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Expression of Interleukin-4 Receptor Alpha in Human Pleural Mesothelioma Is Associated with Poor Survival and Promotion of Tumor Inflammation

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Expression of Interleukin-4 Receptor Alpha in Human Pleural Mesothelioma Is Associated with Poor Survival and Promotion of Tumor Inflammation Published OnlineFirst January 18, 2012; DOI: 10.1158/1078-0432.CCR-11-1808 Clinical Cancer Human Cancer Biology Research Expression of Interleukin-4 Receptor Alpha in Human Pleural Mesothelioma Is Associated with Poor Survival and Promotion of Tumor Inflammation Bryan M. Burt1, Andrew Bader1, Daniel Winter1, Scott J. Rodig2, Raphael Bueno1, and David J. Sugarbaker1 Abstract Purpose: The origin and pathogenesis of malignant pleural mesothelioma (MPM) are closely aligned with inflammation. MPM tumors express interleukin-4 receptor a (IL-4Ra), the principal subunit of the IL-4 receptor. We set out to determine the biologic function and clinical relevance of IL-4Ra in human MPM. Experimental Design: Expression of IL-4Ra by human MPM tumors was determined by quantitative real-time PCR (n ¼ 37) and immunohistochemistry (n ¼ 52). Intracellular cytokine analysis of T-cell– derived IL-4 was carried out on matched tumor and blood samples from eight patients with MPM. Four human MPM cell lines were used to determine the direct effects of IL-4 on MPM tumor cells. Results: High tumor mRNA expression of IL-4Ra was an independent predictor of poor survival in patients with epithelial MPM [HR, 3.13, 95% confidence interval (CI), 1.68–7.15; P ¼ <0.0001]. Ninety- seven percent of epithelial MPM tumors and 95% of nonepithelial MPM tumors expressed IL-4Ra protein by immunohistochemistry, and strong IL-4Ra staining correlated with worse survival in patients with epithelial histology (P ¼ 0.04). A greater percentage of tumor-infiltrating T cells produced IL-4 compared with matched blood T cells (21% Æ 7% vs. 4% Æ 2%, P ¼ 0.0002). In response to IL-4, human MPM cells showed increased STAT-6 phosphorylation and increased production of IL-6, IL-8, and VEGF, without effect on proliferation or apoptosis. Conclusions: Tumor expression of IL-4Ra is inversely correlated with survival in patients undergoing surgical resection for epithelial MPM. Tumor-infiltrating T cells in MPMs are polarized to produce IL-4 and may provide endogenous activation signals to MPM tumor cells in situ. The IL-4/IL-4 receptor axis is a potential therapeutic target in human MPM. Clin Cancer Res; 18(6); 1568–77. Ó2012 AACR. Introduction more aggressive tumors with significantly worse survival Malignant pleural mesothelioma (MPM) is an aggressive (2). tumor that arises from the mesothelial lining of the pleura. It recently has become evident that chronic inflammation It is a unique malignancy whose primary mode of spread is may predispose individuals to develop cancer. Inflamma- invasion of surrounding tissues and whose proclivity for tion promotes the proliferation and survival of malignant distant metastases is low. MPM is a highly fatal tumor that is cells, supports angiogenesis and metastasis, and subverts resilient to single modality treatment with surgery, chemo- immune surveillance and responses to chemotherapy (3). therapy, or radiation. With multimodality therapy in select- MPM, in particular, is a cancer whose origin and pathogen- ed patients, median survival is 19 months (1). Approxi- esis are closely aligned with inflammation. Occupational mately two-thirds of patients with MPM have epithelial exposure to asbestos is identified as the etiology of MPM in histology. The remaining one third of patients have non- 80% of cases (4). Asbestos fibers are thought to initiate a epithelial histology (sarcomatoid or biphasic), which are carcinogenic inflammatory reaction marked by an influx of phagocytes into the tumor and the release of an array of proinflammatory cytokines, such as TNF-a, that promote malignant transformation of the mesothelium via NF-kB– Authors' Affiliations: 1Division of Thoracic Surgery, and 2Department of Pathology, The Brigham & Women's Hospital, Boston, Massachusetts dependent mechanisms (5). In human MPM tumors, ongo- ing inflammation is represented by massive leukocyte infil- Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). trates of T lymphocytes and macrophages whose densities are correlated with survival (6, 7). Corresponding Author: Bryan M. Burt, Division of Thoracic Surgery, Harvard Medical School, Brigham & Women's Hospital, 75 Francis Interleukin-4 receptor a (IL-4Ra) is the major subunit of Street, Boston, MA 02115. Phone: 617-840-7411; Fax: 617-566-6434; the IL-4 receptor. In addition to being present on immune E-mail: [email protected] cells such as B cells, T cells, and macrophages, IL-4Ra is doi: 10.1158/1078-0432.CCR-11-1808 expressed on a variety of cancer cell lines (8), and in situ on a Ó2012 American Association for Cancer Research. number of solid human tumors including non–small cell 1568 Clin Cancer Res; 18(6) March 15, 2012 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst January 18, 2012; DOI: 10.1158/1078-0432.CCR-11-1808 IL-4Ra in Human Pleural MPM were snap frozen on the day of resection. For flow cytometry Translational Relevance experiments, tumor and matched blood samples were Malignant pleural mesothelioma (MPM) is an aggres- obtained fresh and used on the day of surgery. Peripheral sive tumor of the pleura that arises in a background of blood mononuclear cells were isolated by density centrifu- chronic inflammation. The overall survival of patients gation over Ficoll-Paque Plus (GE Healthcare). To isolate with MPM is poor despite multimodality treatment tumor-infiltrating mononuclear cells, tumor tissues were strategies that include surgery, chemotherapy, and radi- minced into fine fragments in cold Hank’s balanced salt ation. In this study, we have identified interleukin-4 solution (HBSS) and digested in RPMI [American Type receptor a (IL-4Ra), the major subunit of the IL-4 Culture Collection (ATCC)] containing 10% fetal calf receptor, as a biomarker with prognostic correlation in serum (FCS; Gibco), 1 mg/mL Collagenase D (Roche Diag- MPM. In patients undergoing resection for MPM, high nostic), and 100 mg/mL DNase (Qiagen) at 37C for 3 tumor IL-4Ra expression was an independent predictor hours. The tissue slurry was passed through 70 and 40 mm of decreased survival. Furthermore, freshly isolated T filters and washed with cold HBSS. The cells were then cells infiltrating human MPM tumors were polarized to layered over a Ficoll-Paque density gradient and centrifuged produce IL-4, and IL-4 had direct biologic effects on at 1,000 Â g for 20 minutes at which time the interface layer MPM tumor cells that included promotion of STAT-6 containing tumor mononuclear cells was harvested. phosphorylation and inflammatory cytokine produc- MPM cell lines. Four human MPM cell lines were main- tion. Therefore, we believe that IL-4 endogenously pro- tained in RPMI media containing 10% FCS (Gibco). H2052 duced in the tumor microenvironment may promote cells (epithelial origin) were obtained from the ATCC. cancer inflammation and progression, and that inter- MS924 (epithelial) and MS428 cells (sarcomatoid) were ruption of the IL-4/IL-4 receptor axis may be a promising kindly provided by Dr. Jonathan A. Fletcher, MD, Depart- therapeutic target in human MPMs. ment of Pathology, Brigham & Women’s Hospital (Boston, MA). JMN cells (biphasic) were a gift from Dr. James Rheinwald (16). Quantitative PCR lung cancer, pancreatic cancer, ovarian cancer, breast can- Quantitative PCR was carried out on MPM tumor tissues cer, prostate cancer, and head and neck cancer (9–12). as previously described (17). Briefly, tumors were selected Furthermore, IL-4 production is augmented in the immune for portions with highest tumor density (>50% tumor cells cells of patients with cancer, and serum and tissue levels of per high-power field) and stored snap frozen. Total RNA (2 IL-4 are correlated with tumor progression in man (13). In mg) was isolated using TRIzol reagent (Invitrogen Life Tech- MPM, Beseth and colleagues have shown IL-4Ra expression nologies) and reverse transcribed using Taq-Man Reverse on mesothelioma tumors from 13 patients using immuno- Transcription reagents (Applied Biosystems). Quantitative histochemistry (14). We set out to determine the clinical real-time PCR (RT-PCR) was carried out using a SYBR-Green significance and biologic function of IL-4Ra in human fluorometric-based detection system (Applied Biosystems) MPM. with a Stratagene MX 3000P device. The primer sequences for IL-4Ra (synthesized by Invitrogen Life Technologies) Materials and Methods used for RT-PCR were as follows: 50-TCATGGAT- Patients, tissues, and cell lines GACGTGGTCAGT-30 (forward) and 50-GTGTCGGAGA- Patients. Human tumor, blood, and effusion were CATTGGTGTG-30 (reverse). PCR amplification of cDNA acquired from patients undergoing extrapleural pneumo- samples was carried out in triplicate using appropriate nectomy (EPP) in accordance with Institutional Review negative controls and the melting curves were in agreement Board policy and following informed consent. Required with the expected amplified fragments. Ribosome 18S RNA criteria for EPP include disease confined to the ipsilateral was used as an internal control for normalization of data hemithorax and sufficient predicted postoperative pulmo- [50-ATGGCCGTTCTTAGTTGGTG-30 (forward) and 30- nary reserve. At our institution, EPP is more commonly CGCTGAGCCAGTCAGTGTAG-50 (reverse)]. carried out over pleurectomy/decortication for MPM and likely provides more complete resection, especially for Immunohistochemistry and tissue microarray patients with bulky disease. Patient cohorts
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