MiR-1 And Restoring The Sensibility Of EGFR-TKI Resistance In NSCLC

Background:Lung cancer is the most common carcinoma worldwide,non-small-cell-lung-cancer represents about nearly 80 percent of lung cancer.Patients with NSCLC harboring EGFR mutations initially respond well to the EGFR-TKIs(gefitinib or erlotinib)and invariably develop acquired resistance to EGFR-TKIs.In the previous study,we found that PI3K(phosphoinositide-3-kinase catalytic subunit alpha)/AKT signaling pathway plays a crucial role in the cell proliferation,migration and invasion.Moreover,PIK3 CA expression negatively correlated with miR-1 expression.Some evidence supports that miRNAs are involved in the EGFR-TKI resistance.Now the mechanisms of miR-1 modulating TKI acquired resistance still remains unknown.Part One Prospective assessment of pemetrexed or pemetrexed plus plantinum in intermittence with EGFR-TKIs in NSCLC patients with acquired resistance to TKI: Retrospective StudyObjective:To assess antitumor efficacy and safety of palliative pemetrexed or pemetrexed-cisplatin followed by gefitinib or erlotinib in 42 NSCLC patients with acquired TKI resistance.Methods:Forty-two patients with lung cancer that responded to TKIs for at least 6 months before developing TKI resistance were enrolled and received sequential pemetrexed or pemetrexed plus platinum followed by gefitinib or erlotinib.Chemotherapy was intravenously administered on day 1,and patients were provided oral EGFR-TKIs between day 8 and 21.The treatment was repeated every 3 weeks for 2 to 4 cycles.At the end of the specified treatment regimen,patients continued to receive TKIs for maintenance until disease progression or unacceptable toxicity.The primary end point was median progression-free survival(m PFS).Secondary end points included the disease control rate(DCR)and adverse events(AEs).Results:Our study showed that mPFS 8.0 months,DCR was 78.6%.The treatment protocol was generally well tolerated.Treatment interruption was required in 2 patients.Grade 3/4 hematologic toxicities included neutropenia(23.8%),leukopenia(16.7%),anemia(4.8%)and thrombocytopenia(4.8%).Common grade 3 non-hematologic toxicities included nausea(7.1%),vomiting(9.5%),anorexia(11.9%),rash(7.1%),fatigue(9.5%),infection(16.7%)and oral mucositis(2.4%).No toxicity-related deaths occurred.Conclusion:For patients with acquired TKI resistance,pemetrexed or pemetrexed plus platinum administration intermittently followed by gefitinib or erlotinib was well tolerated and associated with a fair response.Part Two miR-1 modulates sensitivity of EGFR-TKI resistance via suppressing PIK3 CA in NSCLCObjective:To investigate the effect of miR-1 exerts in the TKI sensitive and resistant cell lines and the mechanisms of restoring the sensitivity of EGFR-TKI resistance.Methods:Transfection of miR-1 mimics or inhibitor was performed in human lung carcinoma PC9G(Gefitinib resistant cell line)or PC9(Gefitinib sensitive cell line),respectively.Quantitative real-time PCR and Western Blot were used to detect miR-1 and PIK3 CA expression.Transwell assay was performed to detect the cell migration.Cell viability assay was assessed using a cell counting kit-8.Cell cycle and apoptosis were detected by flow cytometry.Results:In RT-PCR assay showed that the expression of miR-1 was higher in PC9 cells compared with PC9G cells.The expression of miR-1 was up-regulated in PC9G cells after transfection of miR-1 mimics.Conversely,the expression of miR-1 was down-regulated in PC9 cell after transfection of miR-1 inhibitor.Meanwhile the increase or decrease of miR-1 caused almost no alteration to the expression of PIK3 CA in PC9G cells or PC9 cells,respectively.By Western blot,we found that overexpression of miR-1 significantly suppressed the expression of PIK3CA?Akt and Survivin in PC9G cells,while in PC9 cells,knockdown miR-1 expression dramatically enhanced the expression of PIK3CA?Akt and Survivin.Moreover,overexpression of miR-1 in PC9G cells enhanced the sensibility of EGFR-TKI and inhibited migration.Flow cytometry analysis revealed that overexpression of miR-1 in PC9 cells prohibited the progress of cell cycle.However,transfection of miR-1 mimics did not affect apoptosis of PC9G cells.Conclusion:Our results showed that miR-1 significantly suppressed the growth and migration of TKI resistant cells via down-regulating the expression of PIK3 CA,suggesting that it might be involved with the restoring sensitivity of TKI resistance.Our findings provide a new insight into using miR-1 mimics/PIK3 CA inhibitor–based therapeutic strategies to reverse EGFR-TKI resistance in NSCLC.