Abstract
Background:
Our recent study observed that the expression of ubiquitin D (UBD), a member of ubiquitin-like modifier family, was upregulated in colon cancer parenchymal cells. The present study further investigated the clinical signicance of UBD in colon cancer.
Methods:
Using quantitative PCR, tissue microarray (TMA), western blot analysis and immunohistochemical stain, we evaluated UBD mRNA and protein levels in tumour tissues from patients with colon cancer at different stages and in paired adjacent normal epithelium.
Results:
Immunohistochemical detection of UBD on a TMA containing 203 paired specimens showed that increased cytoplasmic UBD was signicantly associated with depth of cancer invasion, lymph node metastasis, distant metastasis, tumour histologic grade, advanced clinical stage and Ki-67 proliferative index. Patients with UBD-positive tumours had a significantly higher disease recurrence rate and poorer survival than patients with UBD-negative tumours after the radical surgery. Stratification analysis according to tumour stage revealed UBD as an independent predictor for tumour recurrence in patients with stage II and III tumours.
Conclusion:
UBD may contribute to the progression of colon carcinogenesis and function as a novel prognostic indicator of forecasting recurrence of stage II and III patients after curative operations.
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Main
Colon cancer is a major cause of cancer morbidity and mortality and is the third most fatal malignancy worldwide (Jemal et al, 2009). In China and other economically transitioning countries, colon cancer incidence rates have increased over the last 20 years, most likely due to changes in the environment and individual life style and nutritional habits (Zhang et al, 2009). In certain high prevalence regions, colon cancer mortality has become the second leading cause of cancer death (Jiang et al, 2009). Surgical resection is the most widely used treatment for colon cancer. tumour recurrence, the main factor for the failure of colon cancer therapy following radical surgery, negatively impacts patient quality of life and frequently results in patient mortality. At present, risk assessment for colon cancer recurrence is mainly based on tumour node metastasis staging (O’Connell et al, 2004). However, clinical outcomes are quite variable, even among patients diagnosed at the same tumour stage (Galandiuk et al, 1992). Therefore, there is an urgent need to discover and utilize novel factors for predicting tumour recurrence at the time of operation that could assist in implementing individualized, directed therapeutic regimens.
The complicated process of tumour recurrence involves a number of biological changes, including deregulated expression of several oncogenes and tumour suppressor genes within tumour cell subpopulations (Grady and Carethers, 2008). Although several molecular biomarkers, including K-ras, p53 and DPC4, have been evaluated as candidate prognostic indicators in colon cancer (Paul-Samojedny et al, 2005; Duffy et al, 2007), none of these markers has been widely adopted due to conicting literature reports. The protein Ki-67 (also known as MKI67) is a cellular marker for proliferation that is detectable within the cell nucleus during all active phases of the cell cycle (G1, S, G2 and mitosis), but is not expressed in resting cells (Scholzen and Gerdes, 2000). Although Ki-67 expression is widely used as a tumour proliferative index and has been associated with colon cancer treatment response and prognosis (Salminen et al, 2005; Fluge et al, 2009), its clinical utility in predicting disease outcomes remains a topic of investigation.
We recently reported the use of laser capture microdissection and complementary DNA microarrays to explore gene expression profiles in colon cancer parenchymal cells (Fan et al, 1996). Upregulation of UBD mRNA has been observed in hepatocellular, gastrointestinal and gynecological carcinomas (Lee et al, 2003) and expression of both UBD mRNA and protein has been verified in fibroblasts and hepatic cancer cells (Raasi et al, 2001; Lukasiak et al, 2008a). In the present study, we first measured UBD expression in fresh frozen specimens of colon cancer and found that UBD mRNA and protein expression levels were higher in colon tumour tissue than in the surrounding noncancerous mucosa. These data indicated that UBD was upregulated at both transcriptional and post-transcriptional levels. Further validation by immunohistochemistry showed that 66% (135/203) of primary colon cancers had positive UBD protein staining, whereas only 6% (12/203) of normal colonic epithelium were immunoreactive for UBD. These data suggest that UBD might have an important role in the progression of colon carcinogenesis. UBD is reported to have important roles in the regulation of cell mitosis, chromosome instability, apoptosis and immune response (Raasi et al, 2001; Canaan et al, 2006; Lim et al, 2006; Ren et al, 2006).
UBD was reported to be localized in the nuclei of hepatocellular and gastric cancer cells, indicating that it might mediate transcriptional control and tumour development (Lee et al, 2003; Ji et al, 2009). However, in the present study, nuclear localization of UBD was only rarely observed. It has been reported that UBD was closely related to gastric cancer LNM and advanced tumour node metastasis stages (Ji et al, 2009). However, there are currently no published reports on the possible association of UBD expression with the clinicopathological features of colon cancer. Our results revealed significant correlations between tumour UBD overexpression and clinical stage, pT, pN stage, distant metastasis and tumour histological grade. These strong correlations suggest that UBD overexpression might promote tumour invasion and metastasis, and that UBD could possibly be used a biomarker for identification of subsets of colon cancer with a more aggressive phenotype. Our data also showed a significant association between tumour UBD expression and Ki67 index, suggesting that UBD may be involved in the increased proliferation of colon cancer cells.
It has been suggested that UBD expression may be related to other biomarkers used to predict tumour metastasis, such as CD44v6, nm23, MTA1 and matrix metalloproteases (Ji et al, 2009). Our data revealed that UBD levels were higher in tumours with associated LNM than those without LNM. Positive UBD protein expression was signicantly higher in metastatic colon cancer cells within lymph nodes than in matched primary tumours. These data suggested that increased UBD expression might correlate with the invasive behavior and metastatic processes of colon cancer.
The mechanism by which UBD contributes to tumorigenesis is not well elucidated. According to previous reports, UBD is a downstream target of p53 and its promoter is negatively regulated by p53. UBD overexpression in cancers was ascribed to transcriptional upregulation upon the loss of p53 (Zhang et al, 2006), but mutations in the UBD coding sequence have not been reported (Lukasiak et al, 2008b). The proinammatory cytokines interferon-γ and TNF-α can induce UBD expression in conjunction with an immune response within the tumour microenvironment (Lukasiak et al, 2008b). Excessive UBD protein might bind noncovalently to spindle-assembly checkpoint protein MAD2 (Liu et al, 1999; Mapelli et al, 2007), resulting in the inhibition of MAD2 function during the prometaphase stage of the cell cycle and a reduction in cell-cycle time (Lim et al, 2006). These changes could lead to genomic instability and tumour formation (Michel et al, 2001; Ren et al, 2006; Adler et al, 2009). In addition, UBD may influence caspase-dependent apoptosis in HeLa and human renal tubular epithelial cells (Raasi et al, 2001; Ross et al, 2006). Further experimentation is required to define the molecular mechanisms governing the potential role UBD expression in colon cancer progression.
Until now, valid prognostic biomarkers for colon cancer have not been established (Grady and Carethers, 2008). In the present study, patients with a high tumour UBD expression had an increased risk of tumour recurrence and shorter survival. It is interesting that the patients of stage II and III disease and tumours with positive UBD expression were at greater risk for tumour recurrence. The 5-year DFS rates for the negative and positive UBD expression groups were 93 and 80%, respectively, for stage II disease and 70.8 and 36%, respectively, for stage III disease. Although all 10 patients with stage IV disease who were included in the survival analysis had positive tumour UBD expression and post-operative disease recurrence, a statistical relationship could not be established owing to the small sample size. Although lymph node status is different between stage II and III disease and LNM is usually regarded as a poor prognosis factor for colon cancer, whether lymph node involvement should be routinely assessed remains a topic for debate (Johnson et al, 2006; Wang et al, 2008). Also, the current AJCC classification of stage III represents a heterogeneous patient population (Johnson et al, 2006). The present study has delineated the potential utility of assessing UBD expression for predicting recurrence of stage III (lymph node-positive) patients. This information could contribute to clinical decisions and help target drastic therapeutics to patient subgroups with a higher likelihood of disease recurrence and metastasis. Study limitations include the small number of patients with relatively short follow-up time. As increased UBD expression in tumour cells is a phenotypic change that indicates a preneoplastic change, it will be interesting to include colon adenomas with or with out dysplasia or intra mucosal carcinoma in future studies to validate our results showing that increased UBD expression in colon cells may indicate a preneoplastic change.
To the best of our knowledge, this is the first report to show the clinical signicance of tumour UBD expression in colon cancer. UBD was overexpressed in tumour tissue and was associated with aggressive colon cancer phenotypes. We propose that tumour UBD expression may be a clinically useful, prognostic indicator of poor patient survival, independent of tumour stage. Overexpression of UBD in stage II and III colon cancer may correlate with disease recurrence. These preliminary findings need to be verified in a larger, prospective, controlled clinical study.
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Acknowledgements
The project was supported by grants from the Key Basic Research Project of the Science and Technology Commission of Shanghai Municipality (05JC14029); Program for Outstanding Medical Academic Leader of Shanghai Municipality (LJ06024); National High Technology Research and Development Program (‘863’Program) of China (2007AA022003); and the National Natural Science Foundation of China (30700813).
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Statement of translational relevance
This is the first report on ubiquitin D association with human colon cancer progression and tumour recurrence independent of Pathological Tumour-Node-Metastasis staging. Although lymph node metastasis and advanced Pathological Tumour-Node-Metastasis stage are widely accepted prognostic indicators for colon cancer (O’Connell et al, 2004), methods for assessing lymph node involvement and heterogeneity within tumour classifications remain controversies (Johnson et al, 2006; Wang et al, 2008). Ubiquitin D was found to predict tumour recurrence in patients with lymph node-negative cancer (stage II), for whom post-operative chemotherapy is only recommended on the basis of the clinical parameters, as there are currently no validated tissue-based biomarkers of disease recurrence. These findings could be clinically translated to help to select patient sub-groups with higher metastatic potential and offer them more appropriate interventions. A major study limitation is the small number of patients with relatively short follow-up time. Larger, prospective controlled studies are required to confirm the present results.
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Yan, DW., Li, DW., Yang, YX. et al. Ubiquitin D is correlated with colon cancer progression and predicts recurrence for stage II-III disease after curative surgery. Br J Cancer 103, 961–969 (2010). https://doi.org/10.1038/sj.bjc.6605870
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DOI: https://doi.org/10.1038/sj.bjc.6605870
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