Background
Urothelial carcinoma of the bladder (UCB) is one of the major causes of morbidity and mortality in Western countries [
1]. Clinically, radical cystectomy (RC) remains the most common treatment for patients with muscle-invasive UCB or for patients with superficial disease that is at high risk of recurrence and progression. Despite advancement of the surgical technique and the development of novel drugs [
2,
3], approximately 35% of UCB patients will relapse after treatment, and 5-year cancer-specific survival remains at only 50-60% [
4]. It is known that the pathogenesis of UCB is a multistep process that involves multiple genetic changes, including loss of tumor suppressor genes and activation of oncogenes [
5]. Although the molecular and/or genetic alterations of UCB have been widely studied, the discovery of specific molecular markers that are present in UCB cells that could serve as reliable clinical/prognostic factors remains substantially limited to date.
PIN2/TRF1-interacting telomerase inhibitor1 (PinX1) is a newly cloned gene mapped to chromosome 8p23.1 that consists of seven exons in humans and is a region frequently associated with loss of heterozygosity in a variety of human malignancies [
6‐
10]. PinX1 has been identified as a critical component in regulating telomerase activity, and is proposed to be a putative tumor suppressor [
11]. In humans, ectopic overexpression of PinX1 leads to a decrease in both telomerase activity and cancer cell tumorigenicity, whereas suppression of PinX1 expression results in an increase in both telomerase activity and cancer cell tumorigenicity [
11]. Very recently, Chang et al. reported that high significance between a single-nucleotide polymorphism on the PinX1 gene and lower bladder cancer risk [
12]. However, the biological function of PinX1 on UCB tumorigenesis and tumor progression has not been characterized. In this study, we investigated the clinicopathological and prognostic significance as well as the potential role of PinX1 in the development and progression of UCB.
Materials and methods
To prepare of the bladder tissue microarray (TMA), 187 patients with UCB that had undergone RC were selected from the surgical pathology archives of the Department of Pathology of the Sun Yat-Sen University Cancer Center, the First Affiliated Hospital of Sun Yat-Sen University, and Guangdong Provincial People’s Hospital between 1999 and 2008. The median follow-up time was 92 months (range 8–156 months) and the clinicopathological characteristics are summarized in Table
1. Prior patient consent and approval from the Institutional Research Ethics Committee were obtained for the use of these clinical materials for research purposes. The tumor specimens were obtained from the paraffin blocks of 187 primary UCBs. We also obtained 102 samples, in paraffin blocks, of normal bladder mucosa in adjacent non-neoplastic bladder tissue from the same UCB patients. The TMA was constructed according to a method described previously [
13]. In our constructed bladder tissue TMA, three sample cores were selected from each primary UCB and normal bladder tissue. Multiple sections (5-μm thick) were obtained from the TMA block and mounted on microscope slides. Tumor grade and stage were defined according to the criteria of the World Health Organization and the sixth edition of the TNM classification of the International Union Against Cancer (UICC, 2002).
Table 1
Correlation of PinX1 expression in tissue with patients’ clinicopathological variables in 187 cases of UCB
Age(years) | | | | 0.456 |
≤60b | 80 | 33(41.3) | 47(58.8) | |
>60 | 107 | 50(46.7) | 57(53.3) | |
Gender | | | | 0.212 |
Male | 166 | 71(42.8) | 95(57.2) | |
Female | 21 | 12(57.1) | 9(42.9) | |
Tumor multiplicity | | | | <0.001 |
Unifocal | 79 | 17(21.5) | 62(78.5) | |
Multifocal | 108 | 66(61.1) | 42(38.9) | |
WHO grade | | | | 0.844 |
G1 | 46 | 19(41.3) | 27(58.7) | |
G2 | 66 | 29(43.9) | 37(56.1) | |
G3 | 75 | 35(46.7) | 40(53.3) | |
pT status | | | | 0.404 |
pT1 | 35 | 13(64.0) | 22(36.0) | |
pT2 | 95 | 38(54.5) | 57(45.5) | |
pT3 | 37 | 20(50.0) | 17(50.0) | |
pT4 | 20 | 12(37.5) | 8(62.5) | |
pN status | | | | 0.023 |
pN- | 157 | 64(40.8) | 93(59.2) | |
pN+ | 30 | 19(63.3) | 11(36.7) | |
ki-67 index | | | | 0.004 |
≥50% | 95 | 52(54.7) | 43(45.3) | |
<50% | 92 | 31(33.7) | 61(66.3) | |
Immunohistochemistry
Immunohistochemistry (IHC) studies were performed using a standard streptavidin-biotin-peroxidase complex method [
14,
15]. TMA slides were dried overnight at 37°C, dewaxed in xylene, rehydrated with graded alcohol, and immersed in 3% hydrogen peroxide for 20 min to block endogenous peroxidase activity. Antigen retrieval was carried out in a microwave oven with 10 mM citrate buffer (pH 6.0) for 15 min. The slides were incubated with 10% normal goat serum at room temperature for 10 min to reduce nonspecific reactions. Subsequently, the TMA slides were incubated overnight at 4°C with rabbit polyclonal antibody against PinX1 (1:200; Proteintech Group, USA), mouse monoclonal anti-Ki-67 (1:100; Sigma-Aldrich, USA), or mouse monoclonal anti-p16 (1:100; Cell Signaling Technology, USA) and anti-cyclin D1 (1:100; Cell Signaling Technology, USA), overnight at 4°C. After rinsing five times with 0.01 mol/L phosphate-buffered saline (PBS; pH 7.4) for 10 min, primary antibody was detected using a secondary antibody (Envision; Dako, Glostrup, Denmark) for 1 h at room temperature and stained with 3,3-diaminobenzidine (DAB) after washing in PBS again. Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted.
Two independent pathologists blinded to the clinicopathological information performed the analysis of IHC for PinX1. Similar to that observed in other human tissues [
16,
17], positive expression of PinX1 in epithelial cells of bladder tissues was primarily in nuclear pattern. PinX1 immunoreactivity was classified into two groups as previously described [
17]: negative expression, when PinX1 positive cells were less than 50%; and positive expression, when at least 50% of the cells showed positive staining of PinX1. For the Ki-67 labeling index, the proportion of positive cells in the stained sections was evaluated at × 200 magnification and the mean value of 10 representative fields analyzed from each section was recorded. Previous scoring criterions were used for evaluation of the p16 and cyclin D1 IHC staining [
18,
19].
UCB cell lines and cell cultures
The UCB cell lines EJ, T24, and 5637 were cultured in RPMI 1640 (Invitrogen, USA) supplemented with 10% fetal bovine serum (HyClone, USA). All cells were grown in a humidified incubator at 37°C with 5% CO2.
Paired tumor and adjacent tissues
Ten pairs of UCB tissues and matched adjacent, morphologically normal bladder epithelial tissues were frozen and stored in liquid nitrogen until used to compare the expression levels of PinX1 mRNA and protein.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from the 10 pairs of UCB tissue and normal bladder tissue using TRIZOL reagent (Invitrogen, USA). RNA was reverse-transcribed using SuperScript First Strand cDNA System (Invitrogen, USA) according to the manufacturer’s instructions. The PinX1 sense primer was 5'-ATGTCTATGCTGGCTGAA-3', and the antisense primer was 5'-TCTGTGGCTCCTTGCT-3'. For the GAPDH gene, the sense primer was 5'- CCCACATGGCCTCCAAGGAGTA -3', and the antisense primer was 5'- GTGTACATGGCAACTGTGAGGAGG -3'. qRT-PCR was done using SYBR Green PCR master mix (Applied Biosystems, USA) in a total volume of 20 μl on the 7900HT fast Real-time PCR system (Applied Biosystems, USA) as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s. A dissociation procedure was performed to generate a melting curve for confirmation of amplification specificity. GAPDH was used as the reference gene. The relative levels of gene expression were represented as ΔCt = Ctgene- Ctreference, and the fold change of gene expression was calculated by the 2-ΔΔCt Method. Experiments were repeated in triplicate.
Western blotting
Equal amount of whole-cell lysates were resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Pall Corp., USA). This was followed by incubation with primary rabbit polyclonal antibody against human PinX1 (Proteintech Group, USA), mouse monoclonal antibodies to p16 (Cell Signaling Technology, USA), cyclin D1 (Cell Signaling Technology, USA), CDKN2B (Cell Signaling Technology, USA), CCND2 (Cell Signaling Technology, USA), rabbit monoclonal antibodies GADD45A (Santa Cruz Biotechnology, USA), ANAPC2 (Santa Cruz Biotechnology, USA), and CDK5R1 (Santa Cruz Biotechnology, USA), respectively. The immunoreactive proteins were detected with enhanced chemiluminescence detection reagents (Amersham Biosciences, Sweden) according to the manufacturer’s instructions. The membranes were stripped and re-blotted with a mouse monoclonal anti-GAPDH antibody (Santa Cruz Biotechnology, USA) as a loading control.
Construction of the recombinant lentiviral vector
The PinX1 expression construct was generated by subcloning the PCR-amplified human PinX1 coding sequence into the pBABE retroviral vector. The construction of the PinX1 short hairpin RNA (shRNA) lentiviral expression vector and retroviral production and infection have been described previously [
11,
17]. Based on their baseline expression of PinX1, UCB cells were transduced with either pBABE/PinX1 or pSUPER-retro-PinX1-shRNA. EJ and T24 cells showed low expression of PinX1 and they were infected with retroviruses carrying pBABE/PinX1. The 5637 cells showed had high expression of PinX1 and they were infected with retroviruses carrying pSUPER-retro-PinX1-shRNA.
Cell proliferation assay and colony-forming assay
For cell proliferation assays, cells were reseeded in 96-well plates at 2 × 103 cells/well 24 h after transfection and incubated overnight in 100 μL of culture medium. Then, 20 μL of 5 mg/mL 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA) was added to the wells and cells were incubated at 37°C for 4 h. The supernatant was removed, and 150 μL dimethyl sulfoxide was added to the wells. After incubating at 37°C for 15 min, absorbence at 570 nm was measured with a microplate reader (SpectraMax M5, Molecular Devices, USA).
For colony-forming assays, cells were reseeded at 500 or 1000 cells/well in 6-well plates at 24 h after transfection, with medium replacement every three days. After incubating at 37°C for 2–3 weeks, cells were fixed and stained with crystal violet.
Flow cytometry
For cell cycle analysis, cells were collected at the indicated time points. Cells (1 × 106) were washed with PBS and fixed with cold 70% ethanol at 4°C overnight. Then, cells were treated with RNase and stained with propidium iodide (PI, Sigma-Aldrich, USA). The DNA content of the cells was quantified using a flow cytometer (Epics Elite, Beckman Coulter, USA). In total, 10,000 nuclei were examined in the flow cytometer, and DNA histograms were analyzed by ModFit software (Verity Software House, USA).
For apoptosis analysis, cells transfected with above mentioned formulations were stained with annexin V-PE and propidium iodide (PI) 48 h post-transfection using the Annexin V apoptosis detection kit (BD biosciences, USA). The percentage of apoptotic cells was quantified by flow cytometry. Viable cells are both Annexin V-PE and PI negative.
Telomerase activity assay
The telomerase activity was examined when the cells at the 15 passage. Telomerase activity was measured with the TRAPeze telomerase detection kit (Chemicon, USA). PCR products were separated by electrophoresis on a 12.5% nondenaturing polyacrylamide gel, visualized by SYBG green (Invitrogen, USA) staining and semi-quantitated according to the manufacturer’s instruction. Briefly, telomerase activity consists of the intensity of the TRAP product band and the processivity of TRAP ladders.
Telomere lengths analysis
The telomere length was examined when the cells at the 15 passage. Two micrograms of gemonic DNA from tissue extracts were doubly digested with Hinf I and Rsa I overnight at 37°C. The DNA products of enzymes digestion were electrophoresed on 0.8% agarose gel, and transferred onto a nylon membrane for hybridization with digosin-labbed (TTAGGG)3 oligos. The hybridization signal was detected by the AP-conjugated anti-digosin antibodies (Roche Diagnostics, Indianapolis, Indiana, USA) and imaged by CDP-Star (Roche, Switzerland).
In vivo tumorigenicity assays
In total, male BALB/c nu/nu immune deficient mice (6 weeks old, 18–20 g) were purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). The mice were housed in barrier facilities on a 12 h light/dark cycle. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University. Cells (5 × 106 EJ-Vector, 5 × 106 EJ-PinX1, 5 × 106 T24-Vector, 5 × 106 T24-PinX1, 5 × 106 5637-Scramble, 5 × 106 5637-PinX1-shRNA) were suspended in RPMI 1640 medium and injected subcutaneously into the flank of mice. The tumor diameter was measured and the volume (width2 × length × 0.52) calculated every other day. Mice were humanely killed on day 48, and the tumors were dissected and weighed.
Statistical analysis
Data were analyzed using SPSS16.0 software (SPSS Inc.). Significant associations between PinX1 expression and clinicopathological parameters were assessed using a χ2 test. Survival curves were plotted by Kaplan–Meier analysis and compared by the log-rank test. Cox regression analysis was carried out to assess the significance of variables for survival. Data were expressed as mean ± SD, and the t-test was used to determine the significance of differences between two groups. All tests carried out were two-sided. P < 0.05 was considered statistically significant.
Discussion
It has been proposed that the PinX1 gene could be a putative tumor suppressor gene and/or therapeutic target for human cancers [
11,
20,
21]. Although the relationship between the PinX1 gene and human tumors has been studied widely, such as in medulloblastoma, hepatocelllular carcinoma, prostate cancer, and gastric cancer [
15,
22‐
25], the expression and prognostic value of PinX1 protein has not been investigated in UCB. In addition, the molecular mechanisms underlying the potential role of PinX1 in UCB remain unknown. In this study, we examined the expression dynamics status of PinX1 firstly by IHC using a TMA containing a series of UCB and adjacent morphologically normal bladder epithelial tissues. The IHC results demonstrated that negative expression of PinX1 protein in 44.4% of primary bladder tumor, but in only 20.6% of normal bladder epithelial tissues. In addition, western blotting revealed downregulated expression of PinX1 in the majority of UCBs when compared with their adjacent normal bladder epithelial tissues. Furthermore, forced expression of PinX1 in UCB cell lines led to the inhibition of cell proliferation and tumourigenicity in vitro and in vivo, accompanied with G1/S phase arrest, upregulation of p16 expression, downregulation of cyclin D1 expression, as well as the deactivation of telomerase activity. Meanwhile, RNA interference silencing of PinX1 expression induced opposite results. These findings provide evidence for the concept that downregulating the expression of PinX1 may play an important role in the tumorigenic process of UCB.
Further correlation analyses demonstrated that negative expression of PinX1 in our UCB cohort was significantly associated with advanced N classification, higher proliferation index, and tumor multiplicity. Importantly, we found that decreased or depleted expression of PinX1 was associated with poor prognosis and reduced survival periods for UCB patients. Multivariate analysis showed that the loss of PinX1 protein expression could be used as an independent prognostic predictor for UCB patients. Furthermore, in stratified survival analysis, PinX1 expression could differentiate the survival of certain subsets of UCB patients, including patients with grade 1, 2 and 3 tumors and at pT1, pT2, pT3, and pN- stage. Our results indicate that the expression level of PinX1 protein might provide useful information in the evaluation prognosis and follow-up schedule guiding for UCB patients.
PinX1 is an evolutionarily conserved nuclear protein that has been demonstrated to be a telomerase/telomere-interacting factor in humans. Originally, PinX1 was identified as an intrinsic telomerase inhibitor and a putative tumor suppressor because of its binding to and inhibition of telomerase [
20]. Recently, it has been reported that human PinX1 can regulate telomerase activity and suppress tumor growth both in vivo and in vitro [
20,
21]. Overexpression of PinX1 in tumor cells could inhibit telomerase activity, shorten telomeres, and suppress tumor growth, while depletion of endogenous PinX1 increased telomerase activity, elongated telomeres, and enhanced tumorigenicity in telomerase-positive HT1080 cancer cells [
20]. Disruption of the PinX1-dependent telomere maintenance pathway could reduce carcinogenesis and enhance chemotherapeutic sensitivity in telomerase-positive human cancer cells as well [
11]. In the present study, we found that overexpression of PinX1 by transfection of pBABE-PinX1 into EJ and T24 cells significantly reduced cell growth, and arrested cells in the G0/G1 phase via the inhibition of telomerase activity and shortening of telomeres. In contrast, inhibition of PinX1 expression by shRNA transfection in 5637 cells promoted cell growth/proliferation in vitro and vivo via by enhancing telomerase activity and telomere elongating. These findings suggest that PinX1 acts as an intrinsic telomerase inhibitor and arrests cell growth in human UCB.
We showed that PinX1 could prohibit G1/S phase transition, to gain further insight into the downstream molecular events involving PinX1 and UCB growth/proliferation, we compared mRNA expression profiles between T24-PinX1 and T24-Vector cells using a Human Cell Cycle real-time PCR array containing 84 well-known cell cycle related genes. Of the 84 genes, 7 genes were differentially expressed by 2-fold or more (i.e. upregulated: CDKN2A (i.e. p16), CDKN2B (i.e. p15) and GADD45A; downregulated: CCND1 (i.e. cyclin D1), CCND2 (i.e. cyclin D2), ANAPC2 and CDK5R1). Subsequently, protein expression of these seven genes was analyzed by western blotting. Consistent with that of mRNA expression in the real-time PCR array, upregulated p16 expression and downregulated cyclin D1 expression were validated in the protein level following PinX1 overexpression in T24 cells. It was appear that PinX1 regulated the cell cycle and influenced cell growth/proliferation via the regulation of p16 and cyclin D1 expression in the UCB cells we used. Further, the status of p16 and cyclin D1 expression was examined by IHC in a TMA of a large cohort of UCBs. Our analysis demonstrated that there were significant positive correlations between the expression of PinX1 and p16 and between the expression of PinX1 and cyclin D1, which confirmed the results observed in the T24 cells.
The p16 protein acts as an inhibitor of cell proliferation by competitively binding the cyclin-dependent kinase (CDK)4/6 kinases against their regulator cyclin D1 and blocking phosphorylation of the retinoblastoma (Rb) protein, leading to cell cycle arrest [
26]. The p16/cyclin D1 pathway is one of the key signal transduction pathways at the G1/S checkpoint in the cell cycle [
27,
28]. Dysfunction of the proteins involved in the p16 pathway such as deletion of the p16 gene and overexpression of CDKs of cyclin D1 will lead to Rb phosphorylation, subsequent progression of G1/S phase transition and promotion of uncontrolled cell growth/proliferation [
29‐
34]. Song et al. reported that the decrease of p16 cooperated with cyclin D1 and the caused deregulation of G1/S checkpoint, leading to abnormal cell proliferation in nasopharyngeal carcinoma [
35]. These observations, together with the results of our PinX1 functional studies in the UCB cells, suggest that decreased expression of PinX1 in UCB might be involved in the p16/cyclin D1 associated pathway and thus support cancer cell growth/proliferation. Clearly, better understanding of the precise molecular mechanisms of p16 and cyclin D1 regulated by PinX1 may lead to more effective management of UCB growth and/or progression.
Based on previous studies [
20,
21,
26‐
28,
35] and the present study, we propose that PinX1 regulates UCB cell proliferation through at least two distinct mechanisms. In one mechanism, PinX1 influences UCB cell growth/proliferation by binding to telomerase and inhibiting its activity. In the other mechanism, PinX1 inhibites UCB cell growth/proliferation by regulating the expression of the key cell cycle genes for p16 and cyclin D1. More studies are needed to confirm these two mechanisms and to elucidate whether other signaling pathways also contribute to PinX1-mediated cell growth/proliferation in UCB.
In summary, we describe for the first time in this study the protein expression pattern of PinX1 in UCB and adjacent morphologically normal bladder epithelial tissues. Our results provide a basis for the concept that negative expression of PinX1 in UCB may be important in the acquisition of an aggressive and/or poor prognostic phenotype. In addition, the functional studies of PinX1 in this report suggest a potential important role of PinX1 in the control of cell growth/proliferation via the regulation of telomerase activity and the p16/cyclin D1 pathway, an activity that might be responsible, at least in part, for the development and/or ultimately the progression of human UCB.
Competing interest
The authors declare that they have no competing interests.
Authors’ contributions
JYL evaluated the clinical records, carried out the experimental work and drafted the manuscript. DQ, LRH, and YHL contributed for data interpretation and drafted the manuscript. XPT critically revised the manuscript. YJL participated in the statistical analysis and help to draft the manuscript. SJM, YHL, and JXZ help to carry out the immunohistochemistry assays. HFK contributed for critical revision of statistical analysis and of the manuscript. FJZ designed the study and participated in its coordination. YXZ and DX participated in the design of the study, in its analysis and in the interpretation of the data. DX also participated in evaluated the immunohistochemistry results and wrote the manuscript. All authors read and approved the final manuscript.