Investigation On Anti-tumor Activity Of CDK4/6 Inhibitor In Non-small Cell Lung Cancer Cell Lines And The Mechanism Of Overcoming The Third Generation EGFR-TKI Acquired Resistance

Objective: Cyclin dependent kinase(CDK)4 / 6 inhibitor is a specific cyclin dependent kinase inhibitor.Abnormal cell cycle regulation is the basic feature of malignant tumors.To investigate the efficacy of palbociclib,a CDK4 / 6 inhibitor,in non-small cell lung cancer and whether KRAS / LKB1 co-mutation is biomarker of CDK4 / 6 inhibitor in NSCLC.To explore whether CDK4 / 6 inhibitors can reverse the third generation EGFR TKI resistance to osimertinib,and the mechanism of overcoming resistance.Method: CCK8 was used to explore the sensitivity of CDK4 / 6 inhibitor,Palbociclib,in several cell lines of NSCLC,which were H1975 S,H1975OR,H1299,A549,H460,Calu-1,H1299-sh LKB1#407,H1299-sh LKB1#408,H1299-sh LKB1#411.In those cell lines,we explored whether KRAS mutation,LKB1 mutation and KRAS / LKB1 co-mutation were related to the sensitivity of palbociclib.Western blot,Real-Time PCR was used to explore whether the mechanism of drug resistance of H1975 OR was related to the abnormal cell cycle,and to evaluate whether there were differences in genes and proteins such as CDK4,Cdk6,Cyclin D1,RB,CDKN2 A and snail.Flow cytometry was used to detect the differences between H1975 S and H1975 OR after drug application.we first evaluated whether there were differences in the sensitivity of single drug CDK4 / 6 inhibitors between h1975 s and H1975 OR.Secondly,the synergistic effect of CDK4 / 6 inhibitor and osimertinib was evaluated by drug synergy index,and whether the combination of the two drugs could reverse the drug resistance of osimertinib.Finally,Western blot and real time PCR were used to evaluate the changes of cell cycle related proteins and EMT related proteins in single drug CDK4 / 6 inhibitor palbociclib,single drug osimertinib,and combination therapy,and to explore the mechanism of CDK4 / 6 inhibitor combined with osimertinib reversing the third generation EGFR TKI resistance.Result:(1)Western blot showed that the knockdown of LKB1 was successful in h1299-sh LKB1#408 and h1299-sh LKB1#411,but not in h1299-sh LKB1#407.Furthermore,the sensitivity of CDK4 / 6 inhibitor was tested in a variety of lung cancer cell lines.IC50 of palbociclib was 12.93 μM,13.01 μM,29.22 μM,9.478 μM,12.84 μM,11.44 μM,6.604 μMand 10.46 μMin cell lines of H1975 S,H1299,Calu-1,H1299-sh LKB1#408,H1299-sh LKB1#411,H1299-sh LKB1 #407,A549 and H460,respectively.KRAS/LKB1 co-mutation may increase the sensitivity to CDK4 / 6 inhibitor,but it does not reach the difference of more than 10 times.(2)Compared with H1975 S cell line,the sensitivity of H1975 OR cell line to palbociclib was increased,which was 12.94 μMand 4.776 μM,respectively(P < 0.05).In the H1975 OR cell line,the cell survival rate was 81.67%(± 1.29%)with palbociclib(1.25 μM),73.80%(± 5.41%)with osimertinib(2.5 μM),and 40.67%(± 1.13%)with combination(P < 0.05).In terms of mechanism,compared with H1975 S,RT-PCR of H1975 OR showed that the m RNA levels of CDK4,CDK6 and CCND1 were increased,which promoted cell cycle(P values,0.065,0.052 and 0.049),while CDKN1 A,CDKN1B,CDKN2 A and CDKN2 B,which inhibited cell cycle,were significantly decreased(P values,0.041,0.0.12,0.003 and 0.001).Western blot confirmed that CDK4 and p-RB were significantly enhanced in H1975 OR.With the prolongation of the effect of osimertinib on H1975 S,CDK4 and p-RB were enhanced.Palbociclib combined with osimertinib could inhibit the function of CDK4 / 6-Cylin D,control the initial phosphorylation of RB,and then block cells to enter the S-phase through the R point,and control tumor proliferation.(3)In H1975 OR cell line,Western blot confirmed that the expression of snail was increased,but RT-PCR snail m RNA in sensitive cell line h1975 s and resistant cell line H1975 OR had no significant increase,there was no statistical difference.It was further confirmed that the increased expression of snail protein was mainly due to the increased regulation of ubiquitination of dub3.RT-PCR confirmed that there was a significant increase of dub3 m RNA in drug-resistant cell line H1975 OR.CDK4 / 6 inhibitors regulate the expression of dub3 gene to regulate snail and reverse drug resistance.Conclusion: KRAS mutation,LKB1 mutation and KRAS / LKB1 co mutation can not be used as biomarkers related to the efficacy of palbociclib in lung cancer cell lines.The abnormal regulation of cell cycle is involved in the mechanism of resistance of EGFR-TKI.Palbociclib,an inhibitor of CDK4 / 6,combined with osimertinib,reverses resistance by regulating cell cycle,EMT and other pathways,which has further research value in clinical verification.