Academic literature on the topic 'NG2/CSPG4'

Neuron glial antigen 2 (NG2) is a chondroitin sulphate proteoglycan 4 (CSPG4) that occurs in developing and adult central nervous systems (CNSs) as a marker of oligodendrocyte precursor cells (OPCs) together with platelet-derived growth factor receptor α (PDGFRα). It behaves variably in different pathological conditions, and is possibly involved in the origin and progression of human gliomas. In the latter, NG2/CSPG4 induces cell proliferation and migration, is highly expressed in pericytes, and plays a role in neoangiogenesis. NG2/CSPG4 expression has been demonstrated in oligodendrogliomas, astrocytomas, and glioblastomas (GB), and it correlates with malignancy. In rat tumors transplacentally induced by N-ethyl-N-nitrosourea (ENU), NG2/CSPG4 expression correlates with PDGFRα, Olig2, Sox10, and Nkx2.2, and with new vessel formation. In this review, we attempt to summarize the normal and pathogenic functions of NG2/CSPG4, as well as its potential as a therapeutic target.

: Neuro-glia antigen 2/chondroitin sulfate proteoglycan 4 (NG2/CSPG4, also called MCSP, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a large cell-surface antigen and an unusual cell membrane integral glycoprotein frequently expressed on undifferentiated precursor cells in multiple solid organ cancers, including cancers of the liver, pancreas, lungs, and kidneys. It is a valuable molecule involved in cancer cell adhesion, invasion, spreading, angiogenesis, complement inhibition, and signaling. Although the biological significance underlying NG2/CSPG4 proteoglycan involvement in cancer progression needs to be better defined, based on the current evidence, NG2/CSPG4+ cells, such as pericytes (PCs, NG2+/CD146+/PDGFR-β+) and cancer stem cells (CSCs), are closely associated with the liver malignancy, hepatocellular carcinoma (HCC), pancreatic malignancy, and pancreatic ductal adenocarcinoma (PDAC) as well as poor prognoses. Importantly, with a unique method, we successfully purified NG2/CSPG4-expressing cells from human HCC and PDAC vasculature tissue blocks (by core needle biopsy). The cells appeared to be spheres that stably expanded in cultures. As such, these cells have the potential to be used as sources of target antigens. Herein, we provide new information on the possibilities of frequently selecting NG2/CSPG4 as a solid organ cancer biomarker or exploiting expressing cells such as CSCs, or the PG/chondroitin sulfate chain of NG2/CSPG4 on the cell membrane as specific antigens for the development of antibody- and vaccine-based immunotherapeutic approaches to treat these cancers.

Background: Neuron glial antigen 2 or chondroitin sulphate proteoglycan 4 (NG2/CSPG4) is expressed by immature precursors/progenitor cells and is possibly involved in malignant cell transformation. The aim of this study was to investigate its role on the progression and survival of sixty-one adult gliomas and nine glioblastoma (GB)-derived cell lines. Methods: NG2/CSPG4 protein expression was assessed by immunohistochemistry and immunofluorescence. Genetic and epigenetic alterations were detected by molecular genetic techniques. Results: NG2/CSPG4 was frequently expressed in IDH-mutant/1p19q-codel oligodendrogliomas (59.1%) and IDH-wild type GBs (40%) and rarely expressed in IDH-mutant or IDH-wild type astrocytomas (14.3%). Besides tumor cells, NG2/CSPG4 immunoreactivity was found in the cytoplasm and/or cell membranes of reactive astrocytes and vascular pericytes/endothelial cells. In GB-derived neurospheres, it was variably detected according to the number of passages of the in vitro culture. In GB-derived adherent cells, a diffuse positivity was found in most cells. NG2/CSPG4 expression was significantly associated with EGFR gene amplification (p = 0.0005) and poor prognosis (p = 0.016) in astrocytic tumors. Conclusion: The immunoreactivity of NG2/CSPG4 provides information on the timing of the neoplastic transformation and could have prognostic and therapeutic relevance as a promising tumor-associated antigen for antibody-based immunotherapy in patients with malignant gliomas.

Abstract Introduction: Glioblastoma (GBM) is the most frequent treatment resistant and poor prognosis primary brain tumor. Age-adjusted incidence among Caucasians is approx. twice that of Africans. Genetic changes in ethnicity may represent druggable targets for development of new treatments for GBM. Chondroitin sulfate proteoglycan 4 (CSPG4) with sequence homology to neuron-glial-2 (NG2), henceforth CSPG4/NG2, is a transmembrane proteoglycan that is upregulated in GBM, resulting in leaky neovasculature and high cell proliferation, and is independently prognostic for poor survival. Mechanisms regulating CSPG4/NG2 expression remain to be elucidated. Experiments: Tumor-derived DNA whole exome sequences (WES) from mixed American GBM patients (n=300) collected into the TCGA database and blood-derived DNA WES from controls (n = 2504) in the 1000 Genome database were analyzed for mutations in the CSPG4 gene by GATK-3.5. We used prime editing (PE) to introduce a 13-base pair (bp) deletion through design of PE guide RNA (n=27), screening GBM cells (n=14) for CSPG4/NG2 expression, transfecting and expanding PE cells, confirming editing using Sanger sequencing. We functionally characterized the mutation in PE cells and transplanted in orthotopic PDX mouse models compared to wild type (WT) and Cas9 control cells. Western blotting, BrdU proliferation assays, clonogenic- and wound healing assays were used to investigate function. Gelatin zymography assessed CSPG4/NG2 matrix metalloproteinase (MMP) cross-talk during invasion on mixed extracellular matrix- or collagen IV- coated transwells. Results: A novel 13bp frameshift deletion in exon 3 of the CSPG4 gene was identified from WES of GBM samples deposited in TCGA. The mutation was cancer specific (p<0.001). Comparison of frequency in various ethnicities revealed the mutation to be more frequent among African American GBM patients (p=0.019). We generated 3 clones heterozygous for the CSPG4/NG2 deletion in patient derived GBMs. A homozygous deletion was lethal to the cells. The 13bp deletion reduced CSPG4/NG2 expression by approx. 60%, slowed tumor cell proliferation compared to both WT (p<0.001) and Cas9 cells (p<0.0001). PE cells had significantly reduced colony formation (p<0.001), scratch wound healing (p<0.001), had a weaker invasive phenotype when challenged with collagen IV (p<0.001), and reduced cleavage of collagen I by MMP2 that was corroborated by RNAseq gene expression. Reduced growth of PE cells in vivo was verified by multiplex immunohistochemistry on ex vivo brain sections and identified biomarkers for differential tumor development. Tumor growth was reduced in vivo in mice. Conclusions: We identified a novel 13bp deletion in CSPG4/NG2 with phenotypic effect that could be used as a future molecular target in GBM treatment, and present the first successful application of PE in human GBM cells. Citation Format: Victoria Smith Arnesen, Susina Suntharalingam, Żaneta Matuszek, Shahin Sarowar, Stian Knappskog, Stein Atle Lie, David R. Liu, Mohummad Aminur Rahman, Martha Chekenya. A novel 13-basepair deletion in CSPG4/NG2 abrogates protein expression, glioblastoma proliferation and invasion in vitro and in vivo in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3996.

Neuron-glial antigen 2 (NG2, gene name: Cspg4) has been characterized as an important factor in many diseases. However, the pathophysiological relevance of NG2 in liver disease specifically regarding bone marrow mesenchymal stem cell (BMSC) differentiation to myofibroblast (MF) and the molecular details remain unknown. Human liver tissues were obtained from patients with different chronic liver diseases, and mouse liver injury models were induced by feeding a methionine-choline-deficient and high-fat diet, carbon tetrachloride administration, or bile duct ligation operation. NG2 expression was increased in human and mouse fibrotic liver and positively correlated with MF markers α-smooth muscle actin (αSMA) and other fibrotic markers in the liver. There was a co-localization between NG2 and αSMA, NG2 and EGFP (BMSC-derived MF) in the fibrotic liver determined by immunofluorescence analysis. In vitro, TGFβ1-treated BMSC showed a progressive increase in NG2 levels, which were mainly expressed on the membrane surface. Interestingly, there was a translocation of NG2 from the cell membrane into cytoplasm after the transfection of Cspg4 siRNA in TGFβ1-treated BMSC. siRNA-mediated inhibition of Cspg4 abrogated the TGFβ1-induced BMSC differentiation to MF. Importantly, inhibition of NG2 in vivo significantly attenuated the extent of liver fibrosis in methionine-choline-deficient and high fat (MCDHF) mice, as demonstrated by the decreased mRNA expression of fibrotic parameters, collagen deposition, serum transaminase levels, liver steatosis and inflammation after the administration of Cspg4 siRNA in MCDHF mice. We identify the positive regulation of NG2 in BMSC differentiation to MF during liver fibrosis, which may provide a promising target for the treatment of liver disease.

Neuron-glial antigen 2 (NG2, also known as CSPG4) and hyaluronic acid receptor CD44 are chondroitin sulphate proteoglycans actively involved in brain development and its malignant transformation. Here, we aimed to compare prognostic significances of NG2, CD44 and Ki-67 expression in glioblastoma multiforme patients. Totally, 45 tissue samples and 83 paraffin-embedded tissues for 75 patients were analysed. The prognostic values of the genes were analysed using Kaplan–Meier survival curves. Grade III gliomas showed 2-fold difference in NG2 expression between anaplastic astrocytoma and oligoastrocytoma (10.1 ± 3.5 and 25.5 ± 14.5, respectively). For grade IV gliomas, upregulated NG2 expression (21.0 ± 6.8) was associated with poor glioblastoma multiforme prognosis (overall survival < 12 months) compared with glioblastoma multiforme patients with good prognosis (4.4 ± 3.2; overall survival > 12 months). Multivariate survival analysis using Cox proportional hazards model confirmed that high NG2 expression was associated with low survival of the patients (hazard ratio: 3.43; 95% confidence interval: 1.18–9.93; p = 0.02), whereas age (hazard ratio: 1.02; 95% confidence interval: 0.96–1.09; p = 0.42), tumour resection (hazard ratio: 1.03; 95% confidence interval: 0.98–1.08; p = 0.25) and sex (hazard ratio: 0.62; 95% confidence interval: 0.21–1.86; p = 0.40) did not show significant association with prognosis. Although the positive correlation was shown for NG2 and CD44 expression in the glioblastomas (Pearson coefficient = 0.954), Kaplan–Meier and multivariate survival analyses did not revealed a significant association of the increased CD44 expression (hazard ratio: 2.18; 95% confidence interval: 0.50–9.43; p = 0.30) or high Ki-67 proliferation index (hazard ratio: 1.10; 95% confidence interval: 1.02–1.20; p = 0.02) with the disease prognosis. The results suggest that upregulation of NG2/CSPG4 rather than changes in CD44 or Ki-67 expression is associated with low overall survival in glioblastoma multiforme patients, supporting NG2/CSPG4 as a potential prognostic marker in glioblastoma.

Introduction: NG2/CSPG4 is a unique transmembrane chondroitin sulphate proteoglycan molecule expressed as a core protein and a chondroitin sulphate proteoglycan (CSPG) up to 400kD. NG2/CSPG4 mediates the communication between the extracellular and intracellular compartments through interactions with collagen VI, growth factors and the actin cytoskeleton. NG2/CSPG4 affects cell migration, spreading, apoptosis and proliferation processes. NG2/CSPG4 has been shown to be expressed in developing and adult cartilage where less is known of its function. I tested the hypothesis: NG2/CSPG4 is an important regulator of chondrocytes function and has the potential to be a therapeutic target for treatment of diseases of cartilage such as osteoarthritis and chondrosarcoma. To do this, I had the following aims: 1) investigate whether different types of chondrocytes show variation in the form or distribution of NG2/CSPG4 expression and 2) through a knockdown approach develop a model to study the functional roles of NG2/CSPG4 in human chondrocytes. Materials and Methods: JJ012, a chondrosarcoma cell line, chondrocytes derived from human articular cartilage and C20/A4 an immortalised chondrocyte cell line were used. NG2/CSPG4 expression was investigated by RT-PCR western blotting, flow-cytometry and immunocytochemistry. NG2/CSPG4 interaction with Golgi complex and endoplasmic reticulum (ER) was assessed by double immunofluorescence. Biochemical interactions were assessed by immunoprecipitation and mass spectroscopy. For NG2/CSPG knockdown, a viral transduction method was carried out using 5 different constructs. Different functional roles of NG2/CSPG4 were investigated. The role of NG2/CSPG4 in gene regulation was studied by shRNA knockdown of NG2/CSPG4 in JJ012 cells and RTPCR. Results: NG2/CSPG4 mRNA was detectable in all cells tested. Western blotting showed expression of only a 270kD core protein in JJ012 and C20A4 cells. Using two different anti NG2/CSPG4 antibodies human OA chondrocytes were seen to express multiple molecular weight forms differentially recognised with and without chondroitinase ABC pre-treatment. Expression of NG2/CSPG4 in JJ012 cells was predominantly membrane associated whilst in OA chondrocytes and C20A4 cells, additional, predominant punctuate cytoplasmic distribution was evident. In OA chondrocytes NG2/CSPG4 co-localised with the Golgi complex and ER. Immunoprecipitation and mass spectrometry data demonstrated associations between NG2/CSPG4 and both collagen VI and thrombopoietin in OA chondrocytes. A model of NG2/CSPG4 gene knockdown was achieved in JJ012 chondrosarcoma cell line, known as B3. B3 cells spread more and migrate less than JJ012 cells; with a significant difference observed in migration (after 10hours: the closed area was 81.4% for JJ012 and 54.6% for B3). There was no difference in cell adhesion to collagen I, II, VI and fibronectin. EGTA inhibited cell adhesion to fibronectin in dose dependent manner with no significant difference observed between both JJ012 and B3 cells. EDTA reduced adhesion of B3 cells but not JJ012 to fibronectin. A significant difference in cell proliferation was detected with no change in apoptosis. Following NG2/CSPG4 knockdown in JJ012 cells there was no difference in expression of aggrecan, collagen II and SOX-9. In contrast, B3 cells showed a decreased expression of MPP3 and ADAMTS-4, a complete loss of ADAMTS-5 and increased expression of MMP13. Conclusions: I have identified altered expression and multiple forms of NG2/CSPG4 in different types of chondrocytes and shown association of this molecule with type VI collagen and thrombopoietin. Creation of a chondrocyte cell line that has stable knockdown of NG2/CSPG4 allowed further investigation of NG2/CSPG function in chondrocytes. NG2/CSPG4 knockdown reduced the cellular migration and proliferation and increased the chondrocyte spreading. The adhesion mechanism in JJ012 appears to be calcium dependent. Loss of NG2/CSPG4 induced changes in expression of aggrecanases and MMPs. Altered expression or associations of NG2/CSPG4 with extracellular ligands or intracellular signalling cascades may be important in the pathogenesis of OA by regulating proteolytic activity or apoptosis related pathways. NG2/CSPG4 is a potential therapeutic target in degenerative and neoplastic diseases of cartilage.