Molecular and Cellular Pathobiology HAVCR/KIM-1 Activates the IL-6/STAT-3 Pathway in Clear Cell Renal Cell Carcinoma and Determines Tumor Progression and Patient Outcome Thaïs Cuadros 1 , Enric Trilla 3 , Eduard Sarr  o 1 , Maya R. Vil a 1 , Jordi Vilardell 5 , In es de Torres 4 , Mayte Salcedo 4 , Joan L opez-Hellin 1 , Alex S anchez 2 , Santiago Ram on y Cajal 4 , Emilio Itarte 5 , Juan Morote 3 , and Anna Meseguer 1,6,7 Abstract Renal cell carcinoma (RCC), the third most prevalent urological cancer, claims more than 100,000 lives/year worldwide. The clear cell variant (ccRCC) is the most common and aggressive subtype of this disease. While commonly asymptomatic, more than 30% of ccRCC are diagnosed when already metastatic, resulting in a 95% mortality rate. Notably, nearly one-third of organ-confined cancers treated by nephrectomy develop metastasis during follow-up care. At present, diagnostic and prognostic biomarkers to screen, diagnose, and monitor renal cancers are clearly needed. The gene encoding the cell surface molecule HAVCR1/KIM-1 is a suggested susceptibility gene for ccRCC and ectodomain shedding of this molecule may be a predictive biomarker of tumor progression. Microarray analysis of 769-P ccRCC-derived cells where HAVCR/KIM-1 levels have been upregulated or silenced revealed relevant HAVCR/KIM-1–related targets, some of which were further analyzed in a cohort of 98 ccRCC patients with 100 month follow-up. We found that HAVCR/KIM-1 activates the IL-6/STAT-3/HIF-1A axis in ccRCC- derived cell lines, which depends on HAVCR/KIM-1 shedding. Moreover, we found that pSTAT-3 S727 levels represented an independent prognostic factor for ccRCC patients. Our results suggest that HAVCR/KIM-1 upregulation in tumors might represent a novel mechanism to activate tumor growth and angiogenesis and that pSTAT-3 S727 is an independent prognostic factor for ccRCC. Cancer Res; 74(5); 1416–28. Ó2014 AACR. Introduction Renal cell carcinoma (RCC) is the third most prevalent urological cancer. It accounts for approximately 3% of all new cancer cases and the incidence rates for all stages have been rising steadily over the last 3 decades (1–3). The clear cell RCC (ccRCC) is the most common subtype and accounts for approx- imately 80% of all renal cancers. Commonly asymptomatic, one third of ccRCCs are diagnosed when they are already meta- static, resulting in a 95% mortality rate. Moreover, one third of organ-confined cancers treated by nephrectomy develop metastasis during the follow-up (4). Although ccRCC is resistant to chemotherapy, development of new therapies has improved the median survival period of patients with advanced ccRCC, which is about 26 months now (5). Hepatitis A virus receptor/kidney injury molecule 1 (HAVCR/KIM-1) is overexpressed in ccRCC tumors and blocks expression of epithelial differentiation markers when over- expressed in 769-P cells (6). The HAVCR1 gene codes for a type I transmembrane glycoprotein that contains an extracel- lular immunoglobulin-like domain topping a long mucin-like sequence, which is shed and released by metalloproteinases. HAVCR, initially described in primate kidney cells (7), repre- sents the founding member of the HAVCR/KIM/TIM family (8, 9). HAVCR/KIM-1 ectodomain detection in urine represents a diagnostic marker for acute kidney injury (10, 11) and early detection of ccRCC (12, 13). HAVCR/KIM-1 ectodomain shed- ding has been recently correlated with a more invasive phe- notype in vitro and more aggressive tumors in vivo (14). To examine the biologic function of HAVCR/KIM-1 effects in ccRCC, microarray assays on 769-P ccRCC–derived cells, with upregulated or silenced HAVCR/KIM-1 levels, were conducted and relevant HAVCR/KIM-1 targets further analyzed in patients with ccRCC. Results presented in this article provide relevant data to better understand the role of HAVCR/KIM-1 on renal cancer development and progression. Patients and Methods A complete description is given in Supplementary Patients and Methods. Case selection Clinical and pathologic data from 168 patients with renal masses treated with radical or partial nephrectomy for RCC Authors' Affiliations: 1 Fisiopatología Renal, CIBBIM; 2 Statistics and Bioinformatics Unit (UEB), Vall d'Hebron Institute of Research; 3 Servicio de Urología, Hospital Vall d'Hebron; 4 Servicio de Anatomía Patol  ogica, Hospital Vall d'Hebr on; 5 Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica, Facultat de Bioci encies; 6 Departament de Bioqui- mica i Biologia Molecular, Unitat de Bioquímica de Medicina, Universitat Aut onoma de Barcelona, Bellaterra; and 7 Instituto Reina Sofía de Investigaci  on Nefrol  ogica, Fundaci  on Renal  I~ nigo  Alvarez de Toledo, Spain Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Anna Meseguer, Vall d'Hebron Research Institute, Pg. Vall d'Hebron, 119-129, 08035 Barcelona, Spain. Phone: 34-934-894- 061; Fax: 34-934-894-015; E-mail: ana.meseguer@vhir.org doi: 10.1158/0008-5472.CAN-13-1671 Ó2014 American Association for Cancer Research. Cancer Research Cancer Res; 74(5) March 1, 2014 1416 on May 2, 2017. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst January 3, 2014; DOI: 10.1158/0008-5472.CAN-13-1671