The pro-fibrotic miR-143/145 cluster promotes mesenchymal phenotypic plasticity associated with resistance to targeted therapies in melanoma

Because of its intrinsic plasticity and resistance to treatment, melanoma is one of the most aggressive cancers. Due to the MAPK pathway hyperactivation, targeted therapies counteracting this signaling cascade are efficient in most patients harboring BRAFV600E metastatic melanoma. However, innate and acquired resistances constitute major therapeutic challenges. Acquired resistance to MAPK-targeted therapies arises from de novo genetic lesions and non-genetic events such as transcriptional reprogramming and epigenetic changes. Upon MAPK inhibitors exposure, melanoma cells assume functionally different phenotypic states defined by master transcription factors differential activity and fixed by epigenetic events. Among them, the emergence of a poorly differentiated cell state is strongly associated with resistance acquisition and tumor recurrence. Our team has previously shown that melanoma cells switching to a dedifferentiated phenotype in response to MAPK-targeted therapies display features of cancer-associated fibroblasts (CAFs) like extracellular matrix (ECM) remodeling and markers observed in fibrotic diseases, allowing them to generate a drug tolerant microenvironment.This fibrotic state is characterized in vitro and in vivo by increased deposition and altered ECM organization associated with a mechanophenotype regulated by the mechanotransducers YAP and MRTFA. However, post-transcriptional signaling networks that underpin this mesenchymal-like phenotype are still unknown and effective therapeutic treatments to overcome MAPK-targeted therapy resistance are missing. Given the tumorigenic role of ECM in cancer progression and resistance, therapies aimed at “normalizing” the tumorigenic ECM represent promising strategies to overcome non-genetic resistance to MAPK inhibitors. Based on the role of miRNAs in post-transcriptional regulation, I focused on the characterization of a pool of miRNAs, defined as “FibromiRs,” which have been shown to participate in the onset and progression of fibrotic diseases. Their crucial role in the fibrogenic process and the possibility to therapeutically manipulate them make them promising druggable targets to prevent the onset of resistance to MAPK-targeted therapies in melanoma. Starting from a screening designed to compare the expression of “FibromiRs” in MAPK inhibitors resistant mesenchymal melanoma cells compared to therapy-naive parental cells, we have identified the profibrotic miR-143/145 cluster as overexpressed in mesenchymal resistant cells. We then explored the profibrotic function of miR-143/145 cluster in the mesenchymal-like resistant cell state and melanoma phenotypic plasticity. First, we analysed the regulation of miR-143 and miR-145 in melanoma, identifying a negative regulation of the MAPK pathway on its expression and the involvement of signaling pathways typical of the mesenchymal resistant state, such as TGFβ and PDGF signaling, in the activation of their expression. Next, we investigated the function of the cluster in the context of adaptive and acquired resistance, showing its contribution in ECM reprogramming, activation of mechanotransduction pathways, and in driving the switch from a differentiated proliferative phenotype to a dedifferentiated invasive one with decreased sensitivity to MAPK inhibition. We characterized its mechanism of action, identifying FSCN1 as a key target gene of both mature miR-143 and miR-145 in the acquisition of the mesenchymal invasive phenotype. Finally, we tested the cluster as a potential therapeutic target in vitro and in vivo through antisense oligonucleotide-mediated inhibition of its expression or pharmacological modulation combined with MAPK inhibitors administration. Overall, this work highlights the importance of a FibromiR cluster in the acquisition of a dedifferentiated phenotype resistant to MAPK-targeted therapies and proposes new therapeutic strategies based on the inhibition of FibromiRs to overcome such resistance mechanism.