I microRNAs come mediatori di resistenza acquisita agli inibitori tirosin-cinasici dell'EGFR nel carcinoma polmonare non a piccole cellule (NSCLC)

MicroRNAs (miRNAs) are small noncoding RNA playing a major function in regulating gene expression by inhibiting mRNA translation or promoting its degradation. MiRNAs are considered master regulators of almost every cellular process, and the dysregulation of their expression has been implicated in cancer pathogenesis. Recently, miRNAs have been intensively investigated in the context of anti-cancer drug resistance; in particular, increasing evidence ascribing a role to miRNAs in modulating response or resistance to targeted therapies has been coming out over the past few years. Our work aimed at identifying miRNAs potentially involved in acquired resistance to the EGFR tyrosine-kinase inhibitors (TKIs), which are used for treating non-small cell lung cancers (NSCLC) harboring specific sensitizing mutations in the EGFR tyrosine-kinase domain. As experimental model we used a panel of four sensitive NSCLC cell lines rendered resistant to five different EGFR tyrosine-kinase inhibitors (TKIs), namely erlotinib, gefitinib (first generation inhibitors), afatinib, dacomitinib and AZD8931 (new generation inhibitors). The performed analyses showed that the developed mechanisms of resistance are mediated either by already described alterations (ie, EGFR T790M mutation and MET gene amplification), or by new alterations (ie KRAS gene amplification) or by not yet identified players. The global miRNA profiling revealed a cell line-specific pattern of miRNA expression. Looking at the most up-regulated and down-regulated miRNAs in each resistant cell line versus their respective parental cells, two miRNA families resulted markedly deregulated: miR-100/99a/125b cluster was down-regulated in PC9 resistant to erlotinib, gefitinib and afatinib, while miR-200 family down-regulation was observed in all the H4006 resistant cells. These miRNAs have been recently shown to play a role in two processes critical for cancer progression, the epithelial- mesenchymal transition (EMT), and cancer stem cell (CSC) onset and maintenance. These are key events at the base of tumor recurrence and metastasis formation, and emerging evidence shows their implication in development of resistance to targeted therapies. In-depth analyses conducted on our cells revealed that both PC9 and H4006 resistant cells overexpressed stem-cell related markers; moreover, H4006 resistant cells displayed loss of epithelial markers and concomitant acquisition of mesenchymal traits. In light of these findings, we believe that PC9 and H4006 resistant cells may have developed resistance to the EGFR TKIs through the acquisition of stem cell-like features and EMT traits respectively. As a final point, we focused our attention on a representative member of the miR-100/99a/125b cluster, namely miR-100, and investigated its potential involvement in the acquisition of resistance to EGFR TKIs in PC9 cells. Notably, our results show that modulation of miR-100 expression in PC9 parental/resistant cells impacted on drug resistance/sensitivity, suggesting a putative role for miR-100 in mediating acquired resistance to erlotinib, gefitinib and afatinib. A clinical validation of our findings is still needed, but our study could provide novel insights into the clinical application of miRNAs as predictor markers of resistance to EGFR TKIs. Identification of miRNAs able to predict response or resistance to the therapy would represent a powerful tool for lung cancer treatment, since miRNA signatures might contribute to the choice of the best therapeutic approach for patients.