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

NIGRO A; SALVATO I; RICCIARDI L; LAMBERTI MJ; CARAMORI G; CASOLARO V; STELLATO C; DAL COL J

Congreso; 28th Congress of European Association for Cancer Research; 2022

Introduction Advanced non-small cell lung cancer (NSCLC) patients with sensitive mutations in epidermal growth factor receptor (EGFR) are treated with tyrosine kinase inhibitors(TKIs), including gefitinib. However, acquired resistance invariably develops, associated with immune suppressive phenotype involving expression of pro-inflammatory cytokines such as IL-6 and IL-8 and of immunomodulatory factors including CD47 and programmed death ligand 1 (PD-L1). The RNA-binding proteinHuR is an emergent regulator of cancer through the enhancement of mRNA stability/translation of key effector genes by binding to their 3’-untranslated region, including cytokines and PD-L1. Here, we investigated the TKI-mediated modulation of HuR in TKI-sensitive and resistant NSCLC cell lines and, reciprocally, HuR participation in TKI-treatment response. Material and Methods EGFR-TKI-resistant cell lines (HCC827GR and PC9GR) were generated by treatingEGFR-TKI-sensitive HCC827 and PC9 cells with gefitinib; H1975 harboring EGFR-secondary mutation T790M were also used. To study the involvement of HuR in gefitinib treatment, PC9 HuR-KO cell line was generated by CRISPR/Cas9technology. Protein expression was studied by immunoblotting, flow cytometry and ELISA assay. HuR association with 3’-UTR of CD47 and PD-L1 mRNAs was validated by biotin pull-down assay. Results and Discussions Gefitinib treatment reduced HuR expression in TKI-sensitive but not in resistant cells, in which HUR protein is more localized in the cytoplasm. Development of TKI resistance was associated with a significant increase of IL-6 and IL-8 secretion and enhanced expression of CD47 and PD-L1 on cell surface compared to parental cells. Loss of HuR reduced the release of both cytokines in PC9 HuR-KO cells and decreased superficial expression of CD47 without affecting the total level of this protein. Additionally, loss of HuR impaired in vitro TKI-resistance acquisition and migratory ability of PC9 cells and enhanced the apoptotic rate in gefitinib-treated cells, accompanied by down-regulation of the anti-apoptotic protein Bcl-2. Conclusion Overall, our findings indicate that HuR, whose expression pattern is influenced by the acquisition of TKI-resistance, contributes to the adoption of pro-tumor hallmarks in NSCLC, through the modulation of migratory capacity, the promotion of an immunosuppressive tumor micro-environment, the limitation of TKI -induced apoptosis and subsequent acquisition of resistance to target therapy.