The Functional Study Of RICTOR And VAV2 In Gastroesophageal Junction And Esophageal Cancer

Background&Aims:The incidence of gastroesophageal junction cancer is constantly increasing last few years.However,the clinical therapeutic efficacy is limited by the lack of effective treatments.At present,there are few studies for analyzing the genetic characteristics of gastroesophageal junction cancer by integrating genome and transcriptome sequencing data.In our previous study,we analyzed the tumor and non-tumor sequencing data of 89 patients with gastroesophageal junction cancer and found 124 copy number amplified and highly expressed genes.Subsequently,through high content screening based on siRNA library,the gene RICTOR,which has the most significant impact on proliferation,was identified for further study.Methods:In this study,high content screening based on siRNA library was used to screen 124 candidate genes and identify the most significant oncogenes.We used transcriptome sequencing analysis,western blotting and immunohistochemistry to detect the RNA and protein level expression of target gene.We studied the effect of target gene on cell proliferation in vivo and in vitro using CRISPR genome editing and lentiviral infection methods to construct gene overexpression or knockout cell lines.We explored the function and mechanism of the target gene through molecular biology experiments including tumor proliferation,migration,colony formation,and immunoblotting.We analyzed the biological function and potential downstream pathways through phosphorylation proteomics.Results:This study identified RICTOR as the most significant oncogene that promotes tumor proliferation.RICTOR was copy number amplified and highly expressed in a variety of tumors and its expression level is related to the prognosis of patients.RICTOR overexpression significantly promotes tumor proliferation,migration and colony formation in vivo and in vitro.Overexpression of RICTOR can activate p-AKT308 and p-AKT473 through phosphorylation of mTORC2 signaling pathway,thereby promotes the malignant phenotype of tumors.JR-AB2-011 has anti-tumor effects in vitro and in vivo,and its effect is relative to RICTOR expression level.Phosphorylation proteomics further described the biological functions involved in RICTOR and identified downstream phosphorylation sites p-EGFR s1166.These findings are worthy of further study.Conclusion:RICTOR copy number amplification and highly expression promote the phosphorylation of mTORC2 pathway to activate AKT pathway and promote the malignant phenotype of tumors.The RICTOR inhibitor JR-AB2-011 has good anti-tumor effects in vivo and in vitro,and cells with higher expression of RICTOR are more sensitive to the inhibitor.Therefore,inhibiting RICTOR or its downstream pathways are expected to become therapeutic targets for personalized treatment of gastroesophageal junction cancer.Background&Aims:Esophageal squamous cell carcinoma is highly malignant,and radiotherapy is the standard treatment for locally advanced patients.However,due to the presence of radiotherapy resistance,most patients with esophageal cancer do not have a significant therapeutic benefit.In addition,the efficacy and prognosis of radiotherapy for individuals are quite different.Therefore,exploring the radioresistance biomarkers and accurately predicting the sensitivity of radiotherapy are important to achieve precise treatment for esophageal squamous cell carcinoma.Methods:This study constructed the patient-derived(PDX)mouse model of esophageal squamous cell carcinoma.We used PDX model to evaluate the patient's radiotherapy efficacy,combined with the sequencing data,and identified the genes related to radiotherapy resistance.By integrating the copy number data of esophageal squamous cell carcinoma,we found a valuable radioresistance related gene for further study.We detected the expression of target gene in radiotherapy resistance and sensitive patients through transcriptome sequencing and immunohistochemistry data.We constructed the gene knockout or overexpression cell lines in esophageal squamous cell carcinoma,and used clone formation,extreme dilution and comet experiments to explore the function and mechanism of the target gene.Finally,we collected clinical patients to verify the application value of the target gene in the prediction of radiotherapy efficacy.Results:This study found 1369 genes highly expressed in the radiotherapy resistant PDX model.By integrating with the copy number amplification and expression data of 94 patients in esophageal squamous cell carcinoma,we identified the guanine nucleotide exchange factor VAV2 for further study.VAV2 is associated with radioresistance,and knockdown increases radiosensitivity.The mechanisms of VAV2 on radiotherapy efficacy might be mediated through DNA repair.Finally,the clinical data of 31 patients found that VAV2 could act as a biomarker to predict the short-term efficacy of patients with esophageal squamous cell carcinoma.In addition,We found that VAV2 is an important oncogene,which can affect the malignant phenotype of esophageal squamous cell carcinoma.Conclusion:VAV2 is an amplified and highly overexpressed oncogene that mediates radioresistance and affects DNA repair.It could be as a biomarker of radioresistance to predict the short-term efficacy of patients in esophageal squamous cell carcinoma patients.Targeting VAV2 could achieve the dual effect of improving the sensitivity of radiotherapy and the anti-tumor efficacy.It is an important clinical target which can bring new directions for the efficacy prediction and the treatment of cancer.