Expression of TRIP13, ZEB1, and E-cad in Infiltrating Urothelial Carcinoma of Bladder and Their Clinical Significance

Abstract

Background and purpose: Thyroid hormone receptor interacting protein 13 (TRIP13) which plays a carcinogenic role in tumors belongs to AAA+ ATPase superfamily. Zinc finger E-box binding homeobox 1 (ZEB1) and epithelial cadherin (E-cad), the key biomarkers of epithelial-mesenchymal transition (EMT), which aberrant expression may promote tumor invasion and metastasis. The purpose of this study is to investigate the expression of TRIP13, ZEB1, and E-cad in infiltrating urothelial carcinoma of bladder (IUCB) and their clinicopathological significance.

Methods: The expression of TRIP13, ZEB1, and E-cad in 150 cases of IUCB samples were investigated by immunohistochemistry. Clinicopathological, demography, and follow-up data were also collected.

Results: Positive rates of expression of TRIP13 and ZEB1 were significantly higher in IUCB samples when compared with the control bladder samples. And positive rate of expression of E-cad was significantly lower in IUCB than its expression rate in the control group. The positive expression of TRIP13 and ZEB1 were positively associated with tumor stages, local lymph node metastasis (LNM), and tumor node metastasis (TNM) stages. And E-cad expression was negatively associated with tumor stages, LNM, and TNM stages. The results of Kaplan-Meier analysis demonstrated that the patients with TRIP13 and ZEB1 positive expression had an unfavorable overall survival (OS) time when compared with patients with TRIP13 and ZEB1 negative expression. And patients with E-cad positive expression had a favorable OS time when compared with patients with E-cad negative expression. The COX regression analysis indicated that TRIP13, ZEB1, and E-cad, tumor stages, as well as TNM stages were independently factors for OS time in IUCB patients with postoperative therapy.

Conclusions: TRIP13, ZEB1, and E-cad may be considered as potentially promising biomarkers for IUCB patients’ prognosis.

Introduction

Worldwide, bladder cancer is the common cause of cancer incidence and mortality in the urology system, with an approximately 549000 new cases and 200000 deaths in 2018 [1]. Urothelial carcinoma is the leading type of bladder cancers. However, the risk of recurrence and progression of infiltrating urothelial carcinoma of bladder (IUCB) remains high [2]. Thyroid hormone receptor interacting protein 13 (TRIP13) which was originally identified as a protein interacting with human papillomavirus E1 protein belongs to AAA + ATPase superfamily. TRIP13 which is located on chromosome 5p15.33 encodes 432 amino acids. It has been demonstrated that TRIP13 plays an important role in spindle assembly checkpoint pathway and meiotic recombination [3]. Accumulating researcher had demonstrated that overexpression or amplification of TRIP13 should play an important role in many human cancers, such as thyroid cancer [3], colorectal cancer [4], hepatocellular cancer [5], prostate cancer [6], and ovarian cancer [7].

Epithelial-mesenchymal transition (EMT) is characterized that epithelial cells loses their epithelial characteristics, such as cell junctions, morphology, and polarity, acquires mesenchymal characteristics, such as motility and invasiveness. Zinc finger E-box binding homebox 1 (ZEB1), an important EMT-activators, which regulates EMT by repression of epithelial-cadherin (E-cad) expression [8, 9]. ZEB1 is also involved in many human tissue differentiations, such as bone, smooth muscle, and neural tissue. Overexpression of ZEB1 could cause EMT process in various human cancers, such as colorectal cancer, lung cancer, breast cancer, liver cancer, and pancreatic cancer [8–12].

E-cad which can regulate epithelial cell-cell and epithelial cell-stromal cell adhesion belongs to transmembrane glycoprotein superfamily. Down- or lost-expression of E-cad may weaken the adhesion of cell-cell, resulting in the cells easy to move and cells deformation, which leads to cells infiltrate and metastasize. Down- or lost-expression of E-cad is a pivotal role in EMT process and promotes EMT process in various human epithelial cancer.

Overall, accumulating evidence have indicated that expression of TRIP13, ZEB1, and E-cad is closely linked to cancer infiltration and metastasis. However, the correlations between expression of these proteins and IBC have not been widely reported. The purpose of this study is to investigate the expression of these proteins in IUCB and analyze the association among expression of these proteins each other.

Methods

150 cases of IUCB’s patients who were diagnosed in the Department of Pathology of the First Affiliated Hospital of Bengbu Medical College, from January 2013 to December 2015 were considered as experimental group. 150 cases of corresponding “normal bladder mucosa tissues” which came from the same patients were considered as control group. IUCB’s patients who received neoadjuvant chemoradiation treatment or any other anti-cancer treatment were excluded. This study was approved by the ethical committee of Bengbu Medical College and carry out according to the guidelines issued of the Declaration of Helsinki. All IUCB’s specimens were obtained with patients writing consent. Clinicopathological characteristics, demography, and follow-up data of patients were collected at the same time. Overall survival (OS) time was calculated from patients’ surgery date to his or her death date or December 2020. Tumor-node-metastasis (TNM) stages were evaluation by the 8th edition of the guidelines issued by American Joint Committee on Cancer (AJCC). Specific clinicopathological characteristics see Table 1.

Immunohistochemistry

Immunohistochemical staining was performed in accordance with the Elivision™ Plus detection kit instructions. All tissue specimens were fixed in 10% buffered formalin solution and embedded in paraffin, and then cut continuous 4-µm-thick slices. All slices were deparaffinized and dehydrated with xylene and alcohol, then rinsed with phosphate buffer solution (PBS, pH 7.2) for 10 min. Methanol containing 3% hydrogen peroxide solution was used endogenous peroxidase activity of tissues block. 95 ℃ citrate buffer (pH 6.0) for 30 min was used antigen repair. Subsequently, rinsed with PBS several times, all slices were blocked with goat serum for 20 min, then incubated with rabbit polyclonal antibody against human TRIP13, ZEB1, and mouse monoclonal antibody against human E-cad at 4℃ overnight. Reagent A and reagent B were added in sequence, and then developed in diaminobenzidine (DAB) substrate solution and re-dyed with hematoxylin.

Evaluation of immunohistochemical staining

Ten fields randomly at high-power-field (HPF) from different areas of every slice was selected. Two pathologists who were blind on all patients (such as clinicopathological, demography, and follow-up) data independently explained immunostaining results. Immunostaining results was assessed in accordance with the product of immunostaining extent and intensity [13]. For IBC tissues that were positive expression of TRIP13, ZEB1, and E-cad, an average of the result of each slice was taken. When score ≥ 3 was considered positive result in this study.

Results

TRIP13, ZEB1, and E-cad expression in IUCB, and their associations with clinicopathological parameters

The positive immunostaining of TRIP13 was located in the cytoplasm of IUCB cases, that of ZEB1 was located in the cytoplasm, and that of E-cad was located in the cytoplasm and membrane. Overall, positive expression of TRIP13 was 74.7% (112/150) in the control tissues and 6.7% (10/150) in IUCB tissues (see Fig. 1A and 1B). The difference of expression between the two groups was statistically significant (P < 0.001). Expression of TRIP13 was significantly related to tumor stages, LNM, and TNM stages, but not to patients age, gender, smoking and alcohol status, tumor location, and tumor diameter (see Table 2).

The difference of ZEB1 expression is significant between in the control group 8.0% (12/150) and ICUB group 70.0% (105/150; P < 0.001; see Fig. 1C and 1D). Expression of ZEB1 was significantly associated with tumor diameter, tumor stages, LNM, and TNM stages, but not with patients age, gender, smoking and alcohol status, and tumor location (see Table 2).

E-cad expression is 94.0% (141/150) in the control group and 33.3% (50/150) in the tumor group (see Fig. 1E and 1F). The difference of E-cad expression was significant between the two groups (P < 0.001). E-cad expression was negatively correlated with tumor stages, LNM, and TNM stages, but not with patients age, gender, smoking and alcohol status, tumor location, and tumor diameter (see Table 2).

Univariate and multivariate analyzes

As seen in Fig. 2A, the survival curve indicated that OS time of IUCB patients who expressed TRIP13 was more unfavorable than that of patients who not expressed the protein (log-rank = 28.263, P < 0.001). Similar to TRIP13, the survival curve demonstrated that OS time of patients who expressed ZEB1 was also more unfavorable than that of patients who not expressed the protein (log-rank = 49.111, P < 0.001; see Fig. 2B). Inversely to TRIP13, the survival curve suggested that patients who expressed E-cad was more favorable than that of patients not expressed E-cad (log-rank = 31.965, P < 0.001; seen Fig. 2C).

Multivariate analysis demonstrated that positive expression of TRIP13, ZEB1, and E-cad, TNM stages were independently prognostic indicators for IUCB patients (see Table 4).

Associations among the expression of TRIP13, ZEB1, and E-cad in IUCB

The spearman correlation coefficient analysis suggested a negative association between E-cad expression and TRIP13 (r=-0.303, P < 0.001) or ZEB1 (r=-0.463, P < 0.001) expression. There was a positive association between TRIP13 expression and ZEB1 expression (r = 0.555, P < 0.001) (see Table 5).

Discussion

IUCB is the second most malignant tumors of urology system. The main reason for treatment failure is recurrence and metastasis. In this study, through investigating the IUCB and normal tissues of patients, we found that TRIP13 and ZEB1 were highly expressed in IUCB tissues, while E-cad was low expressed. These results are consistent with the previous studies in other cancers [3, 4, 11, 13]. TRIP13 and ZEB1 are highly expressed in tumors [3, 4, 11], E-cad is lowly expressed in tumors [13].

TRIP13 which can ensure accurate cell division is a marker of centromere-specific protein. It has been demonstrated that centromere-specific proteins highly expressed in many tumors which is linked to malignant transformation or instability [3, 14]. In this study,we analyzed positive rate of TRIP13 expression in IUCB tissues and control tissues. We found that positive rate of TRIP13 expression was higher in IUCB tissues than that in the control tissues. And the positive rate of TRIP13 expression was associated with tumor stages, LNM, and TNM stages. The OS survival curve showed that patients with positive expression of TRIP13 had an unfavorable OS time when compared with patients who did not express TRIP13. These results suggested that aberrant expression of TRIP13 should participate in the process of invasiveness and metastasis of IUCB. So, TRIP13 can be considered as a valuable indicator for prediction of IUCB patients’ prognosis.

Epithelial-mesenchymal transition (EMT), which confers the ability of cell migration and invasion, including stem cell characteristics, reducing apoptosis and aging, and promoting immunosuppression, not only plays a key role in the development process, but also participates in tissue healing and carcinogenesis. Zinc finger E-box binding homeobox 1 (ZEB1) is a key inducer of EMT and promotes EMT process by inhibiting the activity of E-cad [15, 16]. The absent expression of E-cadherin leads to the occurrence of EMT [17]. In this study, we found that the expression of ZEB1 in IUCB tissues is higher than that in the control tissues. Furthermore, the positive expression of ZEB1 is significantly associated with tumor diameter, tumor stages, LNM, and TNM stages. Inversely, the expression of E-cad in IUCB tissues is lower than that in the control tissues. And the positive expression of E-cad is negatively related to tumor stages, LNM, and TNM stages. Univariate survival analysis demonstrated that patients with positive expression of ZEB1 had lower OS time when compared with patients who did not express ZEB1. On the contrary, the patients with positive expression of E-cad had a more favorable OS time when compared with patient who did not express E-cad. The above results were consistent with the previous other studies [16, 18, 19]. Aberrant expression of ZEB1 and E-cad should be thought useful and valuable biomarkers for prediction of IUCB patients’ prognosis.

Accumulating evidence have demonstrated that spindle assembly checkpoint is a universal safeguard that assures the integrity of chromosome separation in cell division [20, 21]. TRIP13, which is highly expressed in various human cancers, is thought to result in chromosome instability in many cancers [22–25]. Recently, more and more studies had focused on the effects of TRIP13 in cancer progression, prognosis, and drug resistance. Down- or lost- expression of TRIP13 can inhibit tumor cells proliferation, migration, and invasion, and promote apoptosis and EMT [3]. In this study, the results of that positive expression of E-cad was negatively associated with TRIP13 or ZEB1 expression. And ZEB1 expression was positively correlated with TRIP13 expression. Down expression of E-cad should cause from transcriptional repression through EMT regulators, such as ZEB1 [26]. Therefore, overexpression of TRIP13 and ZEB1 and down-expression of E-cay maybe synergistical cause tumor cell proliferation, migration, invasion, and even metastasis by EMT process. Moreover, the results of multivariate analysis demonstrated that TRIP13, ZEB1, and E-cad expression, tumor stages, as well as TNM stages were independently prognostic indicators for IUCB patients’ OS time.

Conclusions

Study had showed that TRIP13, ZEB1, and E-cad may synergistical affect a serial of processes of IUCB, such as progression, migration, invasion, and metastasis. So, TRIP13, ZEB1, and E-cad could be considered as promising and potentially valuable biomarkers for prediction of metastasis and prognosis for IUCB patients.

Abbreviations

IUCB: infiltrating urothelial carcinoma of bladder; TRIP13: Thyroid hormone receptor interacting protein 13; ZEB1: Zinc finger E-box binding homeobox 1; EMT: epithelial-mesenchymal transition; TNM: tumor-node-metastasis; LNM: lymph node metastasis; OS: overall survival; HPF: high-power-field; PBS: phosphate buffer solution; WHO: World Health Organization; AJCC: American Joint Committee on Cancer; HPF: high-power-field; DAB: diaminobenzidine; ECM: cell-extracellular matrix.

Declarations

Acknowledgments

We thank all staff members at the Department of Pathology of our hospital for assistance with the data collect and project management.

Funding

This work was supported by the Nature Science Key Program of College and University of Anhui Province (No.KJ2016A488).

Availability of data and materials

The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable requests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Tissue samples for diagnostic and research aims were obtained with each patients’ consents and the research was approved by the ethical committee of Bengbu Medical University and performed in accordance with the guidelines of the Declaration of Helsinki.

Competing interests

The authors declare that they have no competing interests.

Author’s contribution

ZL and DCY carried out the design, analysis of pathology and drafted the manuscript. DCY and ZY carried out sample collection and coordination. ZY performed the immunohistochemical staining. All authors read and approved the manuscript.

References

1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2018;68:394–424.
2. Lin F, Yin HB, Li XY, Zhu GM, He WY, Gou X. Bladder cancer cell–secreted exosomal miR–21 activates the PI3K/AKT pathway in macrophages to promote cancer progression. Int J Oncol. 2020;56(1):151–64.
3. Yu L, Xiao Y, Zhou X, Wang J, Chen S, Peng T, Zhu X. TRIP13 interference inhibits the proliferation and metastasis of thyroid cancer cells through regulating TTC5/p53 pathway and epithelial-mesenchymal transition related genes expression. Biomed Pharmacother. 2019;120:109508.
4. Sheng N, Yan L, Wu K, You W, Gong J, Hu L, Tan G, Chen H, Wang Z. TRIP13 promotes tumor growth and is associated with poor prognosis in colorectal cancer. Cell Death Dis. 2018;9:402.
5. Yao J, Zhang X, Li J, Zhao D, Gao B, Zhou H, Gao S, Zhang L. Silencing TRIP13 inhibits cell growth and metastasis of hepatocellular carcinoma by activating of TGF-β1/smad3. Cancer Cell Int. 2018;18:208.
6. Chen F, Liu L, Wang S. Long non-coding RNA NORAD exhaustion represses prostate cancer progression through inhibiting TRIP13 expression via competitively binding to miR-495-3p. Cancer Cell Int. 2020;20:323.
7. Zhou XY, Shu XM. TRIP13 promotes proliferation and invasion of epithelial ovarian cancer cells through Notch signaling pathway. Eur Rev Med Pharmacol Sci. 2019;23:522–9.
8. Manshouri R, Coyaud E, Kundu ST, Peng DH, Stratton SA, Alton K, Bajaj R, Fradette JJ, Minelli R, Peoples MD, Carugo A, Chen F, Bristow C, Kovacs JJ, Barton MC, Heffernan T, Creighton CJ, Raught B, Gibbons DL. ZEB1/NuRD complex suppresses TBC1D2b to stimulate E-cadherin internalization and promote metastasis in lung cancer. Nat Commun. 2019;10:5125.
9. Lindner P, Paul S, Eckstein M, Hampel C, Muenzner JK, Erlenbach-Wuensch K, Ahmed HP, Mahadevan V, Brabletz T, Hartmann A, Vera J, Schneider-Stock R. EMT transcription factor ZEB1 alters the epigenetic landscape of colorectal cancer cells. Cell Death Dis. 2020;11:147.
10. Feldker N, Ferrazzi F, Schuhwerk H, Widholz SA, Guenther K, Frisch I, Jakob K, Kleemann J, Riegel D, Bönisch U, Lukassen S, Eccles RL, Schmidl C, Stemmler MP, Brabletz T, Brabletz S. Genome-wide cooperation of EMT transcription factor ZEB1 with YAP and AP-1 in breast cancer. EMBO J. 2020;39:e103209.
11. Wang XF, Liu JY, Li JL, Wang FZ, Sun BL. ZEB1 causes the production of hsa-microRNA-99b/let-7e/microRNA-125a cluster and promotes invasion of liver cancer cells. Eur Rev Med Pharmacol Sci. 2019;23:1468–75.
12. Al-Ismaeel Q, Neal CP, Al-Mahmoodi H, Almutairi Z, Al-Shamarti I, Straatman K, Jaunbocus N, Irvine A, Issa E, Moreman C, Dennison AR, Emre Sayan A, McDearmid J, Greaves P, Tulchinsky E, Kriajevska M. ZEB1 and IL-6/11-STAT3 signalling cooperate to define invasive potential of pancreatic cancer cells via differential regulation of the expression of S100 proteins. Br J Cancer. 2019;121:65–75.
13. Zhou L, Yu L, Wu S, Feng Z, Song W, Gong X. Clincopathological significance of KAI1 expression and epithelial-mesenchymal transition in non-small cell lung cancer. World J Surg Oncol. 2015;13:324.
14. Roig I, Dowdle JA, Toth A, de Rooij DG, Jasin M, Keeney S. Mouse TRIP13/PCH2 is required for recombination and normal higher-order chromosome structure during meiosis. PLoS Genet. 2010;6:e1001062.
15. Gemmill RM, Roche J, Potiron VA, Nasarre P, Mitas M, Coldren CD, Helfrich BA, Garrett-Mayer E, Bunn PA, Drabkin HA. ZEB1-responsive genes in non-small cell lung cancer. Cancer Lett. 2011;300:66–78.
16. Yang R, Liu Y, Wang Y, Wang X, Ci H, Song C, Wu S. Low PRRX1 expression and high ZEB1 expression are significantly correlated with epithelial-mesenchymal transition and tumor angiogenesis in non-small cell lung cancer. 2021; 100: e24472.
17. BuschEL,McGrawKA SandlerRS. The potential formarkers of epithelial-mesenchymal transition to improve colorectal cancer outcomes: a systematic review. Cancer Epidemiol Biomarkers Prev. 2014;23:1164–75.
18. Zhang M, Han Y, Zheng Y, Zhang Y, Zhao X, Gao Z, Liu X. ZEB1-activated LINC01123 accelerates the malignancy in lung adenocarcinoma through NOTCH signaling pathway. Cell Death Dis. 2020;11:981.
19. Zhao S, Gao X, Zhong C, Li Y, Wang M, Zang S. SNHG3 Knockdown Suppresses Proliferation, Migration and Invasion, and Promotes Apoptosis in Non-Small Cell Lung Cancer Through Regulating miR-216a/ZEB1 Axis. Onco Targets Ther. 2020;13:11327–36.
20. Lu S, Qian J, Guo M, Gu C, Yang Y. Insights into a Crucial Role of TRIP13 in Human Cancer. Comput Struct Biotechnol J. 2019;17:854–61.
21. Chao WC, Kulkarni K, Zhang Z, Kong EH, Barford D. Structure of the mitotic checkpoint complex. Nature. 2012;484:208–13.
22. Zhang G, Zhu Q, Fu G, Hou J, Hu X, Cao J, Peng W, Wang X, Chen F, Cui H. TRIP13 promotes the cell proliferation, migration and invasion of glioblastoma through the FBXW7/c-MYC axis. Br J Cancer. 2019;121:1069–78.
23. Agarwal S, Behring M, Kim HG, Chandrashekar DS, Chakravarthi BVSK, Gupta N, Bajpai P, Elkholy A, Al Diffalha S, Datta PK, Heslin MJ, Varambally S, Manne U. TRIP13 promotes metastasis of colorectal cancer regardless of p53 and microsatellite instability status. Mol Oncol. 2020;14:3007–29.
24. Lu S, Guo M, Fan Z, Chen Y, Shi X, Gu C, Yang Y. Elevated TRIP13 drives cell proliferation and drug resistance in bladder cancer. Am J Transl Res. 2019;11:4397–410.
25. Yost S, de Wolf B, Hanks S, Zachariou A, Marcozzi C, Clarke M, de Voer R, Etemad B, Uijttewaal E, Ramsay E, Wylie H, Elliott A, Picton S, Smith A, Smithson S, Seal S, Ruark E, Houge G, Pines J, Kops GJPL, Rahman N. Biallelic TRIP13 mutations predispose to Wilms tumor and chromosome missegregation. Nat Genet. 2017;49:1148–51.
26. Ma L, Yang R, Gu J, Jiang H, Li H. The expression of AGGF1, FOXC2, and E-cadherin in esophageal carcinoma and their clinical significance. Medicine. 2020;99:e22173.