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

Uranowska,Karolina; Kalic,Tanja; Valtsanidis,Veronika; Kitzwögerer,Melitta; Breiteneder,Heimo; Hafner,Christine

doi:10.3892/or.2021.7965

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

Authors:
- Karolina Uranowska
- Tanja Kalic
- Veronika Valtsanidis
- Melitta Kitzwögerer
- Heimo Breiteneder
- Christine Hafner
View Affiliations

Affiliations: Department of Dermatology, University Hospital St. Poelten, Karl Landsteiner University of Health Sciences, 3100 St. Poelten, Austria, Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria, Department of Pathology, University Hospital St. Poelten, Karl Landsteiner University of Health Sciences, 3100 St. Poelten, Austria
Published online on: February 2, 2021 https://doi.org/10.3892/or.2021.7965
Article Number: 14
Copyright: © Uranowska et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

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.

View Figures

View References

1	Schadendorf D, van Akkooi ACJ, Berking C, Griewank KG, Gutzmer R, Hauschild A, Stang A, Roesch A and Ugurel S: Melanoma. Lancet. 392:971–984. 2018. View Article : Google Scholar : PubMed/NCBI
2	Spagnolo F, Ghiorzo P and Queirolo P: Overcoming resistance to BRAF inhibition in BRAF-mutated metastatic melanoma. Oncotarget. 5:10206–10221. 2014. View Article : Google Scholar : PubMed/NCBI
3	Ascierto PA, Kirkwood JM, Grob JJ, Simeone E, Grimaldi AM, Maio M, Palmieri G, Testori A, Marincola FM and Mozzillo N: The role of BRAF V600 mutation in melanoma. J Transl Med. 10:852012. View Article : Google Scholar : PubMed/NCBI
4	Pratilas CA, Taylor BS, Ye Q, Viale A, Sander C, Solit DB and Rosen N: (V600E)BRAF is associated with disabled feedback inhibition of RAF-MEK signaling and elevated transcriptional output of the pathway. Proc Natl Acad Sci USA. 106:4519–4524. 2009. View Article : Google Scholar : PubMed/NCBI
5	Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, Dummer R, Garbe C, Testori A, Maio M, et al: Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 364:2507–2516. 2011. View Article : Google Scholar : PubMed/NCBI
6	Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, Pochanard P, Kehoe SM, Johannessen CM, Macconaill LE, Hahn WC, et al: Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J Clin Oncol. 29:3085–3096. 2011. View Article : Google Scholar : PubMed/NCBI
7	Griffin M, Scotto D, Josephs DH, Mele S, Crescioli S, Bax HJ, Pellizzari G, Wynne MD, Nakamura M, Hoffmann RM, et al: BRAF inhibitors: Resistance and the promise of combination treatments for melanoma. Oncotarget. 8:78174–78192. 2017. View Article : Google Scholar : PubMed/NCBI
8	Flaherty KT, Infante JR, Daud A, Gonzalez R, Kefford RF, Sosman J, Hamid O, Schuchter L, Cebon J, Ibrahim N, et al: Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med. 367:1694–1703. 2012. View Article : Google Scholar : PubMed/NCBI
9	Johnson DB, Flaherty KT, Weber JS, Infante JR, Kim KB, Kefford RF, Hamid O, Schuchter L, Cebon J, Sharfman WH, et al: Combined BRAF (Dabrafenib) and MEK inhibition (Trametinib) in patients with BRAFV600-mutant melanoma experiencing progression with single-agent BRAF inhibitor. J Clin Oncol. 32:3697–3704. 2014. View Article : Google Scholar : PubMed/NCBI
10	Robert C, Grob JJ, Stroyakovskiy D, Karaszewska B, Hauschild A, Levchenko E, Chiarion Sileni V, Schachter J, Garbe C, Bondarenko I, et al: Five-year outcomes with dabrafenib plus trametinib in metastatic melanoma. N Engl J Med. 381:626–636. 2019. View Article : Google Scholar : PubMed/NCBI
11	Sharma SV, Lee DY, Li B, Quinlan MP, Takahashi F, Maheswaran S, McDermott U, Azizian N, Zou L, Fischbach MA, et al: A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell. 141:69–80. 2010. View Article : Google Scholar : PubMed/NCBI
12	Roesch A, Fukunaga-Kalabis M, Schmidt EC, Zabierowski SE, Brafford PA, Vultur A, Basu D, Gimotty P, Vogt T and Herlyn M: A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth. Cell. 141:583–594. 2010. View Article : Google Scholar : PubMed/NCBI
13	Arozarena I and Wellbrock C: Phenotype plasticity as enabler of melanoma progression and therapy resistance. Nat Rev Cancer. 19:377–391. 2019. View Article : Google Scholar : PubMed/NCBI
14	Das Thakur M, Salangsang F, Landman AS, Sellers WR, Pryer NK, Levesque MP, Dummer R, McMahon M and Stuart DD: Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance. Nature. 494:251–255. 2013. View Article : Google Scholar : PubMed/NCBI
15	Menon DR, Das S, Krepler C, Vultur A, Rinner B, Schauer S, Kashofer K, Wagner K, Zhang G, Rad EB, et al: A stress-induced early innate response causes multidrug tolerance in melanoma. Oncogene. 34:45452015. View Article : Google Scholar : PubMed/NCBI
16	Rolih V, Barutello G, Iussich S, De Maria R, Quaglino E, Buracco P, Cavallo F and Riccardo F: CSPG4: A prototype oncoantigen for translational immunotherapy studies. J Transl Med. 15:1512017. View Article : Google Scholar : PubMed/NCBI
17	Ilieva KM, Cheung A, Mele S, Chiaruttini G, Crescioli S, Griffin M, Nakamura M, Spicer JF, Tsoka S, Lacy KE, et al: Chondroitin sulfate proteoglycan 4 and its potential as an antibody immunotherapy target across different tumor types. Front Immunol. 8:19112017. View Article : Google Scholar : PubMed/NCBI
18	Ross AH, Cossu G, Herlyn M, Bell JR, Steplewski Z and Koprowski H: Isolation and chemical characterization of a melanoma-associated proteoglycan antigen. Arch Biochem Biophys. 225:370–383. 1983. View Article : Google Scholar : PubMed/NCBI
19	Campoli MR, Chang CC, Kageshita T, Wang X, McCarthy JB and Ferrone S: Human high molecular weight-melanoma-associated antigen (HMW-MAA): A melanoma cell surface chondroitin sulfate proteoglycan (MSCP) with biological and clinical significance. Crit Rev Immunol. 24:267–296. 2004. View Article : Google Scholar : PubMed/NCBI
20	Price MA, Colvin Wanshura LE, Yang J, Carlson J, Xiang B, Li G, Ferrone S, Dudek AZ, Turley EA and McCarthy JB: CSPG4, a potential therapeutic target, facilitates malignant progression of melanoma. Pigment Cell Melanoma Res. 24:1148–1157. 2011. View Article : Google Scholar : PubMed/NCBI
21	Stallcup WB and Dahlin-Huppe K: Chondroitin sulfate and cytoplasmic domain-dependent membrane targeting of the NG2 proteoglycan promotes retraction fiber formation and cell polarization. J Cell Sci. 114:2315–2325. 2001.PubMed/NCBI
22	Iida J, Wilhelmson KL, Ng J, Lee P, Morrison C, Tam E, Overall CM and McCarthy JB: Cell surface chondroitin sulfate glycosaminoglycan in melanoma: Role in the activation of pro-MMP-2 (pro-gelatinase A). Biochem J. 403:553–563. 2007. View Article : Google Scholar : PubMed/NCBI
23	Iida J, Meijne AM, Oegema TR Jr, Yednock TA, Kovach NL, Furcht LT and McCarthy JB: A role of chondroitin sulfate glycosaminoglycan binding site in alpha4beta1 integrin-mediated melanoma cell adhesion. J Biol Chem. 273:5955–5962. 1998. View Article : Google Scholar : PubMed/NCBI
24	Yang J, Price MA, Neudauer CL, Wilson C, Ferrone S, Xia H, Iida J, Simpson MA and McCarthy JB: Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms. J Cell Biol. 165:881–891. 2004. View Article : Google Scholar : PubMed/NCBI
25	Yang J, Price MA, Li GY, Bar-Eli M, Salgia R, Jagedeeswaran R, Carlson JH, Ferrone S, Turley EA and McCarthy JB: Melanoma proteoglycan modifies gene expression to stimulate tumor cell motility, growth, and epithelial-to-mesenchymal transition. Cancer Res. 69:7538–7547. 2009. View Article : Google Scholar : PubMed/NCBI
26	Jordaan S, Chetty S, Mungra N, Koopmans I, van Bommel PE, Helfrich W and Barth S: CSPG4: A target for selective delivery of human cytolytic fusion proteins and TRAIL. Biomedicines. 5:372017. View Article : Google Scholar
27	Hoffmann RM, Crescioli S, Mele S, Sachouli E, Cheung A, Chui CK, Andriollo P, Jackson PJM, Lacy KE, Spicer JF, et al: A novel antibody-drug conjugate (ADC) delivering a DNA mono-alkylating payload to chondroitin sulfate proteoglycan (CSPG4)-expressing melanoma. Cancers (Basel). 12:10292020. View Article : Google Scholar
28	Wang Y, Geldres C, Ferrone S and Dotti G: Chondroitin sulfate proteoglycan 4 as a target for chimeric antigen receptor-based T-cell immunotherapy of solid tumors. Expert Opin Ther Targets. 19:1339–1350. 2015. View Article : Google Scholar : PubMed/NCBI
29	Hafner C, Breiteneder H, Ferrone S, Thallinger C, Wagner S, Schmidt WM, Jasinska J, Kundi M, Wolff K, Zielinski CC, et al: Suppression of human melanoma tumor growth in SCID mice by a human high molecular weight-melanoma associated antigen (HMW-MAA) specific monoclonal antibody. Int J Cancer. 114:426–432. 2005. View Article : Google Scholar : PubMed/NCBI
30	Pucciarelli D, Lengger N, Takacova M, Csaderova L, Bartosova M, Breiteneder H, Pastorekova S and Hafner C: Anti-chondroitin sulfate proteoglycan 4-specific antibodies modify the effects of vemurafenib on melanoma cells differentially in normoxia and hypoxia. Int J Oncol. 47:81–90. 2015. View Article : Google Scholar : PubMed/NCBI
31	Yu L, Favoino E, Wang Y, Ma Y, Deng X and Wang X: The CSPG4-specific monoclonal antibody enhances and prolongs the effects of the BRAF inhibitor in melanoma cells. Immunol Res. 50:294–302. 2011. View Article : Google Scholar : PubMed/NCBI
32	Ampofo E, Schmitt BM, Menger MD and Laschke MW: The regulatory mechanisms of NG2/CSPG4 expression. Cell Mol Biol Lett. 22:42017. View Article : Google Scholar : PubMed/NCBI
33	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
34	Campoli M, Ferrone S and Wang X: Functional and clinical relevance of chondroitin sulfate proteoglycan 4. Adv Cancer Res. 109:73–121. 2010. View Article : Google Scholar : PubMed/NCBI
35	Yang J, Price MA, Wanshura LEC, He J, Yi M, Welch DR, Li G, Conner S, Sachs J, Turley EA and McCarthy JB: Chondroitin sulfate proteoglycan 4 enhanced melanoma motility and growth requires a cysteine in the core protein transmembrane domain. Melanoma Res. 29:365–375. 2019. View Article : Google Scholar : PubMed/NCBI
36	Makagiansar IT, Williams S, Mustelin T and Stallcup WB: Differential phosphorylation of NG2 proteoglycan by ERK and PKCalpha helps balance cell proliferation and migration. J Cell Biol. 178:155–165. 2007. View Article : Google Scholar : PubMed/NCBI
37	Luo W, Wang X, Kageshita T, Wakasugi S, Karpf AR and Ferrone S: Regulation of high molecular weight-melanoma associated antigen (HMW-MAA) gene expression by promoter DNA methylation in human melanoma cells. Oncogene. 25:2873–2884. 2006. View Article : Google Scholar : PubMed/NCBI
38	Yadavilli S, Scafidi J, Becher OJ, Saratsis AM, Hiner RL, Kambhampati M, Mariarita S, MacDonald TJ, Codispoti KE, Magge SN, et al: The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma. Oncotarget. 6:12141–12155. 2015. View Article : Google Scholar : PubMed/NCBI