Immunomodulatory effect of gefitinib in epidermal growth factor receptor tyrosine kinase inhibitors sensitive and resistant Non small cell lung cancer: role of the RNA binding protein HuR

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 Cluster of Differentiation 47 (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 through binding to the 3’-untranslated region (UTR) of their mRNAs. In addition to regulating mRNA stability and translation, HuR is also involved in the regulation of the surface localization of several membrane proteins, such as CD47. Specifically, CD47 3’-UTR acts as scaffold to recruit a protein complex containing HuR and SET to translation site. This interaction allows binding of SET to newly-translated CD47 and Rac Family Small GTPase 1 (RAC1) resulting in CD47 translocation to the plasma membrane. 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. Therefore, considering the emerging role of HuR in promoting CD47 localization on the cell surface, we invastigated wheter gefitinib modulates the translocation of CD47 on the cell surface by HuR in our in vitro model. Upon gefitinib treatment, HuR expression detected by immunoblot was decreased in PC9 cells but remained unchanged in PC9GR. Interestingly, HuR increased in cytoplasmic fraction in PC9GR and H1975 cells, while it remained mostly nuclear in PC9 cells. Interestingly, loss of HuR significantly reduced IL-6 and IL-8 release in TKI-untreated and treated PC9 HuR-KO cells compared to parental cell line. Given the significant upregulation of IL-8 and IL-6 in HCC827GR and PC9GR, we investigated HuR involvement in TKI-resistance acquisition. Preliminary results confirmed that loss of HuR suppressed the ability to acquire TKI resistance in vitro and was associated with significantly decrease of IL-8 concentration in the cell supernatant. Furthermore, loss of HuR inhibited cell migration in wound healing assay. Surprisingly, HuR Knockout had no effect on the basal rate of apoptosis, however the apoptotic rate significantly increased in PC9 HuR KO than PC9 cells following exposure to gefitinib. Additionally, pathway enrichment analysis from proteome profiler antibody array data comparing gefitinib-treated versus untreated cells, highlighted that IL-8 signaling was inhibited in the absence of HuR. Flow cytometry analysis showed that EGFR inhibition significantly reduced CD47 surface expression in NSCLC, while the levels of CD47 total protein and mRNA remained unchanged in gefitinib-treated compared to untreated cell lines, suggesting altered protein localization. Biotin pull-down assay identified HuR association with CD47 mRNA. Subsequently we demonstrated that HuR loss significantly reduced CD47 expression on cell surface respect to parental cell lines without affecting total CD47 protein levels. Furthermore, CD47 surface expression remained unchanged after gefitinib treatment in HuR-KO cell line. Collectively, these preliminary data indicated that HuR contributes to the acquisition of gefitinib resistance in NSCLC through increased IL-8 secretion by tumor cells. Furthermore, HuR regulates CD47 transport to the plasma membrane in response to gefitinib treatment. Therefore, based on the obtained results, HuR may represent a novel attractive drugable target for NSCLC therapy.