DEFINITION
Human chondroitin sulfate proteoglycan 4 (CSPG4), originally referred to as high molecular weight-melanoma-associated antigen (HMW-MAA) or melanoma chondroitin sulfate proteoglycan (MCSP), plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. It represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells.
CSPG4 was first identified 30 years ago on human melanoma cells; parallel investigations at that time identified the rat ortholog of CSPG4 termed nerve⁄glial antigen 2 (NG2).
CSPG4 and NG2 are highly conserved, and many of the important concepts regarding the significance and functions of CSPG4 are based on studies from both orthologs.
THE GENE
In human CSPG4 gene is localized on the 5 chromosome (location: 15q24.2 ), in a region of 38,527 bases on the reverse strand. The mRNA encodes for a 2322 aa polypeptide.
Official Symbol: CSPG4 and Name: chondroitin sulfate proteoglycan 4
Other Aliases: HMW-MAA, MCSP, MCSPG, MEL-CSPG, "melanoma-associated chondroitin sulfate proteoglycan", MSK16, NG2
CHEMICAL STRUCTURE AND IMAGES
Biosynthetic and biochemical studies indicated that CSPG4 is a unique glycoproteinproteoglycan complex consisting of an N-linked glycoprotein of 250 kDa and a proteoglycan component of 450 kDa.
The 250-kDa molecule is the core glycoprotein of the proteoglycan, shown to be a chondroitin sulfate proteoglycan by its content of both 2-acetamido-2-deoxy-3-0-(f3-D-gluco-4-enepyranosyluronic acid)-f3-4-0-sulfo-D-galactose (ADi-4S) and 2-acetamido-2-deoxy-3-0-(13-D-gluco-4-enepyranosyluronic acid)-p3-6-0-sulfo-D-galactose (ADi-6S) chondroitin sulfate disaccharides.
CSPG4 is composed of three major structural components: the extracellular domain, the transmembrane region and the cytoplasmic C-terminal domain (CTD).
The extracellular domain contains an N-terminal globular subdomain (D1) consisting of laminin G-type regions (LG) and disulfide bonds. The D2 subdomain consists of 15 CSPG repeats and, in NG2, a single chondroitin sulfate glycosaminoglycan (CS-GAG) chain. This region of the core protein is known tobind certain soluble growth factor ligands, and the CS-GAG is responsible for CSPG4 binding to integrin and matrix metalloproteinases . CS modification is also associated with distinct membrane distribution patterns of CSPG4 on the cell surface. CSPG4 can be expressed with or without CS modification. Proximal to the plasma membrane, the D3 globular subdomain contains sites for N-linked carbohydrate modification, binding sites for lectins (e.g., galectin 3), and proteolytic cleavage by MMPs or other proteases. The transmembrane region of CSPG4 contains a cysteine residue © at position 2230 that may play a role in CSPG4 membrane localization. The CTD contains tyrosine residues (T) that serve as phosphoacceptor sites for PKCa and ERK 1,2 (CSPG4 residues 2252 and 2310, respectively). The proline-rich region (PR) may comprise a non-canonical SH3 protein interaction domain, and the C-terminus contains a 4 residue PDZ domain-binding motif (PDZ) that is responsible for interactions with various PDZ domain-containing binding partners.
Mechanisms of Signal Transduction
Studies on the signaling mechanisms of CSPG4 indicate that it activates two types of signaling pathways: RTK signaling through the MAPK cascade and integrin signaling through (FAK) activation.
Activation of these pathways by CSPG4 leads to regulation of a number of cellular functions that drive tumorigenesis, including cytoskeletal reorganization, adhesion, migration, EMT, growth, survival, and chemoresistance. As CSPG4 does not have intrinsic catalytic activity, we propose that it functions as a type of scaffold protein. Thus, CSPG4 functions to affix signaling molecules in close proximity to one another to form a ‘progression-associated signaling complex’ that facilitates an increased duration and ⁄or intensity of RTK and FAK transduction pathways. We thus have hypothesized that these enhanced signaling functions provided by CSPG4 result in a selective growth and survival advantage when adhesion molecules and growth factors are limiting.
Activation of Integrins in Adhesion, Motility and Survival
CSPG4 interactions with integrins lead to the activation of downstream adhesion-related signaling pathways that contribute to malignant progression. Integrin a4b1 (and other b1 integrin heterodimers) can be superactivated in response to an ECM ligand (fibronectin) when activated in the presence of CSPG4, which results in a high level of activated FAK compared to activation of integrin alone. Antibody clustered CSPG4 co-precipitates with a complex of the small GTPase CDC42, ACK-1, and p130CAS in human melanoma. These factors are implicated in filopodia formation and integrin-mediated cytoskeletal reorganization, consistent with studies demonstrating that CSPG4 localizes to cell surface microspikes (the in vitro equivalent of filopodia). Expression of a full-length CSPG4 in human melanoma cells lacking endogenous CSPG4 expression results in significantly enhanced integrin-mediated spreading and FAK activation. As CSPG4 can activate MMP complexes on melanoma cell surfaces, the collective results implicate CSPG4 as an important contributing factor to localized invasion at the leading edge of invasive primary tumors.
Multiple structural features of CSPG4 have been linked to integrin-mediated cell adhesion and survival.
Both the carboxyl terminal PDZ motif-binding domain and PKCa phosphorylation site within the C-terminal cytoplasmic domain of CSPG4 are important for modulating cell migration. Phosphorylation at T2256 in CSPG4 by PKCa is important for the localization of proteoglycan to lamellipodia at the leading edge of cells, where it co-distributes with b1 integrin. Inhibiting phosphorylation at this site, either by pharmacologic blockade of PKCa or by mutation of the phosphoacceptor site, inhibits migration of CSPG4-transfected U251 human astrocytoma cells. In melanoma, enforced expression of syntenin results in NFkB activation via a FAK-dependent mechanism, which is critical for melanoma migration. Furthermore, melanoma cells plated on fibronectin activate FAK via a PKCa ⁄ syntenin-related mechanism, where syntenin is upregulated by PKCa activation in response to plating on fibronectin. Melanoma cells expressing a truncated CSPG4, which lacks the cytoplasmic domain, are migration deficient compared to cells expressing an intact CSPG4. Taken together, these data suggest a model of outside-in signaling resulting from the coupling of integrins with CSPG4 forming a complex that controls the organization of signaling modules near the plasma membrane.
MAPK-Mediated Growth and Survival
In addition to its control of integrin signaling, CSPG4 ligand complexes also impact on RTK pathways. Early studies demonstrated that CSPG4 impacts the RAS-RAF-MEK-ERK 1,2 pathway by binding to and presenting growth factors (e.g., Bfgf and PDGFaa) to their cognate transmembrane RTK. This capability may have particular consequences to melanoma progression because approximately 60% of human cutaneous melanomas express a constitutively active mutant BRAF (BRAFV600E). Although this mutation contributes to a high level of constitutive ERK 1,2 phosphorylation, expression of BRAFV600E alone is not sufficient to sustain high constitutive activation of ERK 1,2. Studies in multiple melanoma cell lines expressing mutant BRAFV600E retain a requirement for exogenous growth factor stimulation to maximally activate the ERK 1,2 pathway. These results resemble growth factor requirements for sustaining ERK1,2 activation through mutant active Ras and suggest a complexity of pathway regulation that is not yet fully understood. Recent studies also show that BRAFV600E requires expression of a full-length CSPG4 expression sustained and maximal ERK 1,2 activation. Studies utilizing RNA interference or specific monoclonal antibodies that inhibit CSPG4 function in melanoma cells expressing BRAFV600E also show an inhibition in constitutive ERK 1,2 activation. Furthermore, these same CSPG4 antibodies also enhance the effects of BRAFV600E-specific inhibitors in glioblastoma multiforme and melanoma cells, implying that CSPG4-mediated activation of ERK 1,2 may also occur through a BRAF-independent mechanism. Pharmacologic inhibition of ERK 1,2 using MEK 1 inhibitors abrogates the growth and motilitypromoting effects of CSPG4 in BRAFV600E-expressing cells. Furthermore, stable expression of a constitutively active MEK1 can bypass the need for CSPG4 expression in stabilizing high levels of ERK 1,2 activation and can lead to ERK 1,2-mediated growth and motility in the absence of the proteoglycan. Collectively, these data support a model where CSPG4 functions as a membrane scaffold to facilitate the formation of complexes that stabilize both RTKs and integrins. Such complexes would result in enhanced growth factor presentation and increased ECM signaling efficiency, theoretically providing one mechanism for cells to have a selective advantage over cells that lack CSPG4. Furthermore, because CSPG4 acts at a key interface between the tumor microenvironment and a highly penetrant oncogenic mutation in melanoma, it may prove to be an important therapeutic target in combination with BRAFV600E inhibitors for more effective control of this cancer.
CSPG4 Promotes Epithelial to Mesenchymal Transition (EMT)
Epithelial to mesenchymal transition-like changes are well recognized to occur in many primary tumors in experimental tumor models and to signal a transition to a more malignant phenotype. Although the pathological EMT associated with cancer progression does not fully replicate a developmental EMT, it does involve many of the same molecular events including increased motility⁄invasion and the acquisition of a mesenchymal signature, for example, E-cadherin loss, acquisition of mesenchymal cadherins (N-cadherin and CDH11), and increased expression of vimentin and fibronectin. CSPG4 expression in primary melanoma cells leads to a morphologic change consistent with EMT in which the cells appear less differentiated and more mesenchymal. Constitutive expression of CSPG4 also leads to alterations in gene transcription resulting in increased expression of fibronectin and vimentin as well as expression and activation of MET because of increased expression of HGF and loss of E-cadherin, the latter of which is related to increased expression of HGF by CSPG4-expressing cells. Loss of MET expression or function limits CSPG4-mediated increases in growth and motility. Furthermore, the melanocyte lineage-specific transcription factor MITF, which targets the c-Met locus, is induced by CSPG4 through constitutive activation of ERK 1,2.
CSPG4 Expression in Normal and Neoplastic Tissue
CSPG4 is expressed in a number of normal tissues throughout development, suggesting an important role in the development or homeostasis of adult tissues. It is implicated in the development of vascular tissue, as it is expressed by angiogenesis-associated pericytes (both normal and pathologic). The expression of CSPG4 in several pluripotent progenitor cell populations also indicates a role for CSPG4 in tissue development and stem cell niche maintenance.
CSPG4 is detected in stem-like cells associated with the interfollicular epidermis, where it regulates the position and motility of these progenitor cells in their niche.
CSPG4-positive stem cells in the epidermis are important for the renewal of epithelial keratinocytes; loss of these cells is associated with aging of the skin. CSPG4⁄NG2 is also expressed in both fetal and adult articular chondrocytes, bone marrow mesenchymal cells, and smooth muscle cells. CSPG4 ⁄Ng2-expressing pluripotent stem and progenitor cells often lose expression of the proteoglycan as they undergo terminal differentiation; however, this is not always the case.
CSPG4 has been demonstrated to be expressed on melanocytes, although at levels lower than what is seen on most melanomas. Altogether, these data implicate CSPG4 in the maintenance and differentiation of progenitor ⁄stem cell populations in the development of a variety of adult tissues. While the role of CSPG4⁄NG2 in homeostasis is only partially understood, it is noteworthy that embryonic deletion of this gene in mice is not lethal and to date immune-based therapies against this target show no obvious deleterious side effects.
CSPG4 is associated with the progression of melanoma and other cancers including oligodendrocytomas, gliomas, triple-negative breast carcinomas, and squamous cell carcinoma.
CSPG4 and Malignant Melanoma
The link between CSPG4 and melanoma progression was first appreciated as a result of its widespread expression in the majority (70% or greater) of superficial spreading and nodular human melanomas. In superficial spreading and ⁄or nodular melanoma, the core CSPG4 protein is expressed at multiple stages of melanoma progression and is even detected prior to tumor initiation in melanocytes within nevi. In these subtypes of melanoma, CSPG4 is not considered a prognostic factor, because it is expressed prior to the initiation of tumor formation and, indeed, has been detected on melanocytes in vitro. Although not considered a prognostic factor in superficial spreading or nodular melanoma, some studies have suggested that it may be negatively prognostic in acral lentiginous melanomas. Despite its general lack of prognostic significance in melanoma, however, numerous studies in vitro and in vivo using melanoma cell lines in which CSPG4 expression is altered have directly linked the proteoglycan core protein to the development of several phenotypic traits required for tumor progression. For example, expression of CSPG4 in human RGP melanoma cells promotes their anchorage-independent growth and increased motility in vitro.
In addition, enforced expression of this proteoglycan in mouse B16 melanomas enhances experimental metastasis in mice, again illustrating the potential importance of this protein in metastasis formation. Thus, although not prognostic, the data consistently point to an important role for CSPG4 in melanoma progression.
CSPG4 as a Therapeutic Target
While CSPG4 is not an oncogene per se, its expression directly or indirectly enhances activation of multiple signaling pathways associated with oncogenic transformation. As the proteoglycan is multifunctional, it can interact with distinct key oncogenic pathways that may change dynamically during progression. This may be one explanation for the high proportion of human melanomas that retain CSPG4 expression, even at the stage of metastatic lesions. Pathways that are impacted by CSPG4 include survival (PI3K, AKT, and NFjB), adhesion (FAK and integrin function), and growth ⁄ motility (RTK and downstream pathways including ERK 1,2). It is therefore not surprising that numerous reports have implicated CSPG4 as a potential therapeutic target for the treatment of malignant melanoma and other tumors. Several studies in patients or preclinical mouse models have focused on treating CSPG4 as an immune target for the treatment of melanomas. Analyses of clinical trials focused on evaluating the safety and potential efficacy of CSPG4 antibodies: targeting toxin conjugates or anti-idiotype antibodies used to actively generate CSPG4 antibodies in patients. Patients treated with anti-idiotypic antibodies demonstrate a statistically significant increase in survival when segregated according to the presence of serum anti-CSPG4 antibodies generated following treatment.
Anti-anti-idiotypic antibodies mimicking CSPG4 have been shown to induce HLA class 1-restricted CSPG4-specific CTL, suggesting that T-cell based immunotherapy targeted to CSPG4 may also be a viable option for the treatment of melanoma. In support of this hypothesis, an animal study showed efficacy for adoptively transferred CTLs engineered to express antibodies against CD20 and CSPG4 antigens in the treatment of CSPG4-expressing melanomas both blocked growth and promoted regression of CSPG4-expressing tumors. Although CSPG4-specific CD4+ T cells have been detected in the circulation of both healthy subjects and melanoma patients, stimulation of an anti-CSPG4 immune response is not apparently associated with autoimmunity. Collectively, these results support the use of CSPG4 as a target in tumor immunotherapy with a minimum of side effects.
Analyses of experimental tumor models have also shown that anti-CSPG4 monoclonal antibody (mAb)
administered to human melanoma or basal breast tumor xenografts inhibit their growth and metastasis. These particular studies, which are performed in immunocompromised animals, also demonstrated that these anti-CSPG4 mAb blocked CSPG4-regulated signaling pathways. Similarly, the direct injection of lentivirus encoding a CSPG4 shRNA into human melanoma tumor xenografts caused tumor regression, indicating that lowering CSPG4 expression levels also limits tumor growth and ⁄or survival, although the mechanisms for these effects were not defined. These studies did demonstrate, however, that the effect of targeting CSPG4 expression with viral delivery of shRNA or treating the tumors with injection of mAb resulted in reduced blood vessel formation in human melanoma xenografts. As vascular pericytes express CSPG4, the anti-CSPG4 mAb and shRNA may block neoangiogenesis by limiting pericyte growth or viability. Therefore, targeting CSPG4 could have direct effects on tumor cell signaling and also on tumor–stromal cell interactions that promote tumor growth and angiogenesis. Some evidence suggests that CSPG4 may also influence drug resistance. Chemoresistance is a major factor in the successful treatment of melanoma and other cancers. Although tumors in many patients initially respond to therapy, resistance to treatment develops over time and cancer progresses. The development of drug resistance in melanoma appears to be particularly linked to the use of single-target inhibitors, such as those that target BRAFV600E. CSPG4 expression is associated with multidrug resistance in glioblastoma and melanoma tumor experimental models,and this is mediated by its association with integrin-induced activation of PI3K pathways.
The ability of anti-CSPG4 mAb to prolong the growth inhibitory effects of PLX4032, a BRAFV600E inhibitor, on melanoma cell lines in culture provides direct evidence for a role of this proteoglycan in promoting chemoresistance. Taken as a whole, these data provide a rationale for targeting CSPG4 as an adjuvant therapy for melanoma and other select tumors using either an immune-based therapeutic approach or development of CSPG4 small molecule inhibitors.
