Tumor Biol. (2014) 35:11499–11504 DOI 10.1007/s13277-014-2480-1
RESEARCH ARTICLE
Over-expression of TRPM8 is associated with poor prognosis in urothelial carcinoma of bladder Ning Xiao & Lei M. Jiang & Bo Ge & Tian Y. Zhang & Xiao K. Zhao & Xing Zhou
Received: 11 June 2014 / Accepted: 8 August 2014 / Published online: 16 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Transient receptor protein (TRP) channels are frequently associated with tumors and are correlated to patient’s outcome. We firstly investigated TRP channel melastatin 8 (TRPM8) expression in urothelial carcinoma of bladder (UCB) and its correlation with UCB clinicopathological features and additionally evaluated the association between TRPM8 expression and patients’ survival rate to elucidate its role in bladder oncogenesis. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted to examine TRPM8 messenger RNA (mRNA) expression in 36 pairs of freshly frozen UCB tissues and matched noncancerous tissues. Immunohistochemistry (IHC) was performed in 156 archived paraffin-embedded UCB samples to explore the correlation between TRPM8 protein and clinicopathological features. The association between TRPM8 expression and patient’s survival rate was evaluated using the Kaplan–Meier method and multivariate Cox regression analysis, respectively. The expression levels of TRPM8 mRNA in UCB tissues were significantly higher than those in matched noncancerous tissues (P=0.016). Expression of TRPM8 protein in UCB was significantly and positively associated with histological grade (P=0.039) and tumor stage (P=0.037). Significant correlation between high TRPM8 expression and poor cumulative survival of UCB patients was shown using N. Xiao : L. M. Jiang : B. Ge : T. Y. Zhang Department of Urology, The Affiliated Hospital of Guilin Medical College, Guilin, People’s Republic of China N. Xiao : X. Zhou (*) Department of Urology, The Second Affiliated Hospital of Guangzhou Medical University, No. 250, Changgang East Road, Haizhu District, Guangzhou 510260, People’s Republic of China e-mail: [email protected] X. K. Zhao Department of Urology, The Second Xiangya Hospital of Central South University, Changsha, People’s Republic of China
the Kaplan–Meier survival curve (P=0.039). TRPM8 was represented as an independent prognostic biomarker for UCB patients by multivariate Cox regression analysis (P=0.047). The present study provide the convincing evidence for the first time that over-expression of TRPM8 may play a role in the pathogenesis and progression of UCB, and TRPM8 may serve as an independent prognostic biomarker for UCB patients. Keywords TRPM8 . Urothelial carcinoma . Prognosis In 2012, estimated 77,570 new cases of urothelial carcinoma of bladder (UCB) and 15,210 deaths were reported in the USA [1]. Most new cases of UCB are Ta, T1, or carcinoma in situ (CIS), which grouped as non-muscle-invasive bladder carcinoma (NMIBC), and a significant percent of patients with NMIBC will experience recurrence and some will have stage progression after transurethral resection (TUR) and early intravesical chemotherapy [2]. Radical cystectomy (RC) with urinary diversion is the gold standard of treatment for locoregional control of muscle-invasive bladder carcinoma (MIBC), but some serious complications, including excessive bleeding, ileus, metabolic disorders, and pyelonephritis, often occur [3]. Although selected patients with stage IV UCB may benefit from concurrent chemoradiotherapy (CCRT), high mortality can still be seen in patients with advanced UCB [4]. Due to different biological behaviors and tumor heterogeneities, current clinicopathological factor and biomarker of UCB do not predict accurately the clinical outcome of some patients with UCB; therefore, the issue of finding new and powerful molecular makers has to be settled urgently [5]. Recent researches reported that the family of transient receptor potential (TRP) channels is associated with oncogenesis and TRP channels might be potential targets for cancer treatment [6]. In the subfamilies of TRP channels, TRP channel
11500
melastatin 8 (TRPM8) was identified and has been proposed to be the predominant thermoreceptor for cellular and behavioral responses to cold stimuli [7]. Although the aberrant expression of TRPM8 is detected in many tumors [8–11] and is represented as a key protein in central mechanisms of carcinogenesis, some disagreement of their roles in it was raised in different studies [12]. In the present study, we firstly detected the expression profile of TRPM8 messenger RNA (mRNA) in UCB tissues by a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), subsequently assessed the correlation between TRPM8 protein expression and clinicopathological characteristics of UCB, and additionally investigated the influence of TRPM8 expression on the survival of patient with UCB.
Tumor Biol. (2014) 35:11499–11504 Table 1 Sequences of the primers used in this study Primer
Sense (5′–3′)
Antisense (5′–3′)
TRPM8
GAGCTGGATGAGCACA AC TGACTTCAACAGCGAC ACCCA
GAAGTAAGCGAAGACG ATG CACCCTGTTGCTGTAG CCAAA
GAPDH
Materials and methods
Reagent (Invitrogen). RNA was reversely transcribed using SuperScript First Strand cDNA System (Invitrogen) according to the manufacturer’s protocol. The Primer Express Software Version 2.0 was used to designed sequences of qRT-PCR primers (primer sequences are shown in Table 1). The qRTPCR was performed according to standard methods. ΔCt= Ctgene −Ctreference and 2−ΔΔCt method was respectively used to show relative levels and fold change of target gene expression, where Ct represents the threshold cycle of each transcript.
Patients and tissue samples
Immunohistochemistry
The present study was approved by research ethics committees of Guangzhou Medical University, the Central South University, and Guilin Medical College, and the Helsinki Declaration of Human Rights was strictly observed. Written informed consent was obtained from all recruited patients. All tissue samples were obtained from the Second Affiliated Hospital of Guangzhou Medical University, the Second Xiangya Hospital of Central South University, and the Affiliated Hospital of Guilin Medical College. Radical cystectomy was performed in all patients with UCB. The duration of follow-up was calculated from the date of surgery to death or last follow-up, and patients were excluded if they had incomplete medical records or inadequate follow-up. Tumor was staged and graded, respectively, according to the TNM classification and the 1998 WHO classification [13]. For qRTPCR, 36 pairs of fresh UCB tissues and matched noncancerous tissues were collected after surgery between July 2010 and July 2012 and were then immediately frozen in liquid nitrogen and stored at −80 °C until detection of TRPM8 mRNA expression in tissues by qRT-PCR. For immunohistochemical assay, a total of 156 paraffin-embedded UCB samples were obtained from three hospitals between January 2008 and July 2012. One hundred fifty-six patients’ characteristics are summarized in Table 1. None of the patients recruited in the present study had undergone preoperative chemotherapy or radiotherapy.
Immunohistochemistry was performed as described previously [14]. Four random fields of each sample were examined under a compound microscope (Olympus BX51). The intensity of TRPM8 immunostaining was graded by semiquantitative estimation, which was obtained using a composite score by means of multiplying the values of staining intensity and relative abundance of positive cells. Each slide score was gained by two observers on two separate occasions, and the average score was calculated. Intensity grades 0, 1, 2, and 3 represent no, weak, moderate, and strong staining,
Real-time quantitative reverse transcription polymerase chain reaction For mRNA qRT-PCR assay, total RNA was isolated from 36 pairs of UCB tissues and adjacent normal tissues using TRIzol
Fig. 1 TRPM8 mRNA expression in 36 pairs of UCB and matched noncancerous tissues was respectively detected by qRT-PCR assay. The expression level of TRPM8 mRNA in UCB tissue was significantly higher than that in matched noncancerous tissue (3.56±0.50 vs. 1.59± 0.35, P=0.016). UCB urothelial carcinoma of bladder
Tumor Biol. (2014) 35:11499–11504
respectively. For Kaplan–Meier survival analysis, a composite score of more than the median value was considered a high expression and that less than or equal to the median value was considered a low expression.
11501 Table 2 Correlation between TRPM8 protein expression and various clinicopathological variables of UCB Variables
No. (n=156)
High expression (n=84)
34 38
38 46
0.872
27 45
41 43
0.195
37 35
39 45
0.630
74 82
33 39
41 43
0.749
51 105
30 42
21 63
0.039
Results mRNA expression of TRPM8 in UCB and noncancerous tissues
37 119
23 49
14 70
0.037
The expression of TRPM8 mRNA in 36 pairs of UCB and matched noncancerous tissue was detected by qRT-PCR. As shown in Fig. 1, UCB tissues have a significant higher expression of TRPM8 mRNA than matched noncancerous tissues (3.56±0.50 vs. 1.59±0.35, P=0.016).
Immunohistochemistry The expression of TRPM8 protein in 156 pairs of paraffinembedded UCB and matched noncancerous samples was examined by IHC analysis. We found that 53.8 % (84/156) of UCB samples represented as a high expression of TRPM8 protein and 37.2 % (58/156) of noncancerous samples showed high TRPM8 protein expression (Fig. 2). The expression level of TRPM8 protein was significantly higher in UCB samples than in noncancerous samples (P=0.004). Fig. 2 Immunohistological staining of TRPM8 protein in UCB samples. a Typical image of UCB tissue with high expression of TRPM8 protein. b Typical image of matched noncancerous samples with low expression of TRPM8 protein
Gender Male 72 Female 84 Age (years) ≤65 68 >65 88 Tumor number Unifocal 76 Multifocal 80 Tumor size (cm) ≤3.0 >3.0 Grade G1 G2 and G3 T stage Ta–T1 T2–T4
P value
Low expression (n=72)
Statistical analysis SPSS 19.0 was used to perform data analysis. The t test was used to analyze data from UCB tissues and matched noncancerous tissues detected by qRT-PCR. The relationship between TRPM8 expression and clinicopathological parameters was evaluated by chi-square test. The survival rate and comparison of differences in survival curves were determined by Kaplan–Meier analysis and log-rank tests. The significance of multiple predictors of survival was assessed by Cox regression analysis. Differences were considered significant at P0.05).
Association of TRPM8 protein expression and UCB patient’s survival Kaplan–Meier analysis and log-rank tests were used to investigate the relationship between TRPM8 protein expression
11502
Tumor Biol. (2014) 35:11499–11504
Fig. 3 The overall survival rate of patients with UCB estimated according to the TRPM8 expression level in UCB samples (Kaplan–Meier method) with immunohistochemical staining. The patients with high TRPM8 expression inclined to have a significant shorter cumulative survival than those with low TRPM8 expression
and survival of UCB patients. UCB patients with high TRPM8 protein expression were inclined to have a shorter overall survival than those with low TRPM8 protein expression (P=0.039, Fig. 3). Histological grade, tumor stage, and TRPM8 protein expression were significantly associated with overall survival of UCB patients (all P
RESEARCH ARTICLE
Over-expression of TRPM8 is associated with poor prognosis in urothelial carcinoma of bladder Ning Xiao & Lei M. Jiang & Bo Ge & Tian Y. Zhang & Xiao K. Zhao & Xing Zhou
Received: 11 June 2014 / Accepted: 8 August 2014 / Published online: 16 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Transient receptor protein (TRP) channels are frequently associated with tumors and are correlated to patient’s outcome. We firstly investigated TRP channel melastatin 8 (TRPM8) expression in urothelial carcinoma of bladder (UCB) and its correlation with UCB clinicopathological features and additionally evaluated the association between TRPM8 expression and patients’ survival rate to elucidate its role in bladder oncogenesis. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted to examine TRPM8 messenger RNA (mRNA) expression in 36 pairs of freshly frozen UCB tissues and matched noncancerous tissues. Immunohistochemistry (IHC) was performed in 156 archived paraffin-embedded UCB samples to explore the correlation between TRPM8 protein and clinicopathological features. The association between TRPM8 expression and patient’s survival rate was evaluated using the Kaplan–Meier method and multivariate Cox regression analysis, respectively. The expression levels of TRPM8 mRNA in UCB tissues were significantly higher than those in matched noncancerous tissues (P=0.016). Expression of TRPM8 protein in UCB was significantly and positively associated with histological grade (P=0.039) and tumor stage (P=0.037). Significant correlation between high TRPM8 expression and poor cumulative survival of UCB patients was shown using N. Xiao : L. M. Jiang : B. Ge : T. Y. Zhang Department of Urology, The Affiliated Hospital of Guilin Medical College, Guilin, People’s Republic of China N. Xiao : X. Zhou (*) Department of Urology, The Second Affiliated Hospital of Guangzhou Medical University, No. 250, Changgang East Road, Haizhu District, Guangzhou 510260, People’s Republic of China e-mail: [email protected] X. K. Zhao Department of Urology, The Second Xiangya Hospital of Central South University, Changsha, People’s Republic of China
the Kaplan–Meier survival curve (P=0.039). TRPM8 was represented as an independent prognostic biomarker for UCB patients by multivariate Cox regression analysis (P=0.047). The present study provide the convincing evidence for the first time that over-expression of TRPM8 may play a role in the pathogenesis and progression of UCB, and TRPM8 may serve as an independent prognostic biomarker for UCB patients. Keywords TRPM8 . Urothelial carcinoma . Prognosis In 2012, estimated 77,570 new cases of urothelial carcinoma of bladder (UCB) and 15,210 deaths were reported in the USA [1]. Most new cases of UCB are Ta, T1, or carcinoma in situ (CIS), which grouped as non-muscle-invasive bladder carcinoma (NMIBC), and a significant percent of patients with NMIBC will experience recurrence and some will have stage progression after transurethral resection (TUR) and early intravesical chemotherapy [2]. Radical cystectomy (RC) with urinary diversion is the gold standard of treatment for locoregional control of muscle-invasive bladder carcinoma (MIBC), but some serious complications, including excessive bleeding, ileus, metabolic disorders, and pyelonephritis, often occur [3]. Although selected patients with stage IV UCB may benefit from concurrent chemoradiotherapy (CCRT), high mortality can still be seen in patients with advanced UCB [4]. Due to different biological behaviors and tumor heterogeneities, current clinicopathological factor and biomarker of UCB do not predict accurately the clinical outcome of some patients with UCB; therefore, the issue of finding new and powerful molecular makers has to be settled urgently [5]. Recent researches reported that the family of transient receptor potential (TRP) channels is associated with oncogenesis and TRP channels might be potential targets for cancer treatment [6]. In the subfamilies of TRP channels, TRP channel
11500
melastatin 8 (TRPM8) was identified and has been proposed to be the predominant thermoreceptor for cellular and behavioral responses to cold stimuli [7]. Although the aberrant expression of TRPM8 is detected in many tumors [8–11] and is represented as a key protein in central mechanisms of carcinogenesis, some disagreement of their roles in it was raised in different studies [12]. In the present study, we firstly detected the expression profile of TRPM8 messenger RNA (mRNA) in UCB tissues by a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), subsequently assessed the correlation between TRPM8 protein expression and clinicopathological characteristics of UCB, and additionally investigated the influence of TRPM8 expression on the survival of patient with UCB.
Tumor Biol. (2014) 35:11499–11504 Table 1 Sequences of the primers used in this study Primer
Sense (5′–3′)
Antisense (5′–3′)
TRPM8
GAGCTGGATGAGCACA AC TGACTTCAACAGCGAC ACCCA
GAAGTAAGCGAAGACG ATG CACCCTGTTGCTGTAG CCAAA
GAPDH
Materials and methods
Reagent (Invitrogen). RNA was reversely transcribed using SuperScript First Strand cDNA System (Invitrogen) according to the manufacturer’s protocol. The Primer Express Software Version 2.0 was used to designed sequences of qRT-PCR primers (primer sequences are shown in Table 1). The qRTPCR was performed according to standard methods. ΔCt= Ctgene −Ctreference and 2−ΔΔCt method was respectively used to show relative levels and fold change of target gene expression, where Ct represents the threshold cycle of each transcript.
Patients and tissue samples
Immunohistochemistry
The present study was approved by research ethics committees of Guangzhou Medical University, the Central South University, and Guilin Medical College, and the Helsinki Declaration of Human Rights was strictly observed. Written informed consent was obtained from all recruited patients. All tissue samples were obtained from the Second Affiliated Hospital of Guangzhou Medical University, the Second Xiangya Hospital of Central South University, and the Affiliated Hospital of Guilin Medical College. Radical cystectomy was performed in all patients with UCB. The duration of follow-up was calculated from the date of surgery to death or last follow-up, and patients were excluded if they had incomplete medical records or inadequate follow-up. Tumor was staged and graded, respectively, according to the TNM classification and the 1998 WHO classification [13]. For qRTPCR, 36 pairs of fresh UCB tissues and matched noncancerous tissues were collected after surgery between July 2010 and July 2012 and were then immediately frozen in liquid nitrogen and stored at −80 °C until detection of TRPM8 mRNA expression in tissues by qRT-PCR. For immunohistochemical assay, a total of 156 paraffin-embedded UCB samples were obtained from three hospitals between January 2008 and July 2012. One hundred fifty-six patients’ characteristics are summarized in Table 1. None of the patients recruited in the present study had undergone preoperative chemotherapy or radiotherapy.
Immunohistochemistry was performed as described previously [14]. Four random fields of each sample were examined under a compound microscope (Olympus BX51). The intensity of TRPM8 immunostaining was graded by semiquantitative estimation, which was obtained using a composite score by means of multiplying the values of staining intensity and relative abundance of positive cells. Each slide score was gained by two observers on two separate occasions, and the average score was calculated. Intensity grades 0, 1, 2, and 3 represent no, weak, moderate, and strong staining,
Real-time quantitative reverse transcription polymerase chain reaction For mRNA qRT-PCR assay, total RNA was isolated from 36 pairs of UCB tissues and adjacent normal tissues using TRIzol
Fig. 1 TRPM8 mRNA expression in 36 pairs of UCB and matched noncancerous tissues was respectively detected by qRT-PCR assay. The expression level of TRPM8 mRNA in UCB tissue was significantly higher than that in matched noncancerous tissue (3.56±0.50 vs. 1.59± 0.35, P=0.016). UCB urothelial carcinoma of bladder
Tumor Biol. (2014) 35:11499–11504
respectively. For Kaplan–Meier survival analysis, a composite score of more than the median value was considered a high expression and that less than or equal to the median value was considered a low expression.
11501 Table 2 Correlation between TRPM8 protein expression and various clinicopathological variables of UCB Variables
No. (n=156)
High expression (n=84)
34 38
38 46
0.872
27 45
41 43
0.195
37 35
39 45
0.630
74 82
33 39
41 43
0.749
51 105
30 42
21 63
0.039
Results mRNA expression of TRPM8 in UCB and noncancerous tissues
37 119
23 49
14 70
0.037
The expression of TRPM8 mRNA in 36 pairs of UCB and matched noncancerous tissue was detected by qRT-PCR. As shown in Fig. 1, UCB tissues have a significant higher expression of TRPM8 mRNA than matched noncancerous tissues (3.56±0.50 vs. 1.59±0.35, P=0.016).
Immunohistochemistry The expression of TRPM8 protein in 156 pairs of paraffinembedded UCB and matched noncancerous samples was examined by IHC analysis. We found that 53.8 % (84/156) of UCB samples represented as a high expression of TRPM8 protein and 37.2 % (58/156) of noncancerous samples showed high TRPM8 protein expression (Fig. 2). The expression level of TRPM8 protein was significantly higher in UCB samples than in noncancerous samples (P=0.004). Fig. 2 Immunohistological staining of TRPM8 protein in UCB samples. a Typical image of UCB tissue with high expression of TRPM8 protein. b Typical image of matched noncancerous samples with low expression of TRPM8 protein
Gender Male 72 Female 84 Age (years) ≤65 68 >65 88 Tumor number Unifocal 76 Multifocal 80 Tumor size (cm) ≤3.0 >3.0 Grade G1 G2 and G3 T stage Ta–T1 T2–T4
P value
Low expression (n=72)
Statistical analysis SPSS 19.0 was used to perform data analysis. The t test was used to analyze data from UCB tissues and matched noncancerous tissues detected by qRT-PCR. The relationship between TRPM8 expression and clinicopathological parameters was evaluated by chi-square test. The survival rate and comparison of differences in survival curves were determined by Kaplan–Meier analysis and log-rank tests. The significance of multiple predictors of survival was assessed by Cox regression analysis. Differences were considered significant at P0.05).
Association of TRPM8 protein expression and UCB patient’s survival Kaplan–Meier analysis and log-rank tests were used to investigate the relationship between TRPM8 protein expression
11502
Tumor Biol. (2014) 35:11499–11504
Fig. 3 The overall survival rate of patients with UCB estimated according to the TRPM8 expression level in UCB samples (Kaplan–Meier method) with immunohistochemical staining. The patients with high TRPM8 expression inclined to have a significant shorter cumulative survival than those with low TRPM8 expression
and survival of UCB patients. UCB patients with high TRPM8 protein expression were inclined to have a shorter overall survival than those with low TRPM8 protein expression (P=0.039, Fig. 3). Histological grade, tumor stage, and TRPM8 protein expression were significantly associated with overall survival of UCB patients (all P
