- Top of page
- MATERIALS AND METHODS
It is estimated that 73,510 individuals living in the United States will develop urinary bladder cancer in 2012 and 14,880 will die of the disease . Data from comparative studies have demonstrated that bladder cancer affects men three to four times more often than women, while female patients tend to present with more aggressive tumor than male patients [1, 2].
Bladder cancer is one of the first neoplasms recognized to be caused by exposure to carcinogenic compounds, such as industrial chemicals and cigarette smoke. Aromatic amines, well-known industrial bladder carcinogens, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of the most potent and abundant procarcinogens derived from tobacco and its smoke, are glucuronidated in the liver and excreted into the urinary system [3, 4]. It has been shown that UDP-glucuronosyltransferases (UGTs), belonging to the superfamily of major phase II drug metabolism enzymes, play a vital role in catalyzing the glucuronidation of carcinogens, including aromatic amines and NNAL .
Human UGTs are composed of UGT1A, UGT2A, and UGT2B, based on gene sequence homology . In addition to the liver, the expression of UGTs has been detected in other organs, including the aerodigestive tract , gastrointestinal tract , and kidney . It has also been reported that normal bladder expresses all the UGT subtypes except UGT2B17  and that, compared with normal urothelium, UGT1A expression is down-regulated in several bladder cancer tissue samples [11, 12]. Recently, a genome-wide association study revealed a linkage between UGT1A gene locus and bladder cancer susceptibility . Indeed, UGT1A, rather than the UGT2 family, has been suggested to contribute to metabolism of aromatic amines . Down-regulation of Ugt1a, although there is no clear consensus about the functional homology between human UGT and mouse Ugt, was associated with higher incidence of chemically induced bladder cancer in mice . Thus, UGT1A, consisting of nine functional proteins and four pseudogenes generated by alternative splicing, and Ugt1a, consisting of nine functional protein and five pseudogenes , are likely key enzymes involved in bladder carcinogenesis.
Although excessive exposure to industrial chemicals and cigarette smoke may have contributed to male dominance in bladder cancer, men remain at a substantially higher risk of bladder cancer than women even after controlling for these carcinogenic factors . We have shown molecular evidence for this gender-specific difference by implicating androgen receptor (AR) signals in bladder cancer development . There is also increasing evidence suggesting that other steroid hormone receptors are involved in bladder carcinogenesis and cancer progression . Recently, we showed that dihydrotestosterone (DHT) reduced the expression of UGT1A subtypes via the AR pathway in normal urothelial cells . In the previous report , we also showed that UGT1A was down-regulated in high-grade urothelial carcinoma tissues and strong expression of UGT1A correlated with favorable prognosis. However, the results were not conclusive presumably due to a relatively small number of cases (n = 24) including no low-grade tumors. The purpose of the current study is to validate the previous findings in larger patient cohorts with longer follow-up. We also assessed possible associations of UGT1A expression with AR and estrogen receptor (ER) signals in non-neoplastic urothelium and urothelial carcinoma.
- Top of page
- MATERIALS AND METHODS
UGTs are enzymes that contribute to detoxifying bladder carcinogens including aromatic amines and NNAL . A recent genome-wide association study identified UGT1A as one of the susceptibility loci of bladder cancer . It has also been reported, using mouse bladder cancer models, that a chemical carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) reduces Ugt1a expression and that knockout of Nfr2 (nuclear factor-like 2) results in decreased Ugt1a and increased incidence of BBN-induced bladder cancer . Together with other findings demonstrating down-regulation of UGT1A in bladder cancer [11, 12, 19], UGT1A likely plays an important role in prevention of bladder carcinogenesis.
Down-regulation of UGT1A mRNA was first reported in 9 of 10 bladder tumor samples (complete loss in four tumors), compared with matched benign bladder tissues . Then, the same group immunohistochemically stained for UGT1A in 19 bladder tumors and found 6 of the tumors, mostly high-grade and/or invasive, were virtually negative for UGT1A whereas benign tissues consistently expressed it . In our previous study involving 24 high-grade urothelial carcinomas , we showed down-regulation of UGT1A in 13 (54%) tumors as well as an association of strong UGT1A staining with a lower progression rate. In the current study, we analyzed 145 bladder tumors that included PUNLMPs and low-grade carcinomas and found inverse correlations between UGT1A levels versus tumor grade or pT stage. We also showed that decreased UGT1A expression was strongly associated with progression in high-grade non-muscle-invasive tumors and disease-specific mortality in muscle-invasive tumors. In addition, patients with UGT1A-negative muscle-invasive tumor tended to have a risk of disease progression. Multivariate analysis further revealed that loss of UGT1A in muscle-invasive tumors was an independent prognosticator of disease-specific mortality. In contrast, no significant correlations were seen between UGT1A expression status and recurrence/progression of low-grade tumors or progression of high-grade non-muscle-invasive tumors, probably because only few of low-grade and/or non-muscle-invasive tumors were negative or weakly positive for UGT1A. Of note was that, as we recently showed in the identical 68 cases undergoing lymph node dissection , pN status was not a prognostic factor (P = 0.284–0.929) in our cohort. Our results thus suggest that UGT1A has protective effects on not only bladder cancer development but also tumor progression.
Epidemiological and clinical evidence indicates that men have a substantially higher risk of bladder cancer, whereas women with bladder cancer have less favorable prognosis [1, 2]. Recent experimental data have suggested the involvement of AR and ER signaling pathways in bladder tumorigenesis and cancer progression and, therefore, urothelial carcinoma, like prostate and breast cancers, is considered as an endocrine-related neoplasm . Nevertheless, no significant gender difference in the expression of AR, ERα, or ERβ in bladder tumors has been found [21, 24-28]. In the current study, although there were no statistically significant differences in UGT1A expression between male versus female tissues (both benign and malignant bladders), strong positivity of UGT1A was more often detected in male tumors than in female tumors (P = 0.064). It was likely that UGT1A was considerably down-regulated in potentially aggressive tumors from females compared with male tumors, but UGT1A levels in low-grade and/or superficial tumors were similar between genders of the patients. In addition, UGT1A tended to co-express with ERα and to express inversely with ERβ in bladder tumors, especially female tumors, whereas no significant correlation between expressions of UGT1A versus AR was found. In previous studies [21, 25-27], expression levels of AR or ERα alone in bladder cancer exhibited no prognostic significance. However, some of these studies showed an association between higher ERβ expression and poorer patients' outcomes [21, 27]. Furthermore, in a study using specific small interfering RNAs and selective agonists for ERα and ERβ, estrogens were shown to promote bladder cancer cell proliferation through the ER pathways . Based on these findings, we hypothesized that in bladder cancer cells estrogens down-regulated UGT1A which may have a protective role in tumor progression. As expected, E2 repressed UGT1A expression at both mRNA and protein levels in all three bladder cancer lines expressing ERβ, but not ERα, and TAM antagonized the E2 effect. Indeed, up to 76% of bladder tumors were reported to be positive for ERβ, while positive rates of ERα in immunohistochemical studies ranged from 1% to 27% [18, 21, 24, 25, 27, 30, 31]. In AR-positive UMUC3 cells, DHT did not alter the UGT1A level. Thus, estrogen-induced ERβ signals may promote bladder cancer progression via down-regulating UGT1A. Further analyses of UGTs in vitro and in vivo are necessary to determine its biological functions in bladder cancer growth, which may provide new insights into not only prognosis and progression but also novel therapeutic approaches.
We previously proposed that down-regulation of UGT1A by androgens in urothelial cells was a potential mechanism for male dominance in bladder cancer development . We also showed significantly lower levels of Ugt1a in male mouse bladders, which could be augmented by bilateral orchiectomy, than in those from females . In addition, the levels of Ugt1a in castrated males or AR knockout males were still lower, compared with intact female mice. As shown in the liver , our previous results suggested the role of estrogen/ER signals in regulation of Ugt1a in the bladder. In the present study, we further demonstrated that bilateral ovariectomy led to decreases in Ugt1a expression in mouse bladders and estrogen supplement in ovariectomized mice restored the levels of Ugt1a. Consistent with these findings, E2 up-regulated the expression of UGT1A mRNA and protein in SVHUC normal urothelial cells expressing ERβ, but not ERα, which was at least partially abolished by an ER antagonist. Thus, it is likely that both androgens/AR and estrogens/ER signals are able to modulate UGT1A expression in the bladder, which may in turn affect the susceptibility to bladder carcinogens.
It is worth pointing out that bladder cancer is primarily a disease of advanced age. Thus, most of female bladder cancers are diagnosed after menopause in that serum levels of estrogens may not be different from those in male patients. Additionally, as aforementioned, studied have failed to show differential expression of ERs in bladder tumors between genders or ages of the patients [21, 24, 25, 27]. These may not readily support important roles of estrogens and ER signals in bladder carcinogenesis and cancer progression. Although menopausal status of female patients was uncertain in our study, there was no significant difference in UGT1A levels in the younger cohorts compared with their older counterparts, possibly due to a relatively small number of women aged ≤50 (n = 4) or ≤55 (n = 8) yr. However, it is still possible that high levels of UGT1A maintained by estrogens in women until menopause have reduced the exposure to bladder carcinogens excreted in the urine, which subsequently prevents or delays the development of bladder cancer even after menopause. We also showed that UGT1A levels, predominantly in female tumors, were associated with expression status of ERs and the prognosis. While estrogen levels in our cohort of patient were undetermined and might have varied, these results along with our in vitro data suggest the involvement of ER-mediated UGT1A signals in the growth of bladder cancer cells.
In conclusion, we showed down-regulation of UGT1A expression in bladder cancer and its inverse correlations with tumor grades and stages. Loss of UGT1A was an independent prognosticator for cancer-specific mortality in patients with muscle-invasive tumors. In addition, estrogen was found to up-/down-regulate UGT1A expression in normal urothelium/bladder cancer, respectively. These results not only suggest protective roles of UGT1A in both the development and progression of bladder cancer but may also provide potential underlying mechanisms responsible for gender-specific differences in the incidence and outcomes of bladder cancer. Further functional analyses of UGTs in bladder cancer are necessary to determine their biological significance.