Involvement of the RNA-binding protein HuR in promoting the immune suppressive phenotype of lung cancers upon development of target therapy resistance

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

Congreso; World Immune Regulation Meeting XV; 2021

Advanced non-small cell lung cancer (NSCLC) patients with sensitive mutations in epidermal growth factor receptor (EGFR) are eligible for treatment with tyrosine kinase inhibitors (TKIs), including gefitinib. However, acquired resistance invariably develops and is associated with an immune suppressive phenotype involving expression of proinflammatory and tumor resistance-promoting cytokines such as IL-6 and IL-8 and of immunomodulatory factors such as programmed death ligand-1 (PD-L1) and CD47. The RNA-binding protein Human antigen R (HuR) is an emergent regulator of cancer pathogenesis through modulation of mRNA stability/translation of key effector genes, such as proinflammatory cytokines, proliferative and angiogenetic factors through binding to the 3?-untranslated region (UTR) of their mRNAs. Initial evidences indicate also CD47 and PD-L1 as potential HuR targets. The aim of this study is to define HuR role in immune escape mechanisms following TKI-resistance using NSCLC cell lines (PC9 and HCC827 cells with EGFR-sensitizing mutations; H1975 with EGFR-resistant secondary mutation; PC9GR and HCC827GR with in vitro acquired gefitinib-resistance). Moreover, we generated PC9 HuR-KO cell line by CRISPR/Cas9 technology. Upon gefitinib treatment, HuR expression detected by immunoblot was decreased in PC9 cells but remained unchanged in PC9GR. IL-6 and IL-8 secretion detected by ELISA was significantly increased in gefitinib-resistant cells, while basal and TKI-induced IL-6 and IL-8 production was reduced in PC9 HuR-KO cells compared to parental cells. Additionally, loss of HuR impaired in vitro TKI-resistance acquisition and migratory ability of PC9 cells. Flow cytometry analysis showed that surface CD47 expression was increased in PC9GR and HCC827GR, whereas PD-L1 was significantly upregulated only in PC9GR compared to parental cells. Biotin pull-down identified HuR association with 3?-UTR of CD47 and PD-L1 mRNAs in PC9 cell line. In summary, HuR may regulate key factors involved in immune escape following TKI-induced resistance in NSCLC.