MITF/miR-579-3p novel regulatory axis controls BRAF-mutated melanoma decision between cell senescence or progression upon exposure to MAPK inhibitors.

Abstract

Therapy of metastatic melanoma has improved dramatically over the last years thanks to the development of targeted therapies (MAPK inhibitors) and immunotherapies. However, drug resistance continues to be a major limitation to the efficacy of these therapies. Our research group has provided robust evidence as to the involvement of a set of microRNAs in the development of resistance to target therapy in BRAF-mutated melanoma cell lines. Among them, a pivotal role is played by miR-579-3p, whose role as oncosuppressor was discovered by our group some years ago. Here we show that miR-579-3p and the microphthalmia-associated transcription factor (MITF) influence reciprocally their expression through positive feedback regulatory loops. In particular we show that miR-579-3p is specifically deregulated in BRAF-mutant melanomas and that its expression levels mirror those of MITF. Luciferase and ChIP studies show that MITF is a positive regulator of miR-579-3p, which is located in the intron 11 of the human gene ZFR (Zink-finger recombinase) and is co-transcribed with its host gene. Moreover, miR-579-3p, by targeting BRAF is able to stabilize MITF protein thus inducing its own transcription. As a consequence, upon exposure to MAPK inhibitors or, alternatively upon miR-579-3p transfection, the activation of this newly uncovered miR-579-3p/MITF axis induces a potent block of proliferation and senescence of BRAF-mutant melanoma cells. Moreover, the long term development of resistance to MAPKi is able to select cells characterized by the loss of both miR-579-3p and MITF and the same down-regulation is also present in patients relapsing after targeted therapies treatments. Altogether these findings suggest that miR-579-3p/MITF interplay potentially governs the balance between proliferation, senescence and resistance to target therapies in BRAF-mutant melanomas.

KEYWORDS:

Acknowledgements

This study was supported by Italian Association for Cancer Research (AIRC) grants IG19865 to G. Ciliberto and IG24451 to R. Mancini and by the Lazio Innova grants 2018 n. 85-2017-13750 and 2020 n. A0375-2020-36657 to R. Mancini and by Italian Minister of Health grant PRIN 2017 (Prot. 2017HWTP2K) to G. Ciliberto and R. Mancini, by Italian Minister of Health through “Ricerca Corrente” grant L2/1 to P.A. Ascierto. The study was also supported by Lega Italiana per la Lotta ai Tumori (LILT) grant n. 2021U0001637 to L. Fattore. We thank all patients who donated samples for this research.

摘要

转移性黑色素瘤的治疗在过去数年中因靶向治疗（丝裂原活化蛋白激酶MAPK抑制剂）与免疫治疗的发展而取得显著进展。然而，药物耐药仍是制约此类疗法疗效的核心瓶颈。本研究团队已获得充分的实验证据，证实一组微小RNA（microRNA）参与BRAF突变型黑色素瘤细胞系的靶向治疗耐药过程。其中，miR-579-3p发挥关键调控作用——本团队数年前已发现其作为肿瘤抑制因子的功能。本研究证实，miR-579-3p与小眼畸形相关转录因子（MITF, microphthalmia-associated transcription factor）可通过正向反馈调控环路相互影响彼此的表达。具体而言，我们发现miR-579-3p在BRAF突变型黑色素瘤中存在特异性表达失调，且其表达水平与MITF的表达水平呈显著正相关。双荧光素酶报告基因实验与染色质免疫沉淀（ChIP, chromatin immunoprecipitation）研究证实，MITF是miR-579-3p的正向调控因子；该miRNA定位于人类ZFR（锌指重组酶Zink-finger recombinase）基因的第11内含子区域，并与其宿主基因共转录。此外，miR-579-3p可通过靶向BRAF稳定MITF蛋白，进而诱导自身的转录。因此，当细胞暴露于MAPK抑制剂或转染miR-579-3p时，这一新发现的miR-579-3p/MITF调控轴的激活可强效阻滞BRAF突变型黑色素瘤细胞的增殖并诱导其衰老。进一步研究发现，长期接受MAPK抑制剂治疗后产生耐药的细胞株会同时丢失miR-579-3p与MITF的表达；此类下调现象同样存在于靶向治疗后复发的患者样本中。综上，上述研究结果表明，miR-579-3p与MITF的相互作用可能调控BRAF突变型黑色素瘤细胞增殖、衰老与靶向治疗耐药之间的动态平衡。

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致谢

本研究获得意大利癌症研究协会（AIRC, Italian Association for Cancer Research）资助：G. Ciliberto 获得IG19865号项目资助，R. Mancini 获得IG24451号项目资助；同时获得拉齐奥创新计划（Lazio Innova）资助：R. Mancini 获得2018年第85-2017-13750号与2020年第A0375-2020-36657号项目资助；G. Ciliberto与R. Mancini 获得意大利卫生部PRIN 2017项目资助（项目编号：Prot. 2017HWTP2K）；P.A. Ascierto 获得意大利卫生部“当前研究”项目L2/1资助。本研究同时获得意大利癌症防控联盟（LILT, Lega Italiana per la Lotta ai Tumori）资助：L. Fattore 获得2021U0001637号项目资助。我们感谢所有为本研究捐献样本的患者。