Effects And Mechanism Of REC8, A Cancer/Testis Antigens, On Progression Of Colorectal Cancer

Colorectal cancer is one of the common gastrointestinal malignancies with high incidence and high mortality,which seriously threats to national health.However,due to the insidious onset of colorectal cancer,early symptoms are not typical and metastasis has occurred when a number of patients visit.Currently,metastatic colorectal cancer has a poor prognosis in the past 20 years.Currently 1-year and 2-year relative survival rates for tumors diagnosed at a distant stage just only increase to 52.6% and 33.3% respectively.The main treatments of colorectal cancer are surgery and chemotherapy.Survivors are at risk for CRC invasion and metastasis that are the main cause of death in patients with colorectal cancer.Therefore,prevention and treatment of tumor metastasis is an important means to improve the survival rate of patients.DNA ploidy abnormalities are signs of tumor progression and malignancy.Metastasis is a complex process,which is regulated by many factors and the epithelial-mesenchymal transition and tumor stem cells are closely related to tumor metastasis.Chromosomal instability(CIN)is a form of Genomic instability(GIN)that involves frequent cytogenetic changes leading to changes in chromosome copy number(aneuploidy).Chromosomal instability facilitates the acquisition of mutations conferring aggressive or drug-resistant phenotypes during cancer evolution.Currently,aneuploidy is considered to signs of tumor progression and malignancy,which is related with poor prognosis in colorectal cancer,esophageal cancer,stomach cancer,breast cancer,endometrial cancer,non-small cell lung cancer and so on.Epithelial-mesenchymal transition(EMT)has been considered to be a process in which epithelial cells convert to cells in mesenchymal phenotype through specific program.EMT involves Snail,Slug,Twist and a series of pluripotency transcription factors.These transcription factors induce the loss of adhesion connection,the transformation from polygon to fusiform in morphology,the expression of matrix-degrading enzyme,increase motility and enhance apoptosis resistance,in which some can inhibit the expression of E-cadherin which is a key inhibitory factor.EMT is considered an important biological process in tumor metastasis of epithelial cell-derived malignancies.The newly discussed cancer stem cells(CSCs)model proposes that tumors are hierarchically organized and only a small fraction of cells possesses tumor propagation abilities.The recently uncovered link between passage through an EMT and acquisition of stem-like properties indicates that activation of the EMT programs serves as a major mechanism for generating cancer stem cells(CSCs)and both of EMT and CSCs promote tumor metastasis.Malignant tumors aberrantly overexpress various embryonic genes and proto-oncogenes,including a variety of cancer-testis antigens(CTAs).CTAs is immunogenic,which initially only be considered an ideal target for tumor immunotherapy.Emerging evidence indicates that not only CTAs play a role in tumor immunotherapy but also the abnormal expression of CTAs is associated with poor prognosis in a variety of tumors,which play an important role in the occurrence and development of tumors.SSX,CAGE,piwil2 and CT45A1 can raise EMT and tumor metastasis-related genes,some CTA including Piwil2,DNAJB8,CT45A1,MAGE-A,CAGE and SPANX have an important role in the genesis and maintenance of cancer stem cellsREC8,as a member of CTAs,is a cohesin protein required during meiosis for separation of sister chromatids and homologous chromosomes.Studies have shown that the expression and function of REC8 in different tumors are not the same.REC8 is robustly down-regulated by the PI3 K pathway through hypermethylation.The expression of REC8 protein inhibits the proliferation and colony formation of thyroid cancer cells.The expression of REC8 in cutaneous T-cell lymphoma increases.However,the role of REC8 in colorectal cancer remains unclear.Objectives: To investigate the expression of REC8 in colorectal cancer and the relationship between the expression of REC8 in patients with colorectal cancer and the clinicopathological characteristics and explore the mechanism which regulates the proliferation and metastasis of colorectal cancer cells to provide a theoretical foundation for the development of REC8-targeted therapies for colorectal cancer.Methods: Fresh surgically resected specimens,including colorectal cancer tissues and corresponding normal adjacent tissues from 30 patients,were obtained from the Second Hospital of Hebei Medical University.Western blotting and immunohistochemistry was used to detect the expression difference of REC8 in colorectal cancer tissues and normal adjacent tissues.Fisher's exact test was used to analyze the relationship between the expression of REC8 in patients with colorectal cancer and the clinicopathological characteristics.The vector FLAG-REC8 was transfected into HCT116 cells and SW480 cells with the empty vector without REC8 coding sequence(FLAG)as control,then western blotting was used to detect the expression of MMP-9,VEGF-C,PCNA,EMT-associated protein Snail and CDH1,cancer stem cell markers CD133 and CD44 at protein level.DNA ploidy in patients with colorectal cancer was analyzed by flow cytometry.The plasmid FLAG-REC8 was transfected into HCT116 cells and SW480 cells,then flow cytometry was used to detect DNA ploidy.Results:1 The expression of REC8 in colorectal cancerWestern blotting and immunohistochemistry indicated that there is no statistical significance in the expression of REC8 between normal adjacent tissues and colorectal cancer tissues(P=0.066,P=0.761).2 The high expression of REC8 correlated with lymph node metastasis during the clinical progression of CRC.Western blotting indicated that 64%(7/11)CRC patients with lymph node metastasis had much higher expression of REC8,while 5%(1/18)CRC patients without lymph node metastasis had much higher expression of REC8.REC8 expression correlated with lymph node status(P=0.001),clinical stage(P=0.003)in CRC patients,however the analysis data indicated that REC8 expression was not correlated with age(P=0.422),tumor location(P=0.431)and tumor stage(P=0.148).Immunohistochemistry indicated 82%(9/11)CRC patients with lymph node metastasis had much higher expression of REC8,while 33%(5/15)CRC patients without lymph node metastasis had much higher expression of REC8.REC8 expression correlated with lymph node status(P=0.021).Taken together,our analyses revealed that the high expression of REC8 correlated with lymph node metastasis during the clinical progression of CRC.3 REC8 upregulated the expression of MMP-9 and VEGF-C.Western blotting indicated that overexpression of REC8 promotes the expression of MMP-9(P=0.016,P=0.017)and VEGF-C(P=0.013,P=0.009)at protein level in human colon carcinoma cell lines HCT116 and SW480.The analysis data indicated overexpression of REC8 enhanced colon cancer cells invasion and metastasis.4 REC8 did not contribute to the proliferation of colon cancer cells.Western blotting indicated that overexpression of REC8 did not affect the expression of PCNA(P=0.12,P=0.492)at protein level.The analysis data indicated that REC8 did not contribute to the proliferation of colon cancer cells.5 REC8 promoted colorectal cancer cells EMT and the formation of CSCs.Western blotting indicated that the overexpression of REC8 upregulated the expression of Snail(P=0.012,P=0.016)and reduced the expression of CDH1(P=0.022,P=0.043)while promoting the expression of cancer stem cell markers CD133(P=0.004,P=0.012)and CD44(P=0.041,P=0.007).Our analyses revealed REC8 promoted colorectal cancer cells EMT and the formation of CSCs,resulting in enhancing cell invasion and metastasis,thereby promoting progress in colorectal cancer.6 REC8 contributed to chromosomal instability in colorectal cancer.Flow Cytometry indicated that 100%(6/6)patients with aneuploidy CRC had much higher expression of REC8,while 40%(8/20)patients with diploidy CRC had much higher expression of REC8.REC8 expression correlated with DNA ploidy in patients with colorectal cancer(P=0.017).DNA ploidy was detected in the colon cancer cell lines HCT116 transfected plasmid FLAG-REC8 by flow cytometry.The results showed that overexpression of REC8 increased colon cancer cells HCT116 DNA content,resulting in the formation of aneuploidy(P=0.001).The analysis data indicated that REC8 contributed to chromosomal instability in colorectal cancer.Conclusions:1 The high expression of REC8 correlated with lymph node metastasis during the clinical progression of CRC.2 REC8 enhanced colon cancer cells invasion and metastasis.3 REC8 promoted colorectal cancer cells EMT and the formation of CSCs.4 REC8 contributed to chromosomal instability in colorectal cancer.