This article is mentioned in:

Introduction

Colorectal cancer is one of the most common cancers and is the leading cause of cancer-related deaths worldwide (1,2). Colorectal cancer is highly treatable when diagnosed and surgically removed at an early stage. Unfortunately, many patients are diagnosed with (distant) metastasis either during follow-up or at first presentation, 80–90% of which are unresectable (3,4). Despite considerable improvements in the treatment of colorectal cancer over the last few decades, the only option for unresectable metastatic CRC (mCRC) remains palliative systemic treatment (5,6). Recently, targeted and biological therapeutics have made substantial advances in mCRC treatment, and have prolonged overall survival and disease-free survival of patients, with fewer adverse effects than conventional chemotherapy (7,8). Since these therapeutics act on specific target proteins, they are restricted to certain individuals according to their molecular profiles. Therefore, more specific biomarkers, which determine the biological nature and behavior of colorectal cancer, is needed to benefit more patients (8,9).

Ecto-5′-nucleotidase (CD73), a glycosylphosphatidylinositol-linked 70-kDa cell-surface molecule, is expressed on many cell types, including subsets of lymphocytes, endothelial cells and epithelial cells (10). CD73 expression was found abnormally upregulated in many types of cancer tissues, such as colorectal, gastric, liver, ovarian and breast cancer (11–14). Studies have shown that CD73 on tumor cells is associated with tumorigenesis and tumor progression through inhibition of CD4+ T cells and NK cell proliferation, while increasing suppressive immune subsets such as regulatory T cells (Tregs), B cells, and myeloid-derived suppressor cells (15–18). Besides being originally defined as a lymphocyte differentiation antigen, CD73 has been identified as a co-signaling molecule on T cells and as an adhesion molecule required for lymphocyte binding to the endothelium (19,20).

More significantly, CD73 is a critical ectoenzyme in purine metabolism, which hydrolyzes extracellular AMP to adenosine (21). Thus, CD73 is a switch molecule of adenosine-related signaling pathways, which are important in a series of biological events, such as cell survival, proliferation and cell motility (22,23). In this study, we aimed to verify the hypothesis that CD73 promotes human colorectal cancer cell growth through its enzyme and non-enzyme activities, except for immune-related mechanisms.

Materials and methods

Cell culture

The colorectal cancer cell lines (RKO, SW480, HCT-15, LoVo and KM12) used in this study were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (both from Gibco, USA) at 37°C in 5% CO2, under saturated humidity conditions.

Real-time RT-PCR and western blot analysis

Total RNA and proteins were routinely isolated from the cultured cells. Then, mRNA and protein expression levels were determined, respectively by real-time RT-PCR and western blot assays, where the housekeeping gene β-actin was used as control. Primary antibodies against β-actin, CD73, EGFR, β-catenin and cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used at a dilution of 1:500 overnight at 4°C, respectively. HRP-labeled second antibodies (Santa Cruz Biotechnology) were used at a dilution of 1:1,000 for 1 h. To determine whether hydrolase activity of CD73 affects epidermal growth factor receptor (EGFR) expression, similar analyses were performed with cells pretreated with CD73 enzyme activity inhibitor APCP (10 µM) for 1 h or adenosine (20–200 µM) for 24 h.

Recombinant lentiviruses

CD73 interference and overexpressing recombinant lentiviruses were constructed using a 'three plasmid system' (Genechem, China). Interference-interference and overexpression sequences of CD73 were designed according to its gene sequence (gene ID: 4907). The multiplicity of infection (MOI) of the recombinant lentivirus in adherent monolayer colorectal cancer cells was 50. The effects of the recombinant lentivirus were observed at 1 week after infection.

Colony formation assay

Approximately 1×103 colorectal cancer cells infected with the recombinant lentiviruses were plated in 60-mm culture dishes, fixed with ethanol and stained with 0.5% crystal violet 2 weeks later. The clone formation rate (CFR) = clone counts/seeded cell counts × 100%.

Cell Counting Kit-8 (CCK-8) assay

Adherent monolayer colorectal cancer cells in a 96-well plate were treated according to the manufacturer's instructions provided in the CCK-8 assay (Dojindo Laboratories, Gaithersburg, MD, USA). The cells were incubated in 10 µl CCK-8 agents for 1 h at 37°C. Absorbance of each well was quantified at 450 nm 3, 6, 12, 24 and 48 h later, respectively.

Cell cycle analysis

Adherent monolayer colorectal cancer cells were ethanol-fixed and stained with propidium iodide in buffer with RNase A. The DNA content was assessed using flow cytometer (Becton-Dickinson, San Jose, CA, USA). The proliferation index (PI) = [(G2/M + S)/(G0/G1 + G2/M + S)] × 100%.

Tumorigenicity assay

LoVo cells infected with CD73 interference and control lentiviruses were injected subcutaneously into 4-week nude mice (2×106 cells/mouse), respectively. The tumors were measured every 3 days. The mean tumor volume was calculated as follows: 0.5 × a × b2 ('a' is the largest diameter, 'b' is the perpendicular diameter). Tumors were excised and weighed 3 weeks after injection.

Statistical analysis

All analysis of data was performed using SPSS 11.5 software. P<0.05 was considered to indicate a statistically significant result.

Results

CD73 expression in colorectal cancer cell lines

CD73 expression was examined in five human colorectal cancer cell lines (RKO, SW480, HCT-15, KM12 and LoVo) by real-time PCR and western blot analysis (Fig. 1A and B). CD73 expression was higher in the LoVo and KM12 cells with more malignant potential at both the mRNA and protein levels. On contrast, CD73 mRNA and protein expression was lower in the RKO, SW480 and HTC-15 cell lines with moderate malignant potential.

Figure 1 CD73 expression in colorectal cancer cell lines and the effects of recombinant lentiviruses. CD73 expression at both the mRNA (A) and protein (B) level (n=3) was higher in the LoVo and KM12 cells with higher malignant potential than these levels in the other cell lines with moderate malignant potential. Recombinant lentiviruses significantly altered CD73 expression in the colorectal cancer cells (C and D) (n=3, *P<0.05).

Effects of the recombinant lentivirus infection

Recombinant CD73 interference lentiviruses were infected into human colorectal cancer LoVo cells with a high background expression of CD73. Recombinant CD73 overexpression lentiviruses were infected into the RKO cells with a low background CD73 expression. In line with our expectation, both of the recombinant lentiviruses achieved a significant change in CD73 expression at the mRNA and protein levels (Fig. 1C and D).

CD73 promotes colorectal cancer cell growth and cell cycle in vitro

In the colony formation (Fig. 2A) and CCK-8 assays (Fig. 2B), colony formation ability and cell viability of RKO cells were increased after infection with the CD73 overexpression lentivirus. On the other hand, cell viability and colony formation ability of LoVo cells were decreased after infection with the CD73 interference lentivirus. More RKO cells were detected by flow cytometry to enter the S phase from the G0/G1 phase after infection with CD73 overexpression lentivirus, with an increased PI (Fig. 3A). On the contrary, more LoVo cells were found to enter the G0/G1 phase from the S phase after infection with the CD73 interference lentivirus, resulting in decreased PI (Fig. 3B).

Figure 2 Effects of CD73 on the cell growth of colorectal cancer cells. CD73 overexpression lentiviruses promoted the growth of RKO cells while CD73 interference lentiviruses decreased the growth of LoVo cells in the colony formation assay (A) and CCK-8 assay (B).

Figure 3 Effect of CD73 on the cell cycle distribution of colorectal cancer cells. CD73 overexpression lentiviruses promoted the proliferation index (PI) of RKO cells (A) while CD73 interference lentiviruses decreased the PI of LoVo cells (B) in the cell cycle assays. PI = [(G2/M+ S)/(G0/G1 + S + G2/M)] × 100% (n=3, *P<0.05).

CD73 increases EGFR and β-catenin/cyclin D1 expression

The important cell cycle protein cyclin D1, as well as its major transcription regulation molecule β-catenin, were found to be increased in the RKO cells infected with the CD73 overexpression lentivirus (Fig. 4A), while expression levels were decreased after infection of the CD73 interference lentivirus in the LoVo cells (Fig. 4B). EGFR, the critical cell growth signaling pathway molecule and the major target of colorectal cancer biological therapeutics, was also found to be increased after CD73 was overexpressed (Fig. 4A), but decreased after CD73 was silenced (Fig. 4B).

Figure 4 Effects of CD73 on EGFR and β-catenin/cyclin D1 expression in colorectal cancer cells. CD73 overexpression lentiviruses increased EGFR and β-catenin/cyclin D1 expression in the RKO cells (A), while CD73 interference lentiviruses decreased the expression levels in the LoVo cells (B) (n=3, *P<0.05).

The role of adenosine in the effects of CD73 on EGFR expression

Similar to the CD73 overexpression lentivirus, adenosine also increased the expression of EGFR in the RKO cells, but to a lower degree. APCP, a CD73 enzyme activity inhibitor, completely reversed the effects of adenosine and partially the effects of the CD73 overexpression virus (Fig. 5A). In contrast, APCP had a weaker effect than the CD73 interference lentivirus on the expression of EGFR in the LoVo cells. Similarly, adenosine completely reversed the effects of APCP and partially the effects of the CD73 interference virus (Fig. 5B). All these results suggested that the mechanism of the effects of CD73 on EGFR expression was far more marked than adenosine alone.

Figure 5 Role of adenosine in the effects of CD73 on EGFR expression. Adenosine (ade) increased EGFR expression in the RKO cells (A) in a concentration-dependent manner, but at a lower degree than the CD73 overexpression lentiviruses. APCP reversed the effects of adenosine (completely) and CD73 overexpression lentiviruses (partially). On the other hand, APCP exhibited a weaker effect than CD73 siRNA lentiviruses on EGFR expression in the LoVo cells (B). Similarly, adenosine reversed the effects of APCP (completely) and CD73 siRNA lentiviruses (partially) (n=3, *P<0.05).

CD73 promotes colorectal cancer cell growth in vivo

LoVo cells (2×106) infected with the CD73 interference or control lentivirus were injected subcutaneously into athymic nude mice. Tumor growth was obviously slower in the mice injected with the CD73-silenced LoVo cells than the control 20 days after injection (Fig. 6A and B). The weight of the tumors derived from the CD73-silenced LoVo cells was lower than the control (Fig. 6C). The results suggested that CD73 promoted the tumorigenicity of colorectal cancer cells in vivo.

Figure 6 Effects of CD73 on colorectal cancer cell growth in vivo. Slower growth, smaller volume (A and B) and lower weight (C) were observed in nude mice subcutaneously injected with cancer cells treated with CD73 interference lentiviruses when compared with these parameters in nude mice injected with cancer cells treated with the control lentiviruses (n=6, *P<0.05).

Discussion

Metastatic colorectal cancer (mCRC) has a high incidence and mortality rate worldwide, and the effect of surgery remains limited (2,4). Molecular targeted therapy for mCRC has made progress in recent decades. For example, monoclonal antibody (mAb) drugs targeting EGFR (24), such as cetuximab (25) and panitumumab (26), have been recommended to treat mCRC singly or combined with systemic chemotherapy, for prolonging overall and disease-free survival (7,8). Finding more promising biomarkers for targeted therapy has good prospects in mCRC treatment (27,28).

CD73 is a glycosylphosphatidylinositol (GPI) anchored cell surface protein (16). Its expression is commonly upregulated in many types of cancers, such as colorectal, gastric, liver, ovarian and breast cancer (29–31). Studies have proved that CD73 promotes cancer progression through regulating tumor immune surveillance (16,32,33). CD73 is expected to be a promising biomarker for cancer therapy (11,34). Moreover, in this study, we found that CD73 promotes colorectal cancer growth through mechanisms other than immunoregulation.

Firstly, our results showed that CD73 expression was significantly higher in colorectal cancer cells with high malignant potential than in colorectal cancer cells with low malignant potential, which suggest that CD73 expression may be associated with colorectal cancer malignancy. Furthermore, colony formation, CCK-8 and tumorigenicity assays in this study showed that CD73 promoted colorectal cancer cell growth both in vitro and in vivo. Flow cytometry showed that CD73 promoted colorectal cancer growth by regulating more cells into the S phase from the G0/G1 phase, resulting in an improved PI. These findings were consistent with the results of other research in other types of cancers (15,35,36).

Wnt/β-catenin/cyclin D1 is a classic signaling pathway for controlling embryonic growth and development, stem cell self-renewal, tissue structure maintenance and tumor progression (37,38). Cyclin D1 is a critical cyclin for regulating the colorectal cancer cell cycle (39). β-catenin is the major transcription regulator of cyclin D1, the abnormalities of which are commonly found in colorectal cancer (40,41). Our results showed that CD73 promoted both β-catenin and cyclin D1 expression, which well explained the effects of CD73 on colorectal cancer cell growth. EGFR is the critical molecule of many signaling pathways which control cell growth and is the major target of colorectal cancer biological therapeutics (24). Co-expression of CD73 and EGFR has been found in other types of cancers (34,36). Consistently, it was found in this study that CD73 promoted EGFR expression. The exact interactions among CD73, EGFR and β-catenin/cyclin D1 warrant further exploration.

In addition to immunoregulatory effects, CD73 plays a crucial role in adenosinergic signaling through catalyzing AMP into adenosine. Furthermore, CD73 also acts as an adhesion regulating molecule between cancer cells and extracellular matrix (ECM) components, which is not dependent on its enzymatic activity (19,20). In this study, we found that both enzymatic activity and non-enzymatic activity were involved in the regulation of CD73 in colorectal cancer cell growth, since adenosine and CD73 enzyme activity inhibitor APCP could only partly achieve the effects of the CD73 overexpression and interference virus, respectively. These results are supported by many studies in other cancers (42,43), suggesting that adenosine-related pathways and other signaling pathways warrant further study in colorectal cancer growth.

In conclusion, we found that CD73 promotes the growth of human colorectal cancer cells in vitro and in vivo. The effects of CD73 may be related to EGFR and β-catenin/cyclin D1 pathways. Our results suggest that CD73 may be a potential biomarker of colorectal cancer.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (grants no. 81401447).

Abbreviations:

References