Signature in Renal Carcinoma Figure 2. Validation of gene expression findings in clear cell renal cell carcinoma. a. Heatmap generated with qPCR data of DTFs and various genes related to three biological alterations. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in black, as generated by Cluster 3.0. b. IHC of proteins related to normal renal function (KNG1, AQP2, SCNN1B). c. Immune function (TLR2, CXCR4). d. Metabolic function (ENO2, CYP2J2,ALDOB). This pattern of expression is in accord with the microarray findings. Sum scores are shown with n as indicated. *p,0.01 when comparing ccRCC to normal match. doi:10.1371/journal.pone.0010696.g002 undergone an epithelial to mesenchymal transition (EMT). Because we know that ccRCC cells are ladened with lipids [4] and that they histologically resemble adipocytes, we further hypothesized that our ccRCC cells may have under- gone adipogenic transdifferentiation, a type of mesenchymal differentiation. We confirmed lipid accumulation by IHC using oil Red ‘O’ stain (Fig. 4b) and we identified a gene expression signature in ccRCC that is consistent with adipogenic differentiation (Fig. 4a). The 22 genes used for the hierarchical cluster analysis came from the adipogenesis human pathway [12] and literature review [13]. Peroxisome proliferator-activated receptor c (PPARc, NM_015869) and CCAAT/enhancer binding protein b (CEBPb, AL564683) are transcription factors known to be required for adipogenesis and are upregulated in ccRCC. PPARa (BC004162), known to promote lipid degradation and b-oxidation of fatty acids, was downregulated [14];[15];[16]. Adipose differentiation-related protein (ADFP, BC005127), which is a marker of adipocyte differentiation and lipid storage [5], was significantly upregulated and validated by IHC (Fig. 4b). This upregulation of ADFP at the mRNA and protein level in ccRCC cells, is consistent with published data [5]. Angiopoietin-like 4 (ANGPTL4, NM_016109), which increases triglycerides by potent inhibition of lipoprotein lipase [17], was increased in ccRCC tumor tissue by 23-fold and may play a major role in lipid accumulation. Further, ANGPTL4 is upregulated during adipocyte differentiation [18]. Stearoyl-CoA desaturase (SCD, AB032261), which is involved in lipogenesis [19], was also upregulated. GATA2 (AL563460) and GATA3, both known to inhibit adipocyte differentiation [20];[21], were down-regulated and both were validated by IHC (Fig. 4b and [9]). Caveolin-1 (CAV1, NM_001753), which has been linked to lipid droplet biology and neutral lipid storage [22];[23], was significantly upregulated. Fatty acid binding proteins (FABPs) are significantly altered in their expression in ccRCC; FABP5 & 7 (NM_001444 & NM_001446) were upregulated, while FABP1 & 3 (NM_001443 & AI041520) were down-regulated. This pattern of directional alterations of adipogenic gene expression in ccRCC is consistent with adipogenic transdifferentiation. We further validated our findings by compar- ing data from a publicly available gene expression database (GSE#15641, deposited at Gene Expression Omnibus) of early- stage ccRCC against normal renal samples (Fig. S2). Thus, we have discovered a novel adipogenic signature in ccRCC. ccRCC cells undergo induced adipogenic and osteogenic transdifferentiation Using our newly discovered adipogenic ccRCC signature, we performed differentiation experiments on three human ccRCC cell lines to test the adipogenic differentiation capacity of ccRCC in adipogenic media. All ccRCC tumor cells grown in adipogenic media underwent adipogenic transdifferentiation as revealed by oil red ‘O’ staining for lipid droplets (Fig. 5a, 5b). Glycogen accumulation was also observed through the use of Periodic Acid Schiff-Hematoxylin (PASH) stain in adipogenic ccRCC tumor cells (Fig. 5c). This transdifferentiation is unique to ccRCC because adipogenic changes were not seen in controls (tumor cells in renal media, MDCK and patient-matched normal renal epithelial cells in both medias, Fig. 5). After this discovery, we performed osteogenic transdifferentiation experiments on the four cell lines to determine whether or not ccRCC cells were capable of other types of mesenchymal differentiation. Surprisingly, osteogenic differentia- tion, as verified by Alizarin stain, was observed in ccRCC cells grown in osteogenic media, but not in any of the controls (Fig. 6). ccRCC is characterized by EMT molecular markers Genomic profiling identified known molecular markers of EMT [24]. Utilizing data bases and the literature searches we compiled a PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10696