Immune escape mechanisms developed in lung cancer following target therapy resistance: Role of the RNA-binding protein HuR

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

Congreso; CIMT Annual Meeting; 2021

Tyrosine kinase inhibitors (TKIs) including gefitinib are first-line treatment for advanced non-small cell lung cancers (NSCLC) with sensitive mutations in epidermal growth factor receptor (EGFR). However, acquired resistance invariably develops and is associated with an immune suppressive phenotype involving expression of immuno-related proteins, such as programmed death ligand-1 (PD-L1) and CD47, and secretion of proinflammatory and tumor resistancepromoting cytokines. The RNA-binding protein HuR targets these factors in cancer types including NSCLC by binding to 3?-untranslated region (UTR) and modulate mRNA stability/translation. Our aim was to define HuR role in immune escape following TKI-resistance in NSCLC. We investigated in vitro the expression of HuR and its regulatory role on immune checkpoints and cytokine expression, as well as migration of NSCLC cell lines in the absence/presence of gefitinib-induced resistance (PC9 and HCC827 cells with EGFR-sensitizing mutations; H1975 with EGFR-resistant secondary mutation and PC9GR and HCC827GR with in vitro acquired gefitinib-resistance). PC9 HuR-KO cell line was generated by CRISPR/Cas9 technology. Nuclear/cytoplasmic HuR levels were evaluated by Western blot and HuR binding to 3? - UTR of CD47 and PD-L1 mRNA by biotin pull-down assay. Expression of CD47 and PD-L1 was assessed by flow cytometry, IL-6 and IL-8 levels by specific ELISA, cell migration by wound healing assay. Gefitinib treatment reduced HuR expression in PC9 cells but not in PC9GR. While remaining mostly nuclear in PC9 cells, HuR increased in cytoplasmic fraction in PC9GR and H1975 cells. IL-6 and IL-8 release were significantly increased in PC9GR and HCC827GR cell lines while their secretion was significantly reduced in TKI-untreated and treated PC9 HuRKO cells. Additionally, PC9 HuR-KO cells did not acquire TKI-resistance in vitro and wound closure was significantly delayed in these cells compared to PC9 parental cells. Surface CD47 expression was increased in PC9GR and HCC827GR; PD-L1 was significantly upregulated in PC9GR, while it was downregulated in HCC827GR compared to parental cells. Biotin pull-down identified HuR association with CD47 and PD-L1 mRNAs. Our results showed that HuR may regulate key factors involved in immune escape following TKI-induced resistance in NSCLC. Dissecting the contribution of HuR in this context would support the development of pharmacological inhibitors of specific interactions between HuR and its target mRNAs reverting the immune suppressive phenotype promoted by TKI-resistance.