P1.03-46 LncRNA H19 Downregulation Promoted Resistance to EGFR-TKIs Through Regulating AKT and SRC Activating in NSCLC Cells

Abstract

Most NSCLC patients with EGFR activating mutations initially respond to EGFR-TKI treatment. However, acquired resistance to EGFR-TKIs inevitably occurs after treatment for several months. There are evidences that suggest lncRNAs could play important roles in EGFR-TKI resistance, but limited data is available now. In previous study, we found that LncRNA H19 was downregulated in EGFR-TKI-resistant cells. We established two erlotinib-resistant cell lines HCC827-ER and PC9-ER. Real-time quantitative PCR (qPCR) was used to detect H19 levels. We detected gene mutations in the ER cell lines through whole-exome sequencing. We used H19 and H19 shRNA lentiviral expression vector to overexpress or knockdown its expression. MTT cytotoxic and clone formation experiments were performed to assess cell growth inhibition. The apoptosis analysis was evaluated by PARP and caspase-3 degradation. Protein levels in AKT and SRC were examined by western blotting. H19 expression was lower in HCC827-ER and PC9-ER cells than in their parental sensitive (PS) cells. We did not detect mutations in EGFR T790M, PI3KCA, or KRAS by whole-exome sequencing of ER cell lines. MTT and clone formation assays showed H19 knockdown caused resistance to erlotinib in PS cells, whereas H19 overexpression not only enhanced erlotinib sensitivity in PS cells but also recapitulated erlotinib sensitivity in HCC827-ER and PC9-ER cells. Treatment with erlotinib increased PARP and caspase-3 cleavage in H19-ovexpressing ER and PS cells. We also found erlotinib treatment decreased levels of phosphorylated AKT and SRC in PS cells, and further lowered their levels in H19-overexpressing PS cells. On the contrast, erlotinib treatment had no effect on levels of phosphorylated AKT and SRC in H19-knockdown PS cells. Finally, we measured the expression of H19 using qPCR in 20 fresh frozen EGFR-mutant NSCLC specimens. Lower expression of H19 was observed in EGFR-TKI-resistant tissue samples. H19 was downregulated in erlotinib-resistant NSCLC cells. H19 inhibited AKT and SRC activating to restore sensitivity to erlotinib, which could provide new insight into resistance mechanisms to EGFR-TKIs.