comparison between tumor and normal tissue generates a high number of differentially expressed genes, which makes biological interpretation difficult. As a result, we conclude it is important to study gene expression profiles comprehensively by grouping individual genes into gene sets or pathways to extract more sophisticated biological interpretations. We used SigPathway [8], a genome-wide biological pathway analysis package, and discovered three major biologic alterations in early-stage ccRCC that are likely related to a loss of epithelial differentiation. Our findings revealed four known renal develop- mental transcription factors (DTFs) that were significantly down- regulated in ccRCC and likely related to the differentiation changes in renal carcinogenesis. Further analysis revealed a novel molecular signature consistent with adipogenic transdifferentia- tion. This adipogenic molecular signature can explain the ‘‘clear cell’’ RCC morphology noted by cytoplasmic lipid accumulation. We also revealed a gene expression signature consistent with EMT in ccRCC. Based on these findings, we propose a carcinogenic cellular differentiation model of normal renal epithelial cells (NREs) that includes dedifferentiation, EMT, and adipogenic transdifferentiation that results in ccRCC. Results Pathway Signature of ccRCC with three major biological alterations SigPathway analysis of our gene expression data was performed to identify a pathway signature of ccRCC. The top 40 differentially expressed pathways in ccRCC are listed in Table 1. Three biological themes are identified in the pathway signature of ccRCC, that include (1) loss of normal renal function, (2) down-regulation of metabolism, and (3) activated immune pathways. All pathways related to normal renal function are down- regulated and include excretion, sodium ion transport, sodium ion binding, hydrogen transport, proton transport, chloride transport, and voltage-gated potassium channel activity. The excretion pathway is the most significant differentially expressed pathway in ccRCC in terms of overall rank, indicating that loss of normal renal function is the most striking biologic alteration. Other identified pathways are related to ion and electrolyte transport. Our heatmap presents the most noteworthy differentially ex- pressed genes from these pathways (Fig. 1a). The metabolic pathways are predominantly down-regulated and can be classified into 4 groups. (1) The lipid metabolic pathways are at the top of the list and include butanoate metabolism, propanoate metabolism, fatty acid metabolism and benzoate degradation via CoA ligation. (2) Energy metabolic pathways include oxidoreductase activity which acts on CH-OH group of donors, oxidoreductase activity which acts on the CH- OH group of donors, NAD or NADP as acceptor, cellular respiration, oxidoreductase activity, acting on the aldehyde or oxo group of donors, group transfer coenzyme metabolism, oxidative phosphorylation, oxidoreductase activity which acts on the CH-CH group of donors, ATP synthesis, nucleoside phosphate metabolism, and mitochondrial inner membrane. (3) Protein and amino acid metabolic pathways include valine, leucine, and isoleucine degradation, valine degradation, nitrogen compound catabolism, amine catabolism, amino acid catabolism and amino acid biosynthesis. (4) Carbohydrate metabolic pathways include fructose and mannose metabolism and pentose phosphate pathway. We have included the most differentially expressed metabolic genes from these pathways in a heatmap (Fig. 1b). Signature in Renal Carcinoma The immune pathways are significantly upregulated and include response to virus, antigen processing, antigen presentation, antigen presentation of endogenous antigen, regulation of cell activation, antigen processing related to endogenous antigen via MHC class I, and the regulation of lymphocyte activation. We present a heatmap generated by cluster analysis of the immune genes with significant alterations (Fig. 1c). Validation studies Validation at messenger RNA levels was performed by qPCR on several selected genes from the three pathway categories (Fig. 2a) and is consistent with the microarray data. IHC validation of the most significant genes in the three pathway categories was consistent with microarray and qPCR data, compared to patient-matched renal tissues samples (Fig. 2b, 2c, 2d). This includes the decreased expression of kininogen 1 (KNG1, NM_000893), aquaporin 2 (AQP2, AW015506), and sodium channel nonvoltage-gated 1b (SCNN1B, NM_000336) in normal renal function (blue arrows: Fig. 2a and 2b). Increased expression of enolase c (ENO2, NM_001975) and cytochrome P450-family 2-subfamily J-polypeptide 2 (CYP2J2, NM_000775), and decreased expression of Fructose-1,6-bisphosphate aldolase (ALDOB, BF195998) were identified as part of the metabolic alterations (green arrows: Fig. 2a and 2c). Increased expression was observed in toll-like receptor 2 (TLR2, NM_003264) and C- X-C chemokine receptor type 4 (CXCR4, AJ224869), which relates to immune function (red arrows: Fig. 2a and 2d). Validations of altered expressions of KNG1, SCNN1B, ALDOB, and TLR2 are novel and are reported for the first time in ccRCC. ccRCC is characterized by a loss of renal developmental transcription factors The pathway signature showed that ccRCC cells are quite different from normal renal cells, the bulk of which are epithelial cells. This indicates a loss of epithelial differentiation in ccRCC because the top-rated biological alteration is a down-regulation of pathways that are related to normal renal epithelial function. A lack of tubular architecture in ccRCC histology also suggests a loss of epithelial differentiation. We performed a comprehensive analysis to identify significant gene alterations in transcription factors that are known to be important for kidney development and cellular differentiation. We first identified all transcriptional factors in our dataset by using Gene Ontology gene set on transcription (GO: 006350). Statistical analysis with ANOVA was performed to derive a list of significantly altered transcription factors in ccRCC compared to normal renal samples. We then queried the literature for those transcription factors known to regulate renal development and differentiation. Four renal developmental transcription factors—GATA binding protein 3 (GATA3, AI796169), Transcription factor CP2-like 1 (TFCP2L1, AW195353), transcription factor activating protein -2b (TFAP2B, NM_003221) and Doublesex - mab-3 related transcription factor 2 (DMRT2, AF284225)—were significantly down-regulated in ccRCC (Fig. S1) and validated in another independent data set by qPCR consisting of 15 patient-matched normal renal and ccRCC tissues (Fig. 2a: yellow arrows). This data is illustrated by a heatmap and a table that outlines these transcription factors’ roles in renal development and show statistically significant P values for down-regulation in ccRCC (Fig. 3a). Decreased expression of TFCP2L1, TFAP2B, and DMRT2 were also validated by IHC (Fig. 3b). Decreased GATA3 mRNA and protein expression has also been confirmed in a recent publication from our laboratory [9]. PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10696