XIAP as a prognostic marker of early recurrence of... : Chinese Medical Journal (original) (raw)
Transitional cell carcinoma (TCC) of the urinary bladder is the most common cancer of genitourinary tract in China; of them approximately 80% are diagnosed as non-muscular invasive bladder carcinoma. Although non-muscular invasive bladder carcinoma can generally be treated successfully, tumor recurrence is a serious clinical problem. With a strong propensity to recur, more than 70% of patients with non-muscular invasive bladder carcinoma develop a relapse sometime after the organ-preserving operation and a portion of these cases result in the advanced disease with a subsequent poor prognosis.1–3 Unfortunately, very few markers are specific enough to predict the risk of recurrence and the progression of non-muscular invasive bladder carcinoma.4–6
Dysregulation of apoptosis has been implicated in carcinogenesis, tumor progression, resistance of tumor cells to chemotherapy, and tumor recurrence.7,8 The molecular pathways leading to apoptosis are evolutionarily conserved and controlled by those proteins either promoting or inhibiting the activation of a cascade of intracellular cysteine proteases known as caspases. Caspase activity is inhibited by the inhibitor of apoptosis (IAP) family. X-linked IAP (XIAP), a member of the IAP family, whose gene is located at Xq25,9 is a 54 kDa protein that demonstrates significant inhibition of apoptosis. Overexpression of IAPs has been shown to protect tumors cells in vitro from apoptosis induced by different anticancer drugs.10–12 XIAP has been shown to be the most potent caspase-inhibitory IAP family member and is able to inhibit the effector caspases-3, -7, and -9.13–15 It is important to note that IAPs also seems to promote tumor progression in vivo, suggesting that it might be used as the novel prognostic factors.
Overexpression of XIAP was found in bladder cancer and contributes to the resistance of conventional chemotherapeutic drugs.10 Very limited data are available concerning the significance of XIAP in predicting the progression and recurrence of non-muscular invasive bladder cancer. We conducted a prospective study of the expression of XIAP and its potential for predicting the early recurrence in patients with non-muscular invasive bladder cancer.
METHODS
Patients
A total of 258 new cases of bladder cancer were diagnosed in the Institute of Urology, Peking University between June 2002 and August 2005, among them 176 patients with non-invasive bladder cancer underwent organpreserving, transurethral resection of bladder tumors (TUR-BT). Of the 176 patients, 106 were men and 70 were women, aged (63.1±10.7) years (range 41—86).
After surgery all of the tissue samples were confirmed by pathology as non-muscular invasive bladder cancer (pTa —pT1) and informed consent was acquired from each patient. All muscular invasive cases, as confirmed by pathology, were excluded. None of the patients received radiotherapy or chemotherapy before the operation. Formalin-fixed paraffin-embedded samples with bladder cancer and 20 adjacent normal mucosa tissues (as controls) were collected. The fresh frozen samples from 20 patients were also prepared for the mRNA analysis. The tumors were graded according to the Word Health Organization Classification and staged by TNM classification. All patients received the same scheduled adjuvant introvesical management after TUR-BT. Cystoscopies during follow-up were performed at 3, 6, 9, and 12 months in the first year and every 6 months thereafter. The times to first recurrence and progression (histologically proven shift to muscle invasive disease) were recorded.
Immunohistochemistry
Tissue sections (4-μm thick) of the paraffin-embedded samples from bladder cancer tissue and normal mucosa were cut and put on the slides covered with adhesive. After deparaffination and rehydratation, the slides were immersed in 10 mmol/L sodium citrate buffer (pH 6.0) and heated for 5 minutes in a microwave oven at 600 W. After block of endogenous peroxidase activity, nonspecific conjugation was blocked with 5% normal horse serum. Endogenous biotin was blocked with an avidin/biotin blocking kit (SP-2001, Vector Laboratories, USA). The slides were incubated with 1:100 dilution of H62120 monoclonal mouse IgG1 antibody (clone 48, Transduction Laboratories, USA in a humid chamber at 37°C for 60 minutes. Bound antibody was detected by the avidin-biotin complex peroxidase method (ABC Elite Kit, Vector Laboratories, USA). A bladder cancer tissue known to show a strong XIAP immunoreactivity served as the positive control. Negative controls were generated by replacing the primary antibody by an irrelevant monoclonal mouse antibody.
RT-PCR
The RNA was extracted from frozen samples of bladder cancer tissues and normal mucosa tissue using an RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's instructions. For quantitative RT-PCR, fluorescent hybridization probes and the TaqMan PCR core reagents kit with AmpliTaq gold (PerkinElmer Cetus, USA) were used with the ABI prism 7900HT sequence detection system (PerkinElmer, USA). Oligonucleotides as specific primers and TaqMan probes for the XIAP and glutaraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were designed using Oligo 4.0 (International Biosciences, USA). The primers’ sequences are the follows: XIAP: 5′-AGTGGTAGTCCTGTTTCAGCATCA-3′ (forward) and 5′-CCGCACGGTATCTCCTTCA-3′ (reverse), TaqMan probe: 5′-CACTGGCACGAGCAGGGTTTCTTTATACTG-3′; GAPDH: 5′-GAAGGTGAAGGTCGGAGT-3′ (forward) and 5′-GAAGATGGTGATGGGATTTC-3′ (reverse), TaqMan probe: 5′-CAAGCTTCCCGTTCTCAGCC-3′. The reactive conditions for the one-step RT-PCR were as follows: 30 minutes at 48°C (stage 1, reverse transcription), 10 minutes at 95°C (stage 2, RT inactivation and AmpliTaq gold activation), and then 40 cycles of amplification for 15 seconds at 95°C and 1 minute at 60°C (stage 3, PCR). The intensity of the reaction was evaluated from the quantity of total RNA in Raji cells (ng) corresponding to the initial number of PCR cycles to reveal the linear increase in reaction intensity (threshold cycle) for each sample on a logarithmic standard curve. Data on the quantity of XIAP RNA (ng) were normalized using the data for GAPDH in each sample.
Statistical analysis
Chi-square test was used to assess the association of the XIAP expression with clinical and pathological features. Recurrence-free survival was evaluated by Kaplan-Meier curves. Differences between the groups were evaluated by the log rank test. Disease-free survival was defined as the period between surgery and the detection of initial local recurrence. The Cox proportional hazards regression analysis was used to test the association of XIAP expression with clinicopathological variables for the prediction of recurrence. We used the statistical software SPSS 12.0 and P value less than 0.05 was considered statistically significant.
RESULTS
XIAP expression in non-muscular invasive bladder carcinoma
Of the 176 patients, 94 were stage pTa and 82 were stage pT1. Forty-four patients had grade 1 tumors, 74 grade 2 and 58 grade 3 (Table 1).
Clinicopathological characteristics of non-muscular invasive bladder cancer patients with high XIAP expression
XlAP expression was found in 108 cases (61.4%) of the cancer tissues and no expression in 68 (Fig. 1). XIAP staining was not observed in most of the adjacent normal mucosa except for a few of them with a weak staining. These immunohistochemistry results were confirmed by RT-PCR for RNA expression in the samples from the same cases (data not shown).
XIAP protein in the tissue sections of non-muscular invasive bladder cancer (ABC staining, original magnifïcation×400). A: No XIAP expression. B: XIAP is negative in low grade bladder cancer tissue (XIAP-). C: XIAP is positive in high grade bladder cancer tissue (XIAP+).
No significant correlation was found between the XIAP expression rate and the tumor pathological grades, but there seemed to be a trend toward the increased expression levels of XIAP from well (G1) to poorly (G3) differentiated bladder cancer.
Correlation of XIAP expression with cancer recurrence
Patients with non-muscular invasive bladder carcinoma were divided into two groups: XIAP+ group (n=108, XIAP positive tumor) and XIAP" group (n=68, XIAP negative tumor). During a mean follow-up time of 28.6 months (range 6–44 months), 82 (46.6%) patients experienced tumor recurrence, of whom 66 in XIAP+ group (61.1%) and only 16 in XIAP- group (23.5%). Among the 82 patients, 12 developed invasive disease at the time of relapse and all were from XIAP+ group, 14 presented with a pathological up-grading, and 11 with down-grading at the time of relapse. No significant difference about the changes of pathological grade was observed between XIAP positive and negative groups.
The postoperative recurrence-free survival was estimated by Kaplan-Meier analysis. Patients were divided into two groups as described above. Patients in XIAP- group or those with a low pathological grade (grade 1 or 2) had a higher recurrence-free survival compared with those in XIAP+ group (P=0.0015, Fig. 2A) or those with high pathological grade (grade 3) (P<0.001,Fig. 2B) during the follow-up.
Kaplan-Meier analysis for recurrence-free survival in patients with non-muscular invasive bladder cancer. A: recurrence-free survival and XIAP expression (P=0.0015); B: recurrence-free survival and tumor grade (P<0.001).
Multivariate analysis
Multivariate Cox proportional hazards model analysis using the variables of XIAP expression, age, sex, pathological stage and grade, tumor size, and multiplicity showed that XIAP expression, tumor grade, and tumor number were independent predictors for early recurrence (Table 2).
Association of various factors with disease-free survival
DISCUSSION
Suppression of apoptosis contributes not only to tumor progression and resistance to chemotherapy, but also to tumor recurrence. IAP family plays a critical role in the regulation of apoptosis. Recent studies suggest that IAP family member survivin is an important independent prognostic marker in various tumor types.16,17 Increased expression of XIAP has been found in various tumors including bladder cancer. Recent observations also suggest an important role for XIAP as a key regulator of tumor cell susceptibility to anticancer drug.10–12 XIAP levels determine sensitivity or resistance of cells to different chemotherapeutic agents. Whether it can play a role as a prognostic marker for progression and recurrence of non-muscular invasive bladder carcinoma has not been extensively investigated so far. Clinicopathologic markers for superficial bladder cancer recurrence remain the best prognostic predictors in clinical decision making. Some molecular markers, including survivin, hypoxia-inducible factor 1α, p53, have been suggested to have predictive value for recurrence of non-muscular invasive bladder carcinoma.18,19
This study show that 61.4% of non-muscular invasive bladder carcinoma expressed XIAP. Though no significant correlation was found between the XIAP expression rate and tumor pathological grade, there appeared a trend toward the increased expression levels of XIAP from well (G1) to poorly (G3) differentiated bladder cancers. These results were also compatible with results in other cancer types.20
Based on our findings, a question is raised if XIAP expression could be used as a biomarker to predict the recurrence and progression for non-muscular invasive bladder carcinoma. We next will investigate the recurrence-free survival in our study cohort. Multivariate Cox regression analysis reveals that XIAP protein expression is an independent prognostic marker for non-muscular invasive bladder carcinoma. Thus, non-muscular invasive bladder carcinoma patients without XIAP expression have a significantly more favorable outcome compared with those with high XIAP expression. Moreover, these patients have higher risks of earlier recurrence and of developing invasive disease. It is reasonable to assume, therefore, that increased levels of anti-apoptotic XIAP in non-muscular invasive bladder carcinoma contributes to reduced apoptosis susceptibility of tumor cells, thereby providing an important survival advantage during tumor progression. In the present data, the non-muscular invasive bladder carcinoma patients with higher tumor pathological grade have a higher recurrent rate than those with a lower pathological grade. In our study group, all recurrence patients show either a higher pathological grade or XIAP expression or both of them. Multivariate Cox proportional hazards model analysis reveals that XIAP expression, high tumor grade and number are independent predictors for early recurrence and progression of non-muscular invasive bladder carcinoma. In addition, the estimated relative risk of XIAP expression is 3.46, which is higher than low tumor grade (2.94 for grade 1 or 2 vs grade 3) and tumor number (2.05 for single vs multiple tumors). Studies with larger numbers of subjects are needed to confirm these results.
In conclusion, XIAP expression correlates with the early recurrence in patients with non-muscular invasive bladder carcinoma. Patients with XIAP expression bear the high risk of disease progression from superficial to invasive bladder cancer. XIAP might act as a new independent prognostic marker for early recurrence of non-muscular invasive bladder carcinoma and suppressing XIAP might prevent early recurrence and progression and improve the prognosis of non-muscular invasive bladder carcinoma.
REFERENCES
1. Pisani P, Parkin DM, Ferlay J. Estimates of the worldwide mortality from 18 major cancers in 1985. Implications for prevention and projections of future burden. Int J Cancer 1993; 55: 891-892.
2. Heney NM, Ahmed S, Flanagan MJ, Frable W, Corder MP, Hafermann MD, et al. Superficial bladder cancer progression and recurrence. J Urol 1983; 130: 1083-1085.
3. Larsson P, Wijkstrom H, Thorstenson A, Adolfsson J, Norming U, Wiklund P, et al. A population-based study of 538 patients with newly detected urinary bladder neoplasms followed during 5 years. Scand J Urol Nephrol 2003; 37: 195-199.
4. Takahashi T, Hagisawa S, Yoshikawa K, Tezuka F, Kaku M, Ohyama C. Predictive value of N-acetylglucosaminyltransferase-V for superficial bladder cancer recurrence. J Urol 2006; 175: 90-94.
5. Canales BK, Anderson JK, Premoli J, Slaton JW. Risk factors for upper tract recurrence in patients undergoing long-term surveillance for stage ta bladder cancer. J Urol 2006; 175: 74-80.
6. Millan-Rodriguez F, Chechille-Toniolo G, Salvador-Bayarri J, Palou J, Vicente-Rodríguez J. Multivariate analysis of the prognostic factors of primary superficial bladder cancer. J Urol 2000; 163: 73-78.
7. Reed JC. Dysregulation of apoptosis in cancer. J Clin Oncol 1999; 17: 2941-2944.
8. Gaikwad A, Poblenz A, Haridas V, Zhang C, Duvic M, Gutterman J. Triterpenoid electrophiles (avicins) suppress heat shock protein-70 and x-linked inhibitor of apoptosis proteins in malignant cells by activation of ubiquitin machinery: implications for proapoptotic activity. Clin Cancer Res 2005; 11: 1953-1962.
9. Rajcan-Separovic E, Liston P, Lefebvre C, Korneluk RG. Assignment of human inhibitor of apoptosis protein (IAP) genes xiap, hiap-1, and hiap-2 to chromosomes Xq25 and 11q22-q23 by fluorescence in situ hybridization. Genomics 1996; 37: 404-409.
10. Bilim V, Kasahara T, Hara N, Takahashi K, Tomita Y. Role of XIAP in the malignant phenotype of transitional cell cancer (TCC) and therapeutic activity of XIAP antisense oligonucleotides against multidrug-resistant TCC in vitro. Int J Cancer 2003; 103: 29-37.
11. Parton M, Krajewski S, Smith I, Krajewska M, Archer C, Naito M, et al. Coordinate expression of apoptosis-associated proteins in human breast cancer before and during chemotherapy. Clin Cancer Res 2002; 8: 2100-2108.
12. Alas S, Ng CP, Bonavida B. Rituximab modifies the cisplatin-mitochondrial signaling pathway, resulting in apoptosis in cisplatin-resistant non-Hodgkin's lymphoma. Clin Cancer Res 2002; 8: 836-841.
13. Deveraux QL, Leo E, Stennicke HR. Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases. EMBO J 1999; 18: 5242-5250.
14. Huang Y, Park YC, Rich RL. Structural basis of caspase inhibition by XIAP: differential roles of the linker versus the BIR domain. Cell 2001; 104: 781-790.
15. Suzuki Y, Nakabayashi Y, Takahashi R. Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death. Proc Natl Acad Sci U S A 2001; 98: 8662-8667.
16. Ku JH, Kwak C, Lee HS, Park HK, Lee E, Lee SE. Expression of survivin, a novel inhibitor of apoptosis, in superficial transitional cell carcinoma of the bladder. J Urol 2004; 171: 631-635.
17. Schultz IJ, Kiemeney LA, Witjes JA, Schalken JA, Willems JL, Swinkels DW, et al. Survivin mRNA expression is elevated in malignant urothelial cell carcinomas and predicts time to recurrence. Anticancer Res 2003; 23: 3327-3331.
18. Theodoropoulos VE, Lazaris AC, Kastriotis I, Spiliadi C, Theodoropoulos GE, Tsoukala V, et al. Evaluation of hypoxia-inducible factor 1alpha overexpression as a predictor of tumour recurrence and progression in superficial urothelial bladder carcinoma. BJU Int 2005; 95: 425-431.
19. Dekairelle AF, Tombal B, Cosyns JP, Gala JL. Assessment of the transcriptional activity of p53 improves the prediction of recurrence in superficial transitional cell carcinoma. Clin Cancer Res 2005; 11: 4724-7432.
20. Takeuchi H, Kim J, Fujimoto A, Umetani N, Mori T, Bilchik A. X-Linked inhibitor of apoptosis protein expression level in colorectal cancer is regulated by hepatocyte growth factor/C-met pathway via Akt signaling. Clin Cancer Res 2005; 11: 7621-7626.
Keywords:
XIAP; urinary bladder neoplasms; recurrence
© 2007 Chinese Medical Association