Expression of chondroitin sulfate proteoglycan 4 (CSPG4) in melanoma cells is downregulated upon inhibition of BRAF

Abstract

Chondroitin sulfate proteoglycan 4 (CSPG4) is a multifunctional transmembrane proteoglycan involved in spreading, migration and invasion of melanoma. In addition to the activating BRAF V600E mutation, CSPG4 was shown to promote MAPK signaling by mediating the growth-factor induced activation of receptor tyrosine kinases. However, it remains elusive which factors regulate CSPG4 expression. Therefore, the aim of the present study was to examine whether BRAF and MEK inhibitors have an effect on the expression of CSPG4. We exposed a panel of BRAF-mutant CSPG4-positive or -negative melanoma cell lines to BRAF and MEK inhibitors. Protein levels of CSPG4 were analyzed by flow cytometry (FACS), immunofluorescence microscopy (IF), and western blotting. CSPG4 mRNA levels were determined by quantitative PCR (qPCR). The prolonged exposure of cells to BRAF and MEK inhibitors resulted in markedly reduced levels of the CSPG4 protein in permanent resistant melanoma cells as well as decreased levels of its mRNA. We did not observe increasing levels of CSPG4 shedding into the culture supernatants. In addition, patient-derived matched tumor samples following therapy with kinase inhibitors showed decreased numbers of CSPG4-positive cells as compared to pre-therapy tumor samples. Our results indicate that BRAF and MEK inhibition downregulates CSPG4 expression until the cells have developed permanent resistance. Our findings provide the basis for further investigation of the role of CSPG4 in the development of drug-resistance in melanoma cells.

Integrated Analysis of Energy Metabolism Signature-Identified Distinct Subtypes of Bladder Urothelial Carcinoma

“…GPC2 belongs to a six-member human glypican family of proteins and is highly expressed in neuroblastoma ( Li et al, 2017 ). CSPG4 represents an integral membrane chondroitin sulfate proteoglycan, which is highly expressed in human malignant melanoma cells ( Uranowska et al, 2021 ). TPST1 encodes an integral membrane glycoprotein of the trans-Golgi network, catalyzing the tyrosine O-sulfation of soluble and membrane proteins that pass through this compartment.…”

Structural dynamics of receptor recognition and pH-induced dissociation of full-length Clostridioides difficile Toxin B

“…CSPG4 is a highly glycosylated membrane protein and is implicated in cellular signaling pathways, such as the mitogen-activated protein kinase pathway and the focal adhesion kinase (FAK) pathway [ 15 ]. In many cancer cell lines, the expression level of CSPG4 is significantly higher, suggesting a potential new target for the antibody-based immunotherapy of cancers [ 16 , 17 ]. As CSPG4 is expressed in the subepithelial layer of the intestine, it facilitates deeper penetration of TcdB into intestinal tissues [ 12 ].…”

A chondroitin sulfate proteoglycan 4‑specific monoclonal antibody inhibits melanoma cell invasion in a spheroid model

“…It was hypothesized speculate that the downregulation of CSPG4 expression in melanoma colonies may be involved in this observation. As recently demonstrated by the authors, exposure of CSPG4-positive melanoma cells to PLX4032 for up to 14 days led to gradually reduced levels of the CSPG4 protein and decreased levels of its mRNA ( 36 ). Therefore, the exposure of WM164 colonies to PLX4032 for a longer time period has probably led to a downregulation of CSPG4 expression and -as a consequence-to a lower binding of the CSPG4-specific mAb.…”

“…Cell lines and reagents. The human CSPG4-positive melanoma cell lines, WM9, WM35, WM164, 451Lu, and the human CSPG4-negative melanoma cell line, M14, all harboring the BRAF V600E mutation, were previously described (35,36). The cells were maintained in RPMI-1640 medium with 2 mM L-glutamine and 25 mM Hepes (Lonza Group, Ltd.), supplemented with either 5% FBS (WM9, WM35, WM164 and 451Lu) or 10% FBS (M14) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.).…”