Abstract
Background: The cell cycle regulator cyclin D1 (CCND1) is thought to play a major role in the transition of the cell cycle from G1 to S-phase. It is known that cancer cells have unbalanced cell cycle regulation. However, the genetic role of CCND1 in urothelial cancer (UC) is not known. This study was conducted to explore the association between the CCND1 C1722G polymorphism and the susceptibility and progression of UC. Patients and Methods: The CCND1 genotypes of 170 patients and 249 control subjects were determined by polymerase chain reaction-restriction fragment length polymorphism and correlations with the clinical and histopathological data were evaluated. Results: The CCND1 GC or GC + CC genotypes were both more frequently observed in the UC patients than the control individuals (p=0.05 and 0.03, respectively), and people carrying the GC genotype had a 1.6-fold increased risk of UC, compared with those carrying the GG genotype (p=0.05). Also, the GC + CC genotypes had a 1.68-fold higher risk of UC compared with the GG genotype (p=0.03). In addition, the CCND1 genotype was significantly associated with ureter tumor (p=0.005) and advanced tumor status (p=0.019). No association between CCND1 C1722G genotypes and tumor grade, survival and tumor recurrence was found. Conclusion: The C allele of the CCND1 C1722G polymorphism may be a potential predictive and prognostic biomarker for advanced UC, especially ureter tumors of the upper urothelial tract.
Upper tract urothelial cancer (UC) is relatively rare in the West, where a ratio of 3:1:51 is reported for the incidence of UC of the renal pelvis, ureter and bladder, respectively (1). Due to the unusually high incidence of UC of the upper tract in Taiwan with a ratio of renal pelvis to ureter to bladder of about 1:2.08:6.72, it is valuable to study the specifics in Taiwan and then compare with findings in Western populations. The increased incidence of upper tract UC may be associated with arsenic exposure, smoking, analgesics abuse, occupational carcinogens, hypertension, long standing urinary obstructions, infection and Balkan nephropathy (2-7). A recent study has provided evidence that genetic polymorphisms may also predispose to the development of UC (8).
Cyclin D1 (CCND1) is a key regulator of G1-S cell cycle progression and overexpression of cyclin D1 is implicated in the etiology of several carcinomas including transitional cell carcinoma (TCC) of the bladder (9-11). In addition, CCND1 is considered to play an important role in the early stage of urothelial tumorigenesis and has been shown to correlate with early recurrence, tumor differentiation and clinical outcome in bladder cancer (12, 13).
The CCND1 gene is located on human chromosome 11q13. Polymorphism in CCND1 with a common G to A substitution at nucleotide 870 in the splice donor region of exon 4 of the gene has been described in 1995 (14). Several studies have shown that the CCND1 870 AA genotype has an increased risk and influences the outcome for several malignancies including bladder cancer (15-20). In literature, the polymorphism of the CCND1 locus that may affect splicing has been implicated in increased cancer risk or poor outcome (17). The CCND1 G870A has been shown to be related to poor progression in several cancer including UC (19, 20). Wang et al. (18) indicated the possibility that the CCND1 870 AA genotype confers elevated risk of bladder cancer, with more pronounced risk among non-smoking cases and for bladder cancer of higher grade and stage. Ito et al. demonstrated that the CCND1 870 AA genotype was associated with a 3.67-fold and 4.17-fold increased risk of bladder cancer (16). It is known that the CCND1 870 AA genotype influencing the alternatively spliced forms of the CCND1 mRNA and producing variant transcript-b (14). The transcript-b may have a longer half-life since it lacks the PEST (praline-serine-threonine)-rich region for rapid degradation (20), and hence which may alter the normal regulation of the cell cycle. However, another G to C polymorphism at nucleotide 1722 within CCND1 3’ untranslated region (3’ UTR) was seldom investigated (21). The first study reporting an association between CCND1 1722 polymorphism and cancer risk of squamous cell carcinoma of the head and neck was reported by Holley in 2001 (22). However, the influence of CCND1 C1722G on tumorigenesis of other carcinomas has not been reported.
Thus, this study was aimed at exploring the association between the CCND1 C1722G genotype and susceptibility to UC, and the correlation of the CCND1 C1722G genotype with clinicopathological outcomes.
Patients and Methods
Study population and clinicopathological data collection. A total of 170 patients with TCC of UC were recruited at Kaohsiung Medical University medical center between Jan 2006 to Dec 2007, all of whom were diagnosed with UC by pathological examination of specimens obtained by biopsy or surgical resection. The clinical and histopathological information and cigarette smoking history were collected from the patient charts and pathological reports, and the information was reviewed. The tumor stage was assigned according to the TNM staging system (23), and the pathological grade was determined according to the World Health Organization criteria (24). Two hundred and forty nine healthy individuals, matched with the patients age, admitted to the same hospital for a health checkup and who had no previous diagnosis of urological neoplastic disease or other malignancy were enrolled as controls. However, no information on smoking status was obtained from the control subjects. During the recruitment period, all the subjects enrolled provided informed consent and the Human Research Committee of the participating hospital approved this study. This study was also reviewed by the Institutional Review Board (IRB) of Kaohsiung Medical University with the approval number of KMU-IRB-950195.
Genotyping conditions. Genomic DNA for analysis was extracted from blood specimens using proteinase K digestion following phenol-chloroform extraction as described previously (25). Genotyping for CCND1 C1722G of all the subjects was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay as previously described (26-29). The 159 bp fragments containing the polymorphic nucleotide were amplified using the forward primer 5’-CTCTTGGTTACAGTAG CGTAGC-3’ and reverse primer 5’-ATCGTAGGAGTGGGA CAGGT-3’. The following cycling conditions were performed: 5 min of initial denaturation at 95°C, 35 cycles of 30 sec of denaturation at 95°C, 30 sec of annealing at 54°C and 1 min of elongation at 72°C and 7 min of final extension at 72°C. The PCR products were further digested with the restriction enzyme Hae III (New England, Biolabs, Beverly, MA, USA), and then visualized by ethidium bromide stained 3% agarose gel electrophoresis with the help of UV light. On digestion with Hae III, the PCR product arising from the G allele was cut into fragments of 111, 26 and 22 bp, whereas the C allele was cut into fragments of 137 and 22 bp (Figure 1a). Sequences were confirmed by direct sequencing of 10% of the samples, and the results were 100% concordant.
Statistical analysis. To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotype frequencies of CCND1 single nucleotide polymorphisms in the control subjects from those expected under the Hardy-Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson's Chi-square test or Fisher's exact test (when the expected number in any cell was less than five) was used to compare the distribution of the CCND1 genotypes between cases and controls. Cancer risk associated with the genotypes was estimated as odds ratio (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression. The data was recognized as significant when the statistical two-tailed p-value was less than 0.05.
Results
The characteristics of the 170 patients and 249 control subjects are shown in Table I. No significant difference was found between the UC patient and the control groups in the distribution of gender or age (p>0.05).
The CCND1 C1722G genotypes are also presented in Table I. The electrophoregram of PCR-RFLP of CCND1 C1722G is presented in Figure 1b. Both allele distribution frequencies of the patient and the control groups fitted Hardy Weinberg equilibrium. Compared with the CCND1 1722 GG genotype, the patients with the GC genotype had a 1.6-fold increased risk of UC (p=0.05; OR=1.602, 95%CI=0.996-2.578). The patients with the GC+CC genotypes had a 1.68-fold increased risk of UC compared with individuals with the GG genotype (p=0.03; OR, 1.677, 95% CI, 1.046 to 2.687). It seemed that the C allele is a risky genetic factor for UC.
The 170 UC patients comprised subjects suffering from renal pelvic (29, 17.0%), ureter (28, 16.5%), bladder (93, 54.7%) and multi-site tumors (20, 11.8%). The stratification analysis revealed that the CCND1 C1722G genotypes had a significant association with ureter tumors, but not the other tumors (p=0.005; Table II). Again, the C allele seemed to be a genetic risk factor for ureter tumors. The association between CCND1 C1722G genotypes and pathological state and clinical outcome were also examined in this study. Out of the 170 UC patients, 22.9% were of organ-involved advanced tumors (≥pT3) and 60% were of high-grade (G3) tumors. A significant association between CCND1 1722 genotype and advanced tumors was found (p=0.019; Table II), indicating that especially the CC genotype may be associated with tumor aggressiveness. However, no statistically significant differences were found among the different CCND1 C1722G genotypes with tumor grade (p=0.879), survival (p=0.648) or tumor recurrence (p=0.313) (Table II).
(a) Restriction map of CCND1 1722 genotypes. On digestion with Hae III, the PCR product arising from the G allele was cut into fragments of 111, 26 and 22 bp, whereas the C allele was cut into fragments of 137 and 22 bp. (b) Electrophoregram of PCR-RFLP of CCND1 1722. Lane 1, 50 bp MW marker; Lane 2, 159 bp PCR product; Lane 3, GG homozygote; Lane 4, GC heterozygote; Lane 5, CC homozygote.
Since smoking is a well-known environmental factor for UC (30-32), the gene-environment interaction of the CCND1 C1722G genotype and smoking status was also investigated. No differential genetic distribution of the CCND1 C1722G genotypes between the smoking and nonsmoking groups was shown (p=0.153, Table III). Furthermore, the association between the CCND1 C1722G genotype and the tumor grade, tumor stage, survival and recurrence was not influenced by smoking status (Table III).
Discussion
Among the limited reports of the CCND1 C1722G polymorphism, that of Holley et al. (22) was very typical and showed that the CCND1 1722 GC and CC genotype was more frequently observed in the head and neck cancer group than the control group, suggesting that individuals with CCND1 1722 GC or CC genotypes were at a 1.68-fold increased risk of head and neck cancer compared with the GG genotype. However, their sample size was limited with only 69 cases. In the present study all types of upper tract UC were included and the sample size was much larger. The CCND1 1722 variant C allele was found to be associated with an increased risk of ureter tumor, but not bladder cancer or the other types (Table II). Furthermore, the CC genotype in particular was associated with advanced tumors, indicating an important influence of the CCND1 1722 genotype on the tumor aggressiveness (Table III).
Characteristics and CCND1 C1722G genotypes among UC cases and healthy controls.
Characteristics of CCND1 1722 polymorphisms with tumor location, pathological grade and stage in patients with UC.
The most established risk factors for bladder cancer are occupational exposure to certain arylamines and exposure to cigarette smoke. Tobacco consumers have been shown to have a two to five fold higher risk than nonsmokers (19). However, no significant difference was found in the subjects with or without a history of smoking in the present study, indicating that CCND1 1722 genotypes influenced the risk of suffering from UC, independently of tobacco consumption.
A high incidence of upper urinary tract UC has been reported from the endemic area for “blackfoot disease” of Taiwan, and arsenic contaminated water was considered to be the reason for such high prevalence (33, 34). Nevertheless, water hygiene has improved in recent years, and yet the incidence of upper urinary tract UC remains high. A high incidence of upper urinary tract UC also occurs in people who grow-up overseas. Hence factors other than arsenic contamination are suggested to contribute to the unusually high incidence, but so far, no explanation has been found. The present study has revealed that genetic polymorphism in CCND1 C1722G may play an important role in increasing the risk of upper tract UC. However, it is still unclear whether the etiological effect of the CCND1 polymorphism is connected to one or more additional environmental factors in causing the high occurrence of upper tract UC in Taiwan. In addition, how the CCND1 C1722G polymorphism influences protein expression is still not clear since its location is in the 3’UTR.
Smoking-associated risk on the tumor grade, tumor stage, survival and recurrence in patients with variant CCND1 1722 polymorphisms.
As UC cases are rare and it was not easy to collect data within a limited time period, as many age- and gender-matched controls as possible were enrolled to strengthen the analyzing power of the case-control study. The borderline lack of significance of both odds ratios and p-value encouraged us to confirm this preliminary finding with more case samples in the future, and also studies in Western countries are warranted.
In conclusion, the CCND1 1722 CC genotype is associated with a higher risk of UC, and is especially associated with ureter cancer and aggressive tumors. This finding warrants giving attention to individuals with the GC or CC genotype in relation to disease occurrence. Furthermore, the C allele of CCND1 C1722G may be used as an accessory marker for susceptibility and disease progression of UC.
Acknowledgements
The work was supported by a grant from the Kaohsiung Medical University Hospital (KMUH 5D-43).
- Received November 3, 2010.
- Revision received February 2, 2011.
- Accepted February 3, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
