Background
Bladder cancer is one of the most lethal urological malignant tumors worldwide[
1]. Although the treatment of bladder cancer has improved greatly in recent years, the incidence of this disease is gradually increasing [
2]. More than half of the patients with bladder cancer have advanced stage disease with very poor prognosis[
3]. Although some environmental factors and genetic factors have been associated with bladder cancer [
4‐
6], the molecular mechanisms involved in the initiation and progression of bladder cancers remain unclear.
The
BMI1 gene was first isolated as an oncogene that cooperated with c-Myc in generating lymphomas in a murine model[
7,
8]. It is a transcriptional repressor belonging to the Polycomb-group (PcG) family of proteins involved in axial patterning, hematopoiesis, regulation of proliferation, and senescence [
9‐
11]. The
BMI1 gene also was reported to immortalize bone marrow stromal cells and cementoblast progenitor cells, albeit in combination with other oncogenes [
12,
13]. In addition, the
BMI1 gene plays an important role in cell proliferation and tumor progression [
14‐
18]. The
BMI1 gene is widely expressed in diverse human tumors, including lymphomas, non-small cell lung cancer, B-cell non-Hodgkin's lymphoma, breast cancer, colorectal cancer, and neuroblastoma [
14‐
21], and has been shown to be a useful prognostic marker in myelodysplastic syndrome and many cancers, including nasopharyngeal carcinoma and gastric cancer [
14‐
21].
However, there is no published report on the expression of Bmi-1 in bladder cancer. In this study we aimed to explore the expression of Bmi-1 protein and its clinical significance in human bladder cancers.
Methods
Patients and tissue specimens
For RT-PCR and Western blot analysis, we collected 14 paired transitional cell bladder cancers and adjacent normal tissues from the patients who underwent surgery between March 2008 and April 2008. In addition, 137 paraffin-embedded samples of transitional cell bladder cancer and 30 specimens of adjacent normal bladder tissue were collected between 2000 and 2003 for immunohistochemical assay. All tumors were histologically and clinically diagnosed by the cancer center of Sun Yat-sen University. For the use of these clinical materials, prior patient consent, and approval from the institute research ethics committee (Approval number: YP2008063) were obtained.
The disease stage of each patient was classified or reclassified according to the 2002 AJCC staging system[
22]. The 137 patients included 115 males and 22 females from 14 to 72 years (mean, 56 years). Of these patients, 41 patients underwent radical cystectomy, 20 patients underwent partial cystectomy, and 76 patients underwent TURBT (transurethral resection of bladder tumor). After partial cystectomy and TURBT, mitomycin C was used in intravesical therapy as weekly intravesical injection beginning within 24 hours after surgery. Thirty specimens of adjacent normal bladder tissue distant from the tumor were included for these patients, as well. The median follow-up time for overall survival was 56 months for patients still alive at the time of analysis, ranging from 11 to 86 months.
Reverse transcription – PCR analysis
Total mRNA was purified using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), and 1 μg of each sample was reverse transcribed using TIAN Script Kit (TIANGEN, Beijing, China). The BMI1 sense primer was 5'AGCAGAAATGCATCGAACAA-3', and the antisense primer was 5'CCTAACCAGATGAAGTTGCTGA-3'. For the β-actin gene, the sense primer was 5'CCCCTGGCCAAGGTCATCCATGACAACTTT-3', and the antisense primer was 5'GGCCATGAGGTCCACCACCCTGTTGCTGTA-3'. PCR reactions were performed following the cycling parameters on a PTC-200 PCR system (Bio-Rad, Hercules, Calif): 10 min at 94° followed by 28 cycles of 1 min at 94°, 1 min at 55°, 1 min at 72°, and a final cycle at 72°C for 10 min. PCR products were scanned, and quantification was performed by the Quantity One program (Bio-Rad, Hercules, CA).
Western blot analysis
Total proteins were extracted with 1× SDS sample buffer [62.5 mmol/L Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol], and 30 μg of each protein was electrophoretically separated in 12% SDS polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (Millipore). Rabbit polyclonal anti-Bmi-1 (1:800, Upstate Biotechnology, Lake Placid, NY) and anti-Rabbit (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were used to detect Bmi-1 protein. Mouse anti-α-tubulin (1:2000, Sigma) and anti-mouse (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were used to detect α-tubulin. The Western blot bands were scanned and were analyzed by the Quantity One program (Bio-Rad, Hercules, CA).
Immunohistochemistry assay
Immunohistochemistry was performed to examine Bmi-1 expression in 137 human bladder cancers and 30 specimens of adjacent normal bladder tissue. The procedures were performed with classical protocols. In brief, paraffin-embedded specimens were cut into 4-μm sections and baked at 65° for 30 min. The sections were deparaffinized with xylene and rehydrated. Sections were submerged into EDTA antigenic retrieval buffer and microwaved for antigenic retrieval. The sections were then treated with 3% hydrogen peroxide in methanol to quench the endogenous peroxidase activity, followed by incubation with 1% bovine serum albumin to block the nonspecific binding.
The Bmi-1 protein was detected using a mouse monoclonal antibody against Bmi-1 (Upstate Biotechnology, Lake Placid, NY). The specimens were incubated with anti-Bmi-1 antibody (1:100) overnight at 4°. In the negative control, primary antibody was replaced by the non-immune mouse IgG of the same isotype. After washing, the tissue sections were treated with biotinylated anti-mouse secondary antibody followed by further incubation with a streptavidin-horseradish peroxidase complex. The tissue sections were immersed in 3-amino-9-ethyl carbazole, and counterstained with 10% Mayer's hematoxylin, dehydrated, and mounted in Crystal Mount.
The degree of immunostaining of formalin-fixed, paraffin-embedded sections was reviewed and scored by two independent observers. The proportion of cells expressing Bmi-1 varied from 0% to 100%, and the intensity of nuclear staining varied from weak to strong. Cells were scored for intensity of staining on a scale of 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), and 3 (strong staining, brown). Using this method of assessment, we evaluated the expression of Bmi-1 in bladder cancer tissue and in normal bladder tissue. An optimal cutoff value was identified. An intensity score of = 2 with at least 50% of malignant cells positive for Bmi-1 staining was used to classify tumors with high expression, and < 50% of malignant cells with nuclear staining of intensity score of < 2 characterized tumors with low expression of Bmi-1.
Statistical analysis
All statistical analyses were carried out with the SPSS 13.0 statistical software package. In the RT-PCR and Western blot analysis, t-test was used to analyze the significance of mRNA and protein expression between bladder cancers and the adjacent normal tissues. We analyzed the relationship between expression of Bmi-1 protein, clinicopathologic features, and the clinical prognosis. The χ2 test for proportion was used to analyze the relationship between Bmi-1 expression and clinicopathologic characteristics. Survival curves were plotted by the Kaplan-Meier method, and compared by the log-rank test. We determined that the assumption of proportional hazards was met in all Cox regression models. The significance of various variables for survival was analyzed by the Cox proportional hazards model in the multivariate analysis. P < 0.05 was considered statistically significant.
Discussion
At present, the molecular mechanisms of the initiation and progression of bladder cancers are unclear, although many genetic factors have been found to be associated with bladder cancer [
4‐
6]. In this study, we report that Bmi-1 is overexpressed in human bladder cancers. The overexpression of Bmi-1 protein was correlated with tumor classification, recurrence, TNM stage, and prognosis. Patients with higher Bmi-1 expression had shorter survival time, and patients with lower Bmi-1 expression had a longer survival time.
This study demonstrated that there was a significant difference in Bmi-1 expression at both protein and mRNA levels between bladder cancer cells and the adjacent normal bladder tissue. Furthermore, Bmi-1 protein is up-regulated to a much greater extent than is Bmi-1 mRNA in cancer tissue compared with non-cancerous tissues. This observation suggests that dysregulation at the posttranscriptional level might be the major source of Bmi-1 expression in bladder cancers. Immunohistochemistry demonstrated that bladder cancers showed moderate to strong nuclear staining, while adjacent normal tissues showed only weak Bmi-1 expression, or no Bmi-1 expression at all. These results were similar to those of previous studies of other human cancers [
14‐
18]. Furthermore, intense expression of Bmi-1 in bladder cancer correlated with its clinicopathologic features including tumor classification, recurrence, and TNM stage. Previous reports indicated that the
BMI1 gene may be a novel molecular marker to predict the progression and prognosis of breast cancer and myelodysplastic syndrome (MDS)[
19,
20,
23,
24]. Consistent with previous reports of other cancers, over-expression of Bmi-1 protein indicated poor prognosis for patients with bladder cancer. The five-year survival was significantly different between the two groups, and the results showed that the greater the expression of Bmi-1 protein, the lower the survival rate.
The initiation and progression of bladder cancer involve a series of genetic events including activation of oncogenes, and the inactivation of tumor suppressors[
6,
25,
26]. The regulatory mechanism of the Polycomb group proteins relies upon epigenetic modifications of specific histone tails that are inherited through cell division[
10,
27]. Because Bmi-1 is a member of the PcG family, Bmi-1 overexpression could repress the p16Ink4a (
CDKN2A) and p19Arf targets[
17,
20]. In the absence of p16Ink4a, the cyclin D/Cdk4/6 complex can phosphorylate pRB, allowing the E2F-dependent transcription that leads to cell cycle progression and DNA synthesis. Bmi-1-deficient mouse embryonic fibroblasts (MEF) overexpress INK4a/ARF locus-encoded genes
CDKN2A and p19ARF (mouse homologue of human p14ARF) and undergo premature senescence in culture[
28].
Conversely, overexpression of Bmi-1 reduces expression of p16INK4a and p19ARF, and immortalizes MEFs[
28]. Kang[
29] found that Bmi-1 may act through a p16INK4A-independent pathways to regulate cellular proliferation during progression of oral cancer. In addition, MDM2-mediated p53 degradation causes low p53 levels in the absence of p19Arf, thus preventing cell cycle arrest and apoptosis[
30]. The
BMI1 gene also can induce telomerase activity to prevent apoptosis. Other investigators have found that the frequent inactivation of the p14ARF and
CDKN2A genes may be an important mechanism for the dysfunction of p53 and Rb growth regulatory pathways during bladder cancer development [
31‐
34]. However, whether overexpression of
BMI1 results in reduction of p14ARF and
CDKN2A gene expression requires further investigation.
In our experience, the clinicopathologic features of bladder cancer are closely related to its prognosis. In our study, we found that in addition to T classification and TNM stage, Bmi-1 expression is an independent prognostic factor for bladder cancers. In the literature, the
BMI1 gene is reported to be related to the clinicopathologic features and prognosis in other human cancers, including breast cancer, colon cancer, and lung cancer [
19‐
21]. Thus, we believe that the
BMI1 gene probably plays an important role in cell proliferation and tumor progression in bladder cancers. More studies are required to explore the relationships between the
BMI1 gene and other genes such as p14, p16, and
TP53, and its relationship to other molecules that may be associated with bladder cancer.
Conclusion
This study demonstrated overexpression of Bmi-1 in bladder cancers. The overexpression of Bmi-1 protein was correlated with tumor classification, recurrence, clinical stage, and prognosis. Greater expression of Bmi-1 protein predicts a lower degree of differentiation, higher recurrence risk, and poorer prognosis. These results indicate that the BMI1 gene may play an important role in the progression of bladder cancer; however, this study has several limitations. In this study, the old WHO grading system (grades 1, 2, and 3) was used, and lymphovascular invasion was not recorded. In addition, we can say only that Bmi-1 expression is an independent prognostic marker for bladder cancer. It may offer new information that stage and grade cannot provide, but more evidence is required to support this conclusion. Prospective studies, additional cases, and different antibodies would be required to test the relationship between Bmi-1 expression and the clinical biological behavior of bladder cancer.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ZKQ, JAY and YLY were responsible for data collection and analysis, experiment job, interpretation of the results, and writing the manuscript. FJZ, HH, XZ, ZWL and SLB were responsible for conducting the data analysis, reviewing and scoring the degree of immunostaining of sections in cooperation with JAY. MSZ and ZKQ were responsible for experimental design, analysis and interpretation. All authors have read and approved the final manuscript.