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Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma P.K. Singh a , Anupam K. Srivastava a , S.K. Rath b , D. Dalela c , M.M. Goel d , M.L.B. Bhatt e,∗ a
Department of Radiotherapy, King George’s Medical University, Lucknow, Uttar Pradesh 226003, India Genotoxicity Laboratory, Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh 226001, India Department of Urology, King George’s Medical University, Lucknow, Uttar Pradesh 226003, India d Department of Pathology, King George’s Medical University, Lucknow, Uttar Pradesh 226003, India e Department of Radiation Oncology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh 226010, India b c
a r t i c l e
i n f o
Article history: Received 14 August 2014 Accepted 14 August 2014 Available online xxx Keywords: CEP55 Bladder tumorigenesis Cancer-testis Immunotherapy Peptide vaccines TURBT Transitional cell carcinoma
a b s t r a c t Bladder cancer (BC) is one among the most common and lethal urothelial malignancies worldwide. The expression of cancer-testis (CT) antigens in some tumours and restricted expression among normal tissues make CT antigens as attractive vaccine targets. In this context, we evaluated Centrosomal protein 55 kDa (CEP55), which is specifically expressed in normal human testis and various malignancies. Until the expression pattern of CEP55 in transitional cell carcinoma (TCC) of human urinary bladder and its clinical significance are not known. The aim of the present study is to evaluate mRNA/protein expression of CEP55 in TCCs of urinary bladder and correlate its expression with the clinicopathological characteristics of BC patients. In this study, the methods of quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were used to investigate mRNA/protein expression of CEP55 in TCC. Independent Student’s t test, ANOVA and Chi-square (2 ) were used to analyze the data statistically. We observed CEP55 mRNA overexpression in testis and 48.7% of BC patients. Relative mean fold expression of CEP55 mRNA was found to be significantly (p < 0.01) higher in muscle-invasive bladder cancer (MIBC) as compared to non-muscle-invasive bladder cancer (NMIBC) patients (7.88 ± 3.88 vs. 4.75 ± 2.30, p = 0.01). CEP55 protein expression was evaluated using IHC and cytoplasmic staining pattern was recorded in formalin fixed, paraffin-embedded (FFPE) bladder tumour tissues. No significant difference was observed in protein expression of CEP55 between the two groups (NMIBC and MIBC patients) (72.2% vs. 69.0%, p = 0.774). No significant protein expression of CEP55 was observed among adjacent noncancerous tissues (ANCTs) and benign prostatic hyperplasia (BPH) used as control. Our study results suggest that CEP55 mRNA/protein expression was observed is specific to TCC of human urinary bladder and might be used as a diagnostic biomarker and vaccine target in development of BC specific immunotherapy. © 2014 Elsevier GmbH. All rights reserved.
Abbreviations: TCC, transitional cell carcinoma; BC, bladder cancer; BCG, Bacillus Calmette-Guerin; BPH, Benign prostatic hyperplasia; NMIBC, non-muscle-invasive bladder cancer; MIBC, muscle-invasive bladder cancer; RC, radical cystectomy; CTA, cancer testis antigen; CT, cancer-testis; CEP55, centrosomal protein 55; ANCT, adjacent noncancerous tissue; TURBT, transurethral resection of bladder tumour; FFPE, formalin-fixed paraffin embedded; BPH, Benign prostatic hyperplasia; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; IHC, immunohistochemistry; HNSCC, head and neck squamous cell carcinoma; OCSCC, oral cavity squamous cell carcinoma; HCC, hepatocellular carcinoma. ∗ Corresponding author. Tel.: +91 9453428740; fax: +91 522 2258878. E-mail addresses: [email protected], [email protected] (M.L.B. Bhatt). http://dx.doi.org/10.1016/j.imbio.2014.08.014 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Singh, P.K., et al., Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma. Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.08.014
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Introduction Bladder cancer (BC) is one of the major deadly malignancies in the world. BC accounted for approximately 2% of all human malignancies with an estimated 72,570 new cases and 15,210 deaths in United States alone in 2013 (Siegel et al., 2014). Approximately 80% of BCs present as non-muscle-invasive urothelial carcinoma, out of which 70% of them will recur subsequently after initial therapy and 10–20% of them will progress and invade the bladder muscle (Knowles, 2001). Of the patients initially presenting with muscle-invasive bladder cancer (MIBC), 50% will relapse with metastatic disease (Wolff et al., 2005; Williams and Stein, 2004). In spite of various therapeutic options, more than 90% of patients with metastasis die within the first 5 years (Luke et al., 2010). High-grade muscle-invasive disease represents a life-threatening condition and requires timely treatment (Herr et al., 2007; Chang et al., 2003). Standard treatment for MIBC is radical cystectomy (RC) with bilateral pelvic lymphadenectomy, and is justified in patients with life expectancy of greater than 2 years (Singh et al., 2014). Non-muscle-invasive bladder cancer (NMIBC) patients are often treated with a local, organ-sparing transurethral resection of bladder tumour (TURBT) followed by intravesical immunotherapy with attenuated Bacillus Calmette-Guerin (BCG), which by an unknown mechanism reduces the risk of recurring tumours, suggesting that anticancer vaccines could be especially useful for the treatment of BC (Dyrskjot et al., 2012; Simons et al., 2008). Approximately 50% of NMIBC patients at high risk of recurrence remain free of disease after receiving BCG immunotherapy following surgery (Singh et al., 2014). This indicates that immunotherapeutic strategies based on specific tumour-associated antigens (TAAs) in BC might be more effective and associated with less morbidity than BCG (Singh et al., 2014). BC thus represents a unique opportunity to develop and test cancer vaccines that could be used after surgery to prevent tumour recurrence, in an ideal setting of low tumour burden (Singh et al., 2014; Picard et al., 2007). However, the development of cancer specific immunotherapy depends on the identification of relevant target antigens (Fradet et al., 2006). More recently, cancer-testis (CT) antigen has emerged to be a unique group of antigen that could potentially be important antigen targets for antigen-specific cancer immunotherapy (Caballero and Chen, 2009). CT antigens are normally expressed in the testis only, apart from some expressed in the early developing embryo and the placenta (Dyrskjot et al., 2012). However, CT antigens are also expressed in various malignancies including BC (Singh et al., 2014; Dyrskjot et al., 2012; Mengus et al., 2013; Yin et al., 2012). So far, more than 200 CT antigens have been reported in previous studies, which belong to more than 70 distinct subfamilies (Almeida et al., 2009). The development of cancer-specific immunotherapy based on CT antigens has been ongoing for years, with some candidates reaching early-phase clinical trials (Obara et al., 2012; Francois et al., 2009; Sharma et al., 2008; Nishiyama et al., 2001). Furthermore, expression of various CT antigens in BC has been evaluated for potential immunotherapy purposes (Singh et al., 2014; Dyrskjot et al., 2012; Picard et al., 2007; Yin et al., 2012; Bergeron et al., 2009; Kanehira et al., 2007a,b; Sharma et al., 2006, 2003; Kurashige et al., 2001; Patard et al., 1995). In this quest, we evaluated one of the CT antigens, Centrosomal protein 55 kDa (CEP55) that is highly expressed in cancer cells but not in normal cells, except for testis (Inoda et al., 2009). CEP55 is also known by several other names, including FLJ10540, C10orf3 and URCC6 (Tao et al., 2014). CEP55 is overexpressed in human gastric cancer (Tao et al., 2014) nasopharyngeal carcinoma (NPC) (Chen et al., 2012), colorectal cancer (Inoda et al., 2011), oropharyngeal squamous cell carcinoma (Janus et al., 2011), prostate cancer (Shiraishi et al., 2011), head and neck squamous cell carcinoma (HNSCC) (Waseem et al., 2010), lung cancer (Chen et al., 2009a,b)
Table 1 Patient clinciopathological parameters. Clinicopathological characteristics
Age (years, %) ≤45 >45 Sex Male Female Grade Low High Stage Ta T1 T2-T4 Smoking No Yes Tobacco chewers No Yes
(n) %
Real-time-PCR Assay (76)
Immunohistochemistry Assay (65)
24 (31.6%) 52 (68.4%)
17 (26.2%) 48 (73.8%)
67 (88.2%) 9 (11.8%)
63 (96.9%) 2 (3.1%)
33 (43.4%) 43 (56.6%)
23 (35.4%) 42 (64.6%)
3 (3.9%) 33 (43.4%) 40 (52.6%)
6 (9.2%) 23 (35.4%) 36 (55.4%)
34 (44.7%) 42 (55.3%)
29 (44.6%) 36 (55.4%)
36 (47.4%) 40 (52.6%)
33 (50.8%) 32 (49.2%)
and oral cavity squamous cell carcinoma (OCSCC) (Chen et al., 2009a,b), breast carcinoma (Inoda et al., 2009), hepatocellular carcinoma (HCC) (Chen et al., 2007) and colon cancer (Sakai et al., 2006). However, there is little or no information regarding the expression and significance of CEP55 with tumour histological grade, stage, and patient outcome in BC patients. The purpose of the present study is to evaluate the expression levels of CEP55 in BC tissue specimens, and to correlate the results with clinicopathological parameters of BC patients.
Methods Collection of clinical specimens To evaluate CEP55 mRNA expression, 76 freshly frozen BC tissues (male: 67; female: 9) and 7 matched adjacent noncancerous tissue (ANCT) specimens were obtained from histologically proven TCC of urinary bladder patients. All patients had undergone TURBT or RC between March 2006 and April 2011 at Department of Urology, King George’s Medical University (KGMU), Lucknow, India. The mean age of the BC patients used in qRT-PCR assay was 54.75 ± 11.51 years. For evaluation of immunohistochemical (IHC) expression of CEP55, 65 formalin fixed, paraffin-embedded (FFPE) bladder tissues, 12 ANCT specimens and 10 BPH tissues were obtained from the archives of the Pathology Department, KGMU. The mean age of the BC patients was 53.83 ± 11.47 years whereas mean age of BPH patients was 64.14 ± 11.46 years. The 2004 World Health Organization (WHO) bladder tumour classification criteria (low-grade and high-grade) were used in grading of bladder tumours (Eble et al., 2004), whereas pathologic staging was done according to the 2002 tumour-lymph node-metastasis (TNM) classification system (Greene et al., 2002). Demographic and clinical characteristics of BC patients are shown in Table 1. Written informed consent was obtained from all the participants for the use of their clinical samples in this study. Approval of institutional ethics committee was taken before the commencement of study. A unique code was given to all collected tissue specimens and further all investigations were made blind to histopathological or cytologic diagnosis. All clinicopathological information was recorded on a written proforma at the time of recruitment in the study.
Please cite this article in press as: Singh, P.K., et al., Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma. Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.08.014
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RNA extraction and cDNA synthesis Total RNA was extracted from tissue specimens using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, after phenol treatment and drying, RNA pellet was dissolved in RNase-free water and stored at −80 ◦ C till further use. The RNA concentration was quantified and the quality of the RNA was verified by electrophoresis on a 1% denaturing agarose gel. Then cDNA synthesis was carried out using Quantitect® Reverse Transcription Reagent (Qiagen GmbH, Hilden, Germany) as per given instructions. Quantitative real-time PCR analysis The quantitative gene expression levels of CEP55 was measured using SYBR® GreenERTM qPCR SuperMix Universal (Invitrogen), on the LightCycler 480 (Roche Applied Science, Mannheim, Germany) as described previously (Waseem et al., 2010) with the housekeeping gene POLR2A, used as an internal control. The reaction mix consisted of 5 l of 2× SYBR Green Master Mix (Invitrogen), 5 M of each primer, 2 l cDNA and 2 l nuclease-free water to obtain a final volume of 10 l. All samples were analyzed in triplicate. The CEP55 primers used in qRT-PCR were as follows: forward primer of 5 -TGAAGAGAAAGACGTATTGAAACAA-3 and reverse primer of 5 -GCAGTTTGGAGCCACAGTCT-3 . The primer sequence for housekeeping gene POLR2A were as follows: forward primer of 5 -GCAAATTCACCAAGAGAGAC-3 and reverse primer of 5 -CACGTCGACAGGAACATCAG-3 . The optimized amplification protocol consisted of an initial denaturation step of 95 ◦ C for 10 min, followed by 40 amplification cycles at 95 ◦ C for 10 s, annealing at 60 ◦ C for 20 s and elongation at 72 ◦ C for 10 s. At the end of the PCR cycles, melting curve analyses were performed. The fold changes for CEP55 expression levels were calculated using 2−Ct (Livak and Schmittgen, 2001). Immunohistochemistry IHC analysis and evaluation of CEP55 in bladder tumours, ANCTs and BPH tissues was performed using a sensitive EnVision FLEX mini kit High pH (K802321; Dako, Glostrup, Denmark), as described previously (Singh et al., 2014). The CEP55 protein in FFPE tissue sections was detected using a rabbit polyclonal antibody against CEP55 (CEP55 (H-300); sc-134622; 1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 ◦ C.
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expression. CEP55 mRNA expression was observed in higher number of MIBC patients than NMIBC patients (62.5% vs. 33.3%, p < 0.05). In the MIBC patients relative mean fold expression of CEP55mRNA was also found to be significantly (p < 0.01) higher as compared to NMIBC (7.88 ± 3.88 vs. 4.75 ± 2.30, p = 0.01). Furthermore, CEP55 mRNA expression was detected in 48.4% patients with low grade and 48.8% with high grade TCC respectively. In matched BC and their ANCTs, the average expression level of CEP55 mRNA in BC was found to be ∼6-fold higher than those in ANCTs. Among normal tissues CEP55 mRNA expression was detected in testis, placenta, thymus and small intestine. However, overexpression was found only in testis and fold expression level of CEP55 mRNA in other normal tissues was 104-fold lower than that in the testis. The relative mean fold expression of CEP55 mRNA and its association with the clinicopathological characteristics of the BC patients are summarized in Table 2. There was no significant association observed between CEP55 mRNA expression and clinicopathological characteristics such as BC patient’s age, gender, grade and disease stage etc. in both the NMIBC and MIBC patients. Although, high relative mean fold expression of CEP55 mRNA was found in older age patients vs. young patients, males vs. females, advanced stage vs. early stage, high grade vs. low grade, smokers vs. non-smokers and tobacco chewers vs. non-tobacco chewers. Immunohistochemical expression of CEP55 Expression of CEP55 protein was detected in the cytoplasm of urothelial carcinoma cells (Fig. 1). IHC expression of CEP55 was observed in 69.0% (20 of 29) of NMIBC and 72.2% (26 of 36) MIBC patients. Thus IHC expression of CEP55 was found to be positive in 70.8% (46 of 65) BC patients. There is no significant difference was observed in IHC expression of CEP55 between NMIBC and MIBC patients (72.2% vs. 69.0%, p = 0.774). Furthermore, cytoplasmic expression of CEP55 was detected in 69.5% patients with low grade and 71.4% with high grade TCC respectively. No significant expression of CEP55 protein was found among BPH and ANCT tissues used as control (Fig. 1E and F). The IHC expression of CEP55 and its association with the clinicopathological characteristics of the patients are summarized in Table 3. The IHC expression of CEP55 was also not found to be significantly associated with the any of the clinicopathological characteristics of NMIBC and MIBC patients being evaluated. Discussion
Statistical analysis Continuous data were expressed as mean ± SD, while discrete (categorical) data in %. Qualitative variables were shown as percentages and numbers. Comparisons were made between independent groups by independent Student’s t test and ANOVA and the significance of mean difference between the groups were evaluated by Tukey’s post hoc test after adjusting the multiple contrasts for significance. Chi-square (2 ) test was used to evaluate comparisons between categorical groups. All p values were 2-sided and a p-value of less than 0.05 was considered statistically significant. All analyses were performed using SPSS (Windows version 18.0) statistical software packages. Results Quantitative expression of CEP55 mRNA CEP55 mRNA expression was detected in 33.3% (12 of 36) of NMIBC and 62.5% (25 of 40) MIBC patients. Thus an overall 48.7% (37 of 76) BC patients were found to be positive for CEP55 mRNA
BC is the most common malignancy of the urinary tract (Anastasiadis and de Reijke, 2012). The requirement of follow-up and repeated treatment has made BC one of the most expensive cancers in today’s medical practice (Madeb et al., 2007). Despite advances in treatment, the prognosis of BC, especially muscleinvasive tumours, remains poor (Ren et al., 2014). Even with chemotherapeutic treatment, the overall median survival is 1 year advanced TCC patients. This survival is frequently achieved with severe and life-threatening side effects (Perabo and Muller, 2007). Therefore, effective diagnostic biomarkers and novel therapeutic approach is urgently required in BC. Recently, cancer immunotherapy has emerged as a powerful and promising clinical approach for treatment of various malignancies and has shown major success in breast cancer, prostate cancer, melanoma (Matsueda and Graham, 2014). The field of cancer vaccine therapy is currently expected to become the fourth option in the treatment of cancer after surgery, chemotherapy and radiation therapy and a large number of clinical investigations are underway at present (Obara et al., 2012). Several clinical trials of immunotherapy using peptide vaccines derived from CT antigens for various
Please cite this article in press as: Singh, P.K., et al., Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma. Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.08.014
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Table 2 Correlation between relative mean fold CEP55 mRNA expression and clinical characteristics of BC patients. Characteristics Age (years) ≤45 >45 Sex Female Male Grade Low High Smoking No Yes Tobacco No Yes
Non-muscle-invasive Mean ± SD
p value
Muscle-invasive Mean ± SD
p value
p value
Total Mean ± SD
2.00 ± 0.00 (1) 5.00 ± 2.24 (11)
0.384
7.25 ± 2.19 (8) 8.18 ± 4.49 (17)
0.588
6.67 ± 2.69 (9) 6.93 ± 4.04 (28)
0.857
2.00 ± 0.00 (1) 5.00 ± 2.24 (11)
0.228
7.33 ± 1.16 (3) 7.95 ± 4.12 (22)
0.801
6.00 ± 2.83 (4) 6.97 ± 3.84 (33)
0.629
4.78 ± 2.44 (9) 4.67 ± 2.31 (3)
0.946
8.00 ± 2.24 (7) 7.83 ± 4.41 (18)
0.926
6.19 ± 2.81 (16) 7.38 ± 4.28 (21)
0.341
5.63 ± 2.00 (8) 3.00 ± 2.00 (4)
0.057
7.44 ± 3.05 (9) 8.13 ± 4.35 (16)
0.683
6.59 ± 2.69 (17) 7.10 ± 4.47 (20)
0.683
4.40 ± 2.61 (5) 5.00 ± 2.34 (7)
0.677
7.43 ± 3.48 (14) 8.45 ± 4.44 (17)
0.523
6.63 ± 3.48 (19) 7.11 ± 4.04 (18)
0.701
Numbers in parenthesis indicate the number of BC patients.
Table 3 Correlation between CEP55 protein expression and clinical characteristics of BC patients. Characteristics
Age (years) ≤45 >45 Sex Female Male Grade Low High Stage Ta T2–T4 Smoking No Yes Tobacco No Yes
Non-muscle-invasive (n = 29)
Muscle-invasive (n = 36)
Total (n = 65)
p value
Negative, n (%)
Positive, n (%)
p value
Negative, n (%)
Positive, n (%)
p value
Negative, n (%)
Positive, n (%)
2 (22.2%) 7 (77.8%)
4 (20.0%) 16 (80.0%)
0.891
3 (30.0%) 7 (70.0%)
8 (30.8%) 18 (69.2%)
0.964
5 (26.3%) 14 (73.7%)
12 (26.1%) 34 (73.9%)
0.985
0 (0.0%) 9 (100%)
0 (0.0%) 20 (100%)
NA
1 (10.0%) 9 (90.0%)
1 (3.8%) 25 (96.2%)
0.470
1 (5.3%) 18 (94.7%)
1 (2.2%) 45 (97.8%)
0.512
6 (66.7%) 3 (3.3%)
11 (55.0%) 9 (45.0%)
0.555
1 (10.0%) 9 (90.0%)
5 (19.2%) 21 (80.8%)
0.506
7 (36.8%) 12 (63.2%)
16 (34.8%) 30 (65.2%)
0.875
2 (22.2%) 7 (77.8%) 0 (0.0%)
4 (20.0%) 16 (80.0%) 0 (0.0%)
0.891
0 (0.0%) 0 (0.0%) 10 (100%)
0 (0.0%) 0 (0.0%) 26 (100%)
NA
2 (10.5%) 7 (36.8%) 10 (52.6%)
4 (8.7%) 16 (34.8%) 26 (56.5%)
0.950
6 (66.7%) 3 (33.3%)
13 (65.0%) 7 (35.0%)
0.930
3 (30%) 7 (70%)
7 (26.9%) 19 (73.1%)
0.854
9 (47.4%) 10 (52.6%)
20 (43.5%) 26 (56.5%)
0.774
4 (44.4%) 5 (55.6%)
12 (60.0%) 8 (40.0%)
0.436
5 (50.0%) 5 (50.0%)
12 (46.2%) 14 (53.8%)
0.836
9 (47.4%) 10 (52.6%)
24 (52.2%) 22 (47.8%)
0.724
Fig. 1. Immunohistochemical analysis of CEP55 in BC. BC tissues were stained with CEP55 specific rabbit polyclonal antibody. (A) MIBC showing cytoplasmic positivity. (B) MIBC showing strong cytoplasmic expression. (C) NMIBC showing positivity. (D) NMIBC showing positivity. (E) BPH tissue showing negative expression. (F) ANCT showing negative expression.
Please cite this article in press as: Singh, P.K., et al., Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma. Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.08.014
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types of advanced cancers or cancers not responding to standard therapies have been reported recently (Obara et al., 2012; Aruga et al., 2014, 2013). Thus the identification and validation of additional CT antigens is crucial towards development of BC specific immunotherapy. CEP55 overexpression is being observed in a number of human cancers and cell lines, and found to be correlated with specific clinicopathological tumour features (Inoda et al., 2009; Tao et al., 2014; Chen et al., 2012; Inoda et al., 2011; Janus et al., 2011; Shiraishi et al., 2011; Waseem et al., 2010; Chen et al., 2009a,b, 2007; Sakai et al., 2006). However, its expression status and clinical significance in BC remains to be clarified. Moreover, distribution of CEP55 overexpression in a wide range of malignant human tissues makes it a reasonable target for cancer immunotherapy (Inoda et al., 2011). CEP55 derived peptide vaccines have also shown promising results towards treatment of breast and colorectal cancer patients (Inoda et al., 2009, 2011). Thus in the present study, we analyzed the detailed mRNA/Protein expression pattern of CEP55 in human BC towards possible application of CEP55 as a target to develop active BC specific immunotherapy. Significantly, this study is the first to report that CEP55 mRNA/protein expression is upregulated in TCC of urinary bladder. In the present study, we have detected CEP55 mRNA expression in 48.7% BC patients using qRT-PCR. Our study findings derived by qRT-PCR are in agreement with previous observations reporting CEP55 upregulation in a variety of cell lines and tumours, such as breast, colorectal, pancreas, esophageal, gallbladder, and lung carcinomas by using qRT-PCR analysis (Chen et al., 2012; Janus et al., 2011; Waseem et al., 2010; Chen et al., 2009a,b; Sakai et al., 2006). Our study findings are also consistent with the previous studies of CT antigen expression in BC that MAGE-A3 expression was detected in 25–58% of tumours (Picard et al., 2007; Nishiyama et al., 2001; Sharma et al., 2006; Patard et al., 1995) whereas NYESO-1 expression were detected in 32–45% tumours respectively (Dyrskjot et al., 2012; Yin et al., 2012; Sharma et al., 2003; Kurashige et al., 2001). Among normal tissues, CEP55 mRNA was expressed only in the testis, and at a very low level in the thymus, small intestine and placenta. These findings are consistent with the previous observation by Inoda et al. (2009). Furthermore, qRT-PCR analysis demonstrated significant higher fold expression in BC tissues to their ANCT. These results are in agreement with previous published reports (Chen et al., 2012; Janus et al., 2011; Waseem et al., 2010; Chen et al., 2009a,b; Sakai et al., 2006), demonstrating that CEP55 mRNA was highly overexpressed in cancerous tissues compared with their ANCTs, thus our study findings are suggestive that CEP55 mRNA expression might be playing an important role in bladder tumorigenesis. Our study results further conclude that the CEP55 is not only expressed at the mRNA level but is also translated as evident from IHC evaluation of CEP55 in BC tissue specimens. To the best of our knowledge, we have evaluated CEP55 protein expression in BC for the first time by IHC, which revealed cytoplasmic staining pattern in TCC but no significant expression, was observed among ANCT and BPH tissues. Our results are in agreement with previous studies that observed higher protein expression of CEP55 in various tumour tissues and relatively no or little expression in their ANCTs. (Inoda et al., 2009; Tao et al., 2014; Chen et al., 2012; Inoda et al., 2011; Waseem et al., 2010; Chen et al., 2009a,b, 2007; Sakai et al., 2006). Our results are also consistent with previous studies that observed high protein expression of CT antigen in bladder tumour specimens but not in ANCTs (Singh et al., 2014; Mengus et al., 2013; Yin et al., 2012). In the present study, we demonstrated frequent mRNA/protein expression of CEP55 in TCC of urinary bladder patients with different grade and stage. Though its frequent expression was detected among TCC, but no significant association was observed between
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CEP55 protein expression and clinicopathological characteristics such as disease stage, grade and habits of patients. However, it further warrants evaluation of CEP55 in BC in a larger sample size to reach any logical correlation. These results are in accordance with findings from earlier study performed in TCC of bladder (Yin et al., 2012). The present study has few limitations. The tumour samples for the analysis of mRNA expression and protein expression analysis came from different patient. Thus, association between mRNA expression and protein expression was not achieved. Furthermore, the relatively lesser number of patients did not granted to perform subanalyses. Conclusion In conclusion, this study provided the first evidence about upregulation of CEP55 mRNA/Protein expression in BC using qRTPCR, and IHC and renders its diagnostic potential as a molecular marker. Our study findings also conclude that CEP55 might be a potential vaccine candidate for immunotherapy in BC due to its expression among bladder tumours and restricted expression in normal tissues. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements First author (Pankaj Kumar Singh) would like to thank Indian Council of Medial Research (ICMR), New Delhi, India for awarding Senior Research Fellowship (ICMR-SRF) (IRIS ID-2007-04520) under the guidance of corresponding author. References Almeida, L.G., Sakabe, N.J., deOliveira, A.R., Silva, M.C., Mundstein, A.S., Cohen, T., Chen, Y.T., Chua, R., Gurung, S., Gnjatic, S., Jungbluth, A.A., Caballero, O.L., Bairoch, A., Kiesler, E., White, S.L., Simpson, A.J., Old, L.J., Camargo, A.A., Vasconcelos, A.T., 2009. CTdatabase: a knowledge-base of high-throughput and curated data on cancer-testis antigens. Nucleic Acids Res. 37, D816. Anastasiadis, A., de Reijke, T.M., 2012. Best practice in the treatment of nonmuscle invasive bladder cancer. Ther. Adv. Urol. 4, 13. Aruga, A., Takeshita, N., Kotera, Y., Okuyama, R., Matsushita, N., Ohta, T., Takeda, K., Yamamoto, M., 2013. Long-term vaccination with multiple peptides derived from cancer-testis antigens can maintain a specific T-cell response and achieve disease stability in advanced biliary tract cancer. Clin. Cancer Res. 19, 2224. Aruga, A., Takeshita, N., Kotera, Y., Okuyama, R., Matsushita, N., Ohta, T., Takeda, K., Yamamoto, M., 2014. Phase I clinical trial of multiple-peptide vaccination for patients with advanced biliary tract cancer. J. Transl. Med. 12, 61. Bergeron, A., Picard, V., LaRue, H., Harel, F., Hovington, H., Lacombe, L., Fradet, Y., 2009. High frequency of MAGE-A4 and MAGE-A9 expression in high-risk bladder cancer. Int. J. Cancer 125, 1365. Caballero, O.L., Chen, Y.T., 2009. Cancer/testis (CT) antigens: potential targets for immunotherapy. Cancer Sci. 100, 2014. Chang, S.S., Hassan, J.M., Cookson, M.S., Wells, N., Smith Jr., J.A., 2003. Delaying radical cystectomy for muscle invasive bladder cancer results in worse pathological stage. J. Urol. 170, 1085. Chen, C.H., Lu, P.J., Chen, Y.C., Fu, S.L., Wu, K.J., Tsou, A.P., Lee, Y.C., Lin, T.C., Hsu, S.L., Lin, W.J., Huang, C.Y., Chou, C.K., 2007. FLJ10540-elicited cell transformation is through the activation of PI3-kinase/AKT pathway. Oncogene 26, 4272. Chen, C.H., Lai, J.M., Chou, T.Y., Chen, C.Y., Su, L.J., Lee, Y.C., Cheng, T.S., Hong, Y.R., Chou, C.K., Whang-Peng, J., Wu, Y.C., Huang, C.Y., 2009a. VEGFA upregulates FLJ10540 and modulates migration and invasion of lung cancer via PI3K/AKT pathway. PLoS ONE 4, e5052. Chen, C.H., Chien, C.Y., Huang, C.C., Hwang, C.F., Chuang, H.C., Fang, F.M., Huang, H.Y., Chen, C.M., Liu, H.L., Huang, C.Y., 2009b. Expression of FLJ10540 is correlated with aggressiveness of oral cavity squamous cell carcinoma by stimulating cell migration and invasion through increased FOXM1 and MMP-2 activity. Oncogene 28, 2723. Chen, C.H., Shiu, L.Y., Su, L.J., Huang, C.Y., Huang, S.C., Huang, C.C., Yin, Y.F., Wang, W.S., Tsai, H.T., Fang, F.M., Chuang, W.C., Kang, H.C., Hwang, C.F., 2012. FLJ10540 is associated with tumor progression in nasopharyngeal carcinomas and contributes to nasopharyngeal cell proliferation, and metastasis via osteopontin/CD44 pathway. J. Transl. Med. 10, 93.
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Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma P.K. Singh a , Anupam K. Srivastava a , S.K. Rath b , D. Dalela c , M.M. Goel d , M.L.B. Bhatt e,∗ a
Department of Radiotherapy, King George’s Medical University, Lucknow, Uttar Pradesh 226003, India Genotoxicity Laboratory, Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh 226001, India Department of Urology, King George’s Medical University, Lucknow, Uttar Pradesh 226003, India d Department of Pathology, King George’s Medical University, Lucknow, Uttar Pradesh 226003, India e Department of Radiation Oncology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh 226010, India b c
a r t i c l e
i n f o
Article history: Received 14 August 2014 Accepted 14 August 2014 Available online xxx Keywords: CEP55 Bladder tumorigenesis Cancer-testis Immunotherapy Peptide vaccines TURBT Transitional cell carcinoma
a b s t r a c t Bladder cancer (BC) is one among the most common and lethal urothelial malignancies worldwide. The expression of cancer-testis (CT) antigens in some tumours and restricted expression among normal tissues make CT antigens as attractive vaccine targets. In this context, we evaluated Centrosomal protein 55 kDa (CEP55), which is specifically expressed in normal human testis and various malignancies. Until the expression pattern of CEP55 in transitional cell carcinoma (TCC) of human urinary bladder and its clinical significance are not known. The aim of the present study is to evaluate mRNA/protein expression of CEP55 in TCCs of urinary bladder and correlate its expression with the clinicopathological characteristics of BC patients. In this study, the methods of quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were used to investigate mRNA/protein expression of CEP55 in TCC. Independent Student’s t test, ANOVA and Chi-square (2 ) were used to analyze the data statistically. We observed CEP55 mRNA overexpression in testis and 48.7% of BC patients. Relative mean fold expression of CEP55 mRNA was found to be significantly (p < 0.01) higher in muscle-invasive bladder cancer (MIBC) as compared to non-muscle-invasive bladder cancer (NMIBC) patients (7.88 ± 3.88 vs. 4.75 ± 2.30, p = 0.01). CEP55 protein expression was evaluated using IHC and cytoplasmic staining pattern was recorded in formalin fixed, paraffin-embedded (FFPE) bladder tumour tissues. No significant difference was observed in protein expression of CEP55 between the two groups (NMIBC and MIBC patients) (72.2% vs. 69.0%, p = 0.774). No significant protein expression of CEP55 was observed among adjacent noncancerous tissues (ANCTs) and benign prostatic hyperplasia (BPH) used as control. Our study results suggest that CEP55 mRNA/protein expression was observed is specific to TCC of human urinary bladder and might be used as a diagnostic biomarker and vaccine target in development of BC specific immunotherapy. © 2014 Elsevier GmbH. All rights reserved.
Abbreviations: TCC, transitional cell carcinoma; BC, bladder cancer; BCG, Bacillus Calmette-Guerin; BPH, Benign prostatic hyperplasia; NMIBC, non-muscle-invasive bladder cancer; MIBC, muscle-invasive bladder cancer; RC, radical cystectomy; CTA, cancer testis antigen; CT, cancer-testis; CEP55, centrosomal protein 55; ANCT, adjacent noncancerous tissue; TURBT, transurethral resection of bladder tumour; FFPE, formalin-fixed paraffin embedded; BPH, Benign prostatic hyperplasia; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; IHC, immunohistochemistry; HNSCC, head and neck squamous cell carcinoma; OCSCC, oral cavity squamous cell carcinoma; HCC, hepatocellular carcinoma. ∗ Corresponding author. Tel.: +91 9453428740; fax: +91 522 2258878. E-mail addresses: [email protected], [email protected] (M.L.B. Bhatt). http://dx.doi.org/10.1016/j.imbio.2014.08.014 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Singh, P.K., et al., Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma. Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.08.014
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2
Introduction Bladder cancer (BC) is one of the major deadly malignancies in the world. BC accounted for approximately 2% of all human malignancies with an estimated 72,570 new cases and 15,210 deaths in United States alone in 2013 (Siegel et al., 2014). Approximately 80% of BCs present as non-muscle-invasive urothelial carcinoma, out of which 70% of them will recur subsequently after initial therapy and 10–20% of them will progress and invade the bladder muscle (Knowles, 2001). Of the patients initially presenting with muscle-invasive bladder cancer (MIBC), 50% will relapse with metastatic disease (Wolff et al., 2005; Williams and Stein, 2004). In spite of various therapeutic options, more than 90% of patients with metastasis die within the first 5 years (Luke et al., 2010). High-grade muscle-invasive disease represents a life-threatening condition and requires timely treatment (Herr et al., 2007; Chang et al., 2003). Standard treatment for MIBC is radical cystectomy (RC) with bilateral pelvic lymphadenectomy, and is justified in patients with life expectancy of greater than 2 years (Singh et al., 2014). Non-muscle-invasive bladder cancer (NMIBC) patients are often treated with a local, organ-sparing transurethral resection of bladder tumour (TURBT) followed by intravesical immunotherapy with attenuated Bacillus Calmette-Guerin (BCG), which by an unknown mechanism reduces the risk of recurring tumours, suggesting that anticancer vaccines could be especially useful for the treatment of BC (Dyrskjot et al., 2012; Simons et al., 2008). Approximately 50% of NMIBC patients at high risk of recurrence remain free of disease after receiving BCG immunotherapy following surgery (Singh et al., 2014). This indicates that immunotherapeutic strategies based on specific tumour-associated antigens (TAAs) in BC might be more effective and associated with less morbidity than BCG (Singh et al., 2014). BC thus represents a unique opportunity to develop and test cancer vaccines that could be used after surgery to prevent tumour recurrence, in an ideal setting of low tumour burden (Singh et al., 2014; Picard et al., 2007). However, the development of cancer specific immunotherapy depends on the identification of relevant target antigens (Fradet et al., 2006). More recently, cancer-testis (CT) antigen has emerged to be a unique group of antigen that could potentially be important antigen targets for antigen-specific cancer immunotherapy (Caballero and Chen, 2009). CT antigens are normally expressed in the testis only, apart from some expressed in the early developing embryo and the placenta (Dyrskjot et al., 2012). However, CT antigens are also expressed in various malignancies including BC (Singh et al., 2014; Dyrskjot et al., 2012; Mengus et al., 2013; Yin et al., 2012). So far, more than 200 CT antigens have been reported in previous studies, which belong to more than 70 distinct subfamilies (Almeida et al., 2009). The development of cancer-specific immunotherapy based on CT antigens has been ongoing for years, with some candidates reaching early-phase clinical trials (Obara et al., 2012; Francois et al., 2009; Sharma et al., 2008; Nishiyama et al., 2001). Furthermore, expression of various CT antigens in BC has been evaluated for potential immunotherapy purposes (Singh et al., 2014; Dyrskjot et al., 2012; Picard et al., 2007; Yin et al., 2012; Bergeron et al., 2009; Kanehira et al., 2007a,b; Sharma et al., 2006, 2003; Kurashige et al., 2001; Patard et al., 1995). In this quest, we evaluated one of the CT antigens, Centrosomal protein 55 kDa (CEP55) that is highly expressed in cancer cells but not in normal cells, except for testis (Inoda et al., 2009). CEP55 is also known by several other names, including FLJ10540, C10orf3 and URCC6 (Tao et al., 2014). CEP55 is overexpressed in human gastric cancer (Tao et al., 2014) nasopharyngeal carcinoma (NPC) (Chen et al., 2012), colorectal cancer (Inoda et al., 2011), oropharyngeal squamous cell carcinoma (Janus et al., 2011), prostate cancer (Shiraishi et al., 2011), head and neck squamous cell carcinoma (HNSCC) (Waseem et al., 2010), lung cancer (Chen et al., 2009a,b)
Table 1 Patient clinciopathological parameters. Clinicopathological characteristics
Age (years, %) ≤45 >45 Sex Male Female Grade Low High Stage Ta T1 T2-T4 Smoking No Yes Tobacco chewers No Yes
(n) %
Real-time-PCR Assay (76)
Immunohistochemistry Assay (65)
24 (31.6%) 52 (68.4%)
17 (26.2%) 48 (73.8%)
67 (88.2%) 9 (11.8%)
63 (96.9%) 2 (3.1%)
33 (43.4%) 43 (56.6%)
23 (35.4%) 42 (64.6%)
3 (3.9%) 33 (43.4%) 40 (52.6%)
6 (9.2%) 23 (35.4%) 36 (55.4%)
34 (44.7%) 42 (55.3%)
29 (44.6%) 36 (55.4%)
36 (47.4%) 40 (52.6%)
33 (50.8%) 32 (49.2%)
and oral cavity squamous cell carcinoma (OCSCC) (Chen et al., 2009a,b), breast carcinoma (Inoda et al., 2009), hepatocellular carcinoma (HCC) (Chen et al., 2007) and colon cancer (Sakai et al., 2006). However, there is little or no information regarding the expression and significance of CEP55 with tumour histological grade, stage, and patient outcome in BC patients. The purpose of the present study is to evaluate the expression levels of CEP55 in BC tissue specimens, and to correlate the results with clinicopathological parameters of BC patients.
Methods Collection of clinical specimens To evaluate CEP55 mRNA expression, 76 freshly frozen BC tissues (male: 67; female: 9) and 7 matched adjacent noncancerous tissue (ANCT) specimens were obtained from histologically proven TCC of urinary bladder patients. All patients had undergone TURBT or RC between March 2006 and April 2011 at Department of Urology, King George’s Medical University (KGMU), Lucknow, India. The mean age of the BC patients used in qRT-PCR assay was 54.75 ± 11.51 years. For evaluation of immunohistochemical (IHC) expression of CEP55, 65 formalin fixed, paraffin-embedded (FFPE) bladder tissues, 12 ANCT specimens and 10 BPH tissues were obtained from the archives of the Pathology Department, KGMU. The mean age of the BC patients was 53.83 ± 11.47 years whereas mean age of BPH patients was 64.14 ± 11.46 years. The 2004 World Health Organization (WHO) bladder tumour classification criteria (low-grade and high-grade) were used in grading of bladder tumours (Eble et al., 2004), whereas pathologic staging was done according to the 2002 tumour-lymph node-metastasis (TNM) classification system (Greene et al., 2002). Demographic and clinical characteristics of BC patients are shown in Table 1. Written informed consent was obtained from all the participants for the use of their clinical samples in this study. Approval of institutional ethics committee was taken before the commencement of study. A unique code was given to all collected tissue specimens and further all investigations were made blind to histopathological or cytologic diagnosis. All clinicopathological information was recorded on a written proforma at the time of recruitment in the study.
Please cite this article in press as: Singh, P.K., et al., Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma. Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.08.014
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RNA extraction and cDNA synthesis Total RNA was extracted from tissue specimens using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, after phenol treatment and drying, RNA pellet was dissolved in RNase-free water and stored at −80 ◦ C till further use. The RNA concentration was quantified and the quality of the RNA was verified by electrophoresis on a 1% denaturing agarose gel. Then cDNA synthesis was carried out using Quantitect® Reverse Transcription Reagent (Qiagen GmbH, Hilden, Germany) as per given instructions. Quantitative real-time PCR analysis The quantitative gene expression levels of CEP55 was measured using SYBR® GreenERTM qPCR SuperMix Universal (Invitrogen), on the LightCycler 480 (Roche Applied Science, Mannheim, Germany) as described previously (Waseem et al., 2010) with the housekeeping gene POLR2A, used as an internal control. The reaction mix consisted of 5 l of 2× SYBR Green Master Mix (Invitrogen), 5 M of each primer, 2 l cDNA and 2 l nuclease-free water to obtain a final volume of 10 l. All samples were analyzed in triplicate. The CEP55 primers used in qRT-PCR were as follows: forward primer of 5 -TGAAGAGAAAGACGTATTGAAACAA-3 and reverse primer of 5 -GCAGTTTGGAGCCACAGTCT-3 . The primer sequence for housekeeping gene POLR2A were as follows: forward primer of 5 -GCAAATTCACCAAGAGAGAC-3 and reverse primer of 5 -CACGTCGACAGGAACATCAG-3 . The optimized amplification protocol consisted of an initial denaturation step of 95 ◦ C for 10 min, followed by 40 amplification cycles at 95 ◦ C for 10 s, annealing at 60 ◦ C for 20 s and elongation at 72 ◦ C for 10 s. At the end of the PCR cycles, melting curve analyses were performed. The fold changes for CEP55 expression levels were calculated using 2−Ct (Livak and Schmittgen, 2001). Immunohistochemistry IHC analysis and evaluation of CEP55 in bladder tumours, ANCTs and BPH tissues was performed using a sensitive EnVision FLEX mini kit High pH (K802321; Dako, Glostrup, Denmark), as described previously (Singh et al., 2014). The CEP55 protein in FFPE tissue sections was detected using a rabbit polyclonal antibody against CEP55 (CEP55 (H-300); sc-134622; 1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 ◦ C.
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expression. CEP55 mRNA expression was observed in higher number of MIBC patients than NMIBC patients (62.5% vs. 33.3%, p < 0.05). In the MIBC patients relative mean fold expression of CEP55mRNA was also found to be significantly (p < 0.01) higher as compared to NMIBC (7.88 ± 3.88 vs. 4.75 ± 2.30, p = 0.01). Furthermore, CEP55 mRNA expression was detected in 48.4% patients with low grade and 48.8% with high grade TCC respectively. In matched BC and their ANCTs, the average expression level of CEP55 mRNA in BC was found to be ∼6-fold higher than those in ANCTs. Among normal tissues CEP55 mRNA expression was detected in testis, placenta, thymus and small intestine. However, overexpression was found only in testis and fold expression level of CEP55 mRNA in other normal tissues was 104-fold lower than that in the testis. The relative mean fold expression of CEP55 mRNA and its association with the clinicopathological characteristics of the BC patients are summarized in Table 2. There was no significant association observed between CEP55 mRNA expression and clinicopathological characteristics such as BC patient’s age, gender, grade and disease stage etc. in both the NMIBC and MIBC patients. Although, high relative mean fold expression of CEP55 mRNA was found in older age patients vs. young patients, males vs. females, advanced stage vs. early stage, high grade vs. low grade, smokers vs. non-smokers and tobacco chewers vs. non-tobacco chewers. Immunohistochemical expression of CEP55 Expression of CEP55 protein was detected in the cytoplasm of urothelial carcinoma cells (Fig. 1). IHC expression of CEP55 was observed in 69.0% (20 of 29) of NMIBC and 72.2% (26 of 36) MIBC patients. Thus IHC expression of CEP55 was found to be positive in 70.8% (46 of 65) BC patients. There is no significant difference was observed in IHC expression of CEP55 between NMIBC and MIBC patients (72.2% vs. 69.0%, p = 0.774). Furthermore, cytoplasmic expression of CEP55 was detected in 69.5% patients with low grade and 71.4% with high grade TCC respectively. No significant expression of CEP55 protein was found among BPH and ANCT tissues used as control (Fig. 1E and F). The IHC expression of CEP55 and its association with the clinicopathological characteristics of the patients are summarized in Table 3. The IHC expression of CEP55 was also not found to be significantly associated with the any of the clinicopathological characteristics of NMIBC and MIBC patients being evaluated. Discussion
Statistical analysis Continuous data were expressed as mean ± SD, while discrete (categorical) data in %. Qualitative variables were shown as percentages and numbers. Comparisons were made between independent groups by independent Student’s t test and ANOVA and the significance of mean difference between the groups were evaluated by Tukey’s post hoc test after adjusting the multiple contrasts for significance. Chi-square (2 ) test was used to evaluate comparisons between categorical groups. All p values were 2-sided and a p-value of less than 0.05 was considered statistically significant. All analyses were performed using SPSS (Windows version 18.0) statistical software packages. Results Quantitative expression of CEP55 mRNA CEP55 mRNA expression was detected in 33.3% (12 of 36) of NMIBC and 62.5% (25 of 40) MIBC patients. Thus an overall 48.7% (37 of 76) BC patients were found to be positive for CEP55 mRNA
BC is the most common malignancy of the urinary tract (Anastasiadis and de Reijke, 2012). The requirement of follow-up and repeated treatment has made BC one of the most expensive cancers in today’s medical practice (Madeb et al., 2007). Despite advances in treatment, the prognosis of BC, especially muscleinvasive tumours, remains poor (Ren et al., 2014). Even with chemotherapeutic treatment, the overall median survival is 1 year advanced TCC patients. This survival is frequently achieved with severe and life-threatening side effects (Perabo and Muller, 2007). Therefore, effective diagnostic biomarkers and novel therapeutic approach is urgently required in BC. Recently, cancer immunotherapy has emerged as a powerful and promising clinical approach for treatment of various malignancies and has shown major success in breast cancer, prostate cancer, melanoma (Matsueda and Graham, 2014). The field of cancer vaccine therapy is currently expected to become the fourth option in the treatment of cancer after surgery, chemotherapy and radiation therapy and a large number of clinical investigations are underway at present (Obara et al., 2012). Several clinical trials of immunotherapy using peptide vaccines derived from CT antigens for various
Please cite this article in press as: Singh, P.K., et al., Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma. Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.08.014
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Table 2 Correlation between relative mean fold CEP55 mRNA expression and clinical characteristics of BC patients. Characteristics Age (years) ≤45 >45 Sex Female Male Grade Low High Smoking No Yes Tobacco No Yes
Non-muscle-invasive Mean ± SD
p value
Muscle-invasive Mean ± SD
p value
p value
Total Mean ± SD
2.00 ± 0.00 (1) 5.00 ± 2.24 (11)
0.384
7.25 ± 2.19 (8) 8.18 ± 4.49 (17)
0.588
6.67 ± 2.69 (9) 6.93 ± 4.04 (28)
0.857
2.00 ± 0.00 (1) 5.00 ± 2.24 (11)
0.228
7.33 ± 1.16 (3) 7.95 ± 4.12 (22)
0.801
6.00 ± 2.83 (4) 6.97 ± 3.84 (33)
0.629
4.78 ± 2.44 (9) 4.67 ± 2.31 (3)
0.946
8.00 ± 2.24 (7) 7.83 ± 4.41 (18)
0.926
6.19 ± 2.81 (16) 7.38 ± 4.28 (21)
0.341
5.63 ± 2.00 (8) 3.00 ± 2.00 (4)
0.057
7.44 ± 3.05 (9) 8.13 ± 4.35 (16)
0.683
6.59 ± 2.69 (17) 7.10 ± 4.47 (20)
0.683
4.40 ± 2.61 (5) 5.00 ± 2.34 (7)
0.677
7.43 ± 3.48 (14) 8.45 ± 4.44 (17)
0.523
6.63 ± 3.48 (19) 7.11 ± 4.04 (18)
0.701
Numbers in parenthesis indicate the number of BC patients.
Table 3 Correlation between CEP55 protein expression and clinical characteristics of BC patients. Characteristics
Age (years) ≤45 >45 Sex Female Male Grade Low High Stage Ta T2–T4 Smoking No Yes Tobacco No Yes
Non-muscle-invasive (n = 29)
Muscle-invasive (n = 36)
Total (n = 65)
p value
Negative, n (%)
Positive, n (%)
p value
Negative, n (%)
Positive, n (%)
p value
Negative, n (%)
Positive, n (%)
2 (22.2%) 7 (77.8%)
4 (20.0%) 16 (80.0%)
0.891
3 (30.0%) 7 (70.0%)
8 (30.8%) 18 (69.2%)
0.964
5 (26.3%) 14 (73.7%)
12 (26.1%) 34 (73.9%)
0.985
0 (0.0%) 9 (100%)
0 (0.0%) 20 (100%)
NA
1 (10.0%) 9 (90.0%)
1 (3.8%) 25 (96.2%)
0.470
1 (5.3%) 18 (94.7%)
1 (2.2%) 45 (97.8%)
0.512
6 (66.7%) 3 (3.3%)
11 (55.0%) 9 (45.0%)
0.555
1 (10.0%) 9 (90.0%)
5 (19.2%) 21 (80.8%)
0.506
7 (36.8%) 12 (63.2%)
16 (34.8%) 30 (65.2%)
0.875
2 (22.2%) 7 (77.8%) 0 (0.0%)
4 (20.0%) 16 (80.0%) 0 (0.0%)
0.891
0 (0.0%) 0 (0.0%) 10 (100%)
0 (0.0%) 0 (0.0%) 26 (100%)
NA
2 (10.5%) 7 (36.8%) 10 (52.6%)
4 (8.7%) 16 (34.8%) 26 (56.5%)
0.950
6 (66.7%) 3 (33.3%)
13 (65.0%) 7 (35.0%)
0.930
3 (30%) 7 (70%)
7 (26.9%) 19 (73.1%)
0.854
9 (47.4%) 10 (52.6%)
20 (43.5%) 26 (56.5%)
0.774
4 (44.4%) 5 (55.6%)
12 (60.0%) 8 (40.0%)
0.436
5 (50.0%) 5 (50.0%)
12 (46.2%) 14 (53.8%)
0.836
9 (47.4%) 10 (52.6%)
24 (52.2%) 22 (47.8%)
0.724
Fig. 1. Immunohistochemical analysis of CEP55 in BC. BC tissues were stained with CEP55 specific rabbit polyclonal antibody. (A) MIBC showing cytoplasmic positivity. (B) MIBC showing strong cytoplasmic expression. (C) NMIBC showing positivity. (D) NMIBC showing positivity. (E) BPH tissue showing negative expression. (F) ANCT showing negative expression.
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types of advanced cancers or cancers not responding to standard therapies have been reported recently (Obara et al., 2012; Aruga et al., 2014, 2013). Thus the identification and validation of additional CT antigens is crucial towards development of BC specific immunotherapy. CEP55 overexpression is being observed in a number of human cancers and cell lines, and found to be correlated with specific clinicopathological tumour features (Inoda et al., 2009; Tao et al., 2014; Chen et al., 2012; Inoda et al., 2011; Janus et al., 2011; Shiraishi et al., 2011; Waseem et al., 2010; Chen et al., 2009a,b, 2007; Sakai et al., 2006). However, its expression status and clinical significance in BC remains to be clarified. Moreover, distribution of CEP55 overexpression in a wide range of malignant human tissues makes it a reasonable target for cancer immunotherapy (Inoda et al., 2011). CEP55 derived peptide vaccines have also shown promising results towards treatment of breast and colorectal cancer patients (Inoda et al., 2009, 2011). Thus in the present study, we analyzed the detailed mRNA/Protein expression pattern of CEP55 in human BC towards possible application of CEP55 as a target to develop active BC specific immunotherapy. Significantly, this study is the first to report that CEP55 mRNA/protein expression is upregulated in TCC of urinary bladder. In the present study, we have detected CEP55 mRNA expression in 48.7% BC patients using qRT-PCR. Our study findings derived by qRT-PCR are in agreement with previous observations reporting CEP55 upregulation in a variety of cell lines and tumours, such as breast, colorectal, pancreas, esophageal, gallbladder, and lung carcinomas by using qRT-PCR analysis (Chen et al., 2012; Janus et al., 2011; Waseem et al., 2010; Chen et al., 2009a,b; Sakai et al., 2006). Our study findings are also consistent with the previous studies of CT antigen expression in BC that MAGE-A3 expression was detected in 25–58% of tumours (Picard et al., 2007; Nishiyama et al., 2001; Sharma et al., 2006; Patard et al., 1995) whereas NYESO-1 expression were detected in 32–45% tumours respectively (Dyrskjot et al., 2012; Yin et al., 2012; Sharma et al., 2003; Kurashige et al., 2001). Among normal tissues, CEP55 mRNA was expressed only in the testis, and at a very low level in the thymus, small intestine and placenta. These findings are consistent with the previous observation by Inoda et al. (2009). Furthermore, qRT-PCR analysis demonstrated significant higher fold expression in BC tissues to their ANCT. These results are in agreement with previous published reports (Chen et al., 2012; Janus et al., 2011; Waseem et al., 2010; Chen et al., 2009a,b; Sakai et al., 2006), demonstrating that CEP55 mRNA was highly overexpressed in cancerous tissues compared with their ANCTs, thus our study findings are suggestive that CEP55 mRNA expression might be playing an important role in bladder tumorigenesis. Our study results further conclude that the CEP55 is not only expressed at the mRNA level but is also translated as evident from IHC evaluation of CEP55 in BC tissue specimens. To the best of our knowledge, we have evaluated CEP55 protein expression in BC for the first time by IHC, which revealed cytoplasmic staining pattern in TCC but no significant expression, was observed among ANCT and BPH tissues. Our results are in agreement with previous studies that observed higher protein expression of CEP55 in various tumour tissues and relatively no or little expression in their ANCTs. (Inoda et al., 2009; Tao et al., 2014; Chen et al., 2012; Inoda et al., 2011; Waseem et al., 2010; Chen et al., 2009a,b, 2007; Sakai et al., 2006). Our results are also consistent with previous studies that observed high protein expression of CT antigen in bladder tumour specimens but not in ANCTs (Singh et al., 2014; Mengus et al., 2013; Yin et al., 2012). In the present study, we demonstrated frequent mRNA/protein expression of CEP55 in TCC of urinary bladder patients with different grade and stage. Though its frequent expression was detected among TCC, but no significant association was observed between
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CEP55 protein expression and clinicopathological characteristics such as disease stage, grade and habits of patients. However, it further warrants evaluation of CEP55 in BC in a larger sample size to reach any logical correlation. These results are in accordance with findings from earlier study performed in TCC of bladder (Yin et al., 2012). The present study has few limitations. The tumour samples for the analysis of mRNA expression and protein expression analysis came from different patient. Thus, association between mRNA expression and protein expression was not achieved. Furthermore, the relatively lesser number of patients did not granted to perform subanalyses. Conclusion In conclusion, this study provided the first evidence about upregulation of CEP55 mRNA/Protein expression in BC using qRTPCR, and IHC and renders its diagnostic potential as a molecular marker. Our study findings also conclude that CEP55 might be a potential vaccine candidate for immunotherapy in BC due to its expression among bladder tumours and restricted expression in normal tissues. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements First author (Pankaj Kumar Singh) would like to thank Indian Council of Medial Research (ICMR), New Delhi, India for awarding Senior Research Fellowship (ICMR-SRF) (IRIS ID-2007-04520) under the guidance of corresponding author. References Almeida, L.G., Sakabe, N.J., deOliveira, A.R., Silva, M.C., Mundstein, A.S., Cohen, T., Chen, Y.T., Chua, R., Gurung, S., Gnjatic, S., Jungbluth, A.A., Caballero, O.L., Bairoch, A., Kiesler, E., White, S.L., Simpson, A.J., Old, L.J., Camargo, A.A., Vasconcelos, A.T., 2009. CTdatabase: a knowledge-base of high-throughput and curated data on cancer-testis antigens. Nucleic Acids Res. 37, D816. Anastasiadis, A., de Reijke, T.M., 2012. Best practice in the treatment of nonmuscle invasive bladder cancer. Ther. Adv. Urol. 4, 13. Aruga, A., Takeshita, N., Kotera, Y., Okuyama, R., Matsushita, N., Ohta, T., Takeda, K., Yamamoto, M., 2013. Long-term vaccination with multiple peptides derived from cancer-testis antigens can maintain a specific T-cell response and achieve disease stability in advanced biliary tract cancer. Clin. Cancer Res. 19, 2224. Aruga, A., Takeshita, N., Kotera, Y., Okuyama, R., Matsushita, N., Ohta, T., Takeda, K., Yamamoto, M., 2014. Phase I clinical trial of multiple-peptide vaccination for patients with advanced biliary tract cancer. J. Transl. Med. 12, 61. Bergeron, A., Picard, V., LaRue, H., Harel, F., Hovington, H., Lacombe, L., Fradet, Y., 2009. High frequency of MAGE-A4 and MAGE-A9 expression in high-risk bladder cancer. Int. J. Cancer 125, 1365. Caballero, O.L., Chen, Y.T., 2009. Cancer/testis (CT) antigens: potential targets for immunotherapy. Cancer Sci. 100, 2014. Chang, S.S., Hassan, J.M., Cookson, M.S., Wells, N., Smith Jr., J.A., 2003. Delaying radical cystectomy for muscle invasive bladder cancer results in worse pathological stage. J. Urol. 170, 1085. Chen, C.H., Lu, P.J., Chen, Y.C., Fu, S.L., Wu, K.J., Tsou, A.P., Lee, Y.C., Lin, T.C., Hsu, S.L., Lin, W.J., Huang, C.Y., Chou, C.K., 2007. FLJ10540-elicited cell transformation is through the activation of PI3-kinase/AKT pathway. Oncogene 26, 4272. Chen, C.H., Lai, J.M., Chou, T.Y., Chen, C.Y., Su, L.J., Lee, Y.C., Cheng, T.S., Hong, Y.R., Chou, C.K., Whang-Peng, J., Wu, Y.C., Huang, C.Y., 2009a. VEGFA upregulates FLJ10540 and modulates migration and invasion of lung cancer via PI3K/AKT pathway. PLoS ONE 4, e5052. Chen, C.H., Chien, C.Y., Huang, C.C., Hwang, C.F., Chuang, H.C., Fang, F.M., Huang, H.Y., Chen, C.M., Liu, H.L., Huang, C.Y., 2009b. Expression of FLJ10540 is correlated with aggressiveness of oral cavity squamous cell carcinoma by stimulating cell migration and invasion through increased FOXM1 and MMP-2 activity. Oncogene 28, 2723. Chen, C.H., Shiu, L.Y., Su, L.J., Huang, C.Y., Huang, S.C., Huang, C.C., Yin, Y.F., Wang, W.S., Tsai, H.T., Fang, F.M., Chuang, W.C., Kang, H.C., Hwang, C.F., 2012. FLJ10540 is associated with tumor progression in nasopharyngeal carcinomas and contributes to nasopharyngeal cell proliferation, and metastasis via osteopontin/CD44 pathway. J. Transl. Med. 10, 93.
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Please cite this article in press as: Singh, P.K., et al., Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma. Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.08.014
