Patients affected by advanced non-small cell lung cancer (NSCLC) with mutant epidermal growth factor receptor (EGFR) are treated with tyrosine kinase inhibitors (TKIs) such as gefitinib and osimertinib, yet most patients develop disease progression due to acquired resistance. Resistance mechanisms include secondary mutations on EGFR and other membrane receptors responsible for cell proliferation and survival, along with epigenetic alterations critical to tumor recurrence. To this end, increasing data on regulation of RNA metabolism by RNA-binding proteins (RBPs) is supporting their pathological relevance. The RBP Hu antigen R (HuR) is an emergent regulator of cancer, currently considered for therapeutic targeting. HuR enhances mRNA stability/translation of key effector genes involved in all cancer traits including sustained cell proliferation, evasion of cell death and drug response. We investigated the impact of gene ablation and pharmacological inhibition of HuR on cell proliferation and survival in EGFR-TKI resistant NSCLC cells.

EGFR-TKI-resistant cell lines (HCC827GR/PC9GR/PC9OR/H1975OR) were generated by treating HCC827/PC9/H1975 cells with gefitinib or osimertinib. HuR role was studied using PC9- and H1975-HuR-KO cells generated by CRISPR/Cas9 technology and commercially avaible HuR inhibitors KH-3 and SRI-42127. Cell proliferation was evaluated by Cell Counting Kit-8 assay. Cell cycle and apoptosis were analyzed by flow cytometry. Protein expression was analysed by immunoblotting.

In silico analyses of public databases showed significant upregulation of both HuR mRNA and protein levels in primary lung adenocarcinoma tumors compared to normal tissue, with additional increase in samples from patients upon development of EGFR-TKI resistance. Similarly, in vitro generated EGFR-TKI-resistant cell lines showed higher expression of HuR respect to parental cells. Analysis of proliferation of PC9- and H1975-HuR-KO cells indicated that HuR loss significantly reduced cell proliferation with cell cycle arrest in G0/G1 phase associated with p21Cip1/Waf1 upregulation and Cdk2 or Cdk6 downregulation. Pharmacological HuR inhibition by compounds KH-3 and SRI-42127 (2,5 µM, 24 hours) arrested PC9 and PC9GR cells in G1/S phase while in PC9OR, H1975 and H1975OR cells it induced cell accumulation in G2/M phase and modulated the levels of the kinase Aurora A that is a determinant of the acquisition of osimertinib resistance. Regarding cell survival, while HuR gene ablation did not affect cell viability, both HuR inhibitors induced a significant increase of the apoptotic rate in all EGFR-TKI-sensitive and resistant cell lines.

In conclusion, our findings indicate HuR protein as relevant mechanism and potential target in EGFR-TKI resistant NSCLC given its pro-survival and proliferative functions. Study supporting by AIRC