The Treatment Of KRAS Mutant Gastric Cancer And Its Mechanism By Dual-target Combination Therapy

ObjectiveGastric cancer has become the second leading cause of cancer death worldwide due to factors such as high incidence,unsatisfactory prognosis and lack of unique biomarkers and difficulties in personalizing therapeutic drugs.Gastric cancer is usually diagnosed late,and chemotherapy is still the main choice for patients with advanced gastric cancer.RAS mutation is present in 30%of cancers.KRAS mutation is the most frequent RAS mutation in gastric cancer,with an overall median incidence of 11%in gastric cancer.KRAS mutation will over-activates MEK-ERK signaling pathway,induces key cell cycle regulatory proteins CDK and Cyclin D1,promotes tumor cell hyperproliferation,and leads to disordered regulation of cell cycle progression.The curative effect of direct targeted inhibition of KRAS in the treatment of gastric cancer is difficult to achieve the expectation.Targeted inhibition of MEK,the downstream factor of KRAS,leads to the increase of ERK phosphorylation and cyclin D expression,which promotes the proliferation of tumor cells and the disorder of the cell cycle.CDK4/6 is the core driver of the cell cycle.Inhibition of CDK4/6 may activate the MAPK signaling pathway.The combination of targeting CDK4/6 and MEK can effectively regulate the disorder of gastric cancer cell cycle and prevent the activation of MEK signal pathway,effectively inhibiting the proliferation of gastric cancer cells.At present,clinical trials have reported that CDK4/6 inhibitors combined with MEK inhibitors have significant synergistic anti-tumor effects in neuroblastoma,colorectal cancer,non-small cell lung cancer and melanoma,but the experimental therapeutic effect of KRAS mutant gastric cancer is still unclear.Cell senescence is a unique physiological mechanism to inhibit tumor growth and proliferation.Cell senescence may play an important role in inhibiting tumor cell proliferation.This study reported the inhibitory effect of MEK inhibitor combined with CDK4/6 inhibitor on KRAS mutant gastric cancer and its effect on aging,in order to provide a new strategy for the precise treatment of gastric cancer.Methods1.The expression of CDK6 in gastric cancer and adjacent normal tissues was detected by immunohistochemical staining,and the expression of CDK6 in gastric epithelial cells and gastric cancer cells was detected by Western blot and RT-qPCR.GEPIA database was used to study the expression of CDK4 and KRAS in gastric cancer.According to the survival information of 875 patients with gastric cancer from TCGA,EGA and GEO databases by Kaplan Meier plotter,the effects of CDK6,CDK4 and KRAS expression on the prognosis of patients with gastric cancer were analyzed.2.CCK-8 experiment screened the inhibitory concentration of a single drug on gastric cancer cells and the concentration of combined drugs.A clone formation experiment was utilized to observe the effect of drugs on the clone formation of gastric cancer cells.The effect of drugs on the proliferation of gastric cancer cells was detected by flow cytometry.Transwell invasion and migration test and scratch test were utilized to observe the effect of drugs on the invasion and migration of gastric cancer cells.β-Galactosidase staining was used to observe the effect of drugs on the aging of gastric cancer cells.The effect of drugs on the formation of vascular rings in gastric cancer cells was observed by atubule formation experiment.3.Western blot was utilized to detect the effects of MEK inhibitor trametinib and CDK4/6 inhibitor Palbociclib on the protein expressions of p53,p21,p-MEK,MEK and CDK6.4.The BALB/C nude mouse model was constructed with tumor-bearing MKN1 gastric cancer cells.Nude mice were randomly divided into 4 groups.Control,trametinib（3 mg/kg）and/or Palbociclib（150 mg/kg）were administered by gavage for 4 consecutive days a week,and the drug was discontinued for 3 days.The tumor size and weight of mice were recorded the next day.After 21 days,the mice were dissected and the tumor tissue was obtained.Results1.Overexpression of CDK4,CDK6 and KRAS in gastric cancer indicates a poor prognosis.2.The combination of MEK inhibitor and CDK6 inhibitor can significantly inhibit the proliferation,clone formation,invasion,migration and vascular ring formation of KRAS mutant gastric cancer cells,and promote the senescence of KRAS mutant gastric cancer cells.3.The combination of MEK inhibitor and CDK6 inhibitor can significantly increase the expression levels of p53,p21 and p-MEK,and significantly reduce the expression of CDK6,but has little effect on the expression levels of MEK4.The combination of MEK inhibitor and CDK4/6 inhibitor can synergistically inhibit the growth of gastric cancer in vivo.ConclusionsMEK-CDK4/6 dual-target inhibitor has synergistic inhibitory effects on the proliferation,invasion,migration and angiogenesis of KRAS mutant gastric cancer cells,and may induce the senescence of KRAS mutant gastric cancer cells by up-regulating p53 and p21.