Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma Han W. Tun1, Laura A. Marlow2, Christina A. von Roemeling2, Simon J. Cooper2, Pamela Kreinest2, Kevin Wu 3 , Bruce A. Luxon 4 , Mala Sinha4, Panos Z. Anastasiadis2, John A. Copland 2 * 1 Department of Hematology/Oncology, Mayo Clinic, Jacksonville, Florida, United States of America, 2 Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, United States of America, 3 Department of Pathology, Mayo Clinic, Jacksonville, Florida, United States of America, 4 Institute for Translational Science and Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, United States of America Abstract Background: Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer. The purpose of this study is to define a biological pathway signature and a cellular differentiation program in ccRCC. Methodology: We performed gene expression profiling of early-stage ccRCC and patient-matched normal renal tissue using Affymetrix HG-U133a and HG-U133b GeneChips combined with a comprehensive bioinformatic analyses, including pathway analysis. The results were validated by real time PCR and IHC on two independent sample sets. Cellular differentiation experiments were performed on ccRCC cell lines and their matched normal renal epithelial cells, in differentiation media, to determine their mesenchymal differentiation potential. Principal Findings: We identified a unique pathway signature with three major biological alterations—loss of normal renal function, down-regulated metabolism, and immune activation–which revealed an adipogenic gene expression signature linked to the hallmark lipid-laden clear cell morphology of ccRCC. Culturing normal renal and ccRCC cells in differentiation media showed that only ccRCC cells were induced to undergo adipogenic and, surprisingly, osteogenic differentiation. A gene expression signature consistent with epithelial mesenchymal transition (EMT) was identified for ccRCC. We revealed significant down-regulation of four developmental transcription factors (GATA3, TFCP2L1, TFAP2B, DMRT2) that are important for normal renal development. Conclusions: ccRCC is characterized by a lack of epithelial differentiation, mesenchymal/adipogenic transdifferentiation, and pluripotent mesenchymal stem cell-like differentiation capacity in vitro. We suggest that down-regulation of developmental transcription factors may mediate the aberrant differentiation in ccRCC. We propose a model in which normal renal epithelial cells undergo dedifferentiation, EMT, and adipogenic transdifferentiation, resulting in ccRCC. Because ccRCC cells grown in adipogenic media regain the characteristic ccRCC phenotype, we have indentified a new in vitro ccRCC cell model more resembling ccRCC tumor morphology. Citation: Tun HW, Marlow LA, von Roemeling CA, Cooper SJ, Kreinest P, et al. (2010) Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma. PLoS ONE 5(5): e10696. doi:10.1371/journal.pone.0010696 Editor: Chad Creighton, Baylor College of Medicine, United States of America Received January 28, 2010; Accepted April 28, 2010; Published May 18, 2010 Copyright: 2010 Tun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded in part by a Sarah and James Kennedy Career Development Award (H.W.T.), CA104505 (J.A.C.), CA125807 (J.A.C.), CA104505-05S1 (J.A.C.), a generous gift from Susan A. Olde, OBE (J.A.C.), The Scheidel Foundation (J.A.C.) and the David and Lois Stulberg Endowed Fund for Kidney Cancer (J.A.C.). The parties who provided these funds 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. * E-mail: copland.john@mayo.edu Introduction Renal cell carcinoma is the eighth most common cancer and highly lethal, accounting for about 13,000 deaths in the U.S. in 2009 [1]. If detected in early stages, it is potentially curable by surgical resection; however, there is no curative treatment for metastatic RCC. A better understanding of RCC’s biology is essential to improving prognosis. ccRCC represents the most common subtype (83%) of RCC [2]. The cell of origin and cellular differentiation program in ccRCC is not definitively known but ccRCC is commonly thought to arise from renal proximal epithelial cells [3]. The most striking phenotypic feature of ccRCC is its clear cell morphology, which has been linked to lipid and glycogen accumulation [4]. Thus the mechanism behind clear cell morphology is an important component of renal carcinogenesis that has yet to be defined at the molecular level. The gene expression profile of ccRCC has been investigated at the single gene level in multiple studies [5];[6] that have compared ccRCC to normal renal samples and other histological subtypes of RCC. Many statistically significant individual genes have been identified. At present, no comprehensive analysis of ccRCC at the pathway level exists. Since biological processes are mediated by multiple genes working in synchrony and genes are co-regulated, pathway analysis is more conclusive regarding biologic changes. Notably, genes that don’t seem differentially expressed at a statistically significant level may reveal biologically significant changes when studied in ontology groups [7]. Moreover, a PLoS ONE | www.plosone.org 1 May 2010 | Volume 5 | Issue 5 | e10696