BRAF^V600inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells

Abstract

The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAF V600 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 upregulation 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.small RNAs | extracellular vesicles | cancer | noncoding RNAs

Extracellular Vesicles and Epigenetic Modifications Are Hallmarks of Melanoma Progression

“…Moreover, exosomal DNA has been sequenced to detect the presence of BRAF V600 , while high levels of Exo-miR-211 was correlated with reduced sensitivity to BRAF inhibitors in metastatic melanoma, which supports molecular analyses of Exo to predict the responsiveness to targeted agents [30,104]. A potential and very useful application of circulating Exo concerns the isolation of EVs originating from the immune cells.…”

“…Lunavat et al have recently demonstrated that the inhibition of BRAFV600E melanoma cells with vemurafenib is associated with an increase in the release of EVs, which result in specific RNA [30]. The authors have correlated these adaptations with a mechanism of acquired resistance to BRAF inhibitors and suggested that intercellular communication mediated by such EVs is likely to be important for melanoma progression.…”

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

“…However, there is an increasing amount of evidence showing that EVs actually contain more full-length ncRNAs than microRNAs or ncRNA fragments. For instance, Bioanalyzer’s peaks corresponding to intact 18S and 28S rRNAs have been identified in purified EVs 26,61–64 , while full-length YRNAs and other ncRNAs have been identified by sequencing, RT-qPCR and/or Northern blot 31,65 . The use of thermostable group II intron reverse transcriptases (TGIRT-seq) has allowed the identification of full-length tRNAs in EVs, which greatly outnumber tRNA-derived fragments 53,66,67 .…”

Exosomes, the message transporters in vascular calcification

“…It is revealed that some miRs with elevated expression during VC promote osteogenesis via targeting at anti‐calcification proteins or contractile markers, whereas some other miRs with decreased expression suppress osteogenesis of VSMCs through targeting at osteogenic transcription factors (shown in Table ). As described, some of such miRs, including miR‐133b, miR‐204, miR‐211, alter during VC and are also proven to be transported by exosomes to modulate the biological behaviour in various kinds of recipient cells . Such results indicated that exosomes could participate in VC through transporting miRs to influence phenotype transition.…”