Identification of the Chemokine CX3CL1 as a New Regulator of Malignant Cell Proliferation in Epithelial Ovarian Cancer

et al. (2011) Identification of the Chemokine CX3CL1 as a New Regulator of Malignant Cell Proliferation in Epithelial Ovarian Cancer. PLoS ONE 6(7): e21546. doi:10.1371/journal.pone.0021546 Identification of the Chemokine CX3CL1 as a New Regulator of Malignant Cell Proliferation in Epithelial Ovarian Cancer Fran coise Gaudin 0 Salam Nasreddine 0 Anne-Claire Donnadieu 0 Dominique Emilie 0 Christophe Combadie` re 0 Sophie Pre vot 0 Ve ronique Machelon 0 Karl Balabanian 0 Lin Zhang, University of Pennsylvania, United States of America 0 1 UMR_S996, INSERM/Universite Paris-Sud 11, Clamart, France, 2 Service de Microbiologie-Immunologie Biologique, Assistance Publique-Ho pitaux de Paris/Ho pital Antoine-Be cle`re , Clamart, France, 3 UMR_S945 , INSERM/Universite Pierre et Marie Curie, Assistance Publique-Ho pitaux de Paris/H opital de la Pitie -Salpetrie`re, Paris, France, 4 Service d'Anatomie et de Cytologie Pathologiques, Assistance Publique-Ho pitaux de Paris/H opital Antoine-Be cle`re , Clamart , France Background: Little is known about the molecules that contribute to the growth of epithelial ovarian carcinomas (EOC), which remain the most lethal gynecological cancer in women. The chemokine Fractalkine/CX3CL1 has been widely reported to play a biologically relevant role in tumor growth and spread. We report here the first investigation of the expression and role of CX3CL1 in EOC. Results: Epithelial cells from the surface of the ovary and the Fallopian tubes and from benign, borderline and malignant tumors all stained positive for CX3CL1. In tumor specimens from 54 women who underwent surgical treatment for EOC diagnosis, CX3CL1 immunoreactivity was unevenly distributed in epithelial tumor cells, and ranged from strong (33%) to absent (17%). This uneven distribution of CX3CL1 did not reflect the morphological heterogeneity of EOC. It was positively correlated with the proliferation index Ki-67 and with GILZ (glucocorticoid-induced leucine zipper), previously identified as an activator of the proliferation of malignant EOC cells. Hierarchical clustering analysis, including age at diagnosis, tumor grade, FIGO stage, Ki-67 index, CX3CL1, SDF-1/CXCL12 and GILZ immunostaining scores, distinguished two major clusters corresponding to low and high levels of proliferation and differing in terms of GILZ and CX3CL1 expression. GILZ overexpression in the carcinoma-derived BG1 cell line resulted in parallel changes in CX3CL1 products. Conversely, CX3CL1 promoted through its binding to CX3CR1 AKT activation and proliferation in BG1 cells. In a mouse subcutaneous xenograft model, the overexpression of GILZ was associated with higher expression of CX3CL1 and faster tumor growth. Conclusion: Our findings highlight the previously unappreciated constitutive expression of CX3CL1 preceding tumorigenesis in ovarian epithelial cells. Together with GILZ, this chemokine emerges as a regulator of cell proliferation, which may be of potential clinical relevance for the selection of the most appropriate treatment for EOC patients. - Funding: This work was supported by the Ligue contre le cancer (Comite Val dOise), the Institut National de la Sante et de la Recherche Medicale (INSERM), the Universite Paris-Sud, and the European Union FP6 (INNOCHEM, grant number LSHB-CT-2005-518167). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Epithelial ovarian cancer (EOC) constitutes the sixth most common cancer and the fifth leading cause of cancer-related death among women in developed countries [1]. Due to the silent nature of early-stage disease, most women with EOC have disseminated disease (i.e. expansion in the peritoneum and metastasis in the omentum) at the time of diagnosis and present advanced disease, with a five-year survival rate below 30% [2]. Despite the high incidence and mortality rates of EOC, the etiological factors involved in ovarian carcinogenesis remain poorly defined, limiting the efficacy of treatment protocols. The epithelial tumor microenvironment consists of a complex tissue containing several cell types. Most of these cells produce and/or respond to chemokines, which may play key roles in the development and progression of primary epithelial tumors [35]. We have shown, for example, that the a-CXC chemokine Stromal cell-Derived Factor-1 SDF-1/CXCL12 contributes to the immunosuppressive network within the tumor microenvironment, notably by orchestrating the recruitment of pre-DC2s [6]. We have also shown that CXCL12 regulates tumor angiogenesis and that this is critical for tumor growth [7]. By contrast, little if anything is known about the role of the chemokine Fractalkine/ CX3CL1 in EOC, although it has been evidenced to mediate strong cell adhesion [8] and its presence in epithelial tissues is widely documented [910]. CX3CL1 exists in two forms. The membrane-anchored form mediates the firm adhesion of cells expressing its sole receptor, CX3CR1, to the endothelium under physiological flow, through its own intrinsic adhesion function and through integrin activation [1112]. The soluble form is released through cleavage at a site close to the membrane [13]. Like other conventional chemokines, it recruits immune cells bearing CX3CR1, such as T lymphocytes and cytotoxic NK cells, dendritic cells or a large subpopulation of CD14+ monocytes [8]. As a result of both the adhesion and chemoattractant activities of the chemokine, the CX3CL1/CX3CR1 complex may mediate either pro- or anti-tumor effects [14]. Pancreatic ductal adenocarcinoma cells bearing CX3CR1 specifically adhere to CX3CL1expressing cells of neural origin and migrate in response to CX3CL1 produced by neurons and nerve fibers, contributing to perineural dissemination in pancreatic cancer [15]. Prostate cancer cells that express CX3CR1 adhere to human bone marrow endothelial cells and migrate toward a medium conditioned by osteoblasts, which secrete the soluble form of the chemokine contributing to the high likelihood of prostate cancer cells metastasizing to the skeleton [1617]. By contrast, soluble CX3CL1 (sCX3CL1) released in the tumor microenvironment may be an active component of the anti-tumor response [1821], making the vaccination of mice with carcinoma cells modified to produce CX3CL1 a potent anti-tumor response due to the chemoattraction of NK cells [22], or making CX3CL1 expression by colon cancer cells a factor that drastically reduced their metastatic potential [23]. In the present work, we have investigated the expression of CX3CL1 in healthy and malignant ovarian tissues and its role in the proliferation of malignant ovarian epithelial cells. This chemokine was produced by both healthy and malignant ovarian epithelial cells, and its production in EOC was positively correlated to cell prol (...truncated)