TDO2 overexpression correlates with poor prognosis, cancer stemness, and resistance to cetuximab in bladder cancer

Abstract Background Bladder cancer (BC) is the 10th most common cancer in the world. BC with muscle invasion results in a poor prognosis and is usually fatal. Cancer cell metabolism has an essential role in the development and progression of tumors. Expression of tryptophan 2,3‐dioxygenase (TDO2) is associated with tumor progression and worse survival in some other cancers. However, no studies have been performed to uncover the biofunctional roles of TDO2 in BC. Aim This study aim to investigate the clinicopathologic significance of TDO2 in BC. Methods and results TDO2 expression was evaluated by qRT‐PCR and immunohistochemistry in an integrated analysis with the Cancer Genome Atlas (TCGA) and other published datasets. TDO2 overexpression was significantly associated with T classification, N classification, and M classification, tumor stage, recurrence, and basal type, and with the expression of CD44 and aldehyde dehydrogenase 1 (ALDH1) in BC. High TDO2 expression correlated with poor outcome of BC patients. Using BC cell lines with knockdown and forced expression of TDO2, we found that TDO2 was involved in the growth, migration, and invasiveness of BC cells. Moreover, TDO2 was found to be crucial for spheroid formation in BC cells. Importantly, TDO2 promoted BC cells resistance to cetuximab through integration of the EGFR pathway. Conclusion Our results indicate that TDO2 might take an essential part in BC progression and could be a potential marker for targeted therapy in BC.


| INTRODUCTION
Bladder cancer (BC) is the 10th most leading cancer in the world. 1 In Japan, BC is the 8th most leading cancer in men and the 11th leading cancer in women. 2 Almost 70%-80% of the newly diagnosed BC patients present with non-muscle-invasive or early-invasive disease and the rest of patients with muscle-invasive bladder cancer (MIBC). 3 Radical cystectomy is the current standard treatment for MIBC patients without metastasis. 4,5 Although recent innovative gains in the knowledge of new molecular classifications of BC and some remarkable therapeutic strategies have been investigated, the outcome of patients with MIBC remains poor. 5 Therefore, it is necessary to find new biomarkers for therapeutic targets in BC.
Currently, tumor cell metabolism has shown a pivotal role in tumor development and tumor progression and has become a therapeutic target for cancer treatment. 6,7 It was reported that tryptophan metabolites are highly detected in the urine of BC patients. 8 The kynurenine (Kyn) pathway, which included two main enzymes: indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2), takes up almost 95% of tryptophan metabolism. 9 IDO1 is expressed in a variety of tissues, while TDO2 is primarily expressed in the liver. 10 TDO2 overexpression has been described in many cancers including glioblastoma, breast cancer, and lung cancer. [10][11][12] Overexpression of TDO2 enhanced tumor cell survival and associated with worse outcomes in patients with these tumors. [10][11][12] We previously reported that the expression of TDO2 was linked with tumor progression and poor prognosis and cancer stem cells (CSCs) in esophagus squamous cell carcinoma. 13 CSCs, accounting for around 1% of solid tumor cells, are known to be more resistant to conventional treatments than other cancer cells within a tumor. 14,15 However, the clinicopathologic significance of TDO2 in BC remains unknown.
Here, we investigated the expression of TDO2 in BC tissues and studied the relation of TDO2 with the clinicopathologic features and prognosis of BC. We also explored the biofunctional roles of TDO2 in progression, cancer stemness, and drug resistance using BC cell lines with RNA interference (RNAi) knockdown and forced expression of TDO2.

| Tissue samples
A total of 59 primary tumors were obtained from 50 patients diagnosed with MIBC who underwent radical cystectomy at National Hospital Organization Kure Medical Center and Chugoku Cancer Center (Kure, Japan) and from nine patients at Hiroshima University Hospital (Hiroshima, Japan). Informed consent was obtained from each patient.
This study was approved by the Ethical Committee for Human Genome Research of Hiroshima University and the Ethics Committee of Kure Medical Center and Chugoku Cancer Center. The TNM classification system was used to determine tumor staging. 16 We used 9 BC samples and 10 types of normal tissue samples for qRT-PCR. Tumor tissues and nonneoplastic tissues were surgically removed, immediately frozen in liquid nitrogen, and stored at À80 C.

| qRT-PCR analysis
Total RNA was isolated from cells pellet and frozen tissue by using ISOGEN (Nippon Gene, Toyama, Japan). A total of 1 μg RNA was used to synthesis cDNA by the PrimeScript first strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). PCR was performed with a CFX Connect real-time PCR detection system (Bio-Rad) using the SYBR Green PCR Core Reagents Kit (Applied Biosystems; Thermo Fisher Scientific, USA). The 2 ÀΔCT method was used to calculate the relative expression levels, in which ΔCT is the difference in threshold cycle (CT) values between the target gene and Actin Beta (ACTB) served as an internal control. The primer sequences are listed in Data S1.

| Immunohistochemistry and evaluation
The IHC staining procedure was carried out as previously described on 3 μm thick sections. 13 Primary antibody mouse polyclonal anti- to detect protein expression. The expression of TDO2 was evaluated by the expression score, which included the intensity (1+, 2+, 3+) and the percentage (from 0% to 100%) of detected tumor cells. The expression of GATA3 and 34βE12 was evaluated by the percentage of staining cells. IHC staining of CD44 and ALDH1 were carried out as previously described. 17

| Western blot analysis
Cell pellets were lysed in Ripa buffer (50 mM Tris, pH 7.4, 125 mM NaCl, 0.1% NP40, 5 mM EDTA, and protease inhibitor cocktail [cOmplete, Roche]). 18 Western blot procedures were carried out as previously described. 18 The following primary antibodies were used: The primer sequences for qRT-PCR, RNA interference (RNAi), expression vector, proliferation assays, cell migration assays, invasion assays, spheroid colony formation assays, and statistical methods are presented in Data S1.

| TDO2 expression in BC
We investigated the expression of TDO2 in BCs using the TCGA BLCA dataset. As shown in Figure S1a, TDO2 expression in BC tissue was significantly higher than that in normal bladder tissue. We evaluated TDO2 mRNA level in 10 kinds of normal tissue samples and 9 BC samples by qRT-PCR analysis ( Figure 1(A)). The TDO2 mRNA level was highest in the liver compared with that in other normal tissues. BC samples highly expressed TDO2 than normal tissues excepted liver tissue ( Figure S1c).
Next, we conducted immunohistochemistry on the 50 BC tissue samples. Figure 1(B) shows that normal urothelium showed no staining or weakly staining for TDO2. Conversely, the expression of TDO2 was increased in BC and was detected in the cytoplasmic of tumor cells (Figure 1(C)). BC tissues showed heterogeneousness of TDO2 immunoreactivity. The TDO2 expression scores were calculated as detailed in the Methods section, and immunoreactivity images are shown in Figure S2a. The invasive areas and the central or superficial areas presented no difference in TDO2 expression score.
We further used ROC analysis to determine the cut-off value for the TDO2 expression score that correlated with clinicopathologic features ( Figure S2b-e). The cut-off point of the expression score for T, N, and M classification, and stage was determined to be 75 by using the Youden index. 19 TDO2 expression was defined as high if the expression score was above the cut-off value and as low if the score was equal to or below the cut-off value. Next, we investigated the correlation between TDO2 expression, divided by the cut-off point, and the clinicopathologic characteristics of the 50 BCs (Table 1). High TDO2 expression was significantly related to advanced T (p = .002), N (p = .037), M classification (p = .037), tumor stage (p = .002), and recurrence status (p = .002). These data suggested that TDO2 might have a pivotal part in the progression of BC.
Kaplan-Meier analysis revealed that high TDO2 expression BC patients presented with shorter survival than those with low TDO2 expression BC patients (p = .009; Figure 1(D)). Kaplan-Meier analysis results from the BLCA dataset similarly showed unfavorable survival in BC patients with high TDO2 ( Figure S1b). We then performed univariate and Cox regression analysis to investigate the possible role of TDO2 expression as an independent prognostic factor in BC. M classification and TDO2 expression were associated with worse survival in the univariate analysis (Table 2). However, only metastasis was recognized to be an independent prognostic factor in BC in the Cox analysis (Table 2).

| TDO2 expression is associated with basaltype BC and CSC markers in BC
Recently, much large-scale genome sequencing research in BC has revealed the molecular subtypes of BC shown to correlate with clinical progression, patient outcome, and response to treatment. [20][21][22][23] Although there are differences between the classification subtypes determined by each study, central to these classifications is the basal (expressed markers: CK5/6, CK14, CD44) BC type and the luminal (expressed markers: uroplakin, CK20, GATA3) BC type. 20,21,23 We previously showed that TDO2 expression was associated with CD44 expression in esophagus squamous cell carcinoma. 13 Hence, we con- To determine the above gene expression results presented at the protein level, we performed IHC analysis of GATA3 (luminal marker) and 34βE12 (basal marker) in the 50 BC cases ( Figure S3c). Interestingly, we found that TDO2 expression correlated with 34βE12 expression (rho = 0.494, p = 2.6274E-04) but not with GATA3 expression (rho = 0. 253, p = .076) (Figure 2(B)). These data indicated that basaltype BC highly expressed TDO2.
Previous results of BC genomic sequencing analysis revealed that the basal subtype of BC showed more stemness. 20,21,23 The upregulated expression of two stem cell markers CD44 and ALDH1 was also shown to be associated with tumor stage, recurrence, and unfavorable prognosis of BC patients. 24, 25 We then performed IHC and ALDH1 staining (Figure 2(C, D)). TDO2 expression was significantly correlated with CD44 and ALDH1 expression (Table 3).

| TDO2 expression promotes cell proliferation in BC
We further investigated the effect of TDO2 knockdown on cell proliferation. Both qRT-PCR and western blot analysis presented that 253-JBV and UMUC6 cells highly expressed TDO2 (Figure 3(A, B)).   Figure S4b).
To verify the biofunctional roles of TDO2 in BC, we generated a TDO2 overexpression vector. Then, we established stable TDO2 overexpression in RT112 and KMBC2 cells that showed a low level of TDO2 expression (Figure 3(A, B)). As shown in Figure 3(E) and  Figure S5b).

| TDO2 promotes cell migration and invasiveness in BC
High TDO2 expression was associated with tumor metastasis of BC patients. Hence, we evaluated the effect of TDO2 expression on the migration and invasiveness of BC cells. As shown in Figure 4(A, B) and Figure S4c,d, the cell migration and invasive activities of TDO2 siRNA1-transfected and siRNA2-transfected 253-JBV and UMUC6 cells were lower than those of the negative-control siRNA-transfected 253-JBV and UMUC6 cells (p < .05). Following these data, we performed cell migration assays and invasion assays using RT112 and KMBC2 cells with stable TDO2 overexpression. As expected, overexpression of TDO2 also promoted cell migration and invasiveness in the RT112 and KMBC2 cells (Figure 4(C, D), Figure S5c,d).
These data indicated that TDO2 stimulates cell migration and invasive activities in BC cells.

| TDO2 expression involved in spheroid formation
The IHC results revealed the correlation between TDO2 expression and cancer stem cell markers (CD44 and ALDH1) in BC. Spheroid colony formation assays are commonly used to investigate CSC  3.6 | TDO2 induces resistance to cetuximab through integration of downstream signaling of EGFR pathway Several lines of evidence revealed that EGFR may be a novel prognostic biomarker and molecular target in BC. 20,21,28,29 Recently, it was shown that aryl hydrocarbon receptor (AhR), activated by Kyn, a TDO2 metabolites product, bypasses EGFR to activate PI3K/AKT and MEK/ERK signaling. 30 Figure S7a). In contrast, the RT112 and KMBC2 cells with stable expression of TDO2 presented higher levels of phosphorylated Akt and Erk than control cells ( Figure 6(B), Figure S7b).
Hence, we examined the effect of TDO2 expression on the sensitivity to an EGFR-targeted agent in BC cells. Cetuximab, a humanized monoclonal antibody against EGFR, binds to its extracellular domain to inhibit EGFR signaling and is used clinically for certain malignancies. As shown in Figure 6(C, D), inhibition of TDO2 by siRNA1 and siRNA2 in 253-JBV and UMUC6 cells conferred sensitivity to cetuximab. Consequently, RT112 and KMBC2 with stable expression of TDO2 induced resistance to cetuximab ( Figure S7c). However, knockdown of TDO2 did not affect the expression of Vimentin and E-cadherin ( Figure S8a,b). Taken 34 Our results showed that TDO2 was involved in the proliferation, migration, and invasiveness of BC cells. Recently, a study which fully validated the expression of TDO2 by immunohistochemistry and in situ hybridization showed that liver cancer also highly expresses TDO2. 35 These findings suggested that the TDO2-Kyn-AhR axis may have a crucial role in the tumorigenesis of cancer disease.
Currently, the first-line treatment for advanced BC patients is platinum-based chemotherapy. 4,5 In light of the approval of several immunotherapies such as atezolizumab and pembrolizumab (antiprogrammed cell death ligand-1 antibodies), immune-targeted therapy has been become a Category 2A recommendation in the NCCN Guidelines. 5 A previous report showed that TDO2-expressing tumors induced acquired immune tolerance. 10 IDO1/TDO2 inhibitor has been used in human phase I clinical trials. 36 In the present study, we showed that TDO2 could have an essential role in promoting cell survival, cell migration, and invasion of BC cells. Additionally, combining the IDO1 inhibitor with nivolumab is safe and improves response rates in bladder and cervical cancer patients. 37 Interestingly, IDO1 inhibition was also shown to inhibit cell growth, migration, and invasion in BC cells. 38 Taken together, TDO2/IDO1 may serve as an ideal target for the development of targeted therapy.
The EGFR signaling pathway is the most well-known pathway playing pivotal roles in oncogenesis. Transcriptome analysis pointed out that up to 11% of MIBC patients present with EGFR amplification. 20,21 EGFR is overexpressed in up to 74% of BC tissue specimens. 39 Therefore, EGFR could be a potential therapeutic target for BC. There are two main agents against EGFR: monoclonal antibodies against the EGFR including cetuximab and tyrosine kinase inhibitors like gefitinib and erlotinib. 40 EGFR inhibitors inhibit phosphorylation of EGFR, which consequently downregulates phosphorylated AKT and ERK. Therefore, tumor cells begin to harbor resistance to tyrosine kinase inhibitors and anti-EGFR monoclonal antibodies by modulating MEK and ERK signaling. 41 One particular finding in the present work is that ERK and AKT phosphorylation were dramatically inactivated by the knockdown of, and activated by the overexpression of, TDO2 in BC. Furthermore, we showed that inhibition of TDO2 made BC cells sensitive to cetuximab and forced expression of TDO2 made BC cells resistant to cetuximab. Our data corroborate the research findings in non-small-cell lung cancer, that is, activation of AhR by a TDO2 metabolite, Kyn, promotes resistance to EGFR inhibitors by rescuing PI3K/AKT and MEK/ERK signaling. 31 Together, these findings indicated that TDO2 may take part in the activation of EGFR downstream signaling and be involved in the resistance of TKIs in BC.
In conclusion, this study showed that TDO2 overexpression was related to disease progression and unfavorable prognosis in BC patients. In vitro experiments revealed that TDO2 participated in cancer cell proliferation, migration, invasion, and spheroid body formation in BC cells. TDO2 recruited ERK and AKT phosphorylation and made BC cells resistant to cetuximab. Our results demonstrate that TDO2 could be a potential marker for targeted therapy in BC.