title: Anti-chondroitin sulfate proteoglycan 4-specific antibodies modify the effects of vemurafenib on melanoma cells differentially in normoxia and hypoxia.
abstract: Chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, is a potential target for antibody-based immunotherapy. The mechanism by which CSPG4 affects melanoma progression is only partly understood, in particular the involvement of other receptor tyrosine kinases and the tumor microenvironment. We have previously reported on a mimotope-based vaccine against CSPG4 in a human melanoma xenograft model that resulted in reduction of tumor growth. Herein we describe the influence of hypoxia on the response to polyclonal anti-CSPG4-antibodies induced by this vaccine in combination with the BRAF inhibitor vemurafenib to enhance therapeutic efficacy by simultaneously targeting multiple signaling pathways. Melanoma cells were treated with polyclonal anti-CSPG4-antibodies and vemurafenib. Proliferation, migration and invasion were evaluated in a real-time setting in the impedance-based x-CELLigence® system. Western blotting and quantitative PCR arrays were used to determine protein and mRNA expression of hypoxia inducible factor 1α (HIF1α), carbonic anhydrase IX (CAIX) and signaling pathway proteins. A melanoma xenograft model was used to detect HIF1α and CAIX expression in vivo. Hypoxia enhanced the antiproliferative response to vemurafenib. The migration and invasion capacities of vemurafenib-treated melanoma cells were increased, in spite of vemurafenib-decreased expression of HIF1α and CAIX. Polyclonal anti-CSPG4-antibodies reduced the Transwell migration of vemurafenib-treated, BRAF V600E-mutant and CSPG4-expressing melanoma cells in hypoxia. This was associated with the downregulation of phosphorylated AKT, a kinase contributing to tumor cell migration. Our results highlight CSPG4 as a potential target for modulating treatment resistance to vemurafenib induced by the hypoxic microenvironment.
authors.affiliation: Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Daniela
authors.initials: D
authors.lastname: Pucciarelli
authors.affiliation: Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Nina
authors.initials: N
authors.lastname: Lengger
authors.affiliation: Institute of Virology, Department of Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia.
authors.forename: Martina
authors.initials: M
authors.lastname: Takacova
authors.affiliation: Institute of Virology, Department of Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia.
authors.forename: Lucia
authors.initials: L
authors.lastname: Csaderova
authors.affiliation: Institute of Virology, Department of Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia.
authors.forename: Maria
authors.initials: M
authors.lastname: Bartosova
authors.affiliation: Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Heimo
authors.initials: H
authors.lastname: Breiteneder
authors.affiliation: Institute of Virology, Department of Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia.
authors.forename: Silvia
authors.initials: S
authors.lastname: Pastorekova
authors.affiliation: Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Christine
authors.initials: C
authors.lastname: Hafner
mesh_terms: D000818:Animals; D000906:Antibodies / Q000008:administration & dosage* / Q000494:pharmacology; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D011508:Chondroitin Sulfate Proteoglycans / Q000037:antagonists & inhibitors*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D007211:Indoles / Q000008:administration & dosage* / Q000494:pharmacology; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors*; D051379:Mice; D009361:Neoplasm Invasiveness; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000008:administration & dosage* / Q000494:pharmacology; D000077484:Vemurafenib; D023041:Xenograft Model Antitumor Assays; D000099216:Chondroitin Sulfate Proteoglycan 4
chemical_list: D000906:Antibodies; D000951:Antigens, Neoplasm; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; D011508:Chondroitin Sulfate Proteoglycans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007211:Indoles; D008565:Membrane Proteins; D013449:Sulfonamides; D000077484:Vemurafenib; C101390:CSPG4 protein, human; D000099216:Chondroitin Sulfate Proteoglycan 4; C497442:HIF1A protein, human; C089226:CA9 protein, human