Cx43-Associated Secretome and Interactome Reveal Synergistic Mechanisms for Glioma Migration and MMP3 Activation

Extracellular matrix (ECM) remodeling, degradation and glioma cell motility are critical aspects of glioblastoma multiforme (GBM). Despite being a rich source of potential biomarkers and targets for therapeutic advance, the dynamic changes occurring within the extracellular environment that are specific to GBM motility have yet to be fully resolved. The gap junction protein connexin43 (Cx43) increases glioma migration and invasion in a variety of in vitro and in vivo models. In this study, the upregulation of Cx43 in C6 glioma cells induced morphological changes and the secretion of proteins associated with cell motility. Demonstrating the selective engagement of ECM remodeling networks, secretome analysis revealed the near-binary increase of osteopontin and matrix metalloproteinase-3 (MMP3), with gelatinase and NFF-3 assays confirming the proteolytic activities. Informatic analysis of interactome and secretome downstream of Cx43 identifies networks of glioma motility that appear to be synergistically engaged. The data presented here implicate ECM remodeling and matrikine signals downstream of Cx43/MMP3/osteopontin and ARK1B10 inhibition as possible avenues to inhibit GBM

The role of the gap junction protein connexin43 in glioma migration and invasion

Glioblastoma Multiforme (GBM), an aggressive form of adult brain tumor, is difficult to treat due to its invasive nature. A molecular change frequently observed in GBM is a decrease in the expression of the gap junction protein Connexin43 (Cx43); however, how a reduction in Cx43 expression contributes to glioma malignancy is still unclear. The first objective of this thesis was to establish an in vitro human GBM cell model to clearly delineate the role of Cx43 in the migration and invasion phenotype. Characterization of a panel of immortalized high grade human GBM cell lines showed variability in Cx43 protein expression, subcellular localization, gap junctional coupling and migration. For the second objective of my thesis I selected the human GBM cell line U118 from the aforementioned panel and developed a 3D spheroid migration model that mimics the in vivo architecture of tumor cells to quantify migration changes. Down-regulation of Cx43 expression increased migration by reducing cell-ECM adhesion. Using live imaging my findings are the first to show that glioma cells change their migration pattern from collective to single cell when Cx43 is reduced. In addition, reducing Cx43 expression enhanced relative migration by increasing the cell speed and affecting the direction of migration. Subsequently, gap junction intercellular communication (GJIC) played a more prominent role in mediating migration than the cytoplasmic interactions of the C-terminal tail. Taken together my findings reveal an unexplored role of Cx43 in facilitating collective glioma migration. The third objective of this thesis was to assess the role of homocellular and heterocellular gap junctions in glioma invasion using a syngeneic in vivo mouse model. A reduction in invasion was observed when we reduced Cx43 in mouse GL261 glioma cells and deleted it in host astrocytes. Interestingly, blocking the channel in GL261 did not decrease invasion. In summary, a reduction in homocellular gap junction communication increases migration of glioma cells in vitro however when they encounter astrocytes in the brain a lack of heterocellular gap junction communication reduced invasion. This suggests that gap junctions may have opposing roles when formed between glioma cells versus when formed between glioma and astrocytes.Medicine, Faculty ofGraduat

Systematic identification of genes involved in metabolic acid stress resistance in yeast and their potential as cancer targets

A hallmark of all primary and metastatic tumours is their high rate of glucose uptake and glycolysis. A consequence of the glycolytic phenotype is the accumulation of metabolic acid; hence, tumour cells experience considerable intracellular acid stress. To compensate, tumour cells upregulate acid pumps, which expel the metabolic acid into the surrounding tumour environment, resulting in alkalization of intracellular pH and acidification of the tumour microenvironment. Nevertheless, we have only a limited understanding of the consequences of altered intracellular pH on cell physiology, or of the genes and pathways that respond to metabolic acid stress. We have used yeast as a genetic model for metabolic acid stress with the rationale that the metabolic changes that occur in cancer that lead to intracellular acid stress are likely fundamental. Using a quantitative systems biology approach we identified 129 genes required for optimal growth under conditions of metabolic acid stress. We identified six highly conserved protein complexes with functions related to oxidative phosphorylation (mitochondrial respiratory chain complex III and IV), mitochondrial tRNA biosynthesis [glutamyl-tRNA(Gln) amidotransferase complex], histone methylation (Set1C–COMPASS), lysosome biogenesis (AP-3 adapter complex), and mRNA processing and P-body formation (PAN complex). We tested roles for two of these, AP-3 adapter complex and PAN deadenylase complex, in resistance to acid stress using a myeloid leukaemia-derived human cell line that we determined to be acid stress resistant. Loss of either complex inhibited growth of Hap1 cells at neutral pH and caused sensitivity to acid stress, indicating that AP-3 and PAN complexes are promising new targets in the treatment of cancer. Additionally, our data suggests that tumours may be genetically sensitized to acid stress and hence susceptible to acid stress-directed therapies, as many tumours accumulate mutations in mitochondrial respiratory chain complexes required for their proliferation