Targeting MEK And CDK4/6 Induces Cell Cycle Arrest In KRAS-mutant Gastric Cancer Cells

Objective:Gastric cancer(GC)is one of the most common causes of cancer-related deaths,with a KRAS mutation rate of approximately 16%in GC.This mutation leads to the abnormal activation of KRAS protein,promoting the development and progression of GC.Currently,there are no effective treatments for KRAS-mutated GC.The overactivation of the MAPK signaling pathway caused by KRAS mutation leads to abnormal regulation of cyclin-dependent kinases(CDKs)and cell cycle processes,causing proliferation and cell cycle dysregulation in GC.MEK inhibitors directly target the MAPK pathway,inhibiting abnormal signal transduction caused by RAS mutation and achieving the effect of treating cancer.The combination of MEK inhibitor(Trametinib)and CDK4/6 inhibitor(Palbociclib)has shown significant synergistic effects in inhibiting the growth of KRAS-mutated colon cancer.Targeting CDK4/6 in GC mainly works by inhibiting CDKs,blocking the transition of the cell cycle from G1 phase to S phase,and inhibiting the growth and proliferation of cancer cells,Our research has demonstrated that MEK inhibitors can enhance the effect of CDK4/6 inhibitors in KRAS-mutated GC,inducing cell cycle arrest by inhibiting RB protein phosphorylation and the expression of other protein targets it mediates,including inhibiting the G1/S phase transition and promoting apoptosis,thereby inhibiting the development of KRAS-mutated GC.Clarifying the molecular mechanism by which targeting MEK and CDK4/6 induces cell cycle arrest in KRAS-mutated GC will provide a theoretical basis for the precise treatment of KRAS-mutated GC.Methods:1.Conducting clone formation and cell scratch experiments to observe the effects of MEK inhibitors and CDK4/6 inhibitors on the proliferation of KRAS mutant GC cells.2.Using a combination of transcriptome sequencing and proteomic techniques to screen for differentially expressed genes and proteins in KRAS mutant GC cells,further exploring significantly enriched pathways in the combination treatment group of MEK inhibitors and CDK4/6 inhibitors.3.Flow cytometry was used to detect the effect of drugs on the cell cycle of GC cells,and flow cytometry and TUNEL were used to detect cell apoptosis.4.Using Western blot analysis to detect the expression of RB,phosphorylated RB,β-catenin,phosphorylated β-catenin,Axin2,Cyclin D1,BCL2,BAX,and P53 proteins.Using immunofluorescence to detect the localization of β-catenin and phosphorylated RB protein.Results:1.The combination of MEK inhibitors and CDK4/6 inhibitors can significantly inhibit the proliferation of KRAS-mutant GC cells.2.Transcriptome sequencing and proteomic analysis revealed that MEK inhibitors enhance the inhibition of the cell cycle signaling pathway by CDK4/6 inhibitors.3.The combination of MEK inhibitors and CDK4/6 inhibitors can significantly reduce the expression levels of RB,phosphorylated RB,Cyclin D1,BCL2,β-catenin,and P53,while significantly increasing the expression of p-β-catenin,Axin2,and BAX proteins.4.MEK inhibitors can enhance the effect of CDK4/6 inhibitors by inhibiting the expression of the cell cycle protein Cyclin D1,inhibiting excessive phosphorylation of RB,and inducing cell cycle arrest.Conclusions:In vitro experiments have shown that the combination of targeting MEK and CDK4/6 has a stronger inhibitory effect on tumor proliferation than single-targeted drugs.By converting RB to a hypophosphorylated state,cell cycle arrest is induced and apoptosis is promoted.This phenomenon may be related to the inhibition of the Cyclin D1-RB-E2F-BCL2 pathway,which requires further in vivo experiments to validate this hypothesis.