Inactivation of the VHL protein (pVHL) leads to stabilization of hypoxia inducible factors 1a and 2a (HIF1a and HIF2a) (Maxwell et al., 1999). This stabilization leads to up-regulation of HIF target genes involved in cellular proliferation, angiogenesis, erythropoiesi and metabolism. Inactivation of HIF2a by pVHL is necessary and sufficient for the tumor suppressor function of VHL (Kondo et al. 2002, 2003; Zimmer et al., 2004).
‘Clear cell’ renal cell carcinoma is the most prevalent type of renal cell carcinoma (75% of sporadic cases), and it is the sole tumor type developed by patients with VHL disease (Iliopoulos, 2006; Truong and Shen, 2011; Chan-Smutko and Iliopoulos, 2010). Tumors with clear cell histology are characterized by large cells with ‘clear cytoplasm’ resulting from empty vacuoles formed by the fixation of glycogen and lipid droplets contained within the original tumor (Shuch et al. 2015; Truong and Shen, 2011).
Zebrafish larvae with an inactivation of vhl (vhl-/-) mimic several aspects of human VHL disease, including, hyper-vascular lesions of the brain and retina, resembling hemangioblastomas, and erythrocytosis (Van Rooijen et al., 2009, 2010; Metelo et al., 2015). In a recent article published in Disease Models and Mechanisms, Noonan et al. characterized the renal epithelial abnormalities present in vhl-/- zebrafish larvae and identified for the first time a zebrafish model of early stage human ccRCC.
The vhl-/- larval pronephros exhibits several structural abnormalities when compared to their wild-type (wt) siblings (vhl+/- and vhl+/+). These abnormalities, visualized by hematoxylin and eosin staining, include an increased tubule diameter that contains fewer nuclei (seen in transverse histological sections) and cytoplasmic vesicles (Figure 1). Electron microscopy revealed disorganized cilia coating a small, closed lumen. In conjunction with electron microscopy, staining with BODIPY 493/503 lipophilic fluorescence dye highlighted prominent lipid vesicles, whereas periodic acid Schiff staining confirmed increased glycogen content. Strikingly, the overall histological disorganization present in the vhl-/- larval pronephros, including the presence of cytoplasmic lipid vesicles and glycogen, is reminiscent of typical ‘clear cell’ histology.
BrdU and caspase 3 immunofluorescence revealed the presence of aberrant cell proliferation and apoptosis, respectively. These observations are consistent with the fact that ccRCC is characterized by highly proliferative tumor cells with a reduced number of cilia (Schraml et al. 2009). Interestingly, vhl-/- larval pronephric abnormalities were only observed in the proximal, and not distal, pronephros. This corroborates the idea that ccRCC likely originates in the proximal tubule in humans (Rankin et al., 2006; Shuch et al., 2015; Haase, 2005; Chen et al., 2016). For these reasons, the vhl-/- mutant zebrafish may serve as a model of early stage ccRCC.
There is currently no treatment for VHL disease and patients with ccRCC are commonly treated with inhibitors of the vascular endothelial growth factor (VEGF) signaling pathway, resulting in a mere prolongation of disease progression and overall survival (Lonser et al., 2003; Rini and Atkins, 2009). The limited success of these inhibitors is not surprising as VEGF is only one of the many targets downstream of HIF2a. The authors have previously reported the use of a small molecule HIF2a inhibitor, Compound 76, in mammalian cells and the vhl-/- zebrafish model (Zimmer et al., 2008; Metelo et al., 2015). Compound 76 has been shown to rescue several aspects of the vhl-/- zebrafish’s biology, including eythrocytosis, irregular angiogenesis of the brain and retina, cardiomegaly with decreased cardiac contractility, abnormal hematopoiesis and early lethality (Metelo et al., 2015).
Noonan et al. treated the vhl-/- zebrafish larvae with the specific small molecule HIF2a inhibitor Compound 76, noting that it significantly attenuated the proximal pronephric phenotype. Compound 76 treated fish had more organized nuclei, fewer lipid vesicles and a significantly larger, more structured lumen (seen in transverse histological sections). Therefore, the authors concluded that the proximal vhl-/- pronephric phenotype is at least in part Hif2a driven, emphasizing the usefulness of HIF2a inhibitors in the treatment of ccRCC and VHL disease. Establishing a model of ccRCC and VHL disease may accelerate the discovery of novel treatments for VHL-associated tumors, including sporadic renal cell carcinoma.
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Written by: Haley R. Noonan1 and Othon Iliopoulos1,2,8
1 Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02114,USA
2 Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
8 Division of Hematology-Oncology, Department of Medicine, Massachusetts General Hospital, Boston, MA 02142, USA
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