[CANCER RESEARCH 60, 3971–3977, July 15, 2000] STAT5 Activation Is Required for Interleukin-9-dependent Growth and Transformation of Lymphoid Cells 1 Jean-Baptiste Demoulin 2 , Catherine Uyttenhove, Diane Lejeune, Alice Mui, Bernd Groner, and Jean-Christophe Renauld Ludwig Institute for Cancer Research and the Experimental Medicine Unit of the Universite ´ Catholique de Louvain, B-1200 Brussels, Belgium [J-B. D., C. U., D. L., J-C. R.]; Institute for Biomedical Research, D-60596 Frankfurt/Main, Germany [B. G.]; and Department of Surgery, University of British Columbia, Jack Bell Research Centre, Vancouver Hospital and Health Sciences Centre, Vancouver, British Columbia, V6H 3Z6 Canada [A. M.] ABSTRACT Interleukin-9 (IL-9) is a growth factor for T cells and various hemato- poietic and lymphoid tumor cells. IL-9 signaling involves activation of Janus kinase (JAK)1 and JAK3 kinases, and signal transducer and acti- vator of transcription (STAT)1, STAT3 and STAT5. Using a dominant negative form of STAT5 (STAT5), we demonstrated that this factor is an important mediator of IL-9-dependent Ba/F3 cell growth. Mutation of the STAT binding site of the IL-9 receptor (tyr116phe) results in an important decrease in STAT activation and inhibition of proliferation in the presence of IL-9. A small number of cells escape this inhibition, and IL-9-dependent cell lines could be derived. The selected cells required activation of STAT5 for growth, which was blocked by STAT5 expression and enhanced by overexpression of wild-type STAT5. In contrast to parental cells, Ba/F3- Phe116 cells growing in the presence of IL-9 further progress to cytokine- independent tumorigenic clones. These tumorigenic clones exhibited a strong cytokine-independent activation of JAK1 and STAT5, which most likely supports their proliferation. Transfection of a constitutively acti- vated variant of STAT5 promoted the growth of wild-type Ba/F3 cells in the absence of cytokine. Finally, the expression of the proto-oncogene pim-1 was correlated with STAT5 activation and cell growth. Our data suggest that STAT5 is an important mediator of IL-9-driven proliferation and that dysregulation of STAT5 activation favors tumorigenesis of lymphoid cells. INTRODUCTION Abnormal cytokine production accompanies the onset of certain lymphomas and leukemias (1). A large number of such soluble fac- tors, including IL 3 -9, have been implicated in the growth and survival of hematopoietic tumors. IL-9 was first characterized as a T-cell growth factor, but it has little activity on normal T cells, which respond to IL-9 only after long-term activation (2, 3). In contrast, transformed lymphocytes, particularly freshly isolated murine lym- phomas, proliferate on IL-9 stimulation (4). In line with these obser- vations, IL-9 transgenic mice, which express large amounts of IL-9 in most organs, frequently develop T lymphomas and are highly suscep- tible to mutagenic treatment (5). Autocrine or paracrine IL-9 may also stimulate the growth of human acute myeloid leukemias and Hodgkin’s lymphomas, as well as some human T-cell lymphotrophic virus-1-transformed cell lines (6 –10). IL-9 binds to a receptor comprising a specific chain (IL-9R) and the c chain, which is shared by receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (11). IL-9 effects are mediated through the activation of JAK1 and JAK3 tyrosine kinases and STAT transcription factors, namely STAT1, STAT3, and STAT5 (12–15). Activation of the JAK-STAT pathway by cytokines has been studied extensively (16). On ligand binding to hematopoietic receptors, JAK kinases are activated and phosphorylate the cytoplasmic part of the receptor, creating phospho- tyrosine docking sites for STAT factors. Recruited STATs are subse- quently phosphorylated by JAKs, dissociate from the receptor, and form stable dimers that migrate into the nucleus, where they bind to promoter sites and regulate the expression of genes (16). A transac- tivation domain has been located in the COOH terminus of most STATs. Deletion of this domain results in a dominant negative phe- notype (17). STATs are active players in malignant transformation. Fusion pro- teins generated by chromosomal translocation, such as BCR-ABL or TEL-JAK2, and viral oncogenes, for instance v-SRC, HBx, and v-Eyk, are able to activate STATs (16, 18). STAT3 is required for transfor- mation of NIH3T3 cells by v-SRC (19, 20). A constitutively active STAT3 mutant was shown to transform fibroblasts in vitro (21). Many human leukemias and lymphomas are associated with constitutive activation of the JAK-STAT pathway (22–24). In most cases, how- ever, the mechanism that underlies STAT activation in these tumors is unknown. Apoptosis inhibition by STAT3 and STAT5 is well documented, and it is likely that it accounts for their role in oncogenesis (14, 25). The effect of STATs on proliferation is more complex. On the one hand, STAT1, STAT3 and STAT5 have been shown to regulate cell cycle inhibitors, such as p21 waf1 , resulting in cell growth inhibition and differentiation (26, 27). On the other hand, STAT3 and STAT5 play a role in proliferation induced by IL-6 and IL-3, respectively (17, 28). Moreover, hematopoietic cells and T-lymphocytes from STAT5- deficient mice exhibit a decreased response to growth stimuli (29). Two recent studies have suggested that STATs simultaneously regu- late genes that stimulate and inhibit cell growth, the phenotypic outcome depending on the intensity and duration of the expression of both types of genes (26, 28). We have reported that STAT activation by IL-9 correlates with the induction of proliferation and apoptosis inhibition (12, 14). Here, we provide direct evidence that STAT5 plays an important role in IL-9- dependent growth and the malignant transformation of lymphoid cells. MATERIALS AND METHODS Cell Culture and Transfections. The T helper cell line TS1 and the pro-B cell line Ba/F3 were cultured as described in the presence of IL-9 or IL-3, respectively (100 units/ml; Refs. 12, 30). IL-3 was produced by transfected CHO cells (a gift from A. Burgess, Ludwig Institute, Melbourne, Australia). Recombinant human IL-9 was produced in the baculovirus system and was purified as described previously (12). Wild-type and mutated human IL-9R cDNAs were inserted into either the pEFbos/puro plasmid (12) or pEF/myc/cyto plasmid (Invitrogen, Carlsbad, CA), which contain a resistance gene to puromycin or to neomycin, respec- Received 12/3/99; accepted 5/16/00. 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 work was supported in part by the Belgian Federal Service for Scientific, Technical and Cultural Affairs and by the Actions de Recherche Concerte ´es, Communaute ´ franc ¸aise de Belgique, Direction de la recherche scientifique. J-B. D. is a senior research assistant, D. L. is a research assistant, and J-C. R. is a research associate with the Fonds National de la Recherche Scientifique, Belgium. 2 To whom requests for reprints should be addressed, at: Ludwig Institute for Cancer Research, avenue Hippocrate, 74, B-1200 Brussels, Belgium. Phone: 32-2-764-7465; Fax: 32-2-762-9405; E-mail: Jean-Baptiste.Demoulin@bru.licr.org. 3 The abbreviations used are: IL, interleukin; IL-9R, IL-9 receptor; EMSA, electro- phoretic mobility shift assay; STAT, signal transducer and activator of transcription; JAK, Janus kinase; GRR, IFN-response region; SCID, severe combined immunodeficient/ deficiency. 3971 on July 26, 2015. © 2000 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from