BRAFV600 inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells
BRAFV600 inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells
复制标题
DOI：
10.1073/pnas.1705206114
复制
发表时间：
2017-07-18
影响因子：
11.1
通讯作者：
Lotvall, Jan
中科院分区：
文献类型：
--
作者：
Lunavat, Taral R.;Cheng, Lesley;Lotvall, Jan
BRAF抑制剂vemurafenib和dabrafenib可用于治疗携带BRAF（V600）突变的转移性黑色素瘤患者。最初的抗肿瘤反应通常是可见的，但很快就会出现MEK-ERK途径再激活的耐药克隆。近年来，肿瘤来源的细胞外囊泡（EVs）分泌组被认为在癌症中具有重要功能。为了阐明EVs在braf突变型黑色素瘤中的可能功能，我们测定了vemurafenib治疗后从这些癌细胞释放的EVs的RNA含量，包括凋亡小体、微泡和外泌体。我们发现vemurafenib显著增加了释放的ev的总RNA和蛋白质含量，并引起RNA谱的显著变化。RNA测序和定量PCR显示，来自vemurafenib处理的细胞培养物和来自细胞来源和患者来源的异种移植物收获的肿瘤组织的细胞和ev含有独特的mirna，特别是miR-211-5p的表达增加。从机制上讲，BRAF抑制导致miR-211-5p的表达是通过MITF的表达增加而诱导的，MITF调节TRPM1基因，从而激活存活途径。此外，在黑色素瘤细胞中转染miR-211降低了对vemurafenib治疗的敏感性，而在vemurafenib耐药细胞系中抑制miR-211-5p会对增殖产生负面影响。综上所述，我们的研究结果表明，vemurafenib治疗在体外和体内黑色素瘤细胞以及EVs亚群中诱导miR-211-5p上调，这表明EVs可能为了解恶性黑色素瘤的进展提供了一种工具。
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 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.