Glioblastoma (GBM) is the most common, aggressive, and deadly form of primary brain tumor in adults, with a median survival time of only 14.6 months. GBM tumors present with chemo- and radio-resistance and rapid, diffuse invasion, making complete surgical resection impossible and resulting in nearly universal recurrence. While investigating the genomic landscape of GBM tumors has expanded understanding of brain tumor biology, targeted therapies against cellular pathways affected by the most common genetic aberrations have been largely ineffective at producing robust survival benefits. Currently, a major obstacle to more effective therapies is the impact of the surrounding tumor microenvironment on intracellular signaling, which has the potential to undermine targeted treatments and advance tumor malignancy, progression, and resistance to therapy. Additionally, mitochondria, generally regarded as putative energy sensors within cells, also play a central role as signaling organelles. Retrograde signaling occurring from mitochondria to the nucleus allows mitochondria to sense intracellular metabolic stress imposed by the extracellular tumor microenvironment and respond appropriately by relaying their status to the nucleus, thus inducing changes in gene expression and facilitating cellular adaptation.The focus of this work is on the mitochondrial protein coiled-coil-helix-coiled-coil-helix domain-containing protein 2 (CHCHD2) and its capacity to act in a signaling axis between mitochondria and the nucleus to facilitate cellular adaptation and plasticity as GBM cells navigate gradients in the tumor microenvironment. Chapters one, two, and three provide a general introduction of GBM, an analysis of mitochondrial biology in cancer and retrograde signaling, and a review on the current state of the literature surrounding CHCHD2. Chapter four focuses on characterizing the functional capacity of CHCHD2 in GBM cells using CRISPR-Cas9 genome editing techniques. We hypothesized that CHCHD2 knockout would abrogate malignant characteristics such as cell proliferation, sensitivity to therapeutics, and mitochondrial respiration. CHCHD2 gene amplification was found to occur in 9% of GBM tumors, nearly always with EGFR, and was associated with decreased overall survival and progression-free survival. CHCHD2 mRNA levels were increased in grade IV glioma, IDH-wt GBM tumors, and in tumor versus non-tumor tissue. CRISPR-Cas9 derived GBM U87vIII CHCHD2 KO cells displayed decreased OCR and spare respiratory capacity compared to U87vIII CHCHD2 WT cells. The redox state of the glutathione (GSH) pool was more reduced within the mitochondrial matrix of CHCHD2 KO cells compared to WT cells, an effect independent of GSH synthesis, cystine import via xCT, or GPx-1, GPx-2, or GPx-4 levels. The cytosolic GSH pool was not perturbed by CHCHD2 KO. Furthermore, CHCHD2 KO abrogated the increased proliferation and invasion of U87vIII cells in hypoxia (1% O2) compared to standard normoxic culture conditions (20% O2). U87vIII CHCHD2 KO cells displayed increased sensitivity to sulfasalazine, erlotinib, and temozolomide, but not the procaspase activator Pac-1. CHCHD2 exhibited a heterogeneous protein expression pattern among patient-derived cells investigated using western blot, with greater levels observed in more invasive samples. These results indicate that CHCHD2 mediates a variety of GBM cell hallmark characteristics, including cell proliferation, cell invasion in response to hypoxia, and cellular resistance to cytotoxic agents.The C-terminus of CHCHD2 contains redox-sensitive cysteines in twin CX9C motifs that are amenable to thiol/disulfide formation, which in turn determines protein folding and import into mitochondria. Chapter five focuses on defining the relationship between hypoxia and redox stress, CHCHD2 protein folding, and subcellular redistribution of CHCHD2, in order to examine a mechanism by which CHCHD2 signals mitochondrial and cellular status back to the nucleus. We hypothesized that reduction of the C-terminal CHCH domain would induce mitochondrial export of CHCHD2. Total CHCHD2 protein levels were not significantly altered by decreasing oxygen tensions in U87 cells. U87vIII cells exhibited constant levels of total CHCHD2 protein at 20%, 7%, and 4% O2, while incubation in 1% O2 led to a significant decrease in total CHCHD2. Half-life of CHCHD2 was determined to be approximately 3 h in both U87 and U87vIII cells. U87vIII cells constitutively displayed a greater amount of nuclear CHCHD2 compared to isogenic U87 cells at 20% O2. Metabolic stress induced by incubation in 1% O2 for 48 h or serum-free media for 30 min both resulted in increased CHCHD2 detected in nuclei of U87 and U87vIII cells. Oxidative stress induced by the glutathione synthesis inhibitor BSO increased nuclear CHCHD2 in U87vIII cells, while the glutathione precursor NAC did not significantly affect nuclear CHCHD2 levels. Exposure to H2O2 in serum-free media abrogated the amount of nuclear CHCHD2 detected in nuclei of U87 and U87vIII cells. Reductive unfolding of CHCHD2 with the disulfide reductant DTT consistently resulted in the greatest and greatest degree of nuclear accumulation of CHCHD2 in both cell lines within 15-30 min. Ectopic expression of exogenous 3X-FLAG-tagged CHCHD2 in U87vIII cells did not result in increased expression of endogenous CHCHD2. These findings indicate that reductive unfolding of CHCHD2 is responsible for mitochondrial export and nuclear translocation in response to hypoxia, serum deprivation, and redox stress.Finally, in addition to gradients in oxygen, nutrients, and biophysical cues, tumor-associated microglia and macrophages represent a substantial portion of GBM tumors and play a critical role in cultivating a microenvironment that facilitates tumor expansion. Chapter six focuses on examination of the crosstalk between microglia and GBM cells that promotes angiogenesis, cell invasion, and remodeling of the tumor immune landscape. We hypothesized that GBM-microglia crosstalk would facilitate secretion of soluble factors that promote angiogenesis and extracellular matrix remodeling, as well as alter GBM cell glutathione redox balance. The expression of EGFRvIII on U87 cells resulted in an altered profile of secreted angiogenesis-related factors under basal culture conditions. Similarly, primary neonatal murine microglia, but not the human microglia cell line HMC3, exhibited differential angiogenesis-related secretomes when cultured in U87 or U87vIII conditioned media. Primary murine microglia additionally displayed increased cell proliferation when cultured in U87 and U87vIII conditioned media, while HMC3 cells did not. Conditioned media from patient-derived, EGFRvIII-expressing xenograft GBM6 cells led to a striking increase in HMC3 secretion of angiogenesis-related factors, inducing the greatest secretion of CCL2, IGFBP-2, TIMP-4, angiogenin, and IGFBP-3. HMC3 conditioned media decreased expression of the system xc- antiporter and increased glutathione oxidation in mitochondria of U87 cells, while xCT expression and mitochondrial glutathione redox status in U87vIII cells remained stable. These findings indicate that GBM cells stimulate the secretion of cytokines, chemokines, and soluble factors by microglia to cultivate an immunosuppressive microenvironment promoting angiogenesis and cell invasion. Additionally, system xc- expression and, consequently, intracellular glutathione redox balance remained stable in U87vIII cells exposed to HMC3 conditioned media. Together, the studies demonstrate a dynamic crosstalk between GBM cells and microglia that facilitates microenvironment remodeling and has the potential to influence GBM cell glutathione metabolism.
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CHCHD2 and the tumor microenvironment in glioblastoma
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