Theranostic impact of NG2/CSPG4 proteoglycan in cancer

Abstract

NG2/CSPG4 is an unusual cell-membrane integral proteoglycan widely recognized to be a prognostic factor, a valuable tool for ex vivo and non-invasive molecular diagnostics and, by virtue of its tight association with malignancy, a tantalizing therapeutic target in several tumour types. Although the biology behind its involvement in cancer progression needs to be better understood, implementation of NG2/CSPG4 in the routine clinical practice is attainable and has the potential to contribute to an improved individualized management of cancer patients. In this context, its polymorphic nature seems to be particularly valuable in the effort to standardize informative diagnostic procedures and consolidate forcible immunotherapeutic treatment strategies. We discuss here the underpinnings for this potential and highlight the benefits of taking advantage of the intra-tumour and inter-patient variability in the regulation of NG2/CSPG4 expression. We envision that NG2/CSPG4 may effectively be exploited in therapeutic interventions aimed at averting resistance to target therapy agents and at interfering with secondary lesion formation and/or tumour recurrence.

Keywords: CSPG4; NG2; cancer; proteoglycan.

Figure 1

Schematic portrayal of the involvement of NG2/CSPG4 in biological phenomena known to be pivotal in the regulation of tumour growth and spreading. There is solid experimental evidence for a primary role of NG2/CSPG4 in mediating the tumour cells' interactions with their microenvironmental ECM. Through its capability to sequester various growth factors and modulate the activity of their cognate receptors, NG2/CSPG4 directly controls cancer cell proliferation (upper panel). Cells overexpressing NG2/CSPG4 up-regulate signalling pathways associated with cell survival (i.e. the PI-3K-mTOR-Akt1-PTEN axis) and attain a pronounced resistance to both conventional and target therapy drugs, as as well to radiation therapy (middle upper panel). NG2/CSPG4 is a primary pericyte marker and it acts as a primary regulator of angiogenesis. In fact, it serves as a mediator of pericyte activation and thereby as a promoter of angiogenic sprouting (lower middle panel). Its ability to interact with ECM, alongside with the ability to modulate the activity of multiple integrins and metalloproteinases, allow NG2/CSPG4 to be tightly implicated in the control of cancer cell movement and invasion. This includes the entrance and exit of disseminating cancer cells from the haematic and lymphatic circuits (lower panel).

Figure 2

NG2/CSPG4 isoforms are strongly expressed in a variety of tumour types and are effectively exploitable for the identification of circulating cancer cells. (A-H) Examples of NG2/CSPG isoform distribution in selected tumour types known to frequently express high levels of the PG, as provided by immunostainings with propriety monoclonal antibodies and a Human Protein Atlas polyclonal antiserum: high grade cutaneous melanoma (A); ductal breast carcinoma (B, adapted and reprinted with permission from , courtesy Dr. M-F. Huo); adenocarcinoma of stomach (C); colorectal adenocarcinoma (D); chordoma (E; courtesy Dr. E. Tamborini); malignant glioblastoma (F, adapted and reprinted with permission from , courtesy Drs Francesco Girolamo and Daniela Virgintino); lung metastasis of synovial sarcoma (G); and uterine leiomyosarcoma double stained with propriety monoclonal antibodies detecting cancer cell-associated versus pericyte-elective isoforms (H). (I) Detection of circulating cancer cells isolated from a patient with advanced melanoma through CD146-conjugated ferrofluid beads and enumerated and phenotyped by fluorescently labelled anti-CD34/CD45 and anti-NG2/CSPG4 antibodies (adapted and reprinted with permission from , courtesy Dr. L.W.M.M. Terstappen).

Figure 3

Potential of NG2/CSPG4 as a prognostic biomarker. (A) Assessment of the relative NG2/CSPG4 transcript levels in primary versus pulmonary metastatic lesions (the two types of lesions derived from the same individual) in soft-tissue sarcoma patients (n=86) affected by leiomyosarcoma, fibrosarcoma, liposarcoma, pleomorphic sarcoma or synovial sarcoma. A significantly (p<0.003; univariate analysis) enhanced NG2/CSPG4 mRNA expression is detectable in metastatic formations (we have previously reported analogous data; 45, 52). “Healthy tissue” corresponds to the surgical resection margins of the primary lesions. (B) Overall survival rates in soft-tissue sarcoma patients expressing higher (n=65) or lower (n=43) levels of NG2/CSPG4 mRNA in their lung metastases, following standard radiation and chemotherapy treatment and showing a significantly more favorable clinical course in patients with lower NG2/CSPG4 mRNA levels (p<0.03 by univariate analysis; Benassi, Pazzaglia and Perris, unpublished). (C) Overall survival rates of breast carcinoma patients expressing high levels of NG2/CSPG4, as determined by immunolabeling, when compared to patients with lower levels (n= 240). Even if more modestly, clinical outcome was significantly less dismal (p=0.193; univariate analysis) in patients with low NG2/CSPG4 levels (derived and adapted with permission from ; courtesy Dr. M.-F. Hou).

Figure 4

Implication of NG2/CSPG4 in tumour angiogenesis, as evidence in a virally induced mouse model of mammary carcinoma (MMTV-PyMT; derived and adapted with permission from , courtesy Dr. W.B. Stallcup) . (A-D) Primary (P) and secondary (N) lesions (including lymphnodal infiltration; “L”) induced in wild type (A, B) and NG2/CSPG4 null mice (C, D), as shown by Carmine alum staining of mammary glands in the two genotypes. (E-H) Extension of the neovascular network developed in carcinoma lesions arising in wild type (E, F) and NG2/CSPG4 null (G, H) mice (endothelial and pericyte markers, CD31 and αSMA, respectively).