title: Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells.
abstract: BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFβ/SMAD (transforming growth factor-β/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.
authors.affiliation: Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: F
authors.initials: F
authors.lastname: Faião-Flores
authors.affiliation: Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: D K
authors.initials: DK
authors.lastname: Alves-Fernandes
authors.affiliation: Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: P C
authors.initials: PC
authors.lastname: Pennacchi
authors.affiliation: Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: S
authors.initials: S
authors.lastname: Sandri
authors.affiliation: Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Brazil.
authors.forename: A L S A
authors.initials: AL
authors.lastname: Vicente
authors.affiliation: Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Brazil.|Department of Pathology, Barretos Cancer Hospital, Barretos, Brazil.
authors.forename: C
authors.initials: C
authors.lastname: Scapulatempo-Neto
authors.affiliation: Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Brazil.|Department of Surgery Melanoma/Sarcoma, Barretos Cancer Hospital, Barretos, Brazil.
authors.forename: V L
authors.initials: VL
authors.lastname: Vazquez
authors.affiliation: Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, 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, Guimarães, Portugal.
authors.forename: R M
authors.initials: RM
authors.lastname: Reis
authors.affiliation: Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Headington, Oxford, UK.
authors.forename: J
authors.initials: J
authors.lastname: Chauhan
authors.affiliation: Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Headington, Oxford, UK.
authors.forename: C R
authors.initials: CR
authors.lastname: Goding
authors.affiliation: The Department of Tumor Biology, The Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: K S
authors.initials: KS
authors.lastname: Smalley
authors.affiliation: Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: S S
authors.initials: SS
authors.lastname: Maria-Engler
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016922:Cellular Senescence / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D053823:Hedgehog Proteins / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D051741:Kruppel-Like Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology*; D000077484:Vemurafenib; D000071676:Zinc Finger Protein GLI1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000075746:Zinc Finger Protein Gli2
chemical_list: D000970:Antineoplastic Agents; D053823:Hedgehog Proteins; D007211:Indoles; D051741:Kruppel-Like Transcription Factors; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2; D000077484:Vemurafenib; D048493:Proto-Oncogene Proteins B-raf