Subcellular Localization Of RAD21 Modified By P53 Promotes Chemo-resistance Via Regulating Autophagy In Gastric Cancer

Background and objectives:Gastric cancer has become the second leading cause of cancer-related death worldwide.In China,gastric cancer is the third most common malignant diseases.Due to obscure symptoms,most patients with gastric cancer are diagnosed at later stage and chemotherapy still remains the most important option.Platinum,especially the third generation of platinum(oxaliplatin etc.)has been applied widely as a first-line chemotherapeutic drug for gastric cancer in clinical.However,its clinical benefit was suppressed in treating gastric cancer patients result from the existence of tumor intrinsic or acquired platinum-based resistance.In addition,DNA damage repair induced by autophagy is one of the most important mechanisms in this relevant resistance.RAD21 protein is known as DNA double-strand break repair and nuclear-related protein.A large number of studies have shown that the RAD21 gene has an intimate relativity with the sensitivity of chemotherapeutic drug.The expression of RAD21 confers poor prognosis and resistance to chemotherapy in high grade luminal,basal and HER2 breast cancers.Colorectal cancer cells with RAD21 knockdown exhibited enhanced sensitivity to 5-fluorouracil.Therefore,these aforementioned findings strongly underscore RAD21 is a novel target for cancer therapeutics that can potentially enhance the antitumor effects of chemotherapeutic agents acting via regulation of DNA damage.An increasing evidence has supported that autophagy plays a critical role in promoting chemo-resistance or chemo-sensitivity.For example,reducible HMGB1(high mobility group box 1)induces Beclinl-dependent autophagy and promotes tumor resistance to oxaliplatin.Moreover,traditional view considers chemotherapy can directly or indirectly damage DNA which can activate the DNA damage repair signaling pathway,a large number of proteins participating in this signaling pathway such as p53,ATM,PARP1,FOXO3a,mTOR and SIRT1 involved in autophagy regulation.It also provides evidence that RAD21 probably has a close relationship with autophagy.Though a large number of overwhelming evidences have proved that RAD21 is upregulated in several tumors and is link to the sensitivity of chemotherapeutics,its relationship with the sensitivity oxaliplatin in GC remains undefined yet.In this study,we sought to the RAD21 relationship with the sensitivity of oxaliplatin and further clarify the mechanism in gastric cancer.Materials and methods:l.PCR and Western blot assay were used to detect the expression of RAD21 in GC cell lines and GC tissues.2.SiRNA targeting RAD21 mRNA were transfected into BGC823 and MKN45.MTT assay,flow cytometry and plate clone formation assay were applied to detect the effects of siRAD21 in gastric cancer cells,including cell proliferation,apoptosis and sensitivity of oxaliplatin.3.Forty-seven patients with gastric cancer(sensitive to oxaliplatin group)and nine patients with gastric cancer(insensitive to oxaliplatin group)were selected.Immunohistochemistry were used to estimate progress free survival analyses of GC patients with cytoplasmic or nucleoplasmic expression of RAD21.4.Both NLS-GV141-RAD21 and NES-GV141-RAD21 were fused,immunofluorescence assay were used to test its function.5.CoIP and comet assay were used to explore the targeted relationships between P53,RAD21 and drug-resistance-related autophagy.Results:1.The average expression level of RAD21 were significantly up-regulated in GC cell lines and tissues compare to corresponding normal epithelium cell and adjacent non-tumor tissues(p<0.05).2.RAD21 knockdown can increase the sensitivity of oxaliplatin in BGC823 and MKN45 cells.3.The nuclear translocation of RAD21 was related to recurrence rate and clinical stage of GC patients(p<0.05).4.NLS vector would reduce the sensitivity of oxaliplatin compared with NES vector.5.P53 co-localize with RAD21 and mediate the subcellular localization of RAD21.6.SiRAD21 together with the autophagy inhibitor(CQ)can suppressed the proliferation and promoted DNA damage to the greatest extent in the presence of oxaliplatin in GC cells.In the aforementioned process,ATG5(autophagy associated gene 5)had a significantly positive correlation with RAD21 in BGC823 and MKN45 cells(r=0.95,P<0.01;r=0.96,P<0.01).Conclusions:RAD21 are correlated with the sensitivity of oxaliplatin.Furthermore,mechanistically,P53 modify the subcellular localization of RAD21,decreasing the sensitivity of oxaliplatin by autophagy,contributing to the progression of gastric cancer.Taken together,these findings support strategies to target these relevant molecules and pathways for improving the sensitivity of oxaliplatin in gastric cancer patients,particularly making RAD21 an attractive therapeutic target.