Cellular and molecular bases for the pro-tumorigenic role of NG2/CSPG4 proteoglycan

Abstract

CSPG4/NG2 is a unique, variably glycanated transmembrane proteoglycan that has been thoroughly documented to exhibit aberrant expression patterns in solid and haematological tumours. Hence, it has been strongly associated with the pathological traits and progression of several tumours, but the precise molecular mechanisms through which it controls tumour development are not fully unveiled. The aim of my thesis work was therefore to approach the cellular and molecular mechanisms underlying the capability of NG2 to confer to cancer cells a more malignant phenotype. This direct correlation, between ectopic/overexpression of surface NG2 and a higher degree of malignancy, was demonstrated by the diverse tumorigenic behaviour displayed in vitro and in vivo of: immunoselected NG2-expressing and NG2-deficient cell subsets; cells in which their endogenous NG2 was knocked down with siRNAs; or cells engineered to overexpress the full-length or truncated variants of the proteoglycan. In vivo tumorigenesis experiments were supported by comparative in situ analyses of the tumour masses formed by NG2-expressing and NG2 deficient cells, DNA microarray global gene profiling and wide spectrum antibody array-based phospho-proteomic screens. Accentuated malignant behaviour of NG2 expressing cells was corroborated in vitro by: the diverse capacity to grow anchorage independently; the increased survival capabilities; the higher migration rates; and a diverse capability to form cellular aggregates. A substantial part of my studies also addressed the role of the NG2-collagen type VI interaction in the control of malignant behaviour in sarcoma cells. Diversified cellular interactions and migratory abilities were observed between NG2-positive and NG2-negative sarcoma cells with purified Col VI, basement membrane matrices supplemented with Col VI, and cell-free matrices isolated from wild type and Col VI null fibroblasts. The NG2-mediated binding to Col VI triggered activation of convergent cell survival- and cell adhesion/migration-promoting signal transduction pathways, implicating PI-3K as a common denominator and supporting the idea that an NG2-Col VI interplay may govern cancer cell-host microenvironment interactions sustaining sarcoma progression. Further, analysis of biopsies from soft-tissue sarcoma patients demonstrated that enhanced expression of NG2 in pre-surgical primary lesions predicts post-surgical metastasis formation. Thereby it could stratified patients into disease-free survivors and patients destined to succumb from the disease. Cumulatively, the study provides insights into the complex and multivalent role of NG2 in the control of cancer cell behaviour and reinforces its putative effectiveness as a therapeutic target for ablation of malignant cancer cell subsets.