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Elevated expression of ECT2 predicts unfavorable prognosis in patients with colorectal cancer
Biomedicine & Pharmacotherapy 73 (2015) 135–139
Available online at
ScienceDirect www.sciencedirect.com
Original Article
Elevated expression of ECT2 predicts unfavorable prognosis in patients with colorectal cancer Yang Luo, Shao-Lan Qin, Yi-Fei Mu, Zheng-Shi Wang, Ming Zhong, Zheng-Qian Bian * Department of Gastrointestinal Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 May 2015 Accepted 23 June 2015
Epithelial cell transforming sequence 2 (ECT2) is a well-studied guanine nucleotide exchange factor for the Rho family GTPase, which has been demonstrated as an oncogene in many types of human cancers. However, little is known about the prognostic value of ECT2 in colorectal cancer (CRC). In current study, we investigated the expression pattern and underlying clinical significance of ECT2 in CRC. ECT2 expression was detected in 345 CRC specimens by immunohistochemistry, and its correlation with clinicopathologic parameters and prognosis of CRC patients were analyzed. Data from Oncomine database and real-time PCR demonstrated that ECT2 expression was elevated in CRC compared with normal tissues. Among the clinical parameters analyzed, high expression level of ECT2 significantly associated with tumor size (P = 0.020), serum CEA levels (P = 0.000) and TNM stage (P = 0.027). Kaplan– Meier survival analysis showed that patients with high ECT2 expression had a remarkably shorter overall survival. Cox regression analysis revealed that ECT2 expression level was a significant and independent prognostic factor for overall survival rate of CRC patients. These data suggested that ECT2 is an unfavorable biomarker of prognosis in CRC and that ECT2 may be a potential therapeutic candidate for CRC treatment. ß 2015 Elsevier Masson SAS. All rights reserved.
Keywords: ECT2 Colorectal cancer Prognosis Therapy
1. Introduction Colorectal cancer (CRC) is currently one of the most prevalent malignancies in the world [1]. Due to the westernized dietary lifestyle, the incidence of CRC in several Asian countries is rising rapidly [2,3]. Owing to lack of obvious specific symptoms, most CRC is detected at advanced stage with lymph node invasion or distant metastasis and ultimately the prognosis of CRC is poor [4]. The survival rate will be remarkably improved if patients are diagnosed at early stages; however, limited diagnostic procedures and access to colonoscopic screening are the main problems [5]. Therefore, it is beneficial to understand the underlying molecular mechanisms involved in colorectal carcinogenesis and identify the functions of novel biomarkers for better diagnosis and prognosis. Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor (GEF) for Rho family GTPase, which catalyzes the replacement of bound GDP by GTP [6–9]. The C* Corresponding author. Department of Gastrointestinal Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 1630 Dongfang Road, Shanghai 200127, PR China. Tel.: +86 21 683 839 85. E-mail address: [email protected] (Z.-Q. Bian). http://dx.doi.org/10.1016/j.biopha.2015.06.007 0753-3322/ß 2015 Elsevier Masson SAS. All rights reserved.
terminal of ECT2 protein contains Dbl-homology (DH) and pleckstrin homology (PH) domains, which are responsible for GEF catalytic activity, while the N-terminal contains tandem BRCA1-C terminal (BRCT) repeats, which modulates the intracellular localization of ECT2 [9–11]. ECT2 is exclusively distributed in the interphase nucleus in normal cells and becomes hyperphosphorylated and is localized throughout the cytoplasm during mitosis [8]. Besides, through phosphorylation at Thr-341 in G2/M phase, ECT2 changes its formation and stimulates association of the mitotic kinase Plk1 and accumulation of GTP bound RhoA[6,8]. Those characteristics of ECT2 indicate that the mislocalization to the cytoplasm may enhance ECT2 oncogenic potential. Indeed, overexpressed ECT2 is identified in many types of human malignancies, including pancreatic cancer [12], lung cancer [13], glioma [14,15], breast cancer [16], etc. And increased ECT2 activates several Rho-regulated signaling pathway, which ultimately contribute to malignant transformation [10]. However, little is known about the role of ECT2 expression in CRC. In this retrospective study, we examined the expression pattern of ECT2 at both the mRNA and protein level and explored the relationship of ECT2 expression with corresponding clinicopathologic parameters. The results suggested that ECT2 expression was upregulated during malignant transformation in CRC and indicated
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that ECT2 is a reliable indicator for the poor prognosis of CRC patients. 2. Materials and methods 2.1. Clinical tissue samples Consecutive patients with colorectal cancer were recruited from January 2003 to November 2010 at Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. An additional 30 paired freshly frozen CRC tissues and corresponding non-cancerous tissues were also obtained from Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, China. All tissue samples were obtained with informed consent and all procedures were performed in accordance with the Human Investigation Ethical Committee of Ren Ji Hospital. The histology and clinical stages were classified according the definition by the American Joint Committee on Cancer (AJCC). The cases of colorectal cancer were selected in this study only if clinical data were available. The
follow-up time was calculated from the date of surgery to the date of death, or the last known follow-up. None of them had received radiotherapy, chemotherapy, hormone therapy or other related anti-tumor therapies before surgery. Five tissue microarrays containing 345 human colorectal cancer specimens were enrolled in this study. 2.2. Real-time quantitative PCR Total RNA from primary tumor and adjacent non-cancerous frozen tissue samples was extracted with Trizol reagent (Takara, Japan), and reversely transcribed using a PrimeScript RT-PCR kit (Takara, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using an ABI Prism 7500 Sequence Detection System with SYBR Green Master Mix (Takara, Japan). Primer sequences used for ECT2 detection were as follows, forward: 50 -ACTACTGGGAGGACTAGCTTG-30 ; reverse: 50 CACTCTTGTTTCAATCTGAGGCA-30 . The relative expression of was normalized to b-actin RNA (forward: 50 -ACTCGTCATACTCCTGCT-30 ,
Fig. 1. ECT2 expression in CRC at mRNA level. ECT2 expression in Hong Colorectal (A) Gaedcke Colorectal (B) and Skrzypczak Colorectal (C) derived from Oncomine database grouped by normal tissues (1) and colorectal cancer (2). D. Increased ECT2 mRNA expression in 32 pairs of CRC/normal (N) tissues was detected by real-time quantitative PCR. P-values were calculated by paired t-test or Chi2 test.
Y. Luo et al. / Biomedicine & Pharmacotherapy 73 (2015) 135–139 D
reverse: 50 -GAAACTACCTTCAACTCC-30 ). The 2 Ct method was used to quantify the relative ECT2 expression levels and normalized using the b-actin expression. 2.3. Immunohistochemical staining The tissue microarray of CRC were deparaffinized in xylene and rehydrated with graded ethanol. After washing in phosphatebuffered saline (PBS) for three times, the sections were boiled in a microwave for 15 min to unmask antigen epitopes and incubated with 0.3% hydrogen peroxide to neutralize the endogenous peroxidase and then blocked with 10% BSA for 1 h at room temperature. After rinsing three times with PBS, slides were incubated with primary antibody for ECT2 (Proteintech, US) at 4 8C overnight with optimal dilution. After washing three times with PBS, slides were incubated with second antibody labeled by HRP (rabbit) (Proteintech, US) at room temperature for 1 h. Finally, visualization was performed by 3,30 -diaminobenzidine tetrahydrochloride (DAB) and counterstained by hematoxylin. Scoring was conducted according to the percent of positive cells: < 5% scored 0; 6–25% scored 1; 25–50% scored 2; more than 50% scored 3 and staining intensity: no staining scored 0, weakly staining scored 1, moderately staining scored 2 and strongly staining scored 3, respectively. The final score was designated as low or high expression group using the percent of positive cell score staining intensity score as follows: low expression was defined as a total score < 4 and high expression with a total score 4. These scores were determined independently by two senior pathologists.
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Pearson Chi2 test. Survival curves were evaluated using the Kaplan–Meier method, and differences between survival curves were tested by the log-rank test. Cox proportional hazards regression model was used to examine univariate and multivariate hazard ratios for the variables that were dichotomized in this study. P < 0.05 was considered statistically significant.
3. Results 3.1. Expression of ECT in CRC tissues at mRNA and protein levels To investigate the expression pattern of ECT2 in colorectal cancer, we analyzed three independent microarray datasets in CRC research from Oncomine database. As shown in Fig. 1A–C, mRNA expression levels of ECT2 were upregulated in the majority of tumor tissues compared with normal colorectal tissues. Consistent with this data, real-time quantitative PCR (qRT-PCR) performed in present study demonstrated that ECT2 mRNA was significantly upregulated in CRC tissues compared with 30 paired noncancerous tissues (Fig. 1D). Then, the protein expression level of ECT2 in 345 CRC tissue samples was demonstrated by immunohistochemical staining, we found ECT2 protein was highly expressed in 203 (58.8%) of the 345 CRC cases and the immunoreactivity of ECT2 was distributed in both cytoplasm and nucleus of CRC cells (Fig. 2). 3.2. Correlation between ECT2 expression and clinicopathological parameters
2.4. Statistical analysis Statistical analyses and graphical representations were performed using SPSS 16.0 (SPSS Inc.; Chicago, IL, USA) and GraphPad Prism 5 (San Diego, CA) software. Correlation of ECT2 expression with corresponding clinicopathologic parameters was analyzed by
The relationship between the ECT2 expression level and corresponding clinicopathological parameters of the colorectal cancer patients was calculated by Pearson Chi2 test. As shown in Table 1, ECT2 expression was significantly associated with adverse clinicopathological features of CRC, including tumor size
Fig. 2. ECT2 expression in CRC was determined by immunochemistry. A. Positive expression of ECT2. B. Negative expression level of ECT2. Representative images are shown at 200 and 400 magnification, respectively.
Y. Luo et al. / Biomedicine & Pharmacotherapy 73 (2015) 135–139
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Table 1 Relationship between ECT2 expression and clinicopathological features in 345 colorectal cancer patients. Variable
ECT2 (n) Low n = 142
Age 65 years > 65 years Gender Male Female Tumor size 5 cm > 5 cm Tumor location Rectum Colon Serum CEA 5 ng/mL >5 ng/mL Clinical stage 0–I II III IV Histology Mucinous Non-mucinous
P High n = 203
77 (22.32) 65 (18.84)
108 (31.30) 95 (27.54)
0.851
82 (23.77) 60 (17.39)
117 (33.91) 86 (24.93)
0.984
81 (23.48) 61 (17.68)
90 (26.09) 113 (32.75)
0.020
89 (25.80) 53 (15.36)
113 (32.75) 90 (26.09)
0.193
103 (29.86) 39 (11.30)
78 (22.61) 125 (36.23)
0.000
34 45 53 10
(9.86) (13.04) (15.35) (2.90)
26 (7.54) 137 (39.71)
28 67 78 30
(8.12) (19.42) (22.61) (8.70)
0.027
37 (10.72) 145 (42.03)
0.293
Fig. 3. ECT2 is correlated with overall survival rate in CRC patients. Kaplan–Meier survival curves show high expression level of ECT2 was significantly correlated with poor survival of CRC. P-values were calculated by log-rank test.
oncogene in various tumor types [9,13,16–19]. It is well described in the literature that upregulation of ECT2 confers poor clinical outcome in many different types of tumors [14,15,18–20]. However, a comprehensive investigation remains to evaluate the clinical significance and prognostic value of ECT2 in CRC specifically. Here, ECT2 expression pattern and its correlation with clinicopathological parameters and clinical prognosis are reported. Firstly, the data from Oncomine database and our current study collectively confirmed that ECT2 expression was overexpressed in CRC tissues compared with adjacent non-cancerous tissues at the mRNA level. Consistent with previous studies, this observation also indicated that ECT2 might function as an oncogene in CRC [9,11,14,18,20]. Furthermore, ECT2 protein level as analyzed by IHC showed that ECT2 expression was elevated in 58.8% (203/345) of CRC tissues. This finding is consistent with a previous study that ECT2 expression was overexpressed in around 70% of human gastric cancer [20]. Interestingly, apart from the positive staining of ECT2 in the nucleus of CRC cells, strong immunoreactivity of ECT2 in cytoplasm of CRC cells was also observed. Therefore, it is reasonable to suggest that mislocalized ECT2 exhibits oncogenic function in CRC as previous reported in lung cancer [13]. Then the relationship between ECT2 expression and corresponding clinicopathologic parameters was determined. Previous researches in gliomas and osteosarcoma have shown that overexpressed ECT2 promotes tumor malignancy and consequently leads to poor prognosis [14,19]. Here, we revealed a similar phenomenon in which elevated ECT2 expression in CRC was significantly correlated with tumor size, serum CEA levels and TNM stage. Importantly, patients with overexpressed ECT2 had a shorter overall survival rate. Univariate analysis showed that upregulated ECT2 was closely associated with the overall survival rate in CRC patients. Multivariate analysis suggested that ECT2 expression was an independent risk factor for poor outcome of CRC patients. Collectively, these results suggest that ECT2 might be used as a novel prognostic marker for CRC patients. Of note, ECT2 was also
Values in parentheses indicate percentage values. The bold number represents the P-values with significant differences.
(P = 0.020), serum CEA levels (P = 0.000) and TNM stage (P = 0.027). No correlations between ECT2 expression and age, gender, tumor location and histology were found. 3.3. Relationship between ECT2 expression and prognosis in CRC patients To evaluate the prognostic value of ECT2, the overall survival rate of CRC patients was analyzed using Kaplan–Meier method and log-rank test. The result showed that patients with high ECT2 protein expression levels (n = 96) had poorer overall survival than those with low protein expression levels (n = 87) (Fig. 3). Univariate Cox regression analyses showed that ECT2 expression, serum CEA levels and TNM stage were significantly associated with overall survival (Table 2). Furthermore, a multivariate Cox regression analysis suggested that ECT2 expression (P = 0.001) and TNM stage (P = 0.002) were independent predictors of the overall survival in patients with CRC (Table 2). 4. Discussion ECT2 is originally implicated in cellular transformation in human epithelial tumor cells and has been demonstrated as an
Table 2 Univariate and multivariate analysis showing the overall survival in colorectal cancer. Variable
ECT2 Age Gender Tumor size Tumor location Serum CEA TNM stage
Univariate analysis
Multivariate analysis
HR
95% CI
P value
HR
95% CI
P value
2.538 1.216 1.214 0.895 1.461 1.744 1.553
1.554–4.147 0.769–1.922 0.768–1.920 0.876–2.177 0.903–2.361 1.096–2.775 1.175–2.052
0.000 0.402 0.406 0.164 0.122 0.019 0.002
2.357 – – – – 1.448 1.565
1.426–3.894 – – – – 0.902–2.084 1.175–2.084
0.001 – – – – 0.125 0.002
HR: hazard ratio; CI: confidence interval. The bold number represents the P-values with significant differences.
Y. Luo et al. / Biomedicine & Pharmacotherapy 73 (2015) 135–139
demonstrated as an independent risk factor in gastric cancer, thus providing an insight in the role of ECT2 in gastrointestinal tumors. Taken together, our study demonstrated that upregulation of ECT2 is associated with poor survival in CRC patients, indicating that ECT2 may serve as a valuable prognostic marker in CRC and may represent a potential molecular target for the treatment of CRC. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. References [1] M.M. Center, A. Jemal, R.A. Smith, E. Ward, Worldwide variations in colorectal cancer, CA Cancer J. Clin. 59 (2009) 366–378. [2] K.L. Goh, Changing trends in gastrointestinal disease in the Asia-Pacific region, J. Dig. Dis. 8 (2007) 179–185. [3] Y.K. Yee, V.P. Tan, P. Chan, I.F. Hung, R. Pang, B.C. Wong, Epidemiology of colorectal cancer in Asia, J. Gastroenterol. Hepatol. 24 (2009) 1810–1816. [4] H. Brenner, M. Kloor, C.P. Pox, Colorectal cancer, Lancet 383 (2014) 1490– 1502. [5] P. Maisonneuve, E. Botteri, A.B. Lowenfels, Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps, Gastroenterology 135 (2008) 710 [author reply 710–1]. [6] T. Hara, M. Abe, H. Inoue, L.R. Yu, T.D. Veenstra, Y.H. Kang, et al., Cytokinesis regulator ECT2 changes its conformation through phosphorylation at Thr-341 in G2/M phase, Oncogene 25 (2006) 566–578. [7] J.E. Kim, D.D. Billadeau, J. Chen, The tandem BRCT domains of Ect2 are required for both negative and positive regulation of Ect2 in cytokinesis, J. Biol. Chem. 280 (2005) 5733–5739.
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[8] F. Niiya, T. Tatsumoto, K.S. Lee, T. Miki, Phosphorylation of the cytokinesis regulator ECT2 at G2/M phase stimulates association of the mitotic kinase Plk1 and accumulation of GTP-bound RhoA, Oncogene 25 (2006) 827–837. [9] V. Justilien, L. Jameison, C.J. Der, K.L. Rossman, A.P. Fields, Oncogenic activity of Ect2 is regulated through protein kinase C iota-mediated phosphorylation, J. Biol. Chem. 286 (2011) 8149–8157. [10] S. Saito, X.F. Liu, K. Kamijo, R. Raziuddin, T. Tatsumoto, I. Okamoto, et al., Deregulation and mislocalization of the cytokinesis regulator ECT2 activate the Rho signaling pathways leading to malignant transformation, J. Biol. Chem. 279 (2004) 7169–7179. [11] T. Miki, C.L. Smith, J.E. Long, A. Eva, T.P. Fleming, Oncogene ect2 is related to regulators of small GTP-binding proteins, Nature 362 (1993) 462–465. [12] M.L. Zhang, S. Lu, L. Zhou, S.S. Zheng, Correlation between ECT2 gene expression and methylation change of ECT2 promoter region in pancreatic cancer, Hepatobiliary Pancreat. Dis. Int. 7 (2008) 533–538. [13] D. Hirata, T. Yamabuki, D. Miki, T. Ito, E. Tsuchiya, M. Fujita, et al., Involvement of epithelial cell transforming sequence-2 oncoantigen in lung and esophageal cancer progression, Clin. Cancer Res. 15 (2009) 256–266. [14] M. Sano, N. Genkai, N. Yajima, N. Tsuchiya, J. Homma, R. Tanaka, et al., Expression level of ECT2 proto-oncogene correlates with prognosis in glioma patients, Oncol. Rep. 16 (2006) 1093–1098. [15] Y.S. Cheng, C. Lin, Y.P. Cheng, Y.L. Yu, C.T. Tang, D.Y. Hueng, Epithelial cell transformation sequence 2 is a potential biomarker of unfavorable survival in human gliomas, Neurol. India 62 (2014) 406–409. [16] M.C. Srougi, K. Burridge, The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage, PLoS One 6 (2011) e17108. [17] A.P. Fields, V. Justilien, The guanine nucleotide exchange factor (GEF) Ect2 is an oncogene in human cancer, Adv. Enzyme Regul. 50 (2010) 190–200. [18] M. Iyoda, A. Kasamatsu, T. Ishigami, D. Nakashima, Y. Endo-Sakamoto, K. Ogawara, et al., Epithelial cell transforming sequence 2 in human oral cancer, PLoS One 5 (2010) e14082. [19] J. Xu, Q. Yao, Y. Hou, M. Xu, S. Liu, L. Yang, et al., MiR-223/Ect2/p21 signaling regulates osteosarcoma cell cycle progression and proliferation, Biomed. Pharmacother. 67 (2013) 381–386. [20] Y. Jin, Y. Yu, Q. Shao, Y. Ma, R. Zhang, H. Yao, et al., Up-regulation of ECT2 is associated with poor prognosisn gastric cancer patients, Int. J. Clin. Exp. Pathol. 7 (2014) 8724–8731.
Available online at
ScienceDirect www.sciencedirect.com
Original Article
Elevated expression of ECT2 predicts unfavorable prognosis in patients with colorectal cancer Yang Luo, Shao-Lan Qin, Yi-Fei Mu, Zheng-Shi Wang, Ming Zhong, Zheng-Qian Bian * Department of Gastrointestinal Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 May 2015 Accepted 23 June 2015
Epithelial cell transforming sequence 2 (ECT2) is a well-studied guanine nucleotide exchange factor for the Rho family GTPase, which has been demonstrated as an oncogene in many types of human cancers. However, little is known about the prognostic value of ECT2 in colorectal cancer (CRC). In current study, we investigated the expression pattern and underlying clinical significance of ECT2 in CRC. ECT2 expression was detected in 345 CRC specimens by immunohistochemistry, and its correlation with clinicopathologic parameters and prognosis of CRC patients were analyzed. Data from Oncomine database and real-time PCR demonstrated that ECT2 expression was elevated in CRC compared with normal tissues. Among the clinical parameters analyzed, high expression level of ECT2 significantly associated with tumor size (P = 0.020), serum CEA levels (P = 0.000) and TNM stage (P = 0.027). Kaplan– Meier survival analysis showed that patients with high ECT2 expression had a remarkably shorter overall survival. Cox regression analysis revealed that ECT2 expression level was a significant and independent prognostic factor for overall survival rate of CRC patients. These data suggested that ECT2 is an unfavorable biomarker of prognosis in CRC and that ECT2 may be a potential therapeutic candidate for CRC treatment. ß 2015 Elsevier Masson SAS. All rights reserved.
Keywords: ECT2 Colorectal cancer Prognosis Therapy
1. Introduction Colorectal cancer (CRC) is currently one of the most prevalent malignancies in the world [1]. Due to the westernized dietary lifestyle, the incidence of CRC in several Asian countries is rising rapidly [2,3]. Owing to lack of obvious specific symptoms, most CRC is detected at advanced stage with lymph node invasion or distant metastasis and ultimately the prognosis of CRC is poor [4]. The survival rate will be remarkably improved if patients are diagnosed at early stages; however, limited diagnostic procedures and access to colonoscopic screening are the main problems [5]. Therefore, it is beneficial to understand the underlying molecular mechanisms involved in colorectal carcinogenesis and identify the functions of novel biomarkers for better diagnosis and prognosis. Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor (GEF) for Rho family GTPase, which catalyzes the replacement of bound GDP by GTP [6–9]. The C* Corresponding author. Department of Gastrointestinal Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 1630 Dongfang Road, Shanghai 200127, PR China. Tel.: +86 21 683 839 85. E-mail address: [email protected] (Z.-Q. Bian). http://dx.doi.org/10.1016/j.biopha.2015.06.007 0753-3322/ß 2015 Elsevier Masson SAS. All rights reserved.
terminal of ECT2 protein contains Dbl-homology (DH) and pleckstrin homology (PH) domains, which are responsible for GEF catalytic activity, while the N-terminal contains tandem BRCA1-C terminal (BRCT) repeats, which modulates the intracellular localization of ECT2 [9–11]. ECT2 is exclusively distributed in the interphase nucleus in normal cells and becomes hyperphosphorylated and is localized throughout the cytoplasm during mitosis [8]. Besides, through phosphorylation at Thr-341 in G2/M phase, ECT2 changes its formation and stimulates association of the mitotic kinase Plk1 and accumulation of GTP bound RhoA[6,8]. Those characteristics of ECT2 indicate that the mislocalization to the cytoplasm may enhance ECT2 oncogenic potential. Indeed, overexpressed ECT2 is identified in many types of human malignancies, including pancreatic cancer [12], lung cancer [13], glioma [14,15], breast cancer [16], etc. And increased ECT2 activates several Rho-regulated signaling pathway, which ultimately contribute to malignant transformation [10]. However, little is known about the role of ECT2 expression in CRC. In this retrospective study, we examined the expression pattern of ECT2 at both the mRNA and protein level and explored the relationship of ECT2 expression with corresponding clinicopathologic parameters. The results suggested that ECT2 expression was upregulated during malignant transformation in CRC and indicated
136
Y. Luo et al. / Biomedicine & Pharmacotherapy 73 (2015) 135–139
that ECT2 is a reliable indicator for the poor prognosis of CRC patients. 2. Materials and methods 2.1. Clinical tissue samples Consecutive patients with colorectal cancer were recruited from January 2003 to November 2010 at Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. An additional 30 paired freshly frozen CRC tissues and corresponding non-cancerous tissues were also obtained from Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, China. All tissue samples were obtained with informed consent and all procedures were performed in accordance with the Human Investigation Ethical Committee of Ren Ji Hospital. The histology and clinical stages were classified according the definition by the American Joint Committee on Cancer (AJCC). The cases of colorectal cancer were selected in this study only if clinical data were available. The
follow-up time was calculated from the date of surgery to the date of death, or the last known follow-up. None of them had received radiotherapy, chemotherapy, hormone therapy or other related anti-tumor therapies before surgery. Five tissue microarrays containing 345 human colorectal cancer specimens were enrolled in this study. 2.2. Real-time quantitative PCR Total RNA from primary tumor and adjacent non-cancerous frozen tissue samples was extracted with Trizol reagent (Takara, Japan), and reversely transcribed using a PrimeScript RT-PCR kit (Takara, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using an ABI Prism 7500 Sequence Detection System with SYBR Green Master Mix (Takara, Japan). Primer sequences used for ECT2 detection were as follows, forward: 50 -ACTACTGGGAGGACTAGCTTG-30 ; reverse: 50 CACTCTTGTTTCAATCTGAGGCA-30 . The relative expression of was normalized to b-actin RNA (forward: 50 -ACTCGTCATACTCCTGCT-30 ,
Fig. 1. ECT2 expression in CRC at mRNA level. ECT2 expression in Hong Colorectal (A) Gaedcke Colorectal (B) and Skrzypczak Colorectal (C) derived from Oncomine database grouped by normal tissues (1) and colorectal cancer (2). D. Increased ECT2 mRNA expression in 32 pairs of CRC/normal (N) tissues was detected by real-time quantitative PCR. P-values were calculated by paired t-test or Chi2 test.
Y. Luo et al. / Biomedicine & Pharmacotherapy 73 (2015) 135–139 D
reverse: 50 -GAAACTACCTTCAACTCC-30 ). The 2 Ct method was used to quantify the relative ECT2 expression levels and normalized using the b-actin expression. 2.3. Immunohistochemical staining The tissue microarray of CRC were deparaffinized in xylene and rehydrated with graded ethanol. After washing in phosphatebuffered saline (PBS) for three times, the sections were boiled in a microwave for 15 min to unmask antigen epitopes and incubated with 0.3% hydrogen peroxide to neutralize the endogenous peroxidase and then blocked with 10% BSA for 1 h at room temperature. After rinsing three times with PBS, slides were incubated with primary antibody for ECT2 (Proteintech, US) at 4 8C overnight with optimal dilution. After washing three times with PBS, slides were incubated with second antibody labeled by HRP (rabbit) (Proteintech, US) at room temperature for 1 h. Finally, visualization was performed by 3,30 -diaminobenzidine tetrahydrochloride (DAB) and counterstained by hematoxylin. Scoring was conducted according to the percent of positive cells: < 5% scored 0; 6–25% scored 1; 25–50% scored 2; more than 50% scored 3 and staining intensity: no staining scored 0, weakly staining scored 1, moderately staining scored 2 and strongly staining scored 3, respectively. The final score was designated as low or high expression group using the percent of positive cell score staining intensity score as follows: low expression was defined as a total score < 4 and high expression with a total score 4. These scores were determined independently by two senior pathologists.
137
Pearson Chi2 test. Survival curves were evaluated using the Kaplan–Meier method, and differences between survival curves were tested by the log-rank test. Cox proportional hazards regression model was used to examine univariate and multivariate hazard ratios for the variables that were dichotomized in this study. P < 0.05 was considered statistically significant.
3. Results 3.1. Expression of ECT in CRC tissues at mRNA and protein levels To investigate the expression pattern of ECT2 in colorectal cancer, we analyzed three independent microarray datasets in CRC research from Oncomine database. As shown in Fig. 1A–C, mRNA expression levels of ECT2 were upregulated in the majority of tumor tissues compared with normal colorectal tissues. Consistent with this data, real-time quantitative PCR (qRT-PCR) performed in present study demonstrated that ECT2 mRNA was significantly upregulated in CRC tissues compared with 30 paired noncancerous tissues (Fig. 1D). Then, the protein expression level of ECT2 in 345 CRC tissue samples was demonstrated by immunohistochemical staining, we found ECT2 protein was highly expressed in 203 (58.8%) of the 345 CRC cases and the immunoreactivity of ECT2 was distributed in both cytoplasm and nucleus of CRC cells (Fig. 2). 3.2. Correlation between ECT2 expression and clinicopathological parameters
2.4. Statistical analysis Statistical analyses and graphical representations were performed using SPSS 16.0 (SPSS Inc.; Chicago, IL, USA) and GraphPad Prism 5 (San Diego, CA) software. Correlation of ECT2 expression with corresponding clinicopathologic parameters was analyzed by
The relationship between the ECT2 expression level and corresponding clinicopathological parameters of the colorectal cancer patients was calculated by Pearson Chi2 test. As shown in Table 1, ECT2 expression was significantly associated with adverse clinicopathological features of CRC, including tumor size
Fig. 2. ECT2 expression in CRC was determined by immunochemistry. A. Positive expression of ECT2. B. Negative expression level of ECT2. Representative images are shown at 200 and 400 magnification, respectively.
Y. Luo et al. / Biomedicine & Pharmacotherapy 73 (2015) 135–139
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Table 1 Relationship between ECT2 expression and clinicopathological features in 345 colorectal cancer patients. Variable
ECT2 (n) Low n = 142
Age 65 years > 65 years Gender Male Female Tumor size 5 cm > 5 cm Tumor location Rectum Colon Serum CEA 5 ng/mL >5 ng/mL Clinical stage 0–I II III IV Histology Mucinous Non-mucinous
P High n = 203
77 (22.32) 65 (18.84)
108 (31.30) 95 (27.54)
0.851
82 (23.77) 60 (17.39)
117 (33.91) 86 (24.93)
0.984
81 (23.48) 61 (17.68)
90 (26.09) 113 (32.75)
0.020
89 (25.80) 53 (15.36)
113 (32.75) 90 (26.09)
0.193
103 (29.86) 39 (11.30)
78 (22.61) 125 (36.23)
0.000
34 45 53 10
(9.86) (13.04) (15.35) (2.90)
26 (7.54) 137 (39.71)
28 67 78 30
(8.12) (19.42) (22.61) (8.70)
0.027
37 (10.72) 145 (42.03)
0.293
Fig. 3. ECT2 is correlated with overall survival rate in CRC patients. Kaplan–Meier survival curves show high expression level of ECT2 was significantly correlated with poor survival of CRC. P-values were calculated by log-rank test.
oncogene in various tumor types [9,13,16–19]. It is well described in the literature that upregulation of ECT2 confers poor clinical outcome in many different types of tumors [14,15,18–20]. However, a comprehensive investigation remains to evaluate the clinical significance and prognostic value of ECT2 in CRC specifically. Here, ECT2 expression pattern and its correlation with clinicopathological parameters and clinical prognosis are reported. Firstly, the data from Oncomine database and our current study collectively confirmed that ECT2 expression was overexpressed in CRC tissues compared with adjacent non-cancerous tissues at the mRNA level. Consistent with previous studies, this observation also indicated that ECT2 might function as an oncogene in CRC [9,11,14,18,20]. Furthermore, ECT2 protein level as analyzed by IHC showed that ECT2 expression was elevated in 58.8% (203/345) of CRC tissues. This finding is consistent with a previous study that ECT2 expression was overexpressed in around 70% of human gastric cancer [20]. Interestingly, apart from the positive staining of ECT2 in the nucleus of CRC cells, strong immunoreactivity of ECT2 in cytoplasm of CRC cells was also observed. Therefore, it is reasonable to suggest that mislocalized ECT2 exhibits oncogenic function in CRC as previous reported in lung cancer [13]. Then the relationship between ECT2 expression and corresponding clinicopathologic parameters was determined. Previous researches in gliomas and osteosarcoma have shown that overexpressed ECT2 promotes tumor malignancy and consequently leads to poor prognosis [14,19]. Here, we revealed a similar phenomenon in which elevated ECT2 expression in CRC was significantly correlated with tumor size, serum CEA levels and TNM stage. Importantly, patients with overexpressed ECT2 had a shorter overall survival rate. Univariate analysis showed that upregulated ECT2 was closely associated with the overall survival rate in CRC patients. Multivariate analysis suggested that ECT2 expression was an independent risk factor for poor outcome of CRC patients. Collectively, these results suggest that ECT2 might be used as a novel prognostic marker for CRC patients. Of note, ECT2 was also
Values in parentheses indicate percentage values. The bold number represents the P-values with significant differences.
(P = 0.020), serum CEA levels (P = 0.000) and TNM stage (P = 0.027). No correlations between ECT2 expression and age, gender, tumor location and histology were found. 3.3. Relationship between ECT2 expression and prognosis in CRC patients To evaluate the prognostic value of ECT2, the overall survival rate of CRC patients was analyzed using Kaplan–Meier method and log-rank test. The result showed that patients with high ECT2 protein expression levels (n = 96) had poorer overall survival than those with low protein expression levels (n = 87) (Fig. 3). Univariate Cox regression analyses showed that ECT2 expression, serum CEA levels and TNM stage were significantly associated with overall survival (Table 2). Furthermore, a multivariate Cox regression analysis suggested that ECT2 expression (P = 0.001) and TNM stage (P = 0.002) were independent predictors of the overall survival in patients with CRC (Table 2). 4. Discussion ECT2 is originally implicated in cellular transformation in human epithelial tumor cells and has been demonstrated as an
Table 2 Univariate and multivariate analysis showing the overall survival in colorectal cancer. Variable
ECT2 Age Gender Tumor size Tumor location Serum CEA TNM stage
Univariate analysis
Multivariate analysis
HR
95% CI
P value
HR
95% CI
P value
2.538 1.216 1.214 0.895 1.461 1.744 1.553
1.554–4.147 0.769–1.922 0.768–1.920 0.876–2.177 0.903–2.361 1.096–2.775 1.175–2.052
0.000 0.402 0.406 0.164 0.122 0.019 0.002
2.357 – – – – 1.448 1.565
1.426–3.894 – – – – 0.902–2.084 1.175–2.084
0.001 – – – – 0.125 0.002
HR: hazard ratio; CI: confidence interval. The bold number represents the P-values with significant differences.
Y. Luo et al. / Biomedicine & Pharmacotherapy 73 (2015) 135–139
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