STRUCTURAL AND FUNCTIONAL TRAITS OF NG2/CSPG4 PROTEOGLYCAN IN RELATION TO ITS ROLE IN TUMORS

Chondroitin Sulphate Proteoglycan-4 (NG2/CSPG4) is a transmembrane proteoglycan consisting of a N-linked glycoprotein of 280 kDa and a proteoglycan component of about 450 kDa. It is expressed on the surface of various types of immature progenitor cells and its expression decreases with terminal differentiation. NG2/CSPG4 is expressed in several human malignant tumors playing an important role in growth, migration, and metastatic dissemination of tumor cells and it is involved in promotion of tumor progression. Aberrant expression of NG2/CSPG4 on cancer cells and angiogenic vasculature is observed in association with an aggressive disease course in several malignancies. Moreover, NG2/CSPG4 is expressed on the surface of both tumor cells and pericytes, and its distribution in normal tissue can be considered relatively restricted, so to be considered an attractive candidate for simultaneously targeting the malignant and stromal cellular compartments within the tumour. NG2/CSPG4 can trigger rearrangements of the actin cytoskeleton and interacts with components of the extracellular matrix, playing a role in both growth control and motility of cells. The singularity of NG2/CSPG4 structure suggests that it might also be unique in terms of its ability to participate in inter- and intra-molecular interactions. As a membrane-spanning molecule, NG2/CSPG4 has the potential to interact with its cytoplasmic domain, the large extracellular portion, and the glycosaminoglycan chain it carries on the core protein to participate in signaling between the extracellular and intracellular compartments of the cell, guiding the activation of important survival and growth pathways in tumors. Because of its structure, distribution and functions, NG2/CSPG4 has been proposed to promote tumor progression by multiple mechanisms and may represent a putative target for molecular therapy against cancer. To obtain anti-NG2/CSPG4 monoclonal antibodies (mAbs) we performed a systematical production of mAbs with the hybridoma technology, where immunization has been performed employing the extracellular portion of recombinant NG2/CSPG4 protein and from this immunization 63 clones have been selected and they have been characterized. We employed these mAbs to establish the expression patterns and subcellular distribution of NG2/CSPG4 in tumor cells and tissues, and to investigate the existence of NG2/CSPG4 isoforms among different tumors. The reactivity of mAbs against NG2/CSPG4 expressed from different cells or tissues, let us hypothesize that diverse isoforms of NG2/CSPG4 are expressed among the different tumors. As NG2 may represents a putative target for cancer molecular therapy against cancer, we employed our panel of anti-NG2/CPSG4 mAbs as a useful instrument to study in vitro their effect, with the aim to select those able to interfere with the functions that NG2 plays in tumors cells growth, survival, adhesion, migration. NG2 seems to interact with many molecules with its cytoplasmic domain, the large extracellular portion, and the glycosaminoglycan chain it carries on the core protein. In this contests, the last part of this research focus on the study of NG2/CSPG4’s putative interactors, carried on through a proteomic approach with mass spectrometry on tumor cell lines. MALDI-TOF analysis let us confirm some of the interactors already demonstrated in literature, and interestingly we identified some important putative interactors never found before, involved in cell migration. This finding open new ways to our research in target therapy against tumors.