title: Vemurafenib and trametinib reduce expression of CTGF and IL-8 inV600EBRAF melanoma cells.
abstract: Clinical evidence has revealed that while RAS/RAF/MEK/ERK pathway is a crucial component of melanomagenesis, other signaling pathways can also contribute to the malignant growth and development of resistance to targeted therapies. We explored the response ofV600EBRAF melanoma cells derived from surgical specimens and grown in stem cell medium to vemurafenib and trametinib, drugs targeting the activity ofV600EBRAF and MEK1/2, respectively. Cell growth and apoptosis were monitored by real-time imaging system, immunophenotype and cell cycle by flow cytometry, gene expression by quantitative real-time PCR, immunoblotting and enzyme-linked immunosorbent assay. TheV600EBRAF melanoma cell populations were diverse. Differences in morphology, pigmentation, cell cycle profiles, and immunophenotype were observed. At the molecular level, melanoma cells differed in the phosphorylation of ERK1/2, NF-κB, and β-catenin, and expression of several relevant genes, including MITF-M, DKK1, CCND1, BRAF, CXCL8, and CTGF. Despite having different characteristics, melanoma cells responded similarly to vemurafenib and trametinib. Both drugs reduced ERK1/2 phosphorylation and percentages of cells expressing Ki-67 at high level, inhibited expression of CCND1 and induced cell cycle arrest in the Go/G1phase. These expected cytostatic effects were accompanied by increased CD271 expression, a marker of stem-like cells. NF-κB activity was reduced by both drugs, however, not completely abolished, whereas the level of active β-catenin was increased by drugs in three out of six cell populations. Interestingly, expression of IL-8 and CTGF was significantly reduced by treatment with vemurafenib and trametinib. Simultaneous inhibition of NF-κB activity and induction of ERK1/2 phosphorylation revealed that CTGF expression depends on ERK1/2 activity but not on NF-κB activity. Both, the positive effects of treatment with vemurafenib and trametinib such as the newly identified CTGF suppression and undesired effects such as increased CD271 expression suggesting selection of melanoma stem-like cells should be considered in the development of combination treatment for melanoma patients.
authors.affiliation: Department of Molecular Biology of Cancer, Medical University of Lodz, Lodz, Poland.
authors.forename: Mariusz L
authors.initials: ML
authors.lastname: Hartman
authors.affiliation: Department of Molecular Biology of Cancer, Medical University of Lodz, Lodz, Poland.
authors.forename: Michal
authors.initials: M
authors.lastname: Rozanski
authors.affiliation: Department of Molecular Biology of Cancer, Medical University of Lodz, Lodz, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Osrodek
authors.affiliation: Department of Molecular Biology of Cancer, Medical University of Lodz, Lodz, Poland.
authors.forename: Izabela
authors.initials: I
authors.lastname: Zalesna
authors.affiliation: Department of Molecular Biology of Cancer, Medical University of Lodz, Lodz, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Czyz
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D055513:Connective Tissue Growth Factor / Q000235:genetics* / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D016209:Interleukin-8 / Q000235:genetics* / Q000378:metabolism; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D011728:Pyridones / Q000494:pharmacology; D011744:Pyrimidinones / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013449:Sulfonamides / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D000077484:Vemurafenib; D051176:beta Catenin / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C526317:CCN2 protein, human; C508606:CXCL8 protein, human; D007211:Indoles; D016209:Interleukin-8; D016328:NF-kappa B; D011728:Pyridones; D011744:Pyrimidinones; D013449:Sulfonamides; D051176:beta Catenin; D055513:Connective Tissue Growth Factor; D000077484:Vemurafenib; C560077:trametinib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D048049:Extracellular Signal-Regulated MAP Kinases