Abstract
New treatment approaches are urgently needed for children with medulloblastoma (MB) to augment survival and reduce the long-term side effects from the treatment. Malignant tumors often display hypoxic regions where hypoxia-induced transcription factors (HIFs) increase the expression of genes that promote tumor growth (e.g., metabolic adaptation and invasion/metastasis). Based on the known roles of HIF in many solid tumors, we hypothesized that Hypoxia/HIFs contribute to MB cell growth and targeting HIF proteins is a viable strategy for treating MB. To test our hypothesis, first, we exposed MB cell lines (ONS-76; Shh and D556; Group 3) to 1% hypoxia in cell culture. We found increased expression of both HIF1a and HIF2a proteins and activation of a hypoxia-response element driven luciferase reporter assay. Treatment of the cells with 64B, an arylsulfonamide HIF inhibitor abrogated reporter activity and reduced expression of hypoxia-induced genes (GLUT-1, VEGF). Next, we used CRISPR/Cas9 to knockout singly or in combination HIF1a/HIF2a genes. The inhibition of HIF reporter activity by 64B was partially canceled in single HIF-KO cells and completely canceled in double HIFs-KO cells, showing both isoforms were active. Second, to investigate HIFs can be therapeutic targets, we established orthotopic human MB xenografts in mice. We first examined the tumor for the presence of hypoxic areas and used pimonidazole (Hypoxyprobe) injections followed by immunohistochemistry and found hypoxic areas throughout the tumors where tumor cells expressed HIF1a and/or HIF2a. Evaluation of human MB samples for HIF1/2 expression is also ongoing. Next, we plan on injecting mice with cells devoid of HIFs genes and examine their growth. We will also treat the mice with 64B to test whether it has anti-tumor effects and extends survival. In conclusion, these results indicate that HIF activation is part of the pathobiology of MB and HIF proteins may be a target for MB treatment.