miR-1323 Promotes Cell Migration in Lung Adenocarcinoma by Targeting Cbl-b and Is an Early Prognostic Biomarker.

Purpose: MicroRNAs are known to regulate cellular processes in non-small cell lung cancer (NSCLC) cells and predict prognosis. However, identification of specific microRNAs in NSCLC as potential therapeutic targets is controversial. We aim to determine the clinical significance of miR-1323 in the prognosis of patients with lung cancer and the potential mechanism. Patients and methods: A bioinformatics approach was used to screen the importance microRNA in NSCLC through the online GEO database (GSE42425). The relationship between expression level of miR-1323 and overall survival of lung cancer patients was analyzed. Additionally, an independent corhort including 53 NSCLC cases that underwent resection validated the connection between miR-1323 and LUAD patients' overall survival. Next, the function of miR-1323 was studied in vitro by transient transfection. A more in-depth mechanism was studied through luciferase reporter gene experiments. Results: High miR-1323 expression correlated with poor survival in NSCLC patients (P = 0.011), and in lung adenocarcinoma (LUAD) patients (P = 0.015) based on GEO database (GSE42425). In the independent cohort based on our hospital, high miR-1323 expression was associated with LUAD patients (P = 0.025). Moreover, transfection with mimics of miR-1323 showed an increased migratory capacity in LUAD A549 and HCC827 cells. In addition, E3 ubiquitin-protein ligase Casitas B-lineage Lymphoma-b (Cbl-b) was found to be the target genes of miR-1323 and significantly down regulated after mimics of miR-1323 transfection, and high Cbl-b expression predicted better prognosis in NSCLC and LUAD (P = 0.00072 and P = 0.02, respectively). Conclusion: The miR-1323 promoted LUAD migration through inhibiting Cbl-b expression. High miR-1323 expression predicted poor prognosis in LUAD patients.

Suppressed expression of Cbl-b by NF-κB mediates icotinib resistance in EGFR-mutant non-small-cell lung cancer.

Although epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) could greatly improve the prognosis of NSCLC patients harboring activating EGFR mutations, drug resistance still remains a major obstacle to successful treatment. Our previous study found that the EGFR-TKI icotinib could upregulate the expression of Casitas-B-lineage lymphoma protein-B (Cbl-b), an E3 ubiquitin ligase. In the present study, we aimed to clarify the potential role of Cbl-b in the resistance to icotinib, and the underlying mechanisms using EGFR-mutant cell lines. We found that icotinib inhibited the proliferation of mutant-EGFR NSCLC cells (PC9 and HCC827), and upregulated the expression of Cbl-b at both the protein and mRNA levels. Cbl-b knockdown decreased the sensitivity of PC9 and HCC827 cells to icotinib, and partially restored icotinib-inhibited AKT activation in PC9 cells. On the contrary, Cbl-b overexpression could partly reverse the drug resistance in PC9 icotinib-resistant cells (PC9/IcoR). Moreover, overexpressing p65, the main member of transcription factor NF-κB family, reversed the icotinib-mediated upregulation of Cbl-b. Collectively, these data suggest that icotinib could upregulate Cbl-b mediated by NF-κB inhibition, and Cbl-b contribute to the icotinib sensitivity in EGFR-mutant NSCLC cells. This study highlights that low expression of Cbl-b might be the key obstacles in the efficacy of icotinib therapy.

Tyrosine kinase inhibitor-induced IL-6/STAT3 activation decreases sensitivity of EGFR-mutant non-small cell lung cancer to icotinib.

Tyrosine kinase Inhibitors (TKIs) of epidermal growth factor receptor (EGFR) has considerably benefited for non-small cell lung carcinomas (NSCLC) harbor mutations in EGFR. However, the factors attenuating EGFR-TKI efficiency are obstacles to inhibit the proliferation of EGFR-mutant NSCLC cells successfully. Clarifying the insensitivity mechanisms of EGFR-TKI would help to develop new treatment strategy. In this study, the sensitivity of EGFR-mutant NSCLC cell lines, PC9 and HCC827, to icotinib was detected. Similar with other EGFR-TKIs such as gefitinib and erlortinib in previous research, the proliferation of two cell lines was apparently inhibited. However, we surprisingly found that contrast with the suppression of EGFR-AKT/ERK pathway, STAT3 was significantly activated in PC9 cells with the treatment of icotinib, but not in HCC827 cells. Further study confirmed that icotinib concomitantly induced IL-6 secretion and src activation in PC9 cells. Moreover, with the treatment of IL-6 neutralizing antibody or src inhibitor, dasatinib, icotinib-induced phosphorylation of STAT3 was reduced, as well as the sensitivity of PC9 to icotinib was also partially increased. Our results suggest that Src/IL-6/STAT3 bypass pathway is activated to maintain cell survival when the EGFR pathway was inhibited by TKIs, even in some EGFR-mutant NSCLC cells sensitive to TKIs. This finding provides a groundwork for potential combinatorial treatment with TKIs and Src or STAT3 inhibitor to improve icotinib sensitivity.