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

Abstract

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.

Examination of sulfonamide-based inhibitors of MMP3 using the conditioned media of invasive glioma cells

“…NFF-3 is a substrate selective for MMP3 (kcat/Km ¼ 218,000 s À1 M À1 ), and to a much lesser degree, MMP9 (kcat/Km ¼ 10,000 s À1 M À1 ) 54 . Consistent with our MMP3 SDS-PAGE/westernblot datum, the conditioned medium of C6-Cx43 demonstrated robust fluorescence activity compared to low motility C6 cells and unconditioned DMEM 18 . Linear responses of MMP3 activity was robustly measured from 30 to 300 min.…”

“…Cx43 promotes migration/invasion of glioma cells in vitro by wound healing, Transwell TM assay, and brain slices; and in vivo using Cx43 wild-type and knockout mice 12,15,16 . Demonstrating a direct link to secreted factors and glioma motility, the transfer of C6-Cx43 conditioned media was found to be sufficient to stimulate movement of slower-moving cells 18 . Importantly, our previous proteomic analysis of the conditioned media of C6-13 cells demonstrated specific, significant increases in MMP3; with no other members of the MMP family was found to be differentially expressed between high and low motility cells 18 .…”

“…Demonstrating a direct link to secreted factors and glioma motility, the transfer of C6-Cx43 conditioned media was found to be sufficient to stimulate movement of slower-moving cells 18 . Importantly, our previous proteomic analysis of the conditioned media of C6-13 cells demonstrated specific, significant increases in MMP3; with no other members of the MMP family was found to be differentially expressed between high and low motility cells 18 . In support of this view, we first performed SDS-PAGE/western blot analysis of MMP3 within the conditioned media from C6-Cx43, and C6 (parental line) serving as negative controls (Figure 2(A)).…”

“…The C6 glioma line (American Type Culture Collection, ATCC) maintains many characteristics of GBM including cancer stem cell behaviour ability to generate GBMs in rats upon injection in the brain [50][51][52] . The C6 clone stably expressing Cx43 demonstrates enhanced gap junctional intercellular communication, and restricted proliferation, with enhanced migratory potential (scrape-wound assay) when compared to wild-type C6 18,53 . Glioma cells were maintained at 37 C, 95% air and 5% CO 2 in a humidified incubator.…”

“…Identification of genes that are differentially regulated by invasive glioma are of significant interest. Using a model of glioma migration invasion based on the gap junction protein connexin43 (Cx43) [12][13][14][15][16][17] we recently demonstrated increases in the proteinase matrix metalloproteinase-3 (MMP3) within the conditioned media (secretome) 18 . These findings are supported by previous studies demonstrating MMP3 at invasive fronts of GBM tumours, and the reduction of invasion potential with MMP3 loss 19 .…”