title: Vemurafenib resistance increases melanoma invasiveness and modulates the tumor microenvironment by MMP-2 upregulation.
abstract: The BRAF(V600E) mutation confers constitutive kinase activity and accounts for >90% of BRAF mutations in melanoma. This genetic alteration is a current therapeutic target; however, the antitumorigenic effects of the BRAF(V600E) inhibitor vemurafenib are short-lived and the majority of patients present tumor relapse in a short period after treatment. Characterization of vemurafenib resistance has been essential to the efficacy of next generation therapeutic strategies. Herein, we found that acute BRAF inhibition induced a decrease in active MMP-2, MT1-MMP and MMP-9, but did not modulate the metalloproteinase inhibitors TIMP-2 or RECK in naïve melanoma cells. In vemurafenib-resistant melanoma cells, we observed a lower growth rate and an increase in EGFR phosphorylation followed by the recovery of active MMP-2 expression, a mediator of cancer metastasis. Furthermore, we found a different profile of MMP inhibitor expression, characterized by TIMP-2 downregulation and RECK upregulation. In a 3D spheroid model, the invasion index of vemurafenib-resistant melanoma cells was more evident than in its non-resistant counterpart. We confirmed this pattern in a matrigel invasion assay and demonstrated that use of a matrix metalloproteinase inhibitor reduced the invasion of vemurafenib resistant melanoma cells but not drug naïve cells. Moreover, we did not observe a delimited group of cells invading the dermis in vemurafenib-resistant melanoma cells present in a reconstructed skin model. The same MMP-2 and RECK upregulation profile was found in this 3D skin model containing vemurafenib-resistant melanoma cells. Acute vemurafenib treatment induces the disorganization of collagen fibers and consequently, extracellular matrix remodeling, with this pattern observed even after the acquisition of resistance. Altogether, our data suggest that resistance to vemurafenib induces significant changes in the tumor microenvironment mainly by MMP-2 upregulation, with a corresponding increase in cell invasiveness.
authors.affiliation: Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Silvana
authors.initials: S
authors.lastname: Sandri
authors.affiliation: Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Faião-Flores
authors.affiliation: Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Manoela
authors.initials: M
authors.lastname: Tiago
authors.affiliation: Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Paula Comune
authors.initials: PC
authors.lastname: Pennacchi
authors.affiliation: Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Renato Ramos
authors.initials: RR
authors.lastname: Massaro
authors.affiliation: Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Débora Kristina
authors.initials: DK
authors.lastname: Alves-Fernandes
authors.affiliation: Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil.
authors.forename: Gustavo Noriz
authors.initials: GN
authors.lastname: Berardinelli
authors.affiliation: Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil.
authors.forename: Adriane Feijó
authors.initials: AF
authors.lastname: Evangelista
authors.affiliation: Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil; Department of Surgery Melanoma/Sarcoma, Barretos Cancer Hospital, Barretos, Brazil.
authors.forename: Vinicius
authors.initials: V
authors.lastname: de Lima Vazquez
authors.affiliation: Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil; Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; 3B's ⿿ PT Government Associate Laboratory, Braga, Guimarães, Portugal.
authors.forename: Rui Manuel
authors.initials: RM
authors.lastname: Reis
authors.affiliation: Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil. Electronic address: silvya@usp.br.
authors.forename: Silvya Stuchi
authors.initials: SS
authors.lastname: Maria-Engler
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D016209:Interleukin-8 / Q000378:metabolism; D053511:Matrix Metalloproteinase 14 / Q000235:genetics / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D009361:Neoplasm Invasiveness; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors* / Q000235:genetics; D013449:Sulfonamides / Q000494:pharmacology*; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism; D059016:Tumor Microenvironment / Q000187:drug effects; D015854:Up-Regulation; D000077484:Vemurafenib
chemical_list: D000970:Antineoplastic Agents; C508606:CXCL8 protein, human; D058851:GPI-Linked Proteins; D007211:Indoles; D016209:Interleukin-8; D047428:Protein Kinase Inhibitors; C115460:RECK protein, human; D013449:Sulfonamides; C560823:TIMP2 protein, human; D019716:Tissue Inhibitor of Metalloproteinase-2; D000077484:Vemurafenib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14
keywords: MMP-2, BRAF-resistant melanoma; Metalloproteinase; Tumor microenvironment, secretome, 3D skin reconstruction; Vemurafenib