Ecto-5′-nucleotidase promotes invasion, migration and adhesion of human breast cancer cells
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- Wang, L., Zhou, X., Zhou, T. et al. J Cancer Res Clin Oncol (2008) 134: 365. doi:10.1007/s00432-007-0292-z
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Associated with many molecules, metastasis includes cell adhesion to extracellular matrix, migration towards specific direction and invasion into local vessel of distant organs. The purpose of the present study was to evaluate the role of ecto-5′-nucleotidase (eN, ecto-5-NT, CD73) generated extracellular adenosine in biologically malignant behaviors of human breast cancer cell lines.
Materials and methods
Two human breast cancer cell lines, T-47D with lower expression of CD73 and MB-MDA-231 with higher expression of CD73, were used to investigate the functions of CD73. The effects of CD73 over-expression on invasion, migration and adhesion were observed in T-47D transfected with pcDNA-NT5E plasmid. The effects of specific CD73 inhibitor, α, ß-methylene ADP (APCP), were observed in MB-MDA-231 cells.
The results showed CD-73 overexpression increased invasion, migration and adhesion to ECM of the pcDNA-NT5E transfected T-47D cells compared to the saline and mock vector controls. The increased cell mobility of CD-73-overexpressed T-47D cells was blocked by APCP. Adenosine increased the mobility of wild type T-47D cells. APCP inhibited the mobility of the MB-MDA-231 cells.
Taken together, our results indicated that CD73 may facilitate the adhesion, migration and invasion of human breast cancer cells through its enzyme activity of generating adenosine. This study provided a possibly molecular mechanism of metastasis of breast carcinoma.
Recent data suggested that approximately 90% of cancer deaths were attributable to the presence of distant metastasis (Hanahan and Weinberg 2000). Therefore, treatments directly targeting metastatic disease by specifically inhibiting the invasive behavior of tumor cells are extremely attractive. Such approaches might increase complete remission rate by limiting the tumors to original location for radiation and surgical treatments. Acquisition of metastatic capability might be an early event during tumorigenesis and invasive growth because their underlying molecular pathways and abnormal cell behaviors are a prerequisite for the establishment, growth and survival of the primary tumour (Elvin and Garner 2005). The process of tumor metastasis includes multiple steps such as adhesion, migration and invasion.
Statistical data showed that in developed countries, breast cancer is the most common tumor in women, and it is the most considerable cause of cancer-associated morbidity and mortality. Over the last few years, new molecular markers have been discovered as significant targets for diagnosis and therapy. One goal is to identify a specific molecular assay for each breast tumor and then to obtain an individualized therapeutic approach. Today, a variety of molecular factors are being evaluated for potential application in clinical practice to predict the prognosis and response to therapy of breast cancer patients. Expression of CD73 in breast cancer cells may become of the candidates.
CD73, known as ecto-5′-nucleotidase (5′-NT, EC 184.108.40.206), is a glycosyl-phosphatidylinositol-linked 70-kDa molecule. Its enzymatic activity involves catalyzing the dephosphorylation of ribo- and deoxyribonucleotide 5′-monophosphates to their corresponding nucleosides. While the enzyme has broad substrate specificity, it prefers purine ribonucleoside monophosphates. Adenosine 5′-monophosphate (5′-AMP) is the best substrate. CD73 was defined as the lymphocyte differentiation antigen CD73. This surface antigen may regulate the availability of adenosine for the interaction with the cell surface adenosine receptor by converting 5′-AMP to adenosine. CD73 was highly expressed in many human solid tumors, and the level of its expression was associated with tumor neovascularization, invasiveness, and metastasis and with shorter patient survival time (Spychala 2000). In a survey of gene expression among a range of human carcinomas, CD73 was found to be upregulated in carcinomas including those of colon, lung, pancreas and ovary (Su et al. 2001). Recent study suggested that high expression of CD73 may be associated with papillary thyroid carcinoma. Clinically, the strong expression of CD73 in papillary thyroid carcinomas could be a diagnostic aid in the differential diagnosis of thyroid tumors (Kondo et al. 2006). Using CD73 (1E9.28.1) monoclonal antibody, CD73 expressions were observed in breast carcinoma. Moreover, Spychala et al. showed that CD73 was negatively regulated by estrogen receptor-α(ER-α) in a dominant fashion and suggested that CD73 expression and its generation of adenosine may relate to breast cancer progression. Increased expression of CD73 in a subset of ER-negative cells may serve as a novel marker for more aggressive breast carcinoma (Spychala et al. 2004).
Adenosine, a purine nucleoside, acts as a regulatory molecule and is a ubiquitous nucleoside present in all body cells, by binding to specific G-protein-coupled A1, A2A, A2B, and A3 cell surface receptors. Adenosine is a natural metabolite that plays a role in several physiological and pathological processes such as inhibition of platelet aggregation, cardioprotection after ischemia, vasodilation, mast cell activation, and lypolysis. Adenosine may interfere with neutrophil’s adherence to endothelial cells. Adenosine and A2A agonists reduce inflammation in part by reducing adhesion molecule expression and neutrophil adherence to endothelial cells (Okusa et al. 2000). Adenosine also induces a differential effect on tumor and normal cells. Adenosine and AR agonists were recently shown to inhibit growth or induce apoptosis in several types of cancer cells. While inhibiting in vitro melanoma, leukemia and lymphoma cell growth, it stimulated bone marrow cell proliferation. This dual activity was mediated through the A3 adenosine receptor (Fishman et al. 2001; Lu et al. 2003). Adenosine was also able to promote tumor cell migration, stimulate angiogenesis, increase intratumoral blood flow and confer protection against radiotherapy. Adenosine is generated by CD73 enzyme activity. Although adenosine exerts a number of effects that promote tumor expansion (Mujoomdar et al. 2004), the role of CD73 enzyme activity in adhesion, migration and invasion of breast cancer cell lines is unknown. Considering the different effects of adenosine in diverse normal tissues and malignant tumors due to variable distribution of its receptors, we observed the role of adenosine in breast cancer cells, to see whether it promotes tumor cell invasion, migration and adhesion or not. In this study, we developed a transient gene transfection technique in T-47D breast cancer cells which express low levels of CD73 before transfection to investigate the function of CD73. The effects of adenosine on invasion, migration and adhesion in wild type human breast cancer cell T-47D were investigated by pre-treating with adenosine. The concept was further proved in MB-MDA-231 breast cancer cells, which express high level of CD73, using CD73 specific inhibitor.
Materials and methods
Cell lines and culture conditions
Breast carcinoma cell lines T-47D and MB-MDA-231 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). MB-MDA-231 cells were routinely maintained in Dulbecco’s modified Eagle’s medium (Gibco, CA, USA) supplemented 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 and 95% air, penicillin (50 units/ml), streptomycin (50 units/ml) (all of these from Life Technologies, Inc., Burlington, Canada). T-47D human breast cancer cell line was incubated in the same medium plus 0.2 U/ml insulin (Life Technologies, Inc., Burlington, Canada). Cells were split two or three times weekly at a ratio between 1:4 and 1:8.
pcDNA-NT5E plasmid construction and transfection into T-47D cells
To determine the possible role of CD73 in invasion, migration and adhesion of human breast cancer cell lines in vitro, CD73 gene was cut from the vector pBluescript SK(±), then cloned into the unique KpnI and BamHI cloning site of the pcDNA3.0 expression vector. The plasmid pcDNA-NT5E was transfected into T-47D cells using Lipofectamine™ 2000 reagent (Invitrogen, Carlsbad CA, USA) following the protocol offered by the manufacturer. Briefly, parent T-47D cells (3 × 105/well) were plated in 6-well plate and incubated at 37°C with 5%CO2 until 90–95% confluence. Pre-mixed lipofection and plasmid DNA in a ratio 6 (μl): 3 (μg) were added to the wells with gentle swirling for 24–48 h. Clones were selected for invasiveness, motility and adhesion analysis after transfection.
Invasion, migration and adhesion assays in transfected T-47D cell line
Invasion assay was done in 8 μm polyethylene terephthalate filter inserts in Boyden chambers (Becton Dickinson, Bedford, MA, USA). The 8 μm pore inserts were coated with 10 μmol/ml of Matrigel (Becton Dickinson Labware, Bedford, MA) 20 μl at 37°C for 30 min. Breast cancer cells containing transfection pcDNA-NT5E T-47D, mock vector and wild T-47D were trypsinized with 0.25% trypsinase, centrifuged at 1,500 g for 5 min, and suspended in serum-free DMEM medium. 200 μl cells at final concentration of 2 × 105 cells/ml were added to coated filters in triplicate wells. The bottom compartments of the chambers contained 750 μl serum-free medium. The cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2 for 48 h. The matrigel coated on the upper surfaces of the filters was mechanically cleaned with cotton swabs. Cells that migrate through the filters to the opposite side of the filters were fixed with 4% paraformaldehyde (for 20 min at 37°C), and then stained with hematoxylin and eosin, photographed, and counted. Under the inverted microscope, cells in five medium power fields (200×) were counted double blindly by two independent examiners.
Migration assay was determined in the same way described as the invasion assay except that the cells were plated on top of uncoated 8 μm pore polyethylene terephthalate filters in the Boyden chambers. In addition, invasion and migration assays were performed in T-47D cells transfected pcDNA-NT5E plasmid after pre-treating with 3 μM/ml of APCP for 1 h.
Cell adhesion assay
Cell adhesion assay was performed with 96-well tissue culture plates. Plates were pre-coated with 16 μg/ml ECM gel (from mouse sarcoma) diluted with phosphate buffered saline (PBS), 100 μl per well for 4 h at room temperature. Non-specific binding sites in the wells were blocked with 1 mg/ml BSA mixed with PBS for 2 h at room temperature. After the cells grew to approximately 80% confluence in DMEM containing 10% FBS, cells were trypsinized, suspended in serum-free medium at a concentration of 2 × 104 cells/ml and applied 100 μl to each well. The cells were incubated under routine condition as above for 2 h. Following incubation, non-adherent cells were removed by carefully washing three times with PBS and gently rocking. Adherent cells were fixed with 3% paraformaldehyde for 10 min, cleaned with PBS three times, air-dried, and stained with 100 μl 0.5% crystal violet for 5 min. The cells were then washed three times with PBS and lysed with 100 μl ethanol of 1% acetic acid solution and read at A570 on multifunctional reader (Tecan GENios, Zurich, Switzerland). Results were expressed as optical density (OD) at 570 nm ± Standard Error (SE) of three independent experiments. The experimental groups include wild T-47D, T-47D transfected mock vector, T-47D transfected pcDNA-NT5E plasmid, and T-47D transfected pcDNA-NT5E plasmid plus APCP treatment.
Invasion, migration and adhesion assays in T-47D cell treated with adenosine
To study the possible effects of adenosine on invasion, migration and adhesion of wild T-47D cells, cells were treated with adenosine at 100 μM. In human epidermoid carcinoma A431 cells, adenosine evoked a biphasic response in which a concentration of 10 μM produced inhibition of colony formation; however, at concentrations up to 100 μM, this inhibition was progressively reversed (Tey et al. 1992). Our preliminary experiment showed that 100 μM adenosine could reverse inhibition of APCP in T-47D cells transfected with pcDNA-NT5E-CD73 (data not shown). The control group was treated with PBS. Invasion, migration and adhesion assays were conducted as previously described.
Invasion, migration and adhesion assays in MB-MDA-231 cell treated with APCP
The aggressive mammary cell line MB-MDA-231 over-expresses CD73. APCP is specific inhibitor of CD73 enzyme activity. Invasion and migration were examined in MB-MDA-231 cell pre-treated with different concentrations of APCP (0, 3, 6, 9 and 12 μM).
Fibronectin and collagen type IV (Sigma, St Louis, MO, USA) were major ECM proteins. Tumorous cells adhere to ECM before invasion. Additionally, earlier reports suggested that CD73 may interact with ECM proteins (Stochaj et al. 1989). It was also demonstrated that laminin and fibronectin, but not collagen, affected CD73 activity in chicken gizzard. The effect of APCP on the adhesion of breast cancer MB-MDA-231 cells to ECM and its major components. Overnight, 96-well culture plates were coated with 100 μl (10 μg/ml) ECM, fibronectin and collagen type IV, respectively. Cell adhesion to these coated surfaces was observed as described previously.
Mean values were calculated from data obtained from three or more separate experiments and reported as mean ± SEM. The significance of the difference between groups with multiple comparisons was assessed by one-way analysis of variance (ANOVA). P values < 0.05 were considered statistically significant.
CD73transfection increases T-47D invasion, migration and adhesion to ECM
Adenosine increases T-47D invasion, migration and adhesion to ECM
APCP inhibits invasion, migration and adhesion of MB-MDA-231 cells
Metastasis of breast cancer is the main cause for high mortality. If the metastatic mechanism is well known, the cancer patients could be treated before and after cancer metastasis to increase remission rate. Determining the timing of anti-invasive intervention is challenging because the key pathways determining the invasive behavior of tumors are less well clear than those that regulate tumor proliferation.
CD73 is a membrane-bound glycoprotein, which hydrolyzes extracellular nucleotides into bioactive nucleoside intermediates (Zimmermann 1992). It stimulates vasodilation, angiogenesis and cell growth in cancer (Barcz et al. 1998; Khoo et al. 1996; Lelievre et al. 1998; Natori et al. 1992; Rathbone et al. 1992). However, the relationship of CD73 expression, activity, and adenosine generation to tumor cell biology and tumor progression has not yet been determined. Previously published data suggested increased expression of CD73 in breast cancer when compared with nonmalignant surrounding tissues (Canbolat et al. 1996; Thompson et al. 1992; Ujhazy et al. 1994). Preliminary survey of a panel of cancer cell lines showed a highly variable expression of CD73 (Spychala 2000). More detailed analysis of breast cancer cell lines in previous studies revealed a striking negative correlation of CD73 expression with estrogen receptor (ER) status that in turn might be predictive of a more invasive tumor phenotype, as determined in animal models (Bachmeier et al. 2001; Gilles et al. 1998; Manni et al. 2002; Mullen et al. 1996; Sommers et al. 1994). Although CD73 is highly expressed in many tumor cells including breast cancer, its specific function during tumor metastasis is unclear.
Mammary carcinoma frequently metastasizes to specific organs, including the regional lymph nodes, the lung and bone marrow. Several genes encoding adhesion and matrix proteins were up-regulated in tumors that preferentially metastasize to the lymph node. One of the up-regulated genes in lymph-homing tumors was CD73. The expression of CD73 in lymph node metastases was higher than in the primary tumor (Lee et al. 2003). In tumor progression, CD73 may act as a molecule that directed lymph node specific-metastasis. CD73 has also been suggested to serve as a lymphangiogenic marker in the lymphatic spread of colorectal cancer cells to regional lymph nodes. Therefore, it may have a prognostic value for colon cancer patients (Parr and Jiang 2003). Present data revealed that among different melanoma cells, up-regulated expression of CD73 is associated with a highly invasive phenotype (Sadej et al. 2006). In addition, the level of CD73 activity showed significant correlation with tumor grade and location (Eroglu et al. 2000). Previous studies indicated that the CD73 implicated in cell–cell and cell–matrix interactions as protein (Vogel et al. 1991) and involve in drug resistance and tumor-promoting functions (Ujhazy et al. 1996). CD73 has been shown to have a role in regulating lymphocyte adhesion. Antibodies against CD73 was shown to interfere with lymphocyte adhesion to cultured endothelial cell (Airas et al. 1993, 1995; Arvilommi et al. 1997). However, the exact mechanism of CD73 functions has remained unclear. Some data showed that CD73 functions at the activation step in the multi-step adhesion cascade and promotes lymphocyte binding to endothelial cells via a lymphocyte function-associated antigen-1-dependent mechanism (Airas et al. 2000). Therefore, CD73 is likely an important molecule in breast cancer metastasis.
Kamath et al. (2001) classified MB-MDA-231 as highly invasive breast cancer cells while T-47D as weakly invasive on the basis of their relative activities in the chemoinvasion assay. In our previous work, the mRNA expression and enzyme activity of CD73 in such two breast cancer cell lines that differ in invasive and metastatic potential were analyzed by RT-PCR and HPLC. The results indicated the expression of CD73 was positively correlated with the migratory ability of breast cancer cells (Zhou and Zhou 2006). T-47D cells were weakly metastatic (Zajchowski et al. 2001) and had a nearly undetectable expression of CD73, while MB-MDA-231 cell was opposite. The ER(-) breast cancer cell line MDA-MB-231 with higher expression of CD73 expressed A2B adenosine receptor (Panjehpour et al. 2005). The fact that MB-MDA-231 cells derived from advanced metastatic breast cancer cells have significantly higher expression of CD73 compared to weak invasive breast cancer cell T-47D indicated that CD73 is a protein potentially involved in breast cancer metastasis. This study therefore focused on the potential implication of CD73 for breast tumor metastasis. The results showed that the ability of invasion, migration and adhesion of T-47D cells over-expressed with CD73 increased. Previous studies indicated that the CD73 might serve as a marker for migratory nerve cells (Schoen et al. 1988). In vitro, aortic endothelial cells (ECs) from CD73−/− mice display an up-regulation of mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1) (Zernecke et al. 2006). Recent studies with human glioblastoma suggested that CD73 may serve as an adhesion molecule promoting tumor invasiveness (Fenoglio et al. 1997) and in the case of human lymphocytes it may mediate adhesion of lymphocytes to the endothelial layer (Airas et al. 1995). We investigated whether enzyme activity of CD73 promoted invasion, migration and adhesion of T-47D cells over-expressed with CD73. By pre-treating with APCP, the ability of invasion, migration and adhesion of CD73 gene transfected T-47D cells decreased. These results indicated that CD73 facilitated invasion, migration and adhesion of breast cancer cells due to its enzyme activity.
CD73 activities have been found in a variety of neoplastic cells. A highly active enzyme was detected in breast carcinoma, gastric cancer, pancreatic cancer, chronic myelogenous leukaemia, cutaneous T-cell lymphoma, and in Walker 256 carcinoma (Spychala 2000). CD73 could hydrolyze extracellular nucleotides into bioactive nucleoside intermediates (Zimmermann 1992). Being most abundant, adenosine, acting through specific receptors, is implicated in many physiological and pathophysiological processes, including regulation of tumorigenesis (Madi et al. 2003; Merighi et al. 2002; Woodhouse et al. 1998). The concentration of adenosine in the extracellular fluid of solid tumors was higher than normal tissues and tissues around tumor (Blay et al. 1997). A1 receptors mediate motility and chemotactic response to adenosine in melanoma cells. Some experiments revealed that adenosine regulates the CD73 expression and barrier function of vascular endothelial cells by paracrine pathway (Narravula et al. 2000). Extracellular adenosine has been shown to enhance vascular barrier function by activating endothelial A2B receptors (Lennon et al. 1998; Richard et al. 1998) and to interfere with leukocyte-endothelial adhesion via occupancy of A2A receptors on stimulated neutrophils (Cronstein 1994; Sitkovsky 2003). Furthermore, adenosine has been shown to stimulate canine retinal microvascular endothelial cell migration and tube formation (Lutty et al. 1998; Lutty and McLeod 2003). Therefore, it was not surprising that adenosine has been shown to be a crucial factor in determining the cancer cell progression pathway (Merighi et al. 2003).
Thus, we speculate that CD73 could promote invasion, migration and adhesion to ECM of cancer cells through it enzyme activity in generating adenosine. Breast cancer cell lines with high and low expression of CD73 were selected to prove this concept. The results demonstrated that adenosine promote invasion, motility and adhesion of breast cancer cells in vitro.
In order to further investigate the effect of CD73-generated adenosine on invasion, migration and adhesion of breast cancer cells, mammary carcinoma cells MDA-MB-231 which over-express CD73 were selected as a model of aggressive cancer cells. The ability of invasion and migration of MDA-MB-231 cells were significantly inhibited by APCP due to suppression of CD73 enzyme activity. Previous evidences indicated that CD73 purified from chicken gizzard was shown to specifically interact with laminin and fibronectin (Stochaj et al. 1989 ). Both Fibronectin and collagen are fundamental components of ECM. The effects of CD73 on the adhesion of breast cancer cells to fibronectin and collagen were observed, respectively. The results demostrated that adhesion of MDA-MB-231 cells to fibronectin and collagen was inhibited by suppressing CD73 enzyme activity.
In summary, our study found that CD73 promoted invasion, migration and adhesion of breast cancer cells through CD73 enzyme activity in generating adenosine. CD73 may have pro-metastasis activities and may serve as a diagnostic marker and therapeutic target of breast cancer.
We thank Dr. Linda Thompson for providing pBluescript SK(+) plasmid.