BRAF V600 inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells

Proc Natl Acad Sci U S A. 2017 Jul 18;114(29):E5930-E5939. doi: 10.1073/pnas.1705206114. Epub 2017 Jul 6.

Abstract

The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAFV600 mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK-ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in BRAF-mutant melanoma, we determined the RNA content of the EVs, including apoptotic bodies, microvesicles, and exosomes, released from such cancer cells after vemurafenib treatment. We found that vemurafenib significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles. RNA sequencing and quantitative PCR show that cells and EVs from vemurafenib-treated cell cultures and tumor tissues harvested from cell-derived and patient-derived xenografts harbor unique miRNAs, especially increased expression of miR-211-5p. Mechanistically, the expression of miR-211-5p as a result of BRAF inhibition was induced by increased expression of MITF that regulates the TRPM1 gene resulting in activation of the survival pathway. In addition, transfection of miR-211 in melanoma cells reduced the sensitivity to vemurafenib treatment, whereas miR-211-5p inhibition in a vemurafenib resistant cell line affected the proliferation negatively. Taken together, our results show that vemurafenib treatment induces miR-211-5p up-regulation in melanoma cells both in vitro and in vivo, as well as in subsets of EVs, suggesting that EVs may provide a tool to understand malignant melanoma progression.

Keywords: cancer; extracellular vesicles; noncoding RNAs; small RNAs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Drug Resistance, Neoplasm
  • Extracellular Vesicles / drug effects
  • Extracellular Vesicles / genetics
  • Extracellular Vesicles / metabolism*
  • Gene Expression Regulation, Neoplastic / drug effects
  • Humans
  • Indoles / pharmacology*
  • Melanoma / drug therapy
  • Melanoma / genetics*
  • Melanoma / pathology
  • Mice, Inbred NOD
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Microphthalmia-Associated Transcription Factor / genetics
  • Microphthalmia-Associated Transcription Factor / metabolism
  • Mutation
  • Protein Kinase Inhibitors / pharmacology
  • Proto-Oncogene Proteins B-raf / antagonists & inhibitors*
  • Proto-Oncogene Proteins B-raf / genetics
  • Skin Neoplasms / drug therapy
  • Skin Neoplasms / genetics*
  • Skin Neoplasms / pathology
  • Sulfonamides / pharmacology*
  • Up-Regulation / drug effects
  • Vemurafenib
  • Xenograft Model Antitumor Assays

Substances

  • Indoles
  • MIRN211 microRNA, human
  • MITF protein, human
  • MicroRNAs
  • Microphthalmia-Associated Transcription Factor
  • Protein Kinase Inhibitors
  • Sulfonamides
  • Vemurafenib
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf

Supplementary concepts

  • Melanoma, Cutaneous Malignant