Loss of vhl in the zebrafish pronephros recapitulates early stages of human clear cell renal cell carcinoma- Beyond the Abstract

The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in over 90% of sporadic clear cell renal cell carcinomas (ccRCC) (The Cancer Genome Atlas Research Network, 2013). Patients with VHL disease harbor a germline mutation in the VHL gene leading to erythrocytosis and the development of several tumor types serially over their lifetime. Such tumors include ccRCC and hemangioblastomas of the brain, retina, and spinal cord (Maher and Kaelin, 1997).

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

References:

The Cancer Genome Atlas Research Network. (2013). Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature 499, 43-49.

Maher, E. R. and Kaelin, W. G. (1997). von Hippel-Lindau disease. Medicine 76, 381-391.

Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C., Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R. and Ratcliffe, P. J. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271-275.

Kondo, K., Klco, J., Nakamura, E., Lechpammer, M. and Kaelin, W. G., Jr. (2002). Inhibition of HIF is necessary for tumor suppression by the von HippelLindau protein. Cancer Cell 1, 237-246.

Kondo, K., Kim, W. Y., Lechpammer, M. and Kaelin, W. G., Jr. (2003). Inhibition of HIF2alpha is sufficient to suppress pVHL-defective tumor growth. PLoS Biol. 1, E83.

Zimmer, M., Doucette, D., Siddiqui, N. and Iliopoulos, O. (2004). Inhibition of hypoxia-inducible factor is sufficient for growth suppression of VHL−/− tumors.
Mol. Cancer Res. 2, 89-95.

Iliopoulos, O. (2006). Molecular biology of renal cell cancer and the identification of therapeutic targets. J. Clin. Oncol. 24, 5593-5600.

Truong, L. D. and Shen, S. S. (2011). Immunohistochemical diagnosis of renal neoplasms. Arch. Pathol. Lab. Med. 135, 92-109.

Chan-Smutko, G. and Iliopoulos, O. (2010). Familial renal cell cancers and pheochromocytomas. In Principles of Clinical Cancer Genetics: A Handbook from
the Massachusetts General Hospital (ed. D. C. Chung and D. A. Haber), pp. 109-128. US: Springer.

Shuch, B., Amin, A., Armstrong, A. J., Eble, J. N., Ficarra, V., Lopez-Beltran, A., Martignoni, G., Rini, B. I. and Kutikov, A. (2015). Understanding pathologic variants of renal cell carcinoma: distilling therapeutic opportunities from biologic complexity. European Urol. 67, 85-97.

Van Rooijen, E., Voest, E. E., Logister, I., Korving, J., Schwerte, T., SchulteMerker, S., Giles, R. H. and van Eeden, F. J. (2009). Zebrafish mutants in the von
Hippel-Lindau tumor suppressor display a hypoxic response and recapitulate key aspects of Chuvash polycythemia. Blood 113, 6449-6460.

Van Rooijen, E., Voest, E. E., Logister, I., Bussmann, J., Korving, J., van Eeden, F. J., Giles, R. H. and Schulte-Merker, S. (2010). von Hippel-Lindau tumor
suppressor mutants faithfully model pathological hypoxia-driven angiogenesis and vascular retinopathies in zebrafish. Dis. Model. Mech. 3, 343-353.

Metelo, A. M., Noonan, H. R., Li, X., Jin, Y., Baker, R., Kamentsky, L., Zhang, Y., van Rooijen, E., Shin, J., Carpenter, A. E. et al. (2015). Pharmacological HIF2a inhibition improves VHL disease-associated phenotypes in zebrafish model. J. Clin. Invest. 125, 1987-1997.

Schraml, P., Frew, I. J., Thoma, C. R., Boysen, G., Struckmann, K., Krek, W. and Moch, H. (2009). Sporadic clear cell renal cell carcinoma but not the papillary type is characterized by severely reduced frequency of primary cilia. Mod. Pathol. 22, 31-36.

Rankin, E. B., Tomaszewski, J. E. and Haase, V. H. (2006). Renal cyst development in mice with conditional inactivation of the von Hippel-Lindau tumor suppressor. Cancer Res. 66, 2576-2583.

Haase, V. H. (2005). The VHL tumor suppressor in development and disease: functional studies in mice by conditional gene targeting. Semin. Cell Dev. Biol. 16, 564-574.

Chen, F., Zhang, Y., Senbabaoglu, Y., Ciriello, G., Yang, L., Reznik, E., Shuch, B., Micevic, G., De Velasco, G., Shinbrot, E. et al. (2016). Multilevel genomicsbased taxonomy of renal cell carcinoma. Cell Rep. 14, 2476-2489.

Lonser, R. R., Glenn, G. M., Walther, M., Chew, E. Y., Libutti, S. K., Linehan, W. M. and Oldfield, E. H. (2003). von Hippel-Lindau disease. Lancet 361, 2059-2067.

Rini, B. I. and Atkins, M. B. (2009). Resistance to targeted therapy in renal-cell carcinoma. Lancet Oncol. 10, 992-1000

Zimmer, M., Ebert, B. L., Neil, C., Brenner, K., Papaioannou, I., Melas, A.,Tolliday, N., Lamb, J., Pantopoulos, K., Golub, T. et al. (2008). Small-molecule inhibitors of HIF-2a translation link its 5′UTR iron- responsive element to oxygen sensing. Mol. Cell 32, 838-848.