Author Manuscript Published OnlineFirst on September 5, 2018; DOI: 10.1158/0008-5472.CAN-18-0912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Induction of paracrine signaling in metastatic melanoma cells by PPAR γ agonist rosiglitazone activates stromal cells and enhances tumor growth Christine Pich1, Patrick Meylan1, Beatris Mastelic-Gavillet2, Thanh Nhan Nguyen1, Romain Loyon2, Bao Khanh Trang1, Hélène Moser1, Catherine Moret1, Christine Goepfert3, Jürg Hafner4, Mitchell P. Levesque4, Pedro Romero2, Camilla Jandus2, Liliane Michalik1* 1 Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland 2 Department of Oncology, University of Lausanne, Ludwig Cancer Research Center, Lausanne, Switzerland 3 COMPATH, Institute of Animal Pathology, University of Bern, Bern, Switzerland 4 Department of Dermatology, University Hospital Zürich, University of Zürich, Zürich, Switzerland Short running title: RGZ activates paracrine signaling enhancing melanoma growth. Keywords: Thiazolidinediones, PPAR, melanoma, tumor microenvironment *Corresponding author: Dr Liliane Michalik Center for Integrative Genomics University of Lausanne Sorge CH-1015 Lausanne Switzerland Fax: +41 21 692 40 05 [email protected] Conflicts of interest: The authors declare no conflicts of interest. 1 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 5, 2018; DOI: 10.1158/0008-5472.CAN-18-0912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract In addition to improving insulin sensitivity in type 2 diabetes, the thiazolidinedione family of compounds and the pharmacologic activation of their best characterized target PPARγ has been proposed as a therapeutic option for cancer treatment. In this study, we reveal a new mode of action for the thiazolidinedione rosiglitazone that can contribute to tumorigenesis. Rosiglitazone activated a tumorigenic paracrine communication program in a subset of human melanoma cells that involves the secretion of cytokines, chemokines, and angiogenic factors. This complex blend of paracrine signals activated non-malignant fibroblasts, endothelial cells, and macrophages in a tumor-friendly way. In agreement with these data, rosiglitazone promoted human melanoma development in xenografts, and tumors exposed to rosiglitazone exhibited enhanced angiogenesis and inflammation. Together, these findings establish an important tumorigenic action of rosiglitazone in a subset of melanoma cells. While studies conducted on cohorts of diabetic patients report overall benefits of thiazolidinediones in cancer prevention, our data suggest that exposure of established tumors to rosiglitazone may be deleterious. Significance Findings uncover a novel mechanism by which the thiazolidinedione compound rosiglitazone contributes to tumorigenesis, thus highlighting a potential risk associated with its use in patients with established tumors. 2 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 5, 2018; DOI: 10.1158/0008-5472.CAN-18-0912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Thiazolidinediones (TZDs) are synthetic compounds initially described as drug improving insulin sensitivity, which thereby improve the control of glycemia (1). Two members of this family of compounds, rosiglitazone (Avandia) and pioglitazone (Actos) developed in the 90’s, are currently approved for the treatment of type 2 diabetes, a chronic disease characterized by insulin resistance. The best-characterized molecular target of TZDs is the Peroxisome proliferator-activated receptor (PPAR), although all TZD effects are not mediated by PPAR. PPAR is a member of the nuclear hormone receptor family, best known for its key roles in regulating adipocyte differentiation, energy metabolism and insulin sensitivity (2). Even though most of the initial studies on TZDs and PPAR functions focused on these metabolic roles, evidence has accumulated that they are associated with carcinogenesis. TZDs and PPAR activation exhibit anti-cancer effects in a variety of cancer cells and are therefore being considered as potential approaches for chemoprevention and treatment of cancers (3). However, such approaches are currently limited by diverse and sometimes discordant findings. Indeed, despite anti-cancer actions in many cancer cell types (2) and potential efficiency in chemoprevention (3), TZDs and activation of PPAR have shown little therapeutic efficacy in clinical trials performed over the past 15 years (3). Furthermore, retrospective studies conducted in type 2 diabetic patients chronically treated with TZDs support increased risk for developing bladder cancer associated with pioglitazone (1). An increased risk of non-Hodgkin lymphoma and melanoma was also suspected (4). Skin melanoma, a malignant neoplasm of melanocytes, is the most aggressive form of skin cancers, responsible for 80% of skin cancer-related deaths. Melanoma incidence and mortality rates are constantly increasing. In 2012, approximately 232000 new cases of melanoma were diagnosed worldwide (5). Currently, only early-diagnosed, non-invasive melanoma can be cured successfully, when local excision is readily achievable. Upon progression, and once melanoma has become metastatic, it remains a tumor of dismal prognosis in spite of the recent development of immuno- and molecularly targeted therapies (6). Melanoma progression and responses to treatments are driven not only by the malignant cells themselves, but also by impaired reciprocal interactions between the malignant cells and the non-malignant stromal cells of the tumor microenvironment, in particular fibroblasts, endothelial cells, and immune cells. These paracrine interactions are orchestrated by a variety of factors secreted by the melanoma cells, that activate neighboring fibroblasts 3 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 5, 2018; DOI: 10.1158/0008-5472.CAN-18-0912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. and blood vessels, and recruit immune cells, which in turn support tumor progression and metastases (7-10). The co-occurrence of data suggesting tumorigenic effects with data suggesting anti- cancer effects of TZDs indicates that chronic treatment with this family of compounds has context-dependent impacts, for which the underlying molecular and cellular bases and the involvement of PPAR still require clarification. These questions are of importance in regards to the growing interest in PPAR activators as therapeutic options in human cancers (11). The present study explores the basis underlying the debated TZD impact in cancers, using melanoma as a model. Our data reveal that rosiglitazone has tumorigenic effects that were not described previously, and which limit its therapeutic potential in cancer. We show that these tumorigenic actions of rosiglitazone may prevail over its anti-cancer actions in a subset of melanoma, and that therefore, rosiglitazone administration after tumor initiation may be detrimental. 4 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 5, 2018; DOI: 10.1158/0008-5472.CAN-18-0912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Microarray datasets We compared PPARG expression using the GEO2R tool (12) in two melanoma microarray datasets (GSE3189 and GSE46517; (13,14)) selected for reasonably high sample numbers and unequivocal experimental design in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo, accessed on January 19th 2017). GSE3189 data set (13): in the original study, the samples with less than 50% of melanocytes or melanoma cells were excluded. We analysed the samples according to the groups “nevi” (n=18) and “primary melanoma” (n=45) as in the original data set. We transformed and presented the data in log2. A Mann-Whitney test was performed on log2-transformed expression values to assess whether PPAR expression significantly differed between nevi and primary melanomas. GSE46517 data set (14): clinical information was available only for the samples obtained from the Medical University of Vienna, Austria. Among these samples, we excluded those with less than 75% melanocytes or melanoma cells. We also excluded normal skin samples, given that melanocytes only comprise a minimal fraction of all skin cells. In the absence of clinical information, we included in our analysis all the samples obtained from the Memorial Sloan Kettering Cancer Center of New York, NY and from the Brigham and Women’s Hospital, Boston, MA. We then grouped the data into “nevi” (n= 7), “primary melanoma” (n=8) and “melanoma metastases” (n=57), and we transformed and presented the data in log2. A Kruskal-Wallis test was performed followed by a post-hoc analysis using Dunn’s multiple comparisons test on log2-transformed expression values. Cell lines and melanoma cDNA array Human metastatic melanoma cell lines A375 and SkMel28 were purchased from CLS