Involvement of the RNA-binding protein HuR in the acquisition of resistance to EGFR-tyrosine kinase inhibitors in non-small cell lung cancer

NIGRA A; SALVATO I; NAPOLI A; VITALE L; LAMBERTI MJ; RICCIARDI L; CASOLARO V; STELLATO C; DAL COL J

Congreso; SIPMeT - Young Scientists Meeting 2023; 2023

BACKGROUND-AIM Advanced non-small cell lung cancer (NSCLC) patients with sensitive mutations in epidermal growth factor receptor (EGFR) are treated with tyrosine kinase inhibitors (TKIs), such as gefitinib and osimertinib. However, acquired resistance invariably develops, associated with immune suppressive phenotype involving expression of the cytokines IL-6 and IL-8. The RNA-binding protein HuR is an emergent regulator of cancer, currently studied for therapeutic targeting. Indeed, HuR enhances mRNA stability/translation of key effector genes, including cytokines, cell cycle regulators and anti-apoptotic factors. Here, we investigated the role of HuR in supporting NSCLC cell proliferation and survival in response to TKI treatment. METHODS EGFR-TKI-resistant cell lines (PC9GR/H1975OR) were generated by treating PC9/H1975 cells with gefitinib and osimertinib, respectively. Role of HuR was studied using PC9- and H1975-HuR-KO cell lines generated by CRISPR/Cas9 technology. Protein expression was analysed by immunoblotting, confocal microscopy and ELISA assay. Cell cycle and apoptosis were analysed by flow cytometry. RESULTS In silico analyses of public databases showed significant upregulation of both HuR mRNA and protein levels in lung adenocarcinoma primary tumors respect to normal tissue. Gefitinib treatment reduced HuR expression in TKI-sensitive but not in resistant cells, in which HuR protein was more localized in the cytoplasm. Depletion of HuR impaired in vitro TKI-resistance acquisition to both gefitinib and osimertinib in PC9 and H1975 cells, respectively, together with a significant increase of the apoptotic rate induced by the TKIs. Cell proliferation was reduced in PC9- and H1975-HuR-KO cells respect to parental cell lines according to significant cell cycle accumulation in G0/G1 phase, increased levels of p21WAF1 protein and downregulation of Cdk2 and Cdk6. Additionally, loss of HuR protein reduced IL-6 and IL-8 secretion, as well as cell migratory ability, associated with the acquisition of TKI-resistance. CONCLUSIONS Our findings indicate that HuR (i) contributes to the acquisition of TKI-resistance limiting TKI-treatment-induced apoptosis and sustaining cancer cell migration; (ii) promotes the secretion of cancer-associated cytokines favoring an immunosuppressive tumor microenvironment in NSCLC.