Discovery And Biological Study Of MTA2 Sensitizing Gastric Cancer Cells To Olaparib

Gastric cancer is one of the most common malignant cancers worldwide.In 2020,the incidence and mortality of gastric cancer rank fifth(5.6%)and fourth(7.7%)of all cancers around the world respectively.At the same time,in China,the incidence and mortality of gastric cancer rank the second(10.5%)and third(12.4%)respectively,with478,508 newly diagnosed gastric cancer patients in one year.The high incidence and mortality of gastric cancer reflect the need for improvement in diagnosis and clinical therapy of gastric cancer.However,at present,although immunotherapy has achieved great progress,there is still a great need for more targeted drugs in the clinic to enrich the choice of drugs for the treatment of gastric cancer.Poly(ADP-ribose)polymerase(PARP)inhibitor olaparib can selectively kill BRCA-deficient cancer cells and has been approved by FDA for the clinical treatment of BRCA-mutated breast,ovarian,and pancreatic cancer.Clinical trials of olaparib have also been launched in the treatment of gastric cancer.In a randomized phase II clinical trial of olaparib in gastric cancer patients,olaparib combined with paclitaxel showed an improvement in overall survival(OS)of patients.However,the phase III clinical trial of olaparib combined with paclitaxel in gastric cancer did not meet its primary objective to significantly improve the OS.Among the reasons for divergent outcomes of phase II and phase III clinical trials,the lack of effective predictive biomarkers has been widely discussed,and it is concluded that the negative ATM used in clinical trials above is not sufficient to predict the response to PARP inhibitors.Therefore,to explore and find effective predictive biomarkers has become one of the most important directions in research of olaparib in treating gastric cancer.Proteomics is the overall study of the proteome,it is considered to be the large-scale identification,characterization and quantification of proteins in cells,tissues or organisms under given conditions.Proteomics integrates multi-faceted technologies including liquid chromatography,mass spectrometry,genome databases and bioinformatics,it is an effective method to study early disease diagnosis,prognosis,and monitor disease development.Proteomics also plays an important role in drug development,and it is a powerful method to identify drug targets and discover biological mechanisms underlying disease progression.Protein biomarkers can be identified through proteomics to assist in patient selection,thereby improving drug efficacy,avoiding drug resistance and optimizing treatment options.To explore the biomarkers of olaparib for the treatment of gastric cancer,we selected12 gastric cancer cell lines with different gene mutation backgrounds,sources and growth characteristics as experimental models,and tested IC50 of olaparib through cell viability experiments.The proteome profile of cell lines is characterized by non-labeled quantitative proteomics technology.In the results of cell viability experiments,12 gastric cancer cell lines showed significant differences in sensitivity to olaparib,and the differences had nothing to do with the source and growth characteristics of the cell lines,which indicated the difference in sensitivity is most likely caused by the biological difference of gastric cancer cell lines.In the proteome profile of 12 gastric cancer cell lines,7,428 proteins were identified and quantified.After filtering the detection frequency of these proteins in data of cell line and clinical tumor tissue samples separately,3,695high-frequency proteins were reserved and analyzed with the corresponding IC50 value of olaparib by Pearson correlation analysis.In the results,MTA2(metastasis associated1 family member 2)was found,its expression level was positively correlated with the sensitivity of cells to olaparib,indicating that it is very likely to promote the sensitivity of gastric cancer cells to olaparib.For validation,a series of cell viability experiments,colony formation assay and WB detection of γH2AX were performed.In gastric cancer cells knocking down or overexpressing MTA2,we observed a corresponding decrease or increase in the sensitivity of gastric cancer cells to olaparib,thus proving the promoting effect of MTA2 protein on olaparib.In order to explore the potential biological mechanism,we first conducted a proteinprotein interaction experiment and discovered the interaction between MTA2 and DNA replication-related proteins,suggesting that the MTA2 protein may be related to the DNA replication process;the results of cell cycle experiments show that high expression of MTA2 and stimulation of olaparib have consistent effect on cells,which both will promote more cells to arrest in G2/M phase;WB detection of the ATR signaling pathway found that knocking down of MTA2 weakened the induction of cell replication pressure by olaparib.The experiments above verified that the mechanism of MTA2 promoting the sensitivity of gastric cancer cells to olaparib is related to the aggravation of replication stress.Finally,to further explore the relationship between MTA2 and DNA replication,we performed CUT&Tag assay followed by sequencing and found that MTA2 preferentially binds to DNA sequences near replication origins,and the binding can be enhanced by olaparib,thus MTA2 is very likely to be involved in the initiation of DNA replication.Based on biological mechanism discovered above,we speculated that MTA2 may promote the sensitivity of gastric cancer cells to ATR inhibitors and we conducted in vitro and in vivo experiments to validate.The results showed that the overexpressing MTA2 promoted lethality of olaparib,ATR inhibitor and their combination to cancer cells.The main innovations of this study:(1)based on the analysis of proteome data,the potential promotion effect of MTA2 on lethality of olaparib to gastric cancer cells was discovered,which was verified by a series of in vitro experiments;(2)we found that MTA2 promotes the sensitivity of gastric cancer cells to olaparib is related to MTA2’s enhancement of replication stress.In the exploration of olaparib in the treatment of tumors,it is further proved that targeting internally or externally induced replication stress can inhibit tumor cells.The conclusion of this article deepens the understanding of the combination therapy of olaparib;(3)mechanistically,preferential binding of MTA2 to DNA sequence near the DNA replication origins have been found,and it is speculated that MTA2 is very likely to participate in DNA replication process,the relationship between MTA2 and DNA replication process has been explored.