Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells

Cell Death Dis. 2017 Aug 31;8(8):e3029. doi: 10.1038/cddis.2017.406.

Abstract

A majority of patients with BRAF-mutated metastatic melanoma respond to therapy with BRAF inhibitors (BRAFi), but relapses are common owing to acquired resistance. To unravel BRAFi resistance mechanisms we have performed gene expression and mass spectrometry based proteome profiling of the sensitive parental A375 BRAF V600E-mutated human melanoma cell line and of daughter cell lines with induced BRAFi resistance. Increased expression of two novel resistance candidates, aminopeptidase-N (CD13/ANPEP) and ETS transcription factor FLI1 was observed in the BRAFi-resistant daughter cell lines. In addition, increased levels of the previously reported resistance mediators, receptor tyrosine kinase ephrine receptor A2 (EPHA2) and the hepatocyte growth factor receptor MET were also identified. The expression of these proteins was assessed in matched tumor samples from melanoma patients obtained before BRAFi and after disease progression. MET was overexpressed in all progression samples while the expression of the other candidates varied between the individual patients. Targeting CD13/ANPEP by a blocking antibody induced apoptosis in both parental A375- and BRAFi-resistant daughter cells as well as in melanoma cells with intrinsic BRAFi resistance and led to dephosphorylation of EPHA2 on S897, previously demonstrated to cause inhibition of the migratory capacity. AKT and RSK, both reported to induce EPHA2 S897 phosphorylation, were also dephosphorylated after inhibition of CD13/ANPEP. FLI1 silencing also caused decreases in EPHA2 S897 phosphorylation and in total MET protein expression. In addition, silencing of FLI1 sensitized the resistant cells to BRAFi. Furthermore, we show that BRAFi in combination with the multi kinase inhibitor dasatinib can abrogate BRAFi resistance and decrease both EPHA2 S897 phosphorylation and total FLI1 protein expression. This is the first report presenting CD13/ANPEP and FLI1 as important mediators of resistance to BRAF inhibition with potential as drug targets in BRAFi refractory melanoma.

MeSH terms

  • Antineoplastic Agents / therapeutic use
  • Apoptosis / drug effects
  • Apoptosis / genetics
  • CD13 Antigens / antagonists & inhibitors
  • CD13 Antigens / genetics*
  • CD13 Antigens / metabolism
  • Cell Cycle Checkpoints / drug effects
  • Cell Cycle Checkpoints / genetics
  • Cell Line, Tumor
  • Dasatinib / therapeutic use
  • Drug Resistance, Neoplasm / drug effects
  • Drug Resistance, Neoplasm / genetics
  • Ephrin-A2 / genetics*
  • Ephrin-A2 / metabolism
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • Indoles / therapeutic use
  • Melanoma / drug therapy
  • Melanoma / genetics*
  • Melanoma / metabolism
  • Melanoma / pathology
  • Microfilament Proteins / antagonists & inhibitors*
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism
  • Phosphorylation
  • Protein Kinase Inhibitors / therapeutic use
  • Proto-Oncogene Proteins B-raf / antagonists & inhibitors
  • Proto-Oncogene Proteins B-raf / genetics*
  • Proto-Oncogene Proteins B-raf / metabolism
  • Proto-Oncogene Proteins c-akt / genetics
  • Proto-Oncogene Proteins c-akt / metabolism
  • Proto-Oncogene Proteins c-met / genetics
  • Proto-Oncogene Proteins c-met / metabolism
  • Pyridones / therapeutic use
  • Pyrimidinones / therapeutic use
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Receptor, EphA2
  • Receptors, Cytoplasmic and Nuclear / antagonists & inhibitors*
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism
  • Ribosomal Protein S6 Kinases, 90-kDa / genetics
  • Ribosomal Protein S6 Kinases, 90-kDa / metabolism
  • Signal Transduction
  • Skin Neoplasms / drug therapy
  • Skin Neoplasms / genetics*
  • Skin Neoplasms / metabolism
  • Skin Neoplasms / pathology
  • Sulfonamides / therapeutic use
  • Trans-Activators
  • Vemurafenib

Substances

  • Antineoplastic Agents
  • EPHA2 protein, human
  • Ephrin-A2
  • FLII protein, human
  • Indoles
  • Microfilament Proteins
  • Protein Kinase Inhibitors
  • Pyridones
  • Pyrimidinones
  • RNA, Small Interfering
  • Receptors, Cytoplasmic and Nuclear
  • Sulfonamides
  • Trans-Activators
  • Vemurafenib
  • trametinib
  • MET protein, human
  • Proto-Oncogene Proteins c-met
  • Receptor, EphA2
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • Proto-Oncogene Proteins c-akt
  • RPS6KA1 protein, human
  • Ribosomal Protein S6 Kinases, 90-kDa
  • CD13 Antigens
  • Dasatinib