Images were generated using Photoshop (Adobe Systems Inc., San Jose, CA) and Haloxon relative quantification was performed using ImageJ (NIH, Bethesda, MD). Quantitative Real-time PCR Total RNA was prepared from cell using the RNA Aqueous Kit (Ambion). WT1, since WT1 upregulates E-cadherin expression and competes with Snail repression. These findings support a novel paradigm in which WT1 induces an epithelial-mesenchymal hybrid transition (EMHT), characterized by Snail up-regulation with E-cadherin maintenance, a tumor cell differentiation state in which cancer cells keep both EMT and MET characteristics which may Haloxon promote tumor cell plasticity and tumor progression. Introduction Renal cell carcinomas (RCCs) are a heterogeneous group of deadly and treatment-resistant malignancies. The predominant subtype of RCC is clear cell RCC (ccRCC) and is characterized by loss-of-function mutations of the von Hippel-Lindau (encodes a protein of the same name that is a direct oxygen-dependent negative regulator of the -subunits of the transcription factor hypoxia-inducible factor (HIF-). In normoxic conditions, VHL binds to HIF- and promotes its ubiquitylation and subsequent degradation by the proteasome . However, hypoxia prevents VHL binding which stabilizes HIF- and permits the activation of target genes that regulate cellular adaptation to low oxygen . Loss of VHL in ccRCC abrogates oxygen-dependent regulation of HIF resulting in aberrant chronic activation of HIF regardless of cellular oxygenation. The HIF transcriptional program governs many diverse processes that facilitate cancer progression, including angiogenesis, metabolism, proliferation, survival, and metastasis . The Wilms’ tumor gene (gene, which serves as a transcription factor . Conversely, in heart development WT1 has been shown to activate the reverse process, epithelial-to-mesenchymal transition (EMT), in the epicardial cells that generate the cardiovascular progenitor cells which Rabbit polyclonal to TNFRSF10D then differentiate into various adult cardiac cells (coronary smooth muscle, interstitial fibroblasts, cardiomyocytes) . In addition, the highest levels of WT1 expression in adults are found in the podocytes (kidney), Sertoli cells (testis), and mesothelial cells, and all of these cell types share the capacity to readily switch between epithelial and mesenchymal phenotypes , . These observations suggest that WT1 mediates reciprocal transitions between these phenotypes. Transitions in cellular differentiation between epithelial and mesenchymal states are critical not only in organ development and wound healing, Haloxon but also appear to be co-opted during cancer progression. Epithelial cells are typically immobilized within tightly bound layers and exhibit apical-basolateral plasma membrane polarity and extensive cell-cell and cell-matrix adhesions. Vital to epithelial cells is E-cadherin, a Ca2+-dependent cell-cell adhesion molecule that forms the core of the adherens junctions that physically links cells together in close proximity to promote the well-differentiated epithelial phenotype C. In contrast to epithelial cells, mesenchymal cells exhibit an elongated and asymmetric morphology, and form only transient adhesions with neighboring cells . This phenotype promotes the dissolution of tissue integrity and enhances cell motility and invasion , . Loss of E-cadherin expression is a crucial event in the establishment of the mesenchymal phenotype, and transcriptional repressors such as Snail downregulate E-cadherin during EMT C. EMT is thought to occur during the progression of cancer to metastatic disease. This not only confers invasive properties, but also endows tumor cells with stem cell-like characteristics such as self-renewal and therapy-resistance . However, clinical observations have revealed that metastases derived from a variety of carcinoma types often display overtly epithelial differentiation . Recent evidence suggests that disseminated tumor cells may undergo MET in order to better facilitate colonization (i.e. proliferation) at the foreign site , . Although tumor cell differentiation appears to be highly plastic, the cellular and molecular regulators of this phenotypic plasticity are not well known. WT1 expression has been reported to be upregulated in a variety of solid tumors, including in ccRCC where it seems to act as an oncogene , , yet little is known about the pathophysiological consequences of WT1 expression in cancer. In this study, we report that WT1 expression is increased in VHL-deficient ccRCC cells. We show that WT1 directly upregulates Snail while also promoting the expression of E-cadherin. Further, WT1-expressing renal cells exhibited epithelial-like morphological features and epithelial junctions, even in the presence of Snail, while simultaneously also expressing various EMT and MET markers. Our results indicate that WT1 induces features of both Haloxon EMT and MET in ccRCC and suggest that it may regulate phenotypic plasticity by promoting an epithelial-mesenchymal hybrid differentiation state in cancer. Results Knockdown of VHL increases WT1 expression Increased expression of WT1 has been previously reported in ccRCC patient tumor samples . Since functional inactivation of the gene is a common defect in ccRCC, we hypothesized that loss of VHL might be associated with increased WT1 expression. To test this hypothesis, we used two well-characterized isogenic pairs of human.