The Role Of ATXN2L In Metastasis And Sensitivity To Oxaliplatin In Gastric Cancer

Background and objectiveGastric cancer is one of the most common tumors of the digestive system,causing a huge economic burden globally.In Asia,the morbidity of gastric cancer in South Korea,Japan and China remains high.Early screening strategies have been proved to be effective in Japan and South Korea.However,most of the world,including China,still fails to achieve early diagnosis,causing it almost impossible for most patients to be cured by surgical resection solely.Therefore,other remedies play an important part in treating gastric cancer and chemotherapy holds the key role.The emergence of chemotherapy significantly promoted the quality of life and survival time of cancer patients,but the existence of drug resistance often leads to failure in chemotherapy.Overcoming the problem of primary and secondary drug resistance arise in the treatment of gastric cancer is universally concerned to be a major difficulty in the field of modern cancer research.In the past decade,the relationship between neurologic disorders and tumorigenesis has been attracting more and more attention.Both clinical observation and basic research suggested that there exist some connections between the two seemingly unrelated diseases,even common regulatory pathways.The Ataxin family is a group of proteins that cause hereditary Spinocerebellar Ataxias.Abnormal insertion by PolyQ fragments in Ataxin family proteins often results in neurologic disorders.Currently,there were already many researches in Ataxin family's impact on neurological diseases,but there still remains much to be unfolded about its impact on cancer.Recent studies show that Ataxin-3 inhibits the expression of PTEN,enhances the PI3K/Akt signaling pathway and promotes lung cancer,and ATXN2 binding protein 1 inhibits gliomagenesis.ATXN2L(Ataxin-2 like protein)is a homologous protein of ATXN2 with similar structure.There were few reports on ATXN2L till now,which is only known to be highly expressed in immortalized cells such as embryonic tissue,adult testes and hematopoietic stem cells.Previous reports show that ATXN2L over expression promotes the production of stress particles.Although the exact function of ATXN2L is still unclear,there are three sequences between ATXN2 and ATXN2L with high similarity(AA208-352,similarity 60%;AA658-661,similarity 100%;AA895-941,similarity 60%).ATXN2L has almost all the functional domains of ATXN2 base on which,including acidic domain,Sm1,Sm2 and Caspase-3 cleavage sites,suggesting that ATXN2L may also be significant in tumorigenesis like ATXN2.However,the specific mechanism of ATXN2L affecting tumor progression is not yet clear and demands further study.Methods and Results1.ATXN2L is overexpressed in gastric cancer and predicts poor prognosisWe analyzed databases in TCGA and found that the mRNA expression of ATXN2L was higher in gastric cancer than adjacent normal tissue.We extracted the protein from fresh gastric cancer tissues and para-carcinoma tissues.Western blot also confirmed that ATXN2L was higher expressed in the cancer tissues compared to the normal tissues.Furthermore,we collected 48 cases of stage ? and 119 cases of stage?-? gastric cancer with complete follow-up data for immunohistochemical staining.We further analyzed all 167 patients and found that the patients with higher expression of ATXN2L are more likely to develop into later stage,have shorter OS and PFS.2.Silence of ATXN2L affected migration and invasion but not proliferation of gastric cancerqPCR was taken to detect the mRNA expression of ATXN2L of cell lines and showed that the expression of ATXN2L was higher in the cancer cell lines than GES-1.Wound healing essay and Transwell essay showed that migration and invasion of gastric cancer cells were weakened after silence of ATXN2L.The expression of Ecadherin was upregulated but Vimentin was reduced after silence of ATXN2L detected by immunofluorescence.Sliencing ATNX2L did not have impact on proliferation in gastric cancer detected by MTT and EDU assay.3.ATXN2L played an important role in resistance to oxaliplatin of gastric cancer cellsIt was found that the morphology of gastric cancer cells was induced to long shuttle by low concentration of oxaliplatin,but this change was not obvious after silencing ATXN2L.Apoptosis rate was detected by flow cytometry.The results showed that apoptosis induced by oxaliplatin increased after silencing ATXN2L.MTT assay showed that the sensitivity to oxaliplatin of gastric cancer cells was increased after silencing ATXN2L.In colony formation assay,the amount of gastric cancer cell colonies was reduced in the oxaliplatin applied group and silencing ATXN2L apparently widened the difference.ROS detected by flow cytometry was found increased after silencing ATXN2L under the treatment of oxaliplatin.4.Silence of ATXN2L partly reduced the formation of stress granules induced by oxaliplatinGC cells were cultured in time course and concentration gradient.Stained by immunofluorescence antibodies,ATXN2L co-localized with SG marker Ras GTPase-activating protein-binding protein 1(G3BP1)in cytoplasm.As indicated by G3BP1 and ATXN2L,few SG was found in cytoplasm in the oxaliplatin untreated control group.However,when oxaliplatin were added in,formation of SG increased.Western blot and qPCR were respectively used to detect the protein and RNA of ATXN2L.The results showed that the expression of ATXN2L in gastric cancer cells increased with the increase of oxaliplatin concentration and oxaliplatin-stimulated time.Silencing ATXN2L partly reduced the formation of stress granules induced by oxaliplatin.5.Sensitivity of secondary oxaliplatin-resistant cells increased after silence of ATXN2LOxaliplatin-resistant cells constructed in the laboratory were confirmed less sensitive to oxaliplatin than sensitive cells by MTT assay.Expression of ATXN2L was found to be higher in Oxaliplatin-resistant cells than sensitive cells.Apoptosis was detected by flow cytometry.The results showed that apoptosis rate of oxaliplatin-resistant cells induced by oxaliplatin increased after silencing ATXN2L.6.EGF promotes expression of ATXN2L through PI3K/AKT signaling pathwayCluster analysis of two gastric cancer datasets from GEO found that ATXN2L might be associated with EGFR and AKT signaling pathway.Survival analysis of 751 gastric cancer patients from 5 datasets in GEO found that patients with double negative ATXN2L and EGF had longer PFS and OS than patients with double positive ATXN2L and EGF,PFS and OS in patients with either ATXN2L or EGF positive were of moderate level.Western blot and qPCR showed that the expression of ATXN2L increased with concentration of EGF in gastric cancer cells.By additionally applying LY294002,EGF-upregulated ATXN2L expression was dramatically reversed.7.EGF functioned partly through ATXN2LWound healing assay and Transwell assay showed that EGF increased the migration and invasion ability of cells,silencing ATXN2L partially reversed the change.qPCR showed that expression of EGF and EGFR were higher in oxaliplatin-resistant cells than that in sensitive cells.MTT assay found that in oxaliplatin sensitive cell,recombinant human EGF induction greatly elevated cell viability under treatments of different concentration of oxaliplatin.However,silencing the downstream ATXN2L re-sensitize cells to oxaliplatin even under the stimulation of EGF.ConclusionExpression level of ATXN2L in gastric cancer was found higher than para-carcinoma tissues.High expression of ATXN2L in gastric cancer was found to be correlated with later TNM stage and poor prognosis.We also found that ATXN2L had no effect on proliferation;however,silencing ATXN2L could reduce the ability of migration and invasion of gastric cancer cells.Silencing ATXN2L increased sensitivity to oxaliplatin of sensitive gastric cancer cells,Hence,ATXN2L plays a significant role in resistance to oxaliplatin of gastric cancer cells.Silencing ATXN2L partly reduced the formation of stress granules induced by oxaliplatin.Sensitivity of secondary oxaliplatin-resistant cells increased after silencing ATXN2L.ATXN2L affected both primary and secondary oxaliplatin resistance in gastric cancer.Further exploring the reason of high expression of ATXN2L,we found that EGF in tumor microenvironment could promote the expression of ATXN2L.EGF could promote migration and invasion of gastric cancer cells and reduce the sensitivity of gastric cancer cells to oxaliplatin.Silencing ATXN2L partly inhibited the function of EGF in gastric cancer cells.In general,EGF could promote progression of gastric cancer partly by regulating ATXN2L.