reverse transcriptase-polymerase chain reaction analysis, a significantly higher survivin mRNA expression level was ..... JonckheereâTerpstra trend test.
IJC International Journal of Cancer
Two survivin polymorphisms are cooperatively associated with bladder cancer susceptibility Naoko Kawata, Norihiko Tsuchiya, Yohei Horikawa, Takamitsu Inoue, Hiroshi Tsuruta, Shinya Maita, Shigeru Satoh, Yoko Mitobe, Shintaro Narita and Tomonori Habuchi Department of Urology, Akita University Graduate School of Medicine, Akita, Japan
Cancer Genetics
Abnormal survivin expression has been reported to be involved in many types of cancer. A single-nucleotide polymorphism (SNP), C-31G, located in the promoter region of survivin reportedly may alter the mRNA level, while the significance of the nonsynonymous SNP A9194G in exon 4 has not yet been clarified. Here, the association between the two survivin SNPs and bladder cancer susceptibility and progression was investigated in 235 patients with bladder cancer and 346 healthy controls. Regarding the C-31G SNP, subjects with the CC genotype had a significantly higher risk of bladder cancer compared to those with the GG 1 CG genotype [odds ratio (OR) 5 1.85, p 5 0.001]. Regarding the A9194G SNP, the presence of the G allele was associated with a significantly reduced risk with a gene dosage effect (OR 5 0.69, p 5 0.002). Using the C-A haplotype as a reference, the G-G haplotype was associated with a significantly lower risk (OR 5 0.11, p 5 0.00006), indicating the cooperative effect of the two SNPs. Immunohistological evaluation of surgical specimens showed that cancer cells of the C-31G CC genotype had significantly higher nuclear survivin expression than those of the C-31G GG 1 CG genotype. With reverse transcriptase-polymerase chain reaction analysis, a significantly higher survivin mRNA expression level was observed in surgical specimens with an increase in the number of the C-31G C allele (p 5 0.016). These results indicate that the two SNPs have a significant and cooperative influence on bladder cancer susceptibility.
Bladder cancer is the fourth most common cancer in men and the ninth most common cancer in women in the United States,1 and it is the sixth most common cancer in men and the 11th most common cancer in women in Japan.2 The most common type of bladder cancer is transitional cell carcinoma (TCC, also called urothelial carcinoma), and 55–60% of all newly diagnosed bladder cancers are well differentiated or moderately differentiated, superficial (confined to the urothelium or lamina propria) papillary TCCs. Tumor recurrences are observed in majority of these patients after endoscopic resection, with 16–25% of patients developing high-grade tumors. Muscle-invasive or metastatic cancer subsequently develops in 10–20% of patients with superficial papillary TCCs.1 The 5-year survival rate of this variant is Key words: survivin, polymorphism, bladder cancer, susceptibility Abbreviations: aOR: adjusted odds ratio; CI: 95% confidence interval; IAPs: inhibitor of apoptosis proteins; OR: odds ratio; RTPCR: reverse transcriptase-polymerase chain reaction; SNP: singlenucleotide polymorphism; TCC: transitional cell carcinoma Grant sponsor: Japan Society for Promotion of Science, Japan; Grant numbers: 22390302, 19390411, 1965940 DOI: 10.1002/ijc.25850 History: Received 3 Sep 2010; Accepted 23 Nov 2010; Online 7 Dec 2010 Correspondence to: Tomonori Habuchi, Department of Urology, Akita University School of Medicine, Akita, Japan, Tel.: þ81-18-884-6156, Fax: þ81-18-836-2619, E-mail: thabuchi@doc. med.akita-u.ac.jp
C 2010 UICC Int. J. Cancer: 129, 1872–1880 (2011) V
approximately 90%. However, nonpapillary muscle-invasive TCC, which accounts for 20–30% of urothelial malignancies, is invasive at diagnosis and carries a high risk for further invasion and metastasis. At least 50% patients with muscleinvasive cancers generally die within 2 years of diagnosis.3 Concerning the heterogeneous behavior of bladder TCC, several studies have attempted to identify molecular markers that help clinicians in prognosis of patients with TCC. However, successful applications of molecular markers are limited because of the heterogeneity of molecular genetic alterations in bladder cancer.4–6 Survivin is a protein involved in the inhibition of apoptosis and the control of mitotic progression.7 It belongs to a family of proteins known as inhibitors of apoptosis (IAPs), which is a family of antiapoptotic proteins that inhibit initiator (caspase-9) and effector (caspase-3 and -7) caspases and thereby prevent apoptosis.8,9 To date, eight human IAP family members have been identified, and survivin is structurally unique among human IAPs, containing a single baculovirus IAP repeat (BIR) domain and lacking the really interesting new gene (RING) and caspase recruitment domain (CARD) domains.8,10,11 Survivin is abundantly expressed in embryonic tissues and most tumors; however, it is almost absent in normal differentiated cells.10 It is expressed in the G2/M phase of the cell cycle to support the rapidly dividing cell machinery.10,12 Abnormal survivin expression has been reported to be involved in many types of cancer, including bladder cancer.13,14 Several recent studies have identified survivin as a promising biomarker for bladder cancer screening, surveillance and prognosis after treatment as well as a marker of bladder
Kawata et al.
Material and Methods Patients
In a case–control study, a total of 581 subjects, comprising 235 patients with bladder cancer treated at the Akita University Medical Center and 346 age-matched healthy controls attending community-based medical check-ups, were registered. Blood specimens were collected for genomic DNA analysis from March 1984 to December 2008 for patients with bladder cancer and from March 1984 to August 2009 for the controls. All patients had pure or predominant TCC with squamous or adenomatous differentiation of the bladder. C 2010 UICC Int. J. Cancer: 129, 1872–1880 (2011) V
Tumor stage was determined using the 1997 TumorNode-Metastasis (TNM) classification system,24 and tumor grade was classified according to the General Rule for Clinical and Pathological Studies on Bladder Cancer by the Japanese Urological Association and Japanese Society of Pathology.25 The tumor grading system generally conformed to the World Health Organization grading system.26 The clinical stage was further classified into the following two subgroups as described previously27: nonmuscle (stage Ta–T1) and muscle invasive (stage T2–T4). The pathological grade was initially divided into three groups (G1, G2 and G3) and then subdivided into low (G1–G2) and high (G3) grades. In 131 patients who were treated with transurethral resection, the relationship between the recurrence-free survival rate and the two SNP genotypes was evaluated. In 104 patients who were treated with radical cystectomy, the relationship between the disease-specific survival rate and survivin protein expression was evaluated. The controls were recruited at community-based medical health check-ups for common diseases, such as hypertension, anemia and liver disease. Written informed consent was obtained from all patients and controls for the use of the DNA of their peripheral white blood cells and clinical information. Genotyping of C-31G SNP in the survivin promoter and A9194G SNP in exon 4
Polymerase chain reaction-restriction fragment length polymorphism analysis was used to genotype the survivin promoter C-31G SNP and exon 4 A9194G SNP. The primer sequences for the C-31G SNP were as follows: forward, 50 GTT CTT TGA AAG CAG TCG AG-30 and reverse, 50 -GCC AGT TCT TGA ATG TAG AG-30 . The primer sequences for the A9194G SNP were as follows: forward, 50 -GAA GAA AGA ATT TGA GGA AAC CGC-30 and reverse, 50 -AAA CCC TGG AAG TGG TGC AG-30 . PCR was performed in a 15 lL of reaction mixture containing approximately 25 ng of genomic DNA, 1.5 lL of 10 PCR buffer supplied by a manufacturer (Takara, Tokyo, Japan), 2 mM of each dNTP (dATP, dCTP, dGTP and dTTP), 25 mM of MgCl2, 50 pmol of each primer and 0.5 unit of Taq polymerase (Invitrogen, San Diego, CA, USA). After a 10-min initial denaturation step at 95 C, 35 cycles of PCR at 95 C for 30 s, 60 C for 30 s and 72 C for 30 s were performed, followed by a 10-min final extension step at 72 C in a thermal cycler (GeneAmp PCR System 9700; Applied Biosystems, Foster City, CA, USA). After successful PCR amplification with 3.0% agarose gel electrophoresis was confirmed, each PCR product was digested overnight with 3 units of EcoO109I (for C-31G SNP) or SacII (for A9194G SNP; New England Biolabs, Beverly, MA, USA) enzyme at 37 C and was electrophoresed on a 3% agarose gel (NuSieveV CTGV Agarose). For the C-31G SNP, the restriction fragments were 341 bp long for the C allele, and 235 and 106 bp long for the G alleles. For the A9194G SNP, the fragments were 124 bp long for the A allele and 100 and 24 bp long for the G alleles. R
R
Cancer Genetics
cancer detection, recurrence and prognosis.5,13,14 A positive correlation between survivin expression and tumor grade as well as recurrence has been observed.14–17 A study by Shariat et al.16 demonstrated that survivin expression was associated with disease recurrence, disease-specific mortality and all-cause mortality in patients who underwent radical cystectomy for locally advanced bladder cancer. Furthermore, a recent microarray analysis with immunohistological validation showed that survivin expression was a strong independent prognostic factor for response and survival after cisplatin-containing chemotherapy in patients with advanced bladder cancer.17 Therefore, survivin could be one of the most promising diagnostic and prognostic markers in monitoring bladder cancer.5 Several studies have recently explored the possible association between several single-nucleotide polymorphisms (SNPs) of the survivin gene (survivin) and a few types of human cancer.18–22 Among them, one SNP (C-31G) located at the cell-cycle-dependent element/cell-cycle gene homology region (CDE/CHR) repressor binding motif in the promoter region attracted our attention because it has been shown to be associated with altered expression levels of survivin mRNA and protein with in vitro analysis.18 Further, in vitro analyses indicated that C-31G SNP changed cell cycle-dependent transcription by modifying the binding motif of the CDE/CHR repressor.18 Several case–control studies suggested that the C allele may be a risk allele for several types of human cancer,19–22 while a study suggested that this SNP had no significant role in cervical carcinogenesis.23 On the other hand, we identified the nonsynonymous A9194G (E129K; rs2071214) SNP in exon 4 in the SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi? rs¼2071214). The association between SNP and the susceptibility or progression of any type of cancer as well as the biological effect of this amino acid alteration (Lys–Glu) has not yet been clarified. To date, only one study has examined the relationship between polymorphism and lung cancer susceptibility and indicated a negative association.19 In our study, we investigated the association between the two SNPs (C-31G and A9194G) and bladder cancer susceptibility and progression in a Japanese population. Furthermore, the relationship between the promoter C-31G SNP and survivin mRNA and protein expression levels, and the clinical prognostic significance of survivin expression were also investigated.
1873
1874
Cancer Genetics
Immunohistochemical analysis
Seventy bladder specimens obtained during radical cystectomy were subjected to immunohistochemical analysis. The pathological T stage of bladder cancer was T1 in 14 (20.0%), T2 in 19 (27.1%), T3 in 20 (28.6%), T4 in eight (11.4%), and carcinoma in situ (CIS) in nine (12.8%) patients. The pathological grade of bladder cancer was 1–2 in two (2.9%) and 3 in 68 (97.1%) specimens. The specimens were fixed in 10% buffered formalin and embedded in paraffin. Serial sections (4 lm thick) were cut from formalin-fixed, paraffin-embedded sections, deparaffinized in xylene, rehydrated in a graded series of decreasing ethanol concentration, and then rinsed in Tris-buffered saline. For survivin immunostaining, survivin was detected with a rabbit polyclonal antibody ab469 (Abcam, Cambridge, UK). Antigen retrieval treatment was performed at 121 C for 15 min in 0.01 mol/L sodium citric acid (pH 6.0), and endogenous peroxidases were blocked using 0.3% hydrogen peroxide/methanol for 30 min. After washing in phosphate-buffered saline (PBS) for 5 min and 10% bovine serum albumin/PBS for 30 min, the sections were exposed to an anti-survivin polyclonal antibody diluted 1:1,600 overnight at 4 C. After washing in PBS, a secondary antibody conjugated with anti-rabbit IgG was applied, followed by incubation at room temperature for 30 min. After another wash with PBS, tissue sections were developed with diaminobenzidine (Nichirei, Tokyo, Japan) and counterstained with hematoxylin. Assessment of survivin expression and measurement
Survivin expression was assessed by two independent authors, who were blinded to the clinical and pathological data. Briefly, nuclear and cytoplasmic survivin expression was independently classified into four categories according to the percentage of survivin positive cells and staining intensity (0, no staining; 1, less than 50% of cell positivity with any intensity; 2, more than 50% of cell positivity with weak to moderate intensity and 3, more than 50% of cell positivity with strong intensity).28 Nuclear and cytoplasmic survivin staining was evaluated in 70 patients. The survivin score was initially divided into four groups (0, 1, 2 and 3) and then subdivided into the weak- (0, 1) and strong-staining (2, 3) groups. Real-time reverse transcriptase-polymerase chain reaction analysis
We evaluated the relationship between the C-31G genotype and the survivin mRNA expression level by real-time RTPCR. Total RNA was prepared from 41 bladder cancer specimens using the BioRobot AZ1 workstation (Qiagen, Valencia, CA, USA) and EZ1 RNA universal tissue kit (Qiagen) according to the manufacturer’s instructions. First-strand cDNA was synthesized using the PrimeScriptTM First strand cDNA synthesis kit (Takara) and subsequently diluted with nuclease-free water to
