Gene silencing of HIF-2α disrupts glioblastoma stem cell phenotype

Aim: Improved treatment strategies are desperately needed for eradicating cancer stem cells (CSCs), which drive malignancy and recurrence in glioblastoma multiforme. Hypoxic regions within the tumor microenvironment help maintain and promote the proliferation of CSCs. Here, we explored the effects of silencing hypoxia inducible factor-2α (HIF-2α) because of its specificity for CSCs within the hypoxic environment. Methods: Cancer stem cell neurospheres were formed by enriching from both the glioblastoma cell line U87 and from brain tumor stem cells isolated directly from human brain tumors. Silencing of human HIF-2α was performed using both commercial and in-house transfection of a validated short interfering RNA, with all results compared to an established non-silencing control short interfering RNA. Silencing of HIF-2α was established by Western blotting, and phenotypic effects were assayed by cell migration assays, cell viability measurements, and immunofluorescence staining of differentiation markers. Results: Transfection with either our previously reported pH-sensitive, cationic amphiphilic macromolecule-based delivery system or Lipofectamine was similarly effective in silencing HIF-2α. The chemotherapeutic resistance and neurosphere formation were reduced when HIF-2α was silenced. Migratory capacities in the presence of macrophage conditioned media were modulated. HIF-2α silencing was complementary to temozolomide treatment in producing phenotypic rather than cytotoxic effects. Conclusion: HIF-2α silencing under hypoxia inhibited CSC phenotypes while promoting differentiated cell phenotypes and is complementary to existing DNA alkylating treatments in inhibiting glioma CSC activity.