Individuazione di nuovi marker diagnostici e target terapeutici potenzialmente utilizzabili nella pratica clinica trapiantologica per minimizzare il processo di fibrosi polmonare indotta dalle alte dosi di everolimus: risultati di uno studio in vitro di mirnomica e trascrittomica.

Mammalian target of Rapamycin (mTOR) is a serine/threonine kinase member of the PI3K related kinase (PIKK) family that plays an integral role in the coordination of metabolism, protein synthesis, cell growth, and proliferation. It interacts with several proteins to form two distinct complexes named mTOR complex 1 (mTORC1) and 2 (mTORC2). mTORC1 integrates inputs from growth factors, aminoacids, energy status, oxygen promoting anabolic cellular metabolism, stimulating synthesis of proteins, lipids, and nucleotides. It inhibits catabolic processes, such as lysosome biogenesis and autophagy. mTORC2 controls cell survival, cytoskeleton organization, lipogenesis, and gluconeogenesis. The discovery of mTOR is intimately linked to the development of rapamycin, a metabolite produced by the bacterium Streptomyces hygroscopicus with antifungal properties and, in mammalian cellular assays, antitumour and immunosuppressive activity Due to their important antiproliferative and cellular effects, during the last 20 years, many Rapalogs have been developed and used in several clinical settings as immunosuppressive medications Sirolimus (SRL) (Rapamune; Wyeth Pharmaceuticals, New York, NY) was the first mTOR-I approved as an immunosuppressive agent for renal transplantation patients. Everolimus (Certican, Novartis) derived from SRL, contains a 2-hydroxy-ethyl chain in the 40th position that makes the drug more hydrophilic than SRL and increases oral bioavailability by approximately 10–16%. Like other antineoplastic/immunosuppressive drugs, mTOR-I may induce several dose-related renal and systemic side effects. Some of them are manageable, whereas in other cases they require drug withdrawal. Among them, pneumonitis or interstitial lung disease with a range of pulmonary histopathologic changes (including alveolar hemorrhage, pulmonary alveolar proteinosis, focal fibrosis, bronchiolitis obliterans organizing pneumonia) have been largely reported in clinical records and they have been associated with worsened patients’ clinical outcomes and drug discontinuation Although the underlying mechanism is not fully clarified, this condition could be in part caused by epithelial to mesenchymal transition (EMT) of airway cells. Our group has recently demonstrated that high doses of EVE are able to induce a reprogramming of gene expression in several epithelial cell lines (airway, renal epithelial proximal tubular and hepatic cells) with a consequent loss of their phenotype (junctions and apical-basal polarity) and the acquisition of mesenchymal traits increasing the motility and enabling the development of an invasive and pro-fibrotic phenotype. The pro-fibrotic attitude of EVE has also been confirmed in vivo through the estimation of an arbitrary pulmonary fibrosis index score in renal transplant patients chronically treated with this drug. In this patients’ subset, high blood trough level of EVE was associated with high rate of pulmonary signs of fibrosis Therefore, we employed, for the first time, a high throughput approach combining a transcriptomic with a miRNome analysis to study the capability of EVE to induce pro-fibrotic changes in primary bronchial epithelial cells. Primary bronchial epithelial cells (BE63/3) were treated with EVE (5 and 100 nM) for 24 hours. EMT markers (α-SMA, vimentin, fibronectin) were measured by RT-PCR. Transepithelial resistance was measured by Millicell-ERS ohmmeter. mRNA and microRNA profiling were performed by Illumina and Agilent kit, respectively. Only high dose EVE increased EMT markers and reduced the transepithelial resistance of BE63/3. Bioinformatics showed 125 de-regulated genes that, according to enrichment analysis, were implicated in collagen synthesis/metabolism. Connective tissue growth factor (CTGF) was one of the higher up-regulated mRNA. Five nM EVE was ineffective on the pro-fibrotic machinery. Additionally, 3 miRNAs resulted hyper-expressed after 100 nM EVE and able to regulate 31 of the genes selected by the transcriptomic analysis (including CTGF). RT-PCR and western blot for MMP12 and CTGF validated high-throughput results. However, all together, our results suggested that high doses of EVE, through the activation of a multi-factorial biological/cellular machinery, may lead to pulmonary fibrosis and underlined potential pathogenetic, diagnostic biomarkers and targets for future pharmacological interventions to introduce in the “day by day” clinical practice. Finally, at a clinical point of view, we confirm that, whenever possible, the dose of EVE should be the minimized in patients with early signs of lung toxicity.