The Effect And Underlying Mechanisms Of Ecto-5’-nucleotidase(CD73) In The Invasion And Metastasis Of Colorectal Cancer Cells

Introduction Colorectal cancer[1]is the second most common type of malignancy in theWestern world, and metastasis is the main death cause[2]. In CRC development, apreneoplastic polyp gradually accumulates genetic changes that allow uncontrolledproliferation and cell survival, followed by invasive and metastatic properties typical ofcarcinoma[1][2]. Although causes and genetic bases of tumorigenesis vary greatly, key eventsrequired for metastasis are similar, including alteration of adhesion ability, enhancement ofmotility, and secretion of proteolytic enzymes to degrade extracellular matrix[3]and vascularbasement membrane; all these steps are orchestrated by a plethora of signaling events[3][4].Over the last few years, scientists have been working hard to discover the new molecularmarkers as significant targets for diagnosis and therapy. One goal is to identify a specificmolecular assay for each colon tumor and then to obtain an individualized therapeuticapproach. Presently, CD73expression has been associated with different prognosis of somedifferent tumor patients in a few studies[5][6][7][8], and it has been postulated to play animportant role in carcinogenesis, as adenosine promotes tumor progression andCD73-expressing cancer cell lines are more aggressive[9][1][2][3][4][5][6]Besides, CD73is also also think as a novel target for cancer immuthery[7][8][9][10]. However, the role of CD73expression in human colon cancer has not been fully investigated, and there is still littleinformation available on whether CD73has a direct effect on invasion and metastasis ofhuman colon cancer cells. Thus we want to investigate whether the expression of CD73incolon cancer cells may become one of the new molecular candidates to predict the prognosisand response to therapy of colon cancer patients or not. We therefore describe our data thatCD73may promote metastasis by facilitating the migration, adhesion and invasion of humancolon cancer cells.Ecto-5′nucleotidase[11]is a membrane-bound glycoprotein that functions to hydrolyzeextracellular nucleotides into bioactive nucleoside intermediates[12]. Surface bound CD73converts AMP to adenosine. Adenosine, acting through G-protein coupled receptors(i.e.adenosine A1, A2A, A2B, and A3receptors), has been known to control multiple cardio andcerebro protective pathophysiological events including vasodilation, stimulation ofangiogenesis, cytoprotection, and anti-inflammation[13]. In addition, adenosine could mediatecell signaling in physiological, as well as, pathological conditions, including stimulatingtumor growth and angiogenesis and inhibiting cytokine synthesis, adhesion of immune cellsto the endothelial wall, and inhibiting the function of T-cells, macrophages, and natural killercells[14][15]. Although adenosine exerts a number of effects that promote tumor expansion, the role of CD73enzyme activity in adhesion, migration and invasion of cancer cell has not beenfully investigated. In addition to its enzymatic function, CD73has been suggested to have arole in T-cell signaling and cell adhesion[1][2][3]. CD73may serve as an adhesive molecule andinteract with extracellular matrix glycoprotein, such as fibronectin and laminin, to producecancer-invasive properties[4].Ecto-5’-nucleotidase[5], the final enzyme in the extracellular pathway for the completehydrolysis of ATP to adenosine, is expressed in many different tissues[6]. Recently, significantoverexpression of ecto-5’-nucleotidase has been found in many human solid tumors,including melanoma[7][8], breast cancer[9][10], papillary thyroid carcinomas[11], and prostatecancer[12]. All these factors implicate the crucial role of CD73in tumorigenesis. Furthermore,some studies reported the level of its expression has been associated with tumorneovascularization, invasiveness, metastasis, and with tumor grade and localization[13].However, the role of CD73expression in human colon cancer is still limited, and there is stilllittle information available on whether CD73has a direct effect on invasion and metastasis ofhuman colon cancer cells. In the present study, dynamics of CD73was detected by usingWestern blotting and realtime PCR. Then, tissue microarrays[4]for two independent CRCcohorts were constructed, and immunohistochemical (IHC) analysis was performed toexamine the expression of CD73. Besides, the prognostic significance of CD73expression in CRC was analyzed. Finally, the findings further evaluated and confirmed that CD73canpromote migration, adhesion to fibronectin, invasion of colon cancer cells in vitro as well asexperimental lung and liver matastasis in vivo.Objectives To evaluate the expression of CD73and its prognostic significance inhuman colorectal cancer, and further investigate its role in promoting colorectal cancer cells(CRC) invasion and metastasis.Methods Firstly, immunohistochemistry, immunobloting and real time RT-PCR wereused to detect the expression levels of CD73in human colorectal cancer (CRC) tissues andcells. the value of CD73and clinico-pathological features in CRC patients were analysised byKaplan-Meier method, log-rank test, and multivariate Cox regression.Secondly, The role of CD73in the regulation of CRC invasion and metastasis wasinvestigated using both specific inhibitor and short interference RNA(siRNA)–mediateddownregulation of CD73in CRC cell lines Lovo and CD73overexpression plasimids waswas than transfected in CRC cell lines RKO. In vitro wound healing assay,transwell migration/invasion assays, cell adhesion assay and xenografts transplation assay were used to tedectthe migration,invasion, adhesion and matestasis of CRC cell lines both in vitro and in vivo.Thirdly, liquid scintillation assay, enzyme-labeled assay, Western blot and real time PCRwere used to detect the active protein levels of A1R、A2AR、A2BR、A3R signaling pathway.Results Firstly, CD73expression was observed more hightly on the membrance ofcancer cells compared to endothelial cells adjacent to normal colon mucosa distant fromcancer. In all, CD73expression was observed in57.1%（226/396）of colon cancersrepresented in the membrane of neoplastic cells and the malignant epithelial cells of thecolorectal adenomas and adjacent adenocarcinomas. The expression of CD73was muchhigher in CRC tumors with lymph node and distant metastasis than that without metastasis.Additionally, a large increase CD73mRNA expression was observed in the biopsies ofhuman primary colorectal tumors with lymph node and distal metastasis as compared withthat of primary colorectal carcinomas without metastasis by using realtime PCR andwesternblot. The number of high CD73expression increased with higher tumor burdendefined by tumor staging (p=0.020) and Lymph node metastasis (p=0.004), but not byhistopathological grading (p=0.233), age, gender, tumor location. The survival of CRCpatients with high CD73expression was significantly shorter than that of the patients with low expression by Kaplan-Meier survival analysis, specifically in patients who hadearly-stage tumor.The CD73mRNA and protein expression were low in CRC cell lines RKO,DLD1, whereas those were high in CRC cell lines Lovo, KM12detected by realtime PCR andWestern blot analysis.Secondly, we got satisfactory transfection efficiency and successfully acquiredLovo-mock, Lovo-GFU-RNAi1, Lovo-GFU-RNAi2, RKO-mock and RKO-PGC-FU.Both transwell migration assay and wound-healing assay showed that the closure ofLovo-GFU-RNAi was significantly more slowly than that of Lovo and Lovo Mock.(p<0.05).Transwell invasion assay also showed that the number of Lovo cells passed through matrigelwas much more as compared with Lovo-GFU-RNAi1and Lovo-GFU-RNAi cells (p<0.01).Conversely, following transfection with an CD73cDNA expression construct, RKO coloncancer cells, which express low endogenous levels of CD73, demonstrated a significantincrease in cellular migration and invasion. We therefore tested whether the expression ofCD73would affect the ability of colorectal cancer cells to adhere to plastic that was coatedwith Matrigel or collagen. As compared with wild cells group and mock cell group, celladehesion was significantly decreased when endogenous CD73expression was silenced inboth Lovo and KM12cells.Whereas expressing CD73in RKO cells exhibited an increase inadhesion, from1.5-fold to6-fold on Matrigel after60min As compared with wild or mockgroup.Enhanced vascular invasion and penetration of Lovo control cancer cells into adjacentnormal tissue were oberserved in the Lovo control group and it increased the number ofmetastatic nodules in the lungs. Hematoxylin and eosin staining (H&E) also showed thatCD73led to more massive metastasis in the lungs and livers of the mice bearing Lovo orLovo-mock xenografts as compared with Lovo-GFP-CD73RNAi1and Lovo-GFP-CD73RNAi2group. More importantly, Kaplan-Meier analysis demonstrated that the survival of themice xenografted with Lovo-mock cells injection reduced compared with Lovo CD73RNAigroup Collectively, these data suggest that CD73can enhances lung metastases of coloncancer xenografts.Thirdly, CD73is coexpression withA2AR and A2BR. To address the contribution of A2BRin the migration and invasion-promoting effects of CD73, we treated the Lovo-RNAi1andLovo-RNAi2cells with BAY-60-6583, the specific A2BR agonist, the cell migration and invasion increased significantly. But treating with other adenosine receptor agonists, did notalter the cell migration and invasion. All these data show that CD73promote CRC migrationand invasion at least partly through adenosine A2Breceptor stimulation.Conclusions Firstly, CD73is abnormally highly expressed in CRC, and highlyexpressed in colorectal cancer with with lymph node and distant metastasis than that withoutmetastasis. High CD73expression in CRC could be a novel independent factor forunfavorable prognosis.Secondly, CD73plays an important role invasion and metastasis of CRC cells.Thirdly, CD73promote invasion and metastasis of colorectal carcinoma throughadenosine A2B receptor stimulation.