TDO2 Was Downregulated in Hepatocellular Carcinoma and Inhibited Cell Proliferation by Upregulating the Expression of p21 and p27

Background Tryptophan-2,3-dioxygenase (TDO2) converts tryptophan into kynurenine in the initial limiting step of the kynurenine pathway. During the past decade, the overexpression of TDO2 has been found in various human tumors. However, the role of TDO2 in hepatocellular carcinoma is controversial, and we sought to clarify it in this study. Methods Western blot analysis and immunochemistry were used to detect the expression of TDO2 in human tissue specimens. The effect of TDO2 on cell proliferation in vitro was assessed using CCK8 and colony formation assays, and a xenograft mouse model was used to detect the effect of TDO2 on tumor growth in vivo. Flow cytometry was used to assess the cell cycle status. Results Low TDO2 expression was found in HCC and was associated with poor prognosis and adverse clinical outcomes. Conversely, TDO2 could restrain the proliferation of HCC cells in vivo and in vitro. Furthermore, TDO2 upregulated the expression of p21 and p27, inducing cell-cycle arrest. Conclusions The loss of TDO2 expression in HCC was correlated with a poor prognosis and adverse clinical outcomes. At the same time, TDO2 could restrain the growth of HCC in vivo and in vitro. The results indicate that TDO2 is a potential biomarker and therapeutic target for HCC.


Introduction
As the sixth most commonly diagnosed type of cancer worldwide, hepatocellular carcinoma (HCC) imposes a heavy burden on global health [1]. HCC is an aggressive cancer and often diagnosed at an advanced stage, which results in a poor prognosis because surgical resection is only effective in the early stage [2]. Although combining immune checkpoint inhibitors with antivascular endothelial growth factor (VEGF) antibodies demonstrated a promising survival benefit for some patients with advanced HCC, this combined treatment is not effective for all advanced HCC patients [3,4]. Hence, it is vital to explore and understand the complex pathogenesis of HCC to develop new therapeutic targets and biomarkers for the early diagnosis and treatment of HCC.
Tryptophan-2,3-dioxygenase (TDO2) converts tryptophan into kynurenine in the initial limiting step of the kynurenine pathway [5]. Under physiological conditions, TDO2 is only expressed in the liver, where it is responsible for tryptophan metabolism. However, TDO2 can also be expressed in other tissues, such as the placenta, epididymis, and testes [6,7]. During the past decade, TDO2 was found to be overexpressed in various tumors, such as glioma, breast cancer, basal cell carcinoma, melanoma, cervical cancer, and colorectal cancer [5,[8][9][10][11][12][13]. As the depletion of tryptophan and the production of its downstream metabolites can restrain the proliferation of immune cells, the overexpression of TDO2 in tumors was considered to promote tumor immune escape and stimulate cancer metastasis [5,12,14]. However, the role of TDO2 in hepatocellular carcinoma is controversial. Some studies reported that TDO2 is highly expressed in hepatocellular carcinoma [15,16], while others implied that TDO2 in HCC tumors is expressed at lower levels than in adjacent tissues [5,17].
In this study, we firstly used UALCAN (http://ualcan .path.uab.edu) to conveniently access clinical data from TCGA for an analysis of the differences in TDO2 mRNA levels between normal samples and HCC as well as their relationship to clinicopathological parameters [18]. The results revealed lower TDO2 mRNA levels in HCC than in normal tissues. We then examined TDO2 protein expression in a clinical tissue microarray (TMA) and tissue specimens of HCC using immunohistochemical (IHC) staining and western blot analysis, which revealed lower TDO2 protein levels in tumor specimens than in normal tissues. At the same time, Kaplan-Meier survival analysis showed that the loss of TDO2 expression was associated with shorter survival of HCC patients. Furthermore, we also analyzed the roles of TDO2 in HCC cell lines both in vivo and in vitro. Overexpression of TDO2 restrained the proliferation of HCC cells, resulting in cell-cycle arrest in the G1 phase. Additionally, TDO2 was found to upregulate the expression of p21 and p27 while reducing CDK2 and CDK4 expression.

Patients and Methods
2.1. Patients. Primary HCC tissues and adjacent matched normal tissues from a total of 116 patients who underwent tumor resection at the Hepatic Surgery Center, Tongji Hospital of Huazhong University of Science and Technology (HUST) (Wuhan, China) between 2012/2/16 and 2014/4/1 were used in this study. In addition, 40 paired fresh specimens of HCC tissues and adjacent normal tissues from the specimen collection were used for western blot analysis. Overall survival (OS) and disease-free survival (DFS) was defined as the time from the first surgery to death or recurrence/metastasis, respectively.
2.3. Immunohistochemistry. Paraffin sections of 4 μm thickness were used for TDO2 detection. Xylene and a gradient of ethanol solutions were used to dewax and gradually hydrate the tissue sections. For antigen retrieval, the slides were heated in a steam cooker for 10-15 min in 0.01 mol/l citrate buffer (pH 6.0). Then, the slides were washed with PBS (pH 7.4), and endogenous peroxidases were blocked by incubation in 1 × TBS with 0.3% H 2 O 2 for 20 min at room temperature. Then, the primary anti-TDO2 polyclonal antibody was added to the samples and incubated at 4°C overnight. On the next day, 1 × TBS was used to wash the slides thoroughly, and a fresh substrate solution containing diaminobenzidine was used to visualize the antibody binding. Two independent pathologists who were blinded to the patient information assessed the immunohistochemical staining. Each specimen was assessed and scored according to the percentage of stained cells (0-5% = 0, 5-25% = 1, 26-50% = 2, 51-75% = 3, and 76-100% = 4) and intensity of positive tumor cells (none = 0, weak = 1, intermediate = 2, and strong = 3) and summed up to calculate the immunostaining score of TDO2 expression. The low-and high-level expressions were judged based on overall scores of <6 and ≥6, respectively.

Flow
Cytometry. Propidium Iodide Staining Solution (BD Pharmingen™, New Jersey, USA) was used for flow cytometry. Cells were trypsinized and suspended at 1 × 10 5 cells/ml, followed by staining with a fluorescent antibody and propidium iodide (PI) at room temperature in the dark for 30 min. After incubation, the cells were centrifuged at 200 g and resuspended for analysis on a FACS instrument (BD Bioscience, San Jose, CA).

Cell
Lines and Culture Conditions. HLF cells were obtained from our laboratory, while HEK293 and HepG2 cells were ordered from the China Center for Type Culture Collection (CCTCC, Wuhan, China). All cells were cultured at 37°C in Dulbecco's Modified Eagle's Medium (DMEM) (high glucose) (HyClone, USA) supplemented with 10% fetal bovine serum (Bovogen, Argentina) in a humidified atmosphere comprising 5% CO 2 .   3 BioMed Research International well of a 6-well plate was seeded with 1000 cells, which were cultured for 2 weeks at 37°C. The resulting colonies were fixed with 20% methanol and stained using 0.1% crystal violet, followed by manual counting under a conventional optical microscope. All experiments were done in triplicate. were randomly divided into 2 groups (n = 6 per group) and injected subcutaneously with 1:5 × 106 tumor cells in 100 μl of serum-free DMEM into the flank. Every 3 days, the development of the tumors was observed, and the mice were sacrificed after the indicated number of days. The tumors were then removed, photographed, weighed, and measured.
2.9. Statistical Analysis. Statistical analyses were conducted using SPSS 22.0 (IBM Corp., USA). The χ 2 test was used to evaluate the statistical significance of the correlation between clinicopathological variables and TDO2 expression.   [18]. As shown in Figures 1(a) and 1(b), the expression of TDO2 was upregulated in most cancers, but it was downregulated in cholangiocarcinoma, hepatocellular carcinoma, and pancreatic adenocarcinoma (Figures 1(a) and 1(b)). As TDO2 is only expressed in the liver under physiological conditions, we wished to explore the expression of TDO2 in HCC. We used 40 paired fresh HCC specimens and adjacent normal tissues for western blot and tissue microarray analyses of the expression of TDO2. The results indicated that TDO2 expression was downregulated in HCC (Figures 2(a)-2(d)). Furthermore, we used UALCAN to analyze the relationship between TDO2 expression and clinicopathological characteristics in HCC, which revealed that low TDO2 expression was correlated with a low tumor grade, advanced cancer stage, and cancer metastasis (Figures 1(c)-1(e)). Then, TDO2 expression in tumors from 116 HCC patients was assessed by microarray IHC analysis. Subsequently, the patients were divided into low-and high-expression groups according to the immunostaining score to explore the relationship between TDO2 expression and clinical outcomes. The results of regression analysis indicated that low TDO2 expression was associated with tumor size (P = 0:011), tumor stage (BCLC stage P = 0:019 and TNM stage P = 0:027), tumor differentiation (P = 0:038), and tumor recurrence (P = 0:041). However, low TDO2 expression was not associated with age, sex, ALT level, AST level, serum AFP level, tumor number, and tumor capsule (Table 1). Additionally, Kaplan-Meier survival analysis was used to investigate the relationship between the expression of TDO2 and overall survival (OS) or disease-free survival (DFS). The results demonstrated that patients with low TDO2 expression had shorter OS (P = 0:0384) (Figure 2(e)) and DFS (P = 0:0194) (Figure 2(f)).

Overexpression of TDO2 Inhibited Cell Proliferation In
Vitro. Since the loss of TDO2 expression was correlated with adverse clinical outcomes and a poor prognosis in HCC, it is possible that TDO2 acts as a tumor suppressor. Accordingly, we wanted to investigate the effect of TDO2 on tumorigenesis in HCC cell lines and used lentiviral vectors to transduce HLF and HepG2 cells to establish stable cell lines with TDO2 overexpression. The expression of TDO2 was detected by WB (Figures 3(a) and 3(b)). The CCK8 assay indicated that TDO2 overexpression restrained the proliferation of HLF and HepG2 cells (Figures 3(c) and 3(d)). At the same time, the overexpression of TDO2 in HLF and HepG2 cells reduced their colony formation ability (Figures 3(e) and 3(f )). Taken together, these results show that TDO2 restrained the proliferation of HCC cell lines in vitro.

Overexpression of TDO2 Induced Cell Cycle Arrest and
Upregulated the Expression of p21 and p27 In Vitro. As the results of the CCK8 and colony formation assays demonstrated that the overexpression of TDO2 could restrain the proliferation of HCC cell lines, flow cytometry and western blot analyses were used to explore the effects of TDO2 on the cell cycle and related proteins, respectively. The results of flow cytometry demonstrated that the overexpression of TDO2 increased the percentage of cells in the G1 phase in both the HLF and HepG2 HCC cell lines, leading to cell cycle arrest (Figures 4(a)-4(d)). Furthermore, the results of western blot analysis showed that the overexpression of TDO2 enhanced the expression of p21 and p27 while reducing the expression of CDK2 and CDK4 (Figures 4(e) and 4(f)). In conclusion, the results indicate that TDO2 might induce cell cycle arrest via the upregulation of p21 and p27 expression.

TDO2 Overexpression Inhibited Tumor Growth In Vivo.
In order to further verify the influence of TDO2 on tumor growth in vivo, we subcutaneously injected HLF and HepG2 cells stably transfected with the TDO2 expression vector or an empty vector into nude mice. At 21 days after subcutaneous injection, the growth and weight of tumors comprising the TDO2-overexpressing stable cell lines were reduced compared to those of the empty vector group (Figures 5(a)-5(d)).

Discussion
TDO2 is a heme-containing enzyme responsible for the oxidation of tryptophan [5]. Under physiological conditions,

BioMed Research International
TDO2 is only expressed in the liver, where it is responsible for tryptophan metabolism [19]. Since TDO2 was firstly reported to be overexpressed in glioma, where it plays a tumor promoting role, increasing numbers of studies reported that TDO2 was overexpressed in other tumors, such as breast cancer, basal cell carcinoma, melanoma, cervical cancer, and colorectal cancer, where it is also involved in tumor development [5,10,13,14]. However, the role of TDO2 in HCC is controversial. Some studies implied that TDO2 was downregulated in HCC [5,17], while other studies indicated that TDO2 may be overexpressed in HCC [15]. In this study, we firstly used UALCAN to analyze the relative TDO2 mRNA levels in normal samples and HCC tumors and found that TDO2 mRNA expression in HCC samples was lower than in healthy tissues. Furthermore, we used WB and IHC to detect the expression of TDO2 in 40 paired fresh specimens of HCC and a tissue microarray, respectively, which confirmed that TDO2 was downregulated in HCC. At the same time, the loss of the TDO2 expression was correlated with a poor prognosis and adverse clinical outcomes. These results implied that TDO2 may function as a tumor suppressor in HCC, which is in contrast to the current viewpoint that TDO2 functions as a tumor promoter. However, it should be noted that TDO2 is normally only  BioMed Research International expressed in the liver where it is responsible for tryptophan metabolism. This in turn may indicate that its role in liver cancer is different from other tumors. Alternatively, the inconsistency of antibody staining and the cutoffs used to assign patients to high or low TDO2 expression groups might account for this discrepant finding.
As the loss of TDO2 was correlated with a poor prognosis and adverse clinical outcomes in HCC, we further explored the effect of TDO2 on HCC cell proliferation. We found that TDO2 could restrain the proliferation of HCC line cells and induce cell-cycle arrest by enhancing the expression of p21 and p27 while decreasing CDK2 and CDK4 expression. As most studies on the roles of TDO2 in tumors focus on tumor immune evasion, there are relatively few studies on the effect of TDO2 on the proliferation of tumor cells. As the depletion of tryptophan and accumulation of kynurenine can restrain the proliferation of T cells, TDO2 expression was also found to restrain the proliferation of T cells [5]. At the same time, IDO1, the isozyme of TDO2, was reported to induce cell cycle arrest by upregulating the expression of p27 via the kynurenine-AHR axis [20]. Accordingly, TDO2 might upregulate the expression of p21 and p27 via the kynurenine-AHR axis, but this conjecture needs further investigation.
Under normal physiological conditions, TDO2 is only expressed in the liver, while the expression of TDO2 is lost in HCC. At the same time, the expression of TDO2 is upregulated in most cancer types [9,10,13,14]. Hence, the underlying mechanisms and effects to the loss of TDO2 expression in HCC merit further attention. As the promoter of the tdo2 gene contains two glucocorticoid-responsive elements (GREs), TDO2 can be upregulated by glucocorticoids [21]. However, plasma glucocorticoid levels were reported to be practically the same in hepatocarcinoma patients and healthy controls or portal hypertension patients without hepatocarcinoma [22]. It is also possible that the abnormal expression of 11β-HSD1 and 11β-HSD2 in HCC, two critical enzymes that regulate the activity of glucocorticoids in the liver, accounts for the loss of TDO2 expression, but this hypothesis needs further investigation [23,24].

Conclusions
In summary, our data demonstrate that TDO2 expression is reduced in HCC, and low TDO2 expression was correlated with a poor prognosis and adverse clinical outcomes. At the same time, TDO2 overexpression could restrain the proliferation of HCC cells in vivo and in xenograft mouse models in vitro. TDO2 overexpression induced cell cycle arrest by upregulating the expression of p21 and p27. These data indicate that TDO2 might serve as a biomarker for HCC.

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
The studies involving human participants were reviewed and approved by the Research Ethics Committee of Tongji Hospital of Huazhong University of Science and Technology (China).

Consent
The patients/participants provided their written informed consent to participate in this study.