Background
Liver cancer is the fifth most common form of cancer, with a third cause of cancer associated death worldwide [
1]. Liver cancer is an aggressive disease with poor overall survival [
2]. Only 20 % of liver cancer patients are still alive one year after diagnosis in the United Kingdom [
3]. Despite the high incidence of liver cancer, there is a startling lack of therapeutic interventions. Of all diagnosed liver cancer patients, most patients take a cure of surgery combining with chemotherapy or immunotherapy. And most patients have a remaining lifespan of 4–6 months due to the advance of disease [
4]. Therefore, there is an urgent demand to explore the underlying mechanism of tumor progression and discover innovative targets to eliminate tumor cells.
Tumor development is regulated by a diverse of factors, including mutations of anti-/oncogenes, tumor microenvironment, cancer stem cells, and abnormal tumor metabolic pathways. Notably, upregulation of Trp metabolism induced by IDO/TDO enzymes represents one of the most important pathways for tumor occurrence and development [
5]. IDO1 and TDO2 enzymes catalyze Trp oxidation to Kyn, which are frequently observed in tumor tissues and tightly correlated with the poor prognosis in patients [
6,
7]. Kyn synthesis has been demonstrated to promote tumorigenicity and growth by activating pro-survival signaling pathways, including PI3K/AKT and Wnt/β-catenin signals [
8]. Compelling studies have suggested that IDO1 expression in tumor cells was linked to sustained tumor growth and the presence of immunosuppression [
9]. Meanwhile, upregulation of IDO1 was observed in a series of tumor types in response to the infiltration reaction [
10]. Despite the pro-tumor effects of IDO1, the specific role of TDO2 in cancer remains controversial.
IL-6 is a member of the IL-6 family, consisting of polypeptide cytokines and acting as both pro-inflammatory and anti-inflammatory molecules. Enhanced expression of IL-6 was observed in most human liver cancer tissues, in which it was produced in an autocrine manner in tumor cells [
11]. Increasing evidence suggested that IL-6 plays an important role to drive tumor progression in several tumor types [
12]. Mechanistically, IL-6 mediated the activation of pro-survival signals, especially the JAK/STAT3 signaling pathway, to facilitate tumor growth and distant metastasis [
13]. Recent studies have implicated that cytosolic transcription factor AhR is involved in Kyn associated tumor progression, and could transcriptionally induce the expression of IL-6 [
14]. However, the potential role of IL-6 in tumor-associated Trp metabolism still remains incompletely understood.
In our study, we now demonstrated that elevated expression TDO2 mediated liver cancer progression through upregulation of Trp metabolism. Constitutive TDO2 expression gave rise to the metabolic production of Kyn, resulting in the activation of AhR. In mechanism, we found that activation of AhR contributed to the upregulation of IL-6, which further promotes liver cancer development through a STAT3 and NF-kB/TIM4 dependent manner. Inhibition of AhR signals exhibited improved tumor suppressive effects, which described novel sight for liver cancer therapy.
Methods
Cell line and reagents
Human liver cancer cell lines SMC-7721 and HepG2 cells were obtained from the Key Laboratory of Hubei Province for Digestive System Disease (China) and cultured in RMPI-1640 complete culture medium (Gibco, USA) containing 10 % fetal bovine serum (Gibco, USA) at 37 °C in a humidified atmosphere of 5 % CO2. Doxorubicin (Dox), Kyn, human recombinant IL-6, and 5-Fluorouracil (5-FU) were purchased from Sigma (USA). AhR inhibitor PDM2 and NF-kB inhibitor JSH-23 were purchased from Selleck (USA).
Tumor tissues collection from patients
The paraffin sections of tumor tissues were obtained from Fujian Medical University Union Hospital. Samples were divided into two groups, high stage (H-S, stage T2–T4) and low stage (L-S, stage T1a, and T1b) according to the pathological diagnosis. All patients agreed to participate in the study and were informed with written consent. The clinical experiments were performed according to the Declaration of Helsinki. This study was approved by the Ethics Committee of the Fujian Medical University Union Hospital.
Cell proliferation
Tumor cells (3000 cells per well) treated in advance were seeded in a 96-well plate and incubated with 10 µl per well Cell Counting Kit-8 (CCK-8, Solarbio, China) reagent after 24, 48, and 72 h, respectively. Cell proliferation rates were detected using a microplate reader (BD, USA) at 450 nm.
SMC-7721 and HepG2 cells were seeded in six-well plates (500 cells per well) and cultured with RMPI-1640 complete culture medium at 37 °C for 10 days. Colonies were fixed with methyl alcohol and stained with 4 % crystal violet solution. The colony numbers in each group were washed before counting.
Western blotting
Tumor cells were collected and lysed in RIPA buffer (Byotime, China) with protease and phosphatase Inhibitor Cocktails (Biyuntian, China). Aliquots of 10 µg protein samples were separated by electrophoresis on 10 % SDS-polyacrylamide gels and transferred to a methanol‐pretreated PVDF membrane (Thermo, USA). Then samples were blocked with 5 % BSA in TBST and incubated with anti‐TDO2 (1:1000, Abcam, UK), anti-IDO1 (1:1000, Abcam, UK), anti‐AhR (1:1000, Abcam, UK), anti-STAT3 (1:1000, Abcam, UK), anti-TIM4 (1:1000, Abcam, UK) and anti‐β‐actin (1:1000, Abcam, UK) primary antibodies. Samples were visualized with an HRP‐conjugated secondary antibody (1:1000, Thermo, USA) and western blotting analysis system (Tanon, China).
Transfection and plasmids
For TDO2 overexpression, cDNAs for human TDO2 were obtained from Ruibo Inc, China, The TDO2 cDNAs were inserted into pLVX-EF1α-IRES-Puro lentiviral vector (Takara, Japan) for stable overexpression in SMC-7721 and HepG2 cells. The overexpression of TDO2 was examined by western blotting.
Immunohistochemical staining
Paraffin-embedded tumor tissue sections were cut. After dewaxing and rehydration, sections were treated with antigen retrieval solution for 15 min at 100 °C. Subsequently, samples were blocked with 5 % BSA in PBST for 30 min. Then slides were incubated with anti-TDO2 (1:200, Abcam, UK), anti-IDO1 (1:200, Abcam, UK), anti-IL-6 (1:200, Abcam, UK), anti-Kyn (1:200, Abcam, UK), anti-pSTAT3 (1:200, Abcam, UK), anti-NF-kB (1:200, Abcam, UK) antibodies overnight at 4 °C. After that, samples were incubated with HRP-conjugated secondary antibody (1:1000, Thermo, USA), developed by 3,3-diaminobenzidine solution and counterstained with hematoxylin. The intensity of protein expression in immunohistochemistry was determined by Image-Pro Plus 6.0 software. 8 tumor tissues from 8 patients were included in each group. 10 fields in each tumor tissue were analyzed and the mean of 10 values was determined as the expression intensity of this tumor tissues.
Animal protocols
Female 6–8-week-old NOD-SCID mice were obtained from Huafukang (Beijing, China). Mice were bred in-house and fed on a standard animal chow diet, and raised in the SPF room. The animal protocol of this study was approved by the Institutional Animal Care and Use Committee of Fujian Medical University Union Hospital. The animal studies were conducted following the Public Health Service Policy and complied with the WHO guidelines for the humane use and care of animals.
For tumorigenic analysis, 1 × 105 SMC-7721 cells resuspended in 50 µl PBS were injected subcutaneously (n = 10 in each group), and the tumorigenic rates were calculated on day 20. For tumor volume and survival analysis, 106 SMC-7721 cells were injected into NOD-SCID subcutaneously (n = 6 in each group). When the tumor size reached 600 mm3, mice were treated with PBS, DOX (5 mg/kg), 5-FU (10 mg/kg), PDM2 (5 mg/kg), or combining treatment twice a week. Tumor volume and survival of mice were recorded every day (end point, day 80). The calculation formula of tumor volume is: tumor volume = length × width 2/2.
Statistical analysis
The overall survival of liver cancer patients was downloaded from
https://www.cbioportal.org/. All data were compared using either Student’s t-test, one-way or two-way analysis of variance with Tukey multiple comparison posttest in GraphPad Prism 8 (USA) to determine statistical significance between different groups. The survival was analyzed by Kaplan–Meier survival analysis. *p < 0.05; **p < 0.01; ns, no significant difference. Each experiment was performed three times independently.
Discussion
Constitutive IDO/TDO expression is a key mechanism to promote tumor sustained growth and immunosuppression in patients, which represents an ideal target for tumor therapy [
9,
17,
18]. Previous clinical studies have suggested the IDO1 inhibitors, such as 1-methyl-tryptophan, exhibited remarkable tumor suppressive effects in patients with solid tumors [
19,
20]. Despite the pro-tumor efficacy of IDO1 in tumor development and therapy, the role of TDO2, an IDO1 isozyme, remained poorly investigated. In this study, we identified a novel mechanism in which TDO2, through Trp/Kyn metabolism, activated AhR/IL-6/STAT3/NF-kB signaling and promoted liver cancer progression. Comparing tumor tissues from clinical liver cancer patients and TCGA database, we examined the TDO2 activity and the downstream metabolites, that promoting liver cancer progression. Using liver cancer cell lines and subcutaneous mice models, we demonstrated that Kyn produced by TDO2/Trp metabolism activated AhR/IL-6 pathway in liver cancer, eventually resulting in upregulation of the NF-kB/TIM4/STAT3 signals and cancer development. These findings described novel mechanistic sight and strengthened the concept for Trp metabolism associated with tumor progression.
The Trp metabolite Kyn has been defined as AhR ligands in several tumor types and immune cells [
14,
21]. Notably, Liu and her colleagues reported that Kyn derived from tumor repopulating cells mediated the activation of AhR in T cells to suppress the inflammatory response in the tumor microenvironment [
22]. And the Kyn produced by Trp catabolizing enzyme IDO in tumor cells has been demonstrated to facilitate AhR activation and tumor growth in an immune independent manner [
14,
23]. Our study further provided evidence to suggest the TDO2 was equally capable of producing Kyn to mediate the AhR pathway activation in liver cancer cells.
To our knowledge, this is the first study to define a signaling pathway by which TDO2 facilitated cancer cell proliferation via an autocrine IL-6 manner. Compelling reports have demonstrated that IDO promoted the IL-6 expression in tumor cells [
24‐
26], and in turn, IL-6 was reported to upregulate IDO expression through a JAK/STAT signaling pathway [
27,
28]. Notably, the expression of AhR has been shown to mediate the IL-6 expression in ovarian cancer [
29], and Kyn derived from Trp metabolism could efficiently promote the activation of AhR signals in a diverse of tumor cells [
21]. Our study further identified the correlation between those findings, in which we demonstrated that TDO2 mediated the IL-6 production through Trp/Kyn/AhR signaling.
The expression of IL-6 has been demonstrated to correlate with the tumor development in several tumor types [
12,
30,
31]. Mostly, autocrine or paracrine IL-6 regulated tumor progression and promoted the conversion of non-stem cancer cells into cancer stem-like cells through JAK/STAT3 signaling pathway [
31‐
33]. However, increasing evidence suggested that T-cell immunoglobulin domain and mucin domain 4 (TIM4) and NF-kB are involved in the tumor progression induced by IL-6 in non-small cell lung cancer [
23]. Here, our study showed that constitutive STAT3 activation was controlled by autocrine IL-6. Notably, upregulation of TIM4 and NF-kB was observed in IL-6 treated liver cancer cells and tumor tissues from patients. It was likely that both STAT3 and NF-kB signals were required for IL-6 induced liver cancer progression, though the specific underlying mechanism remains unclear. However, blockade of AhR directly suppressed the IL-6 secretion and tumor development induced by TDO2. More importantly, the combination of AhR inhibitor PDM2 and chemotherapy revealed strengthened tumor suppressive effects in subcutaneous tumor-bearing mice, which descried an innovative approach for clinical tumor therapy targeting Trp metabolism.
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