce Apoptosis and Cell Cycle Arrest 1 Guilian Niu, Kenneth H. Shain, Mei Huang, Rajani Ravi, Atul Bedi, William S. Dalton, Richard Jove, and Hua Yu 2 Immunology Program [G. N., H. Y.], Clinical Investigations Program [K. S., W. D.], and Molecular Oncology Program [M. H., R. J.], H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33613; Department of Oncology, University of South Florida College of Medicine, Tampa, Florida 33612; and Johns Hopkins Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287 [R. R., A. B.] Abstract Gene therapy of B16 tumors with a dominant-negative signal trans- ducer and activator of transcription (Stat3) variant, designated Stat3, results in inhibition of tumor growth and tumor regression. Although only 10 –15% of the tumor cells are transfected in vivo, the Stat3-induced antitumor effect is associated with massive apoptosis of B16 tumor cells, indicative of a potent bystander effect. Here, we provide evidence that blocking Stat3 signaling in B16 cells results in release of soluble factors that are capable of inducing apoptosis and cell cycle arrest of nontrans- fected B16 cells. RNase protection assays using multi-template probes specific for key physiological regulators of apoptosis reveal that overex- pression of Stat3 in B16 tumor cells induces the expression of the apoptotic effector, tumor necrosis factor-related apoptosis-inducing li- gand. These in vitro results suggest that the observed in vivo bystander effect leading to tumor cell growth inhibition is mediated, at least in part, by soluble factors produced as a result of overexpression of Stat3 in tumor cells. Introduction Effective cancer gene therapies require the killing of genetically untransduced tumor cells (“bystander” cells) concomitant with genet- ically transduced tumor cells. Because transfection efficiency is one of the rate-limiting steps for gene therapy, the efficacy of any cancer gene therapy depends heavily on bystander effects. Several studies have demonstrated that p53 gene therapy results in antiangiogenic effects in vivo, thereby enhancing its antitumor activity (1–3). Herpes simplex virus-thymidine kinase-based gene therapy relies on the spreading of toxin converted from prodrug by thymidine kinase- expressing tumor cells to adjacent tumor cells via gap junctional intercellular communication (4). The bystander effect exerted by FasL 3 -based gene therapy involves the activation of neutrophils via Fas receptor (5, 6). Activated neutrophils in turn kill both FasL- transfected and parental tumor cells (5, 6). These studies suggest that, in the absence of high efficiency of in vivo gene transfection, the antitumor efficacy of a cancer gene therapy is greatly influenced by the strength of its associated bystander effects. STATs are latent cytoplasmic transcription factors that function as intracellular effectors of cytokine and growth factor signaling path- ways (7). STAT proteins were originally defined in the context of normal cell signaling, where STATs have been implicated in control of cell proliferation, differentiation, and apoptosis (8, 9). Recently, a number of studies have demonstrated that aberrant STAT signaling may participate in development and progression of human cancers. In contrast to normal cells in which STAT activation is rapid and transient, in diverse human cancers, including breast cancer, multiple myeloma, lymphomas, leukemias, head and neck carcinoma (10 –17) and melanoma, 4 STAT family proteins, especially Stat3, are consti- tutively activated. These findings raise the possibility that Stat3 may serve as a molecular target for novel cancer therapy. Recent studies have indicated that inhibition of Stat3 activity in human tumor cells induces apoptosis and/or growth arrest in vitro. Stat3 is a dominant-negative Stat3 variant, which is a truncated form of Stat3 that contains the dimerization and DNA-binding domain but lacks the transactivation domain (18). As a consequence, Stat3 can bind DNA but cannot transactivate gene expression, thus blocking Stat3 signaling in a trans-dominant negative fashion in most cases. Blocking Stat3 signaling by Stat3 in human myeloma cells down- regulates IL-6-induced expression of the antiapoptotic gene, Bcl-X L , resulting in a dramatic sensitization of cells to Fas-mediated apoptosis in vitro (16). In human head and neck squamous carcinoma cells, interrupting Stat3 signaling by antisense oligonucleotides abrogates transforming growth factor- induced oncogenic growth of these cells (11). Similar to multiple myeloma, disrupting Stat3 signaling in head and neck cancer cells inhibits Bcl- X L expression and induces apo- ptosis (17). Constitutive activation of Stat3 in human breast cancer cells correlates with elevated EGF receptor and c-Src activity (19). Blocking Stat3 activity by dominant-negative Stat3 in breast cancer cells leads to apoptosis (19). Our recent results also demonstrate that inhibiting Stat3 activity by Stat3 causes growth inhibition in human melanoma cells. 5 These findings raise the possibility that targeting Stat3 may result in antitumor responses in vivo in a wide variety of human cancers. We have demonstrated recently that in vivo transgenic expression of Stat3 in murine B16 tumors results in tumor regression involving massive apoptosis of tumor cells, despite relatively low transfection efficiencies (10 to 15%; Ref. 20). To begin elucidating the cellular and molecular mechanisms underlying the Stat3-mediated bystander ef- fects observed in vivo, we performed in vitro studies. Our results show that inhibition of Stat3 signaling in B16 cells leads to production of soluble factors that are capable of inducing both apoptosis and cell cycle arrest. Consistent with the finding that soluble factors are involved in the bystander effects, induction of mRNA encoding the apoptosis effector, TRAIL, is detected in Stat3-transfected B16 cells. Received 9/12/00; accepted 2/28/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This research was supported by NIH Grants CA75243 (to H. Y.), CA55652 (to R. J.), and CA77859 (to W. D.) and by the Dr. Tsai-Fan Yu Cancer Research Endowment. 2 To whom requests for reprints should be addressed, at Immunology Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612. 3 The abbreviations used are: FasL, Fas ligand; STAT, signal transducer and activator of transcription; IL, interleukin; EGF, epidermal growth factor; EGFP, enhanced green fluorescence protein; TRAIL, TNF-related apoptosis-inducing ligand; EMSA, electro- phoretic mobility shift assay; FACS, fluorescence-activated cell sorting; PE, phyco- erythrin; RPA, RNase protection assay; IRES, internal ribosomal entry site. 4 G. Niu, T. Bowman, M. Huang, R. Jove, and H. Yu, unpublished results. 5 G. Niu and H. Yu, unpublished results. 3276 Research. on January 12, 2016. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from