L’espressione della cellular retinol binding protein-1 nel carcinoma del polmone

Background: Lung adenocarcinoma and lung squamous cell carcinoma are the most common histological subtype and the leading cause of cancer mortality in worldwide. However, the identification of genetic alterations in NSCLC has transformed patients’ treatment. Retinoids promote cancer cell differentiation and death and their trafficking and action is mediated from specific cytoplasmic and nuclear receptors, respectively. Cellular Retinol Binding Protein-1 (CRBP1) is a 15-kDa protein a key component of the retinoid pathway regulating retinol bioavailability and transcriptional activities in normal and neoplastic tissue. Furthermore, microRNAs (miRNAs) have been found to regulate cancer progression by participating in the process of tumour cell growth, migration, invasion and apoptosis. Aim: We investigated the relationship between CRBP1, RARα and EGFR/pEGFR through bioinformatics analysis of miRNAs and genes expression in CRBP1+A549 cells and, preliminary, in lung adenocarcinoma patients with CRBP1High expression to deepen the possible role of CRBP1 in the pathology of lung adenocarcinoma and also the effect of CRBP1 transfection in H460 human NSCLC cell line, normally not expressing CRBP1. Methods: Protein expression, gene, miRNAs and Copy Number Variation (CNV) analysis, were performed in CRBP1+A549 cells and in a small cohort of lung adenocarcinoma and squamous cell carcinoma patients with CRBP1High expression. H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBP1 gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans Retinoic Acid (atRA) and retinol (ROL). Akt-related gene expression was analysed by using western blot and Signosis array and results analysed by one-way analysis of variance (ANOVA) or by T-student test. Results: Immunoprecipitation studies showed an interaction between RARα and CRBP1, EGFR/pEGFR in CRBP1+A549 compared to CRBP1-A549 cells. Furthermore, CRBP1 did not affect the Ubiquitin-Proteasome System in vitro and no EGFR accumulation was observed after MG132 treatment in those cells. CRBP1 overexpression in A549 cells modulated different miRNAs and genes expression. In silico analysis between miRNAs and genes deregulated in CRBP1+A549 cells disclosed several miRNA-gene target interactions, two of which (hsa-miR-148a-3p and hsamiR-1915-3p) bind TP53 and EGFR genes, respectively. Our in vivo results showed that CRBP1High in lung adenocarcinoma patients modulated miRNAs expression and influenced gene CNV. Among them, only miR-4286 correlated its expression with the chromosome 8 region in which we observed one copy in CSMD1 gene. Finally, the intersection between miRNAs deregulated in CRBP1+A549 cells and CRBP1High lung adenocarcinoma patients showed three miRNAs in common: miR-1915, miR-148a and miR-4286. Those miRNAs were implicated in different cancer pathways, in particular, miR-148a, miR-1915 and miR-4286 bound TP53, PLIN1, EGFR, RARα, CREB1 and FASN genes, respectively, suggesting that lower expression of miR-1915 did not repress EGFR and RARα expression, maintaining so active. On the other hand, H460 CRBP1+ cells showed reduced proliferation and viability in basal condition and after atRA treatment when compared to empty-transfected H460 cells. Reduced proliferation in H460 CRBP1+ cells is associated to the downregulation of pAkt/pErk/pEGFR-related genes. In particular, gene array documented the downregulation of Akt and Stat-3-related genes, including mTOR, Akt1, Akt2, Akt3, Foxo1, p27, Jun. This suggests that the reduced proliferation and viability of H460 cells due to the restoration of CRBP1+ expression (both in basal condition and after atRA treatment), is probably due to the downregulation of Akt-related gene level. Conclusion: Our study provides new insights concerning CRBP1 potential role as a biological therapeutic target in lung adenocarcinoma. For this purpose, we suggest a hypothetical mechanism which CRBP1 influences lung adenocarcinoma proliferation in vitro. Further studies are needed to better clarify how those CRBP1-related intracellular pathways contribute to counteract NSCLC progression in order to suggest a potential tool to improve efficacy of retinoid anti lung cancer adjuvant therapy.