Synergistic Induction Of Autophagy In KRAS Mutant Gastric Cancer Cells By Targeting CDK4/6 And MEK

Background and Objective:Gastric cancer（GC）is one of the most common malignant tumors in the world,ranking fifth in cancer incidence and fourth in mortality.The main treatment methods for early gastric cancer are endoscopic resection,surgical treatment for advanced gastric cancer,and continuous targeted chemotherapy or tumor immunotherapy for advanced gastric cancer.KRAS is recognized as the most commonly mutated oncogene in human cancer and is associated with poor prognosis,with approximately 3%-13%of gastric cancers containing KRAS mutations.Studies have reported that direct targeting of the key downstream MAPK signaling pathway of KRAS has limited inhibitory effect on gastric cancer cells.Our previous study found that targeting CDK4/6 and MEK could synergistically act on KRAS-mutated gastric cancer cell cycle regulation and MAPK signaling pathway,effectively inhibiting the proliferation of gastric cancer cells,but some cells still survived.Autophagy is a pathway involved in protein and organelle degradation in lysosomes and plays an important role in maintaining cell homeostasis.Studies have reported that autophagy plays an important role in KRAS mutant cancer cells.Whether targeting CDK4/6 and MEK can synergically induce autophagy in KRAS-mutated gastric cancer cells remains unclear.In this study,autophagy of KRAS mutant gastric cancer cells was synergically induced by targeting CDK4/6 and MEK and its therapeutic effect in KRAS mutant gastric cancer cells.The purpose of this study was to synergically induce autophagy of KRAS mutant gastric cancer cells and its therapeutic effect in KRAS mutant gastric cancer cells by targeting CDK4/6 and MEK.The aim is to find a new strategy for the treatment of KRAS mutant gastric cancer.Method:1.Two KRAS mutant gastric cancer cell lines,AGS and SNU1,were divided into blank control group,MEK inhibitor Trametinib group,CDK4/6 inhibitor Palbociclib group and Trametinib+Palbociclib group,respectively.Cell proliferation and scratch healing were observed by colony formation assay and cell scratch assay.2.The expression of autophagy marker protein LC3 in each group was detected by immunofluorescence assay.Western blotting and reverse transcription-quantitative polymerase chain reaction（RT-q PCR）were used to detect the expression levels of autophagy marker proteins LC3II/LC3I,Beclin-1,P62 and ATG5 and their m RNA in each group.3.The ATP level in each group was measured by ATP detection experiment,and the expression levels of P-AMPK,AMPK,P-m TOR and m TOR in each group were detected by Western Blot.RT-q PCR was used to detect the expression levels of AMPK and m TOR m RNA in each group.4.Cell Counting Kit-8（CCK8）assay was used to detect the half inhibitory concentration(IC₅₀)of autophagy inhibitor Chloroquine（CQ）on KRAS mutant gastric cancer cells and the survival activity of cells in each group after adding CQ and without adding CQ.Then the cells were divided into blank control group,Trametinib+Palbociclib group and CQ+Trametinib+Palbociclib group.Colony formation assay and cell scratch assay were used to observe the cell proliferation and scratch healing of each group.Result:1.Targeting CDK4/6 and MEK synergistically inhibit the proliferation of KRAS mutant gastric cancer cells.2.Targeting CDK4/6 and MEK synergistically induces autophagy in KRAS mutant gastric cancer cells.3.Targeting CDK4/6 and MEK induces autophagy in KRAS mutant gastric cancer cells via AMPK/m TOR pathway.4.Targeting CDK4/6 and MEK enhanced the inhibitory effect of autophagy inhibitor（CQ）on KRAS mutant gastric cancer cell proliferation.Conclusion:1.Targeting CDK4/6 and MEK induces autophagy in KRAS mutant gastric cancer cells through AMPK/m TOR pathway.2.The combination of autophagy inhibitors and dual targets further inhibited the proliferation of KRAS mutant gastric cancer cells.Innovation:In this study,autophagy inhibitor combined with targeted CDK4/6 and MEK was found to effectively inhibit the proliferation of KRAS mutant gastric cancer cells.