MAPK-induced miR-29 targets MAFG and suppresses melanoma progression

The tumor suppressive miR-29 family of microRNAs is encoded by two clusters, miR-29b1~a and miR-29b2~c, and is regulated by several oncogenic and tumor suppressive stimuli. Here we investigated whether oncogenic MAPK hyperactivation regulates miR-29 abundance and how this signaling axis impacts melanoma development. Using mouse embryonic fibroblasts and human melanocytes, we found that oncogenic MAPK signaling stimulates p53-independent and p53-dependent transcription of pri-miR-29b1~a and pri-miR-29b2~c, respectively. Expression analyses revealed that while pri-miR-29a~b1 remains elevated, pri-miR-29b2~c levels decrease during melanoma progression. Using a rapid mouse modeling platform, we showed that inactivation of miR-29 in vivo accelerates the development of frank melanomas and decreases overall survival. We identified MAFG as a relevant miR-29 target that has oncogenic potential in melanocytes and is required for growth of melanoma cells. Our findings suggest that MAPK-driven miR-29 induction constitutes a tumor suppressive barrier by targeting MAFG, which is overcome by attenuation of miR-29b2~c expression. The human immortalized melanocytes cell lines Hermes1, Hermes2, Hermes3A and Hermes4B were obtained from the Functional Genomics Cell Bank at St George’s, University of London, UK, and cultured in RPMI media supplemented with 10% FBS, 10ng/mL hSCF, 200nM TPA, 200pM Cholera Toxin and 10nM Endothelin-1 at 37˚C in a humidified atmosphere containing 10% CO2. SK-MEL28 cells were purchased from ATCC; WM164, WM35, WM793 and 1205Lu cells were a gift from M. Herlyn from the Wistar Collection of Melanoma cell lines. All melanoma cell lines were cultured in RPMI containing 5% FBS at 37˚C in a humidified atmosphere containing 5% CO2. Hermes1 cell line was transduced with either pLGH or BRAF-V600E expressing lentiviral constructs to mimic melanoma initiation (1). Prior to RNA isolation, all growth factors were withdrawn from media of melanocytes cell lines and melanoma cells were cultured in RPMI 10% FBS for 24 hours. RNAseq was performed to compare changes in gene expression during melanoma initiation (1) or between melanocytes and melanoma cells (2).