publication that uses 177 ccRCC samples and demonstrates a wound-healing signature in ccRCC that the authors associated with EMT [50]. The authors concluded that concomitant loss of differentiation and acquisition of a wound-healing signature was associated with EMT. Moreover, this signature was highly prognostic for poor survival. We have revealed a gene expression signature and IHC findings that are consistent with adipogenesis in ccRCC. Moreover, our differentiation experiments have validated that ccRCC cells can undergo adipogenic transdifferentiation. It is well known that clear cell morphology is quickly lost when ccRCC cells are grown in standard cell culture media. In adipogenic media, ccRCC cells become adipogenic and redevelop clear cell morphology accumu- lating lipids, recapitulating the in vivo phenotype. It is interesting that in vitro adipogenic differentiation also leads to glycogen accumulation, which is another biological event responsible for clear cell morphology. These findings indicate that the clear cell morphology is a phenotypic marker of EMT, since adipogenic differentiation is a mesenchymal event. Our in vitro differentiation experiments reveal a pluripotent mesenchymal stem cell-like quality in ccRCC cells with the capacity to undergo adipogenic and osteogenic transdifferentia- tion. This stem cell-like quality belongs to the tumor cell population but not to the patient-matched normal renal epithelial cell population. Previous studies show that cells induced to undergo EMT can acquire stem cell-like qualities [51]. We believe that the stem cell-like qualities observed in ccRCC are directly linked to EMT. Because ccRCC cells are capable of pluripotent differentiation in vitro and undergo adipogenic differentiation preferentially in vivo in human patients, it is likely that the adipogenic process is an important component of renal carcino- genesis. It is also possible that preferential adipogenic differenti- ation may be due to prevailing ecological factors in the kidney. Heuristically, we think that cellular differentiation experiments using differentiation media is useful for determining mesenchymal stem cell-like qualities in cancer cells. In addition, the stem cell-like characteristic of ccRCC may be responsible for its notorious drug resistance [52]; [53]. Recently, active compounds, such as Salinomycin, with cytotoxicity against stem cells were identified and used in cells with induced EMT changes and stem cell-like features [54]. Pathway and cellular differentiation changes in ccRCC may be partly mediated by a loss of four developmental transcription factors identified in our study. Since transcription factors typically regulate multiple genes, dramatic attenuation of an expression of four DTFs could have a major impact on biological function in ccRCC. One of the DTFs, GATA3, may play a crucial role in ccRCC, as it can potentially regulate all three biologic alterations identified in ccRCC: loss of renal epithelial differentiation, adipogenic differentiation, and immune activation. GATA3 is important for renal development [55]; [56] and is necessary for nephric duct morphogenesis in the pro/mesonephric kidney [57]. A novel mutation in the GATA3 gene that results in haplodefi- ciency causes HDR syndrome (hypoparathyroidism, deafness, renal dysplasia) [58]; [59]. GATA3 also inhibits adipogenesis [20]; [21] and regulates immune function [60]. Because TFCP2L1 controlled genes are down-regulated in ccRCC and it is known to mediate renal epithelial cell development and tubule formation, loss of TFCP2L1 may play an important role in epithelial de- differentiation. TFAP2B is a member of the AP-2 family of transcription factors that functions as both a transcriptional activator and repressor. It appears to be required for proper terminal differentiation and function of renal tubular epithelia [61] and is integral to the survival of renal epithelial cells during renal Signature in Renal Carcinoma development [62]. DMRT2 is the top-ranking gene in our ccRCC gene expression dataset with the highest discriminant power based on our analysis using Fisher discriminant analysis (FDA) (Data not shown). This transcription factor is important for myogenesis and sex determination [63]. DMRT2 is also expressed in the urogenital tract [64] and its mutations are associated with renal abnormalities [65]. Based on our findings, we propose a model (Fig. 8) for aberrant cellular differentiation in ccRCC. Developmentally, kidneys are mesenchymal in origin and develop by biological processes, which includes mesenchymal epithelial transition (MET) [66]. We believe this process is reversed in ccRCC, which results in EMT and dedifferentiation. In our model, normal renal epithelial cells experience dedifferentiation and EMT as well as preferential adipogenic differentiation to develop an adipogenic phenotype in ccRCC. As a result of EMT and dedifferentiation, we postulate that ccRCC cells possess stem cell-like qualities. Since our samples came from patients with early-stage ccRCC, it appears that EMT plays a prominent role in renal carcinogenesis. This finding is in line with the important role of EMT pathogenesis of cancer [67]. Our present findings shed new light on the biology of ccRCC and have implications for future research. We showed a comprehensive pathway signature and identified an adipogenic transdifferentiation signature in ccRCC. We revealed that clear cell morphology is a marker of adipogenic transdifferentiation and EMT. We identified four DTFs, which may play a role in ccRCC due to loss of expression. Furthermore, our in vitro differentiation experiments may be useful as an assay for determining the pluropotency of cancer cells that have undergone EMT. With an implication for cell models used in ccRCC research, we showed that adipogenic media can restore the clear cell morphology in ccRCC cell lines. Since these cells exhibit the characteristic ccRCC phenotype, we suggest that they may be another relevant in vitro model to study ccRCC. Materials and Methods This study was approved by the Mayo Institutional Review Board Committee. The tissues used in this study were either de- identified or were archival tissues and thus patient consent is not necessary. Study Samples The tissue samples for the microarray study consisted of 10 patient-matched normal renal cortex and ccRCC tissues, five from stage I and five from stage II ccRCC. Both early-stage I and II tumors were localized disease; stage I tumors were less than 7 cm and stage II tumors were greater than 7 cm. We saw little to no difference in gene expression between the two tumor stages. Validation by real time quantitative PCR (qPCR) was performed using an independent set of 15 early-stage ccRCC patient-matched normal and renal samples. Another independent set of patient samples was used to measure protein levels by immunohistochem- istry (IHC) in tissue arrays comprised of 50 paired early-stage ccRCC patient-matched normal and renal samples for immunohistochem- ical (IHC) validation. An n value was assigned for each IHC that indicated scorable samples from the tissue arrays. Rarely were all array samples intact, which allowed all 50 samples to be read. The pathologic diagnosis was confirmed by a central pathology review and was approved by the institutional review board. Microarray Protocols DNA microarray experiments were performed using the Affymetrix HG-U133 set, which consisted of HG-U133a and PLoS ONE | www.plosone.org 11 May 2010 | Volume 5 | Issue 5 | e10696