NG2 Proteoglycan Enhances Brain Tumor Progression by Promoting Beta-1 Integrin Activation in both Cis and Trans Orientations

Abstract

By physically interacting with beta-1 integrins, the NG2 proteoglycan enhances activation of the integrin heterodimers. In glioma cells, co-localization of NG2 and α3β1 integrin in individual cells (cis interaction) can be demonstrated by immunolabeling, and the NG2-integrin interaction can be confirmed by co-immunoprecipitation. NG2-dependent integrin activation is detected via use of conformationally sensitive monoclonal antibodies that reveal the activated state of the beta-1 subunit in NG2-positive versus NG2-negative cells. NG2-dependent activation of beta-1 integrins triggers downstream activation of FAK and PI3K/Akt signaling, resulting in increased glioma cell proliferation, motility, and survival. Similar NG2-dependent cis activation of beta-1 integrins occurs in microvascular pericytes, leading to enhanced proliferation and motility of these vascular cells. Surprisingly, pericyte NG2 is also able to promote beta-1 integrin activation in closely apposed endothelial cells (trans interaction). Enhanced beta-1 signaling in endothelial cells promotes endothelial maturation by inducing the formation of endothelial junctions, resulting in increased barrier function of the endothelium and increased basal lamina assembly. NG2-dependent beta-1 integrin signaling is therefore important for tumor progression by virtue of its affects not only on the tumor cells themselves, but also on the maturation and function of tumor blood vessels.

Structural deciphering of the NG2/CSPG4 proteoglycan multifunctionality

“…Glycanated variants concentrate in filopodial extensions, including the finest protrusions conventionally denoted "microspikes," where they seem to colocalize with b1, a3, and a4 integrin subunits, ezrin, and neural precursor cell expressed, developmentally downregulated 9 ( Fig. 4 and Table 1) (17,23,(43)(44)(45)(46)(47). However, NG2/CSPG4 molecules lacking GAG chains have also been noted in microclustered configurations confined to putative lipid rafts.…”

“…4). Furthermore, in membrane microprotrusions, the intracellular domain of NG2/CSPG4 is preferentially phosphorylated in Thr 2314 by a PKCadependent mechanisms and this phosphorylation event seems crucial in directing the PG, along with integrin a3b1 (and possibly other b1-class integrins), into these cell membrane domains (43,44,47). Whether NG2/CSPG4, in its phosphorylated or unphosphorylated state, acts as a promoter of the cell membrane protrusion leading to formation of filopodial/invadopodial extensions would need to be defined by further investigations.…”

“…A concomitant macroclustering is noted in the lamellopodium, contiguously with dynamic focal adhesion placques and putative areas of cell-substrate contact. Intriguingly, in such areas, the cytoplasmic domain of NG2/CSPG4 is differentially phosphorylated on its more C-terminal threonine residue Thr 2256 (43,44,47) and the PG undergoes extensive stonin-1promoted internalization and endocytic intracellular trafficking ( Fig. 4).…”

“…In mammalian species, at least 3 different integrins, including a2b1, a3b1, and a4b1 (Fig. 4), have thus far been co-immunoprecipitated with the NG2/CSPG4 from cells exhibiting a PG-integrin coaction in the context of cell attachment and migration (35,39,45,47,124,125,(127)(128)(129)(130). Aside from possible CS-mediated interactions of NG2/ CSPG4 with the a4 integrin subunit (35), a more consolidated evidence for an integrin interaction with the core protein of the PG remains to be fully documented.…”

Differential Expression of Cancer-Associated Fibroblast-Related Proteins in Ductal Carcinoma in situ According to Molecular Subtype and Stromal Histology

“…The functional pathways of genes of which expression increased in CAFs of HER-2 positive breast cancer were actin cytoskeleton signaling and integrin signaling. It has been reported that actin cytoskeleton signaling is associated with NG2 [29] and PDGFR [30], while integrin signaling is associated with S100A4 [31][32][33] and NG2 [34][35][36]. It has also been reported that molecular features that induce disease progression are different in each intrinsic molecular subtype of DCIS [24] and have an important role in the tumor microenvironment and progression of DCIS [37,38].…”