Regulation of Bi‐organellar CHCHD2 in Response to Hypoxia is Dependent upon EGFR Molecular Subtype in Glioblastoma

Glioblastoma (GBM) is the most common and malignant form of brain cancer in adults, with a median survival time of 12–15 mo. Tumors are heterogeneous and characterized by hypoxic foci. Amplification, overexpression, and mutation of the receptor tyrosine kinase (RTK) EGFR are prevalent in GBM, and the constitutively active EGFR vIII mutant shows the poorest clinical outcomes. However, RTK inhibitors have failed clinically against GBM, with EGFR subtype poorly predicting outcome. We are investigating compensatory signaling pathways active within GBM tumor margins as a possible source of resistance to molecular subtype therapies. Previous studies have reported coiled‐coil‐helix‐coiled‐coil‐helix domain‐containing protein 2 (CHCHD2) as a bi‐organellar protein localized predominantly in the mitochondrial inner membrane but also present in the nucleus where it acts as a transcription factor. We hypothesized that this co‐localization enables GBM cells to adapt to hypoxic stress in the tumor microenvironment, particularly via mitochondrial retrograde signaling. We examined CHCHD2 localization by immunocytochemistry in U87 wt and U87 vIII (constitutively active EGFR vIII mutant) GBM cells subjected to 24, 48, and 72 h hypoxic (1% O 2 ) incubation. Time‐lapse imaging revealed significantly greater nuclear accumulation of CHCHD2 in U87 vIII cells compared to U87 wt cells at all hypoxic time points. We next used engineered gliomas formed by microfluidic templating as a model of the GBM microenvironment. RT‐PCR and Western blot analyses revealed increased CHCHD2 gene expression but decreased CHCHD2 protein levels in U87 wt cells in 72 h hypoxic incubation compared to normoxic conditions. No changes in CHCHD2 gene expression or protein content were observed in U87 vIII cells after 72 h hypoxic incubation. Using the aforementioned engineered gliomas, we observed hypoxia significantly increased GBM invasion and pERK1/2 activation in both EGFR wt and EGFR vIII variants. These data indicate more stable levels of CHCHD2 and greater translocation to the nucleus in U87 vIII cells under hypoxic stress, thus impacting downstream genes and pathways and potentially contributing to compensatory oncogenic signaling capable of overcoming RTK inhibition. Additional work will focus on subcellular fractionation and mitochondrial isolation to measure CHCHD2 protein levels in nuclei and mitochondria. EGFR wt and EGFR vIII variant GBM cells with CRISPR‐silenced CHCHD2 are being created to determine the extent to which differential mitochondrial‐to‐nuclear translocation of this bi‐organellar protein in hypoxic U87 wt and U87 vIII cells contributes explicitly to their enhanced invasion potential and therapeutic resistance. Support or Funding Information Funding: RO1CA197488