Abstract
Background/Aim: Ubiquitin-conjugating enzyme H10 (UBCH10) is required in the cell-cycle transition from metaphase to anaphase. Therefore, we investigated whether its expression level in cancerous esophageal lesions affected prognosis of patients with esophageal squamous-cell carcinoma. Materials and Methods: Paraffin-embedded tissue samples from 121 patients with esophageal squamous cell carcinoma were stained with antibody to UBCH10 for immunohistochemical analysis. Results: UBCH10 was expressed in cancerous and dysplastic lesions, but not in normal tissue. Patients were grouped according to expression: High (N=33) or low (N=88), depending on the staining pattern. There were significant differences between the groups in terms of invasion into lymphatic vessels, number of metastatic lymph nodes, TNM classification, and stages, as well as in survival: the 50% survival rate in the high expression group was 2.3 years, whereas it was 9.9 years for the low-expression group (p<0.0001). Even with multivariate adjusting for stage 0 to stage IV using the Cox proportional hazard model, patients belonging to the high-expression group had a poor prognosis (Hazard ratio=2.5; 95% Confidence Interval=1.3-4.5; p=0.004). Conclusion: High protein expression of UBCH10 is a marker of poor prognosis in esophageal squamous cell carcinoma.
Esophageal squamous cell carcinoma is an aggressive type of cancer, with a poor prognosis (1, 2), although survival continues to improve (3). Novel molecular-targeting agents are desired to further improve the prognosis of patients with esophageal cancer. Recently, a variety of candidate molecules have been listed by our and other groups, such as endothelin (4), Cluster of differentiation 147, metalloproteinase-2 (5), vascular-endothelial growth factor (6), Fas expression (7), C-X-C chemokine receptor 4 (8), Mut-L-homologon-1/Mut-S-homologon-2 (9), endoglin (10); these have not yet succeeded in drug development.
To screen for key molecules to aid the prediction of prognosis of patients with esophageal cancer, we carried-out and reported gene expression profiling in esophageal cancer: we found that RNA expression of ubiquitin-conjugating enzyme was up-regulated in patients with a poor outcome (11). Ubiquitin is a small (8 kDa) protein that is transferred first to the ubiquitin-activating enzyme E1, then to the ubiquitin-conjugating enzyme E2, and finally to the target protein by E3 ubiquitin ligase, which leads to the formation of a poly-ubiquitin chain that is recognized and destroyed by the 26S proteasome (12). This ubiquitin–proteasome system plays an important role in cell-cycle regulation through timely and specific degradation of cyclins and kinase inhibitors (13, 14). Thus, aberrant ubiquitination may trigger not only oncogenic transformation of cells but also cancer progression to more advanced stages. Recently, it was shown that the RNA of one of the ubiquitin-conjugating enzymes, UBCH10, was highly expressed in a variety of human tumors but not in normal tissues: ectopic expression of UBCH10 promoted cell growth and malignant transformation (15, 16). In turn, decreased UBCH10 expression, triggered by small interfering RNAs, significantly reduced the growth of cancer cells (17). UBCH10 has been cloned as a cyclin-selective ubiquitin carrier protein, which is required in the cell-cycle transition from metaphase to anaphase by destroying cyclin A and cyclin B (18, 19) in association with the anaphase-promoting complex (20). These findings suggest that overexpression of UBCH10 may not merely be a consequence of the accelerated cell-cycle progression non-specifically observed in cancer cells but in fact an essential pathway in their development.
Recently, higher expression of UBCH10 was investigated in a variety of malignancies, such as carcinoma of the thyroid (21), breast (22), ovary (23) and colorectum (24). However, to our knowledge there have been no reports to demonstrate an association between levels of UBCH10 protein expression and clinical prognosis of patients with esophageal cancer.
Thus, we were motivated to study associations between expression patterns of UBCH10 protein in paraffin-embedded specimens of esophageal squamous-cell carcinoma and, retrospectively, the patient's prognosis.
Materials and Methods
Patients. Between February 1995 and November 2003, 147 patients with esophageal tumors and treated with videoscopic or open surgery were listed on file in the Department of Pathology, Clinical Service, Jikei University Hospital. Patients treated with endoscopic mucosal resection were not included. Of the 147 patients, 26 were excluded from this study: paraffin-embedded specimens were not available from seven patients, 10 patients had adenocarcinoma, six patients had carcinoma in situ, and three were lost to follow-up during the first year after surgery. Thus 121 patients with esophageal squamous-cell carcinoma for whom specimens and clinical information could be obtained were included in this study.
Clinical information was abstracted from surgical and clinical charts. Based on preoperative staging diagnosed by endoscopic biopsy and multidetector row computed tomography and endoscopic ultrasonography, nine patients with clinical stage III and IV cancer were treated with chemoradiotherapy starting two months before surgery. Patients were admitted and underwent chemotherapy (cisplatin 4 mg/m2/day five days/week for four weeks plus 5-fluorouracil 750 mg/m2/day for 24 h by continuous infusion for 28 days) and local radiation (40 Gy, 2 Gy/day for five days/week for four weeks) before operation. TNM classification and stages were determined by pathological findings according to the Japanese Society of Esophageal Diseases' Guidelines for the Clinical and Pathologic Studies on Carcinoma of the Esophagus (49). Some patients received radiotherapy to improve their quality of life during disease recurrence. Patients were periodically (every one to three months) examined on an outpatient basis to make sure disease had not relapsed; examinations consisted of standard tests, including endoscopy and computed tomography of the chest and abdomen.
Pathological specimens. Specimens were obtained by endoscopic mucosal resection or surgery. Formalin-fixed, paraffin-embedded specimens of esophageal tumors were retrieved from the Department of Pathology and processed for conventional histological assessment by hematoxylin staining. Only patients with squamous cell carcinoma of the esophagus confirmed by two or more board-certified pathologists were included. Patients with all other forms of carcinoma, including partly adenomatous lesions, were excluded. All specimens were free of cancer invasion of the tumor margin and cases of carcinoma in situ were excluded. Histological features and the extent of the lesions, invasion into lymphatic or blood vessels, intra-mural metastasis, and lymph node metastasis were evaluated. Pathological diagnosis and classification were made according to the Japanese Society of Esophageal Diseases' Guidelines for the Clinical and Pathologic Studies on Carcinoma of the Esophagus (49). Lymphatic vessel invasion was regarded as definite when cancer cells were detected in thin-walled, endothelium-lined spaces containing no red blood cells and occasional lymph fluid. Similarly, blood vessel invasion was defined by the presence of cancer cells and red blood cells within round or ovoid endothelium-lined spaces surrounded by a layer of smooth muscle (25-27). Intramural metastasis was defined as evidence of tumor within the esophageal wall that was not directly related to the primary tumor (28, 29). Histologic grades of differentiation were assigned according to whether a tumor was well, moderately, or poorly differentiated (30).
Immunohistochemical staining for UBCH10. UBCH10 protein was detected with the rabbit polyclonal antibody against UBCH10 (1:150 dilution; CHEMICON International, Temecula, CA, USA) (31), which was visualized using the Histofine Simple Stain PO(M) kit (Nichirei, Tokyo, Japan) according to the instruction manual (32). Briefly, endogenous peroxidase was blocked with 10% hydrogen peroxide for 10 minutes. Antigen retrieval was carried out by incubation in 0.01 M citrate buffer (pH 6.0) at 95°C for 30 minutes. To reduce nonspecific binding, the sections were incubated with 10% goat serum for 30 minutes at room temperature. Polyclonal antibody to UBCH10 was incubated with sections at 4°C overnight. For each case, a corresponding section was incubated with nonimmune rabbit serum as a negative control. The nuclei were counterstained with Mayor's hematoxylin.
The staining in both cancerous and dysplastic lesions was classified into the following four patterns by the percentage of positive cells in the lesion: no staining, 0% positive cells in the lesion; spotty staining, more than 0% and fewer than 10% positive cells; partial staining, at least 10% and fewer than 30% positive cells; and diffuse staining 30% and more positive cells. The examiners were blinded to patients' clinical information when assigning staining patterns. Two investigators evaluated the staining levels independently, and discordant evaluations were adjusted using connected microscopes.
Statistical analysis. Student's t-test, the Mann–Whitney U-test and the chi-square test were used to evaluate the differences between high and low UBCH10 expression groups in cancerous and dysplastic lesions. Survival curves of the patients were compared using the Kaplan–Meier method and analyzed by the log-rank test. Cox proportional hazard models were fitted for multivariate analysis, and hazard ratios (HRs) and 95% confidence intervals (95% CIs) were computed. The significance of adding UBCH10 grouping to staging as a prognostic factor was evaluated by a likelihood ratio test. All statistical analyses were performed using STATA 8.0 (STATA Corporation, College Station, TX, USA). A value of p less than 0.05 was considered to be statistically significant.
Results
Patients' characteristics. Patients ranged in age from 43 to 83 years (mean±SD, 60±8 years) and there were more men (n=104) than women (n=17). All patients were followed from day 16 after surgery up to a maximum of 3787 days (median=1148 days). Forty-seven patients died of esophageal carcinoma and 11 of other causes, including other types of cancer. Thus, 63 patients were alive and 58 had died at June 15th 2009.
Patients' characteristics divided by staining pattern of cancerous/dysplastic tissue for Ubiquitin-conjugating enzyme H10.
Immunohistochemistry for UBCH10 protein expression. Cancerous lesions, dysplastic lesions around cancerous lesions, and normal esophageal mucosal tissue were stained for UBCH10. UBCH10 protein was located in the cytoplasms or nucleus. The distribution of UBCH10-positive cells in dysplastic lesions was similar to that of proliferating cells. The contrast between positive and negative cells was quite clear; thus, we divided staining patterns into either cancerous or dysplastic lesions and into three categories: diffuse, partial and spotty (Figure 1). Forty-six (38%) cancerous lesions stained with UBCH10 antibody exhibited a diffuse pattern, whereas 75 (62%) had a partial or spotty pattern; none had no staining. In dysplastic tissue, a diffuse or partial pattern was observed in 74 (61%) lesions and a spotty pattern in 47 (39%). In contrast, normal esophageal mucosa was not stained with UBCH10 antibody (not shown). Therefore, we classified the 121 patients into two groups: the high UBCH10 expression group (n=33), which exhibited both a diffuse pattern in cancerous tissue and diffuse/partial pattern in dysplastic lesions; and the low UBCH10 expression group (N=88), which included all other patients.
UBCH10 expression and clinicopathological variables. Clinicopathological variables were compared between high and low UBCH10 groups (Table I). Compared with to the low UBCH10-expression group, there was more invasion into lymphatic vessels, more frequent metastatic lymph nodes, more advanced node and metastasis stage in TNM classification, and more advanced clinical stage in the high-expression group, but no increase in other factors, including invasion into vascular vessels and tumor infiltration in TNM classification. Nine patients underwent chemoradiotherapy as a preoperative treatment, which did not affect UBCH10 expression.
UBCH10 expression and relapse-free survival. Firstly, Kaplan–Meier survival curves were created based on tumor stage (0, n=22; I, n=16; II, n=33; III, n=34; IV, n=16) for the 121 patients (Figure 2). As expected, patients with more advanced cancer had a shorter median relapse-free survival period: 50% survival in stage II, 8.1 years; stage III, 3.4 years; stage IV, 1.5 years (log-rank test: p<0.0001). Next, Kaplan–Meier survival curves were drawn based on UBCH10 expression (Figure 3). Patients with high UBCH10 expression did not survive as long (log-rank test: p<0.0001): 50% survival in high UBCH10 expression group was 2.3 years, whereas it was 9.9 years in the low UBCH10 expression group. Finally, by stratifying each stage, Kaplan–Meier survival curves were drawn based on high and low UBCH10 expression. Only in stage III was the difference between high and low expression of UBCH10 significant (log-rank test: p=0.0080): 50% survival for patients with high UBCH10 expression was 2.9 years, whereas it was 8.3 years for those with low UBCH10 expression (Figure 4).
The Cox hazard model. Cox regression analysis was performed to determine whether the prognostic significance of grouping by high- and low-UBCH10 expression pattern improved the accuracy of staging in predicting relapse-free survival. Even with multivariate adjusting by stage, patients belonging to the high UBCH10 expression group had a poor prognosis (HR=2.5; 95% CI=1.3-4.5; p=0.004). Grouping by UBCH10 expression levels significantly improved the model of ordinal staging alone (log-likelihood test: p=0.0041) (Table II).
Ubiquitin-conjugating enzyme H10 (UBCH10) protein expression in cancerous and dysplastic lesions in esophageal squamous-cell carcinoma. Staining patterns were divided into three: diffuse: ≥30% positive cells; partial: ≥10% and <30% positive cells; and spotty: <10% positive cells in the lesion), which allowed the classification of 121 patients into Imo groups: the high UBCH10 expression group (N=33), with both diffuse staining in cancerous tissue and diffuse/partial staining in dysplastic lesions; and the low UBCH10 expression group (N=88), which included all other patients. ×100.
Discussion
In this study, we have demonstrated an association between high levels of UBCH10 protein expression in esophageal tissues and poor prognosis in patients with esophageal squamous cell carcinoma. Our detection of UBCH10 protein in cancerous lesions and some dysplastic lesions surrounding cancerous tissue, but not in normal tissue, is consistent with previous reports that UBCH10 is detectable on cancer cells but not normal cells (15, 16). Increases in ubiquitin, small ubiquitin-related modifier 1, ubiquitin-conjugating enzyme 9 and p53, as well as decreased levels of p27 (KIP1), were observed in dysplastic lesions and carcinoma in situ in cystitis induced by low-dose ionizing radiation (33). Moreover, differential gene expression profiles in rats with Barrett's esophagus and esophageal adenocarcinoma induced by gastro-duodenoesophageal reflux showed that the ubiquitin-conjugating enzyme was up-regulated (34). Thus, aberrant ubiquitination might already be initiated in the early stages of malignant transformation.
Cox hazard model by Ubiquitin-conjugating enzyme H10 expression level and staging.
In the present study, invasion into lymphatic vessels, as well as metastatic lymph nodes and higher N stage were more frequently observed in the high UBCH10 expression group. In esophageal squamous cell carcinoma, lymph node metastasis is the most important prognostic factor (35). These results suggest that overexpression of UBCH10 in cancer cells might accelerate migration via the lymphatic stream. Although we have shown that levels of free ubiquitin and multi(poly)-ubiquitin chain consisting of ubiquitin monomers linked to each other covalently are up-regulated in colorectal cancer and correlate with the pathological findings (36), there are few reports showing an association between pathological findings and the expression of ubiquitin-conjugating enzymes in cancer. Currently, TNM staging is the best way to predict the prognosis of patients with esophageal carcinoma (37). However, the combination of immunohistochemical examination for UBCH10 with ordinal TNM staging has significantly improved diagnostic accuracy.
Kaplan–Meier survival curves based on cancer stage.
Genomic amplifications in chromosome 20q have been reported in a variety of cancer types (38). In a study of 41 primary tumors of esophageal squamous cell carcinoma, patients with 20q gain including 20q12-13 exhibited lymph node metastasis, as well as extensive lung metastasis, pleural effusion and liver metastasis, and a poorer prognosis compared to patients with no 20q gain (39). The International Radiation Hybrid Mapping Consortium mapped the UBCH10 gene to chromosome 20q13.12 (sts-T86744). These two facts and our results reported here suggest that a gain in chromosome 20q13.12 in esophageal squamous cell carcinoma might lead to up-regulation of UBCH10 gene expression and is thus associated with a poor prognosis.
The main future goals are not only to predict patients' prognoses, but also to develop a drug that specifically targets ubiquitin-conjugating enzymes and their downstream pathways. Recently, the proteasome inhibitor bortezomib was shown in clinical trials to be effective for multiple myeloma (40, 41), as well as non-Hodgkin's lymphoma and mantle-cell lymphoma (42, 43). Although bortezomib is not effective for all kinds of carcinomas (44-48), research into the mechanisms of the ubiquitin/proteasome system in malignant transformation should be continued, as it is an extraordinary and important field.
Kaplan–Meier survival curves based on high and low expression of Ubiquitin-conjugating enzyme H10 protein.
Kaplan–Meier survival curves based on high and low expression of Ubiquitin-conjugating enzyme H10 protein for patients with stage III cancer only.
In conclusion, these results suggest that high UBCH10 protein expression might be useful as a marker of poor prognosis in esophageal squamous cell carcinoma.
Acknowledgements
This study was supported by a Grant in Aid for Scientific Research (Japan. Research Project Number:17591442)
- Received December 15, 2013.
- Revision received January 19, 2014.
- Accepted January 20, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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