Identification Of Molecular Drivers Of Human Hemangioblastoma

Hemangioblastoma of the central nervous system is the most common manifestation of Von Hippel-Lindau (VHL) disease, an autosomal dominant inherited cancer syndrome. The underlying molecular mechanism of hemangioblastoma is not known. To discover the molecular drivers of hemangioblastomas, 17 hemangioblastoma human tissue cases were submitted to MIP (molecular inversion probe) analysis to assess copy number alteration. Surprisingly, a focal loss of chromosome 17q was observed in 15 of 17 cases. Further analysis discovered a specific deletion involving HNF1B (Hepatocyte Nuclear Factor 1 homeobox B), a nuclear transcription factor. To elucidate the molecular effects of HNF1B loss, we used three different sequences of shRNA to knock down Hnf1b in mouse embryonic fibroblast (MEF) cells. Loss of Hnf1b resulted in significantly fewer cells arrested in G2/M in response to nocodazole treatment. Cell cycle related protein levels such as cyclin B1, cyclin D1 and phospho-Tyr15-Cdk1 were correspondingly changed. Of note, the cyclin dependent kinase inhibitor Cdkn1b (p27) was remarkably suppressed while another cyclin dependent kinase inhibitor, p21, was not affected at all. To elucidate the mechanism of Hnf1b dependent p27 regulation, the proteasome inhibitor MG132 was used to treat Hnf1b knockdown cells, which failed to increase p27 levels. An in vitro ubiquitination assay demonstrated that Hnf1b did not affect p27 ubiquitination. Both results indicated that Hnf1b regulation of p27 is not via posttranslational ubiquitin-proteasome system. Real time-polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) experiments demonstrated that Hnf1b regulated p27 at the mRNA level and specific consensus binding regions were identified. We then showed that p27 overexpression by lenti-virus infection in Hnf1b knockdown cells normalized the percentage of G2/M arrested cells. Hemangioblastoma are highly vascularized tumors. We discovered a dramatic up-regulation of vascular endothelial growth factor (Vegf) by enzyme-linked immunosorbent assay in cells with Hnf1b knockdown. These findings provided further evidence that loss of Hnf1b is one of the major drivers of the hemangioblastoma disease phenotype. Taken together, our findings for the first time identified HNF1B as a major gene loss in hemangioblastoma patients, and provide molecular evidence that Hnf1b transcriptionally regulates cell cycle protein p27, and drives the proangiogenic phenotype. This novel study sheds insights into molecular mechanisms of hemangioblastoma and provides tools to develop prevention and treatment strategies for the disease. Citation Format: Mianen Sun, Federico Monzon, Lijun Zhou, Xiande Liu, Zhiyong Ding, Xuesong Zhang, Shanshan Bai, Peter German, Sarathi Kalra, In Young ‎ Park, Ruhee Dere, Tia Berry, Xuefei Tong, Cheryl Walker, Nizar Tannir, Surena Matin, Gregory Fuller, Ian McCutcheon, Eric Jonasch. Identification of molecular drivers of human hemangioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2224. doi:10.1158/1538-7445.AM2014-2224