Clin Transl Oncol DOI 10.1007/s12094-015-1428-2
CLINICAL GUIDES IN ONCOLOGY
Clinical significance of ECT2 expression in tissue and serum of gastric cancer patients H.-B. Wang1 • H.-C. Yan2 • Y. Liu3
Received: 15 May 2015 / Accepted: 9 October 2015 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2015
Abstract The ECT2 (epithelial cell transforming sequence 2) oncogene acted as a guanine nucleotide exchange factor for RhoGTPases, and regulates cytokinesis; thus, it may play a role in the pathogenesis of gastric cancer. In this study, we investigated the expression ECT2 gene in tissues and serum of gastric cancer patients to explore its clinical significance. ECT2 mRNA expression levels in tissues and serum were examined by RT-PCR, and ECT2 protein expression in tissue was evaluated by Western blot, and was further validated by immunohistochemistry and enzyme-linked immunosorbent assay at serum level. ECT2 level was significantly increased in the GC tissues and serum compared to normal control. ECT2 expression was positively correlated with the histologic differentiation, stages of TNM, and lymph node metastasis in GC (P \ 0.05). Our results suggest that ECT2 plays an important role during GC progression and it may become a new diagnostic marker and therapeutic molecular target for management of GC. Keywords Gastric cancer Epithelial cell transforming sequence 2 Guanine nucleotide exchange factors
H.-B. Wang and H.-C. Yan have contributed equally to this work. & H.-B. Wang [email protected] 1
Department of Oncology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, China
2
Department of Oncology, The Second People’s Hospital of Lianyungang, Lianyungang, Jiangsu, China
3
Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical University, Xuzhou, Jiangsu, China
Introduction Gastric cancer (GC) is an aggressive neoplasm associated with a very poor prognosis. Worldwide, GC is the fourth most prevalent malignancy and the second leading cancer cause of death [1]. Although the early diagnosis and treatment of gastric cancer has improved in recent years, GC still remains among the leading causes of death from cancer in China [2]. The development and progression of gastric cancer involves a number of genetic alterations of tumor suppressor and tumor-related genes [3]. In recent years, a series of GC related gene have been found and been studied in depth, but obviously this theory cannot cover the entire process of carcinogenesis of GC. So it is critically important to understand the molecular mechanism of GC. ECT2 (epithelial cell transforming sequence 2) was found by Miki et al., in epithelial cells, and is located on chromosome 3q26. Human ECT2 encodes a polypeptide of 883 amino acids, with a predicted molecular weight of approximately 104 kDa [4]. ECT2 is a guanine nucleotide exchange factor (GEF) for Rho family GTPases related to cytokinesis [5–8]. The C-terminal protein of ECT2 catalyzes guanine nucleotide exchange on the Rho family GTPases, while the N-terminal contains a domain related to cell cycle regulator/checkpoint control proteins GEFs catalyze the exchange of GDP for GTP, thereby activating the RhoGTPases in signal transduction [9]. The RhoGTPases have been implicated in the malignant phenotype of human cancers as a result of their participation in aberrant signaling in tumor cells [10–12]. To date, ECT2 is considered as an oncogene in human cancer [13], and it has been showed to up-regulate in a variety of human tumors including lung cancer [14], glioma [15], hepatocellular carcinoma [16], pancreatic
123
Clin Transl Oncol
cancer [17], etc. However, few studies have analyzed the role of ECT2 expression in GC. In the current study, the expression of ECT2 in gastric carcinoma tissues and serum was analyzed and the correlation between ECT2 levels and the clinicopathological characteristics of gastric carcinoma was investigated, to determine the possible use of ECT2 as a diagnostic marker and therapeutic molecular target for management of GC.
Patients and methods Patients and sample collection 50 cases of primary gastric carcinoma patients, who had undergone a gastrectomy at the Affiliated Hospital of Xuzhou Medical University between April 2014 and September 2014, were involved in our study. 34 cases were male and 16 cases were female, with a mean age of 60 years. None of the patients had received chemotherapy or radiotherapy prior to the surgery and no additional malignancies were evident. All patients were informed of the nature of the study and provided written informed consent, and this study was approved by the ethics committee of Xuzhou Medical University. Peripheral venous blood (10 ml) was collected from each patient prior to the surgery and stored at -80 °C for the RT-PCR and enzyme-linked immunosorbent assay (ELISA). Tumor tissue samples were obtained immediately from the resected specimens and the adjacent tissue was collected from a distance of [5 cm from the tumor. For immunohistochemistry, the tissue samples were fixed with 10 % formalin, dehydrated with gradient alcohol, cleared with xylene and embedded in paraffin, and cut into serial sections at a thickness of 4 lm. In addition, peripheral serum from 30 healthy individuals was randomly selected to serve as controls. Classification of the tumors was conducted according to the tumor-node-metastasis (TNM) staging system issued by the Union Internationale Contrele Cancer (UICC) in 2010. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of ECT2 expression in tissues and serum Total RNA was extracted using Trizol (for tissues) (Invitrogen Inc, USA) or RNA extraction kit (Bioteke Biotech Co., LTD, Beijing, China) followed by complementary DNA synthesis using the reverse kit (Tiangen Biotech Co., LTD, Beijing, China). PCR was performed using Taq polymerase (Tiangen Biotech Co., LTD, Beijing, China) under the following conditions: 94 °C for 3 min; 30 cycles of 94 °C for 30 s, 55 °C (for b-actin) or 58 °C (for ECT2)
123
for 30 s and 72 °C for 60 s; followed by 72 °C for 5 min. b-actin served as an internal control. The PCR product lengths for ECT2 and b-actin were 326 and 285 bp, respectively, and were sequenced by Sangon Biotechnology Co., LTD (Shanghai, China). The primers for the PCR were as follows: Sense, 50 -GCCTTGCTTGTGAGGCCA CCAA-30 and anti-sense, 50 -TCCACTGAGAGCCGTG GGATGTCA-30 for ECT2; sense, 50 -GCCTTGCTTGT GAGGCCACCAA-30 , and anti-sense, 50 -TCCACTGA GCCGTGGGATGTCA-30 for b-actin. Each PCR product was examined by agarose gel electrophoresis and images were captured using a digital camera (PowerShot A2400; Canon Inc., Tokyo, Japan). The protein band intensities were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). Western blot of ECT2 Total proteins were extracted using RIPA lysis buffer from Beyotime Biotechnology (Nanjing, China), and the protein concentration was quantified using the BCA Protein Assay Kit (Beyotime Biotechnology, Nanjing, China). Total proteins were separated on 10 % SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5 % skimmed-milk powder in Tris-buffered saline with tween-20 (TBS-T), incubated with ECT2 antibody (1:400; Bioss Biotech Co., LTD, Beijing, China), and then incubated with anti-rabbit IgG secondary antibody (1:10,000; Sigma, St. Louis, MO, USA). Then they were subjected to quantitative immunoblot using the Odyssey imaging system (Li-Cor, USA). Immunohistochemical staining of ECT2 Routine hematoxylin and eosin (H&E) staining was conducted prior to immunohistochemical staining for serial sections. Paraffin-embedded tissues were dewaxed in xylene, rehydrated by serial concentrations of ethanol, and then rinsed in phosphate buffer solution (PBS) followed by treatment with 3 % H2O2 to refrain endogenous peroxidase. After being heated in a microwave for 15 min to retrieve the tissue antigen, the sections were incubated with 10 % normal goat serum at room temperature for 10 min to block non-specific reactions. This was followed by a PBS wash and incubation with rabbit polyclonal antibody against human ECT2 (1:400, Bioss Biotech Co., LTD, Beijing, China) for 12 h at 4 °C, and biotinylated goat antirabbit serum IgG was used as secondary antibody. After a PBS wash, the sections were developed in diaminobenzidine (DAB) substrate. The sections were then counterstained in hematoxylin for 2 min and then dehydrated in ethanol and xylene before being mounted. Sections were re-prepared by immunohistochemical staining. The
Clin Transl Oncol
staining results of colon cancer tissue sections which were already known to be ECT2 positive were regarded as positive control and PBS instead of primary antibodies was as negative control. ELISA of ECT2 The concentration of the ECT2 protein in the peripheral serum was measured using a double-antibody sandwich ELISA method (Shanghai Westang Biological Technology, Ltd, Shanghai, China) according to the manufacturer’s instructions. The optical density value was measured at a wavelength of 450 nm and a standard curve was determined to calculate the concentration of ECT2 protein. Statistical analysis Statistical analysis was performed using SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA). Student’s t test was applied for data analysis between the two groups, v2 was applied for numeration data, and a Pearson correlation coefficient was used to analyze the correlation between gene expression and the protein level. P \ 0.05 was considered to indicate a statistically significant difference.
Fig. 1 ECT2 expression in tumor tissue and adjacent tissue in gastric cancer patients. a RT-PCR showed stronger band intensity in tumor tissue (T) compared with adjacent tissue (NT). b Densitometric analysis result shows that the expression of ECT2 in tumor tissue is significantly increased comparing with the adjacent tissue (*P \ 0.05)
Results ECT2 expression levels in tissue samples Semi-quantitative RT-PCR (Fig. 1a) identified ECT2 expression in 70.0 % (35/50) of the gastric carcinoma tissues, 40.0 % (20/50) of the adjacent tissues. The mean level of ECT2 mRNA in the gastric carcinoma tissues (1.475 ± 0.047) was significantly higher than in the adjacent tissues (0.467 ± 0.034; P \ 0.05; Fig. 1b). Western blot showed that ECT2 level in gastric carcinoma tissues was higher than those in the adjacent normal tissues and the mean of ECT2/b-actin (gray scale value) was (1.275 ± 0.034) versus (0.414 ± 0.026), and there was significant difference (P \ 0.01; Fig. 2). H&E (Fig. 3) and immunohistochemical (Fig. 4) staining for ECT2 were observed in the majority of gastric carcinoma tissues. The expression rate of ECT2 was 76.0 % (38/50) in the gastric carcinoma tissues and 36.0 % (18/50) in the adjacent tissues. ECT2 expression level in preoperative serum Semi-quantitative RT-PCR (Fig. 5) identified ECT2 mRNA expression in 54.0 % (27/50) of the gastric carcinoma serum, 0 % (0/30) of the healthy volunteers.
Fig. 2 Western blot show ECT2 expression in tumor tissue (T) and adjacent tissue (NT) in gastric cancer patients (**P \ 0.01)
The mean preoperative serum ECT2 protein level in gastric carcinoma patients (1096.243 ± 214.332 lg/ml) was significantly higher than those of the control group (789.204 ± 79.709 lg/ml; P \ 0.05). Correlation between tissue ECT2 level and clinicopathological features of gastric carcinoma ECT2 mRNA expression was significantly associated with histological differentiation (P = 0.004), TNM stage (P = 0.001), and lymph node metastasis (P = 0.001), and there were no correlations between ECT2 mRNA expression and gender (P = 0.064), age (P = 0.355) (Table 1).
123
Clin Transl Oncol
Fig. 3 Hematoxylin and eosin staining of ECT2 in tumor tissue (T) and adjacent tissue (NT) in gastric cancer patients (magnification, 9100)
Fig. 4 Immunohistochemical staining of ECT2 in tumor tissue (T) and adjacent tissue (NT) in gastric cancer patients (magnification, 9400)
Fig. 5 ECT2 expression in gastric carcinoma and healthy volunteers
123
The correlation between ECT2 protein expression and clinicopathological features was also analyzed. The ECT2 protein level in patients with low differentiation (1.324 ± 0.036), an advanced TNM stage (1.474 ± 0.016), and lymph node metastasis (1.371 ± 0.032) was significantly higher than that in the patients at high differentiation (1.135 ± 0.072), an early TNM stage (1.042 ± 0.028), and exhibiting no lymph node metastasis (1.003 ± 0.037), respectively (P \ 0.05). However, there was no significant correlation between the ECT2 protein level and other clinicopathological characteristics, such as the gender and age (Table 1).
Clin Transl Oncol Table 1 Correlation between tissue ECT2 levels and clinicopathological features
Feature
Cases (n)
Positive expression of ECT2 mRNA (n (%))
P value
0.064
ECT2 protein (mean ± SE)
P value
1.259 ± 0.043
0.495
Gender Male
34
21 (61.8)
Female
16
14 (87.5)
1.310 ± 0.055
Age (year) B60
25
16 (64.0)
[60
25
19 (76.0)
0.355
Well
13
5 (38.5)
Poor
37
30 (81.1)
23 27
10 (43.5) 25 (92.6)
0.001**
0.001**
1.255 ± 0.050
0.554
1.296 ± 0.047
Differentiation 0.004**
1.135 ± 0.072
0.014*
1.324 ± 0.036
TNM stage I–II III–IV
1.042 ± 0.028 1.474 ± 0.016
0.001**
1.003 ± 0.037
0.001**
Lymphatic metastasis No
13
4 (30.8)
Yes
37
31 (83.8)
1.371 ± 0.032
* P \ 0.05; ** P \ 0.01
Correlation between preoperative serum ECT2 level and clinicopathological features of gastric carcinoma The expression of ECT2 mRNA in preoperative serum of gastric carcinoma was associated with TNM stage and lymph node metastasis (all P \ 0.01), and there were no correlations between ECT2 mRNA expression and gender, age, and histological differentiation (all P [ 0.05), (Table 2). The serum concentration of ECT2 protein level in patients with an advanced TNM stage (1256.316 ± 137.420) and lymph node metastasis (1170.813 ± 190.704) was significantly higher than that in the patients at an early TNM stage (908.332 ± 107.834) and exhibiting no lymph node metastasis (884.007 ± 111.953), respectively (P \ 0.05). However, there was no significant correlation between the ECT2 protein level and other clinicopathological characteristics, such as the gender, age, and histological differentiation (Table 2). Correlation between ECT2 expression level in gastric carcinoma tissues and preoperative serum ECT2 level The correlation between ECT2 mRNA level in gastric carcinoma tissues and the preoperative serum was identified to be statistically significant (r = 0.594; P = 0.000). The correlation between ECT2 protein level in gastric carcinoma tissues and the preoperative serum was identified to be statistically significant (r = 0.810; P = 0.000).
Diagnostic value of serum ECT2 level for gastric carcinoma To explore the diagnostic performance and the optimal cutoff value of serum ECT2 for gastric carcinoma, we carried out ROC curve analysis. With the healthy population as reference, the optimal discrimination of gastric carcinoma was defined at a cut-off value of 924.618 ng/l with a sensitivity of 68.0 % and specificity of 93.4 %. The area under the curve (AUC) value was 0.915 (Fig. 6).
Discussion As we known, most clinically patients with gastric cancer are in advanced, at this time, treatment options are limited [18]. Despite the advance in diagnosis and treatment, the prognosis for advanced gastric cancer is poor, the overall 5-year survival rate is low at 40 % [19], and it is predominantly attributed to their capacity to invade and metastasize. With the development of molecular biology techniques, gene therapy of gastric cancer has made great progress and is expected to become new avenues. Therefore, it is crucial to develop more target of molecular targeted therapy of gastric cancer. The small RhoGTPases is known to play important roles in essential cellular processes such as the regulation of actin cytoskeleton, gene transcription, cell motility, cell adhesion, and cytokinesis [20]. The RhoGTPases cycle between a GTP bound active state and a GDP bound
123
Clin Transl Oncol Table 2 Correlation between serum ECT2 levels and clinicopathological features
Feature
Cases (n)
Positive expression of ECT2 mRNA (n (%))
P value
0.151
ECT2 concentration, (mean ± SD (ng/l))
P value
1069.632 ± 213.320
0.204
Gender Male
34
16 (47.1)
Female
16
11 (68.8)
1152.792 ± 211.983
Age (year) B60
25
14 (56.0)
[60
25
13 (52.0)
0.777
Well
13
5 (38.5)
Poor
37
22 (59.4)
23 27
4 (17.4) 23 (85.2)
0.001**
0.001**
1125.006 ± 207.543
0.328
1065.084 ± 221.586
Differentiation 0.199
997.152 ± 573.766
0.052
1131.014 ± 208.494
TNM stage I–II III–IV
908.332 ± 107.834 1256.316 ± 137.420
0.001**
884.007 ± 111.953
0.001**
Lymphatic metastasis No
13
2 (15.4)
Yes
37
25 (67.6)
1170.813 ± 190.704
** P \ 0.01
Fig. 6 Receiver operating characteristic (ROC) curve analysis of serum ECT2 levels for the diagnosis of gastric carcinoma
inactive state, and have been implicated in the malignant phenotype of many human cancers as a result of their participation in aberrant signaling in tumor cells and overexpression in human tumors [21]. ECT2 is a member of the Dbl family of guanine nucleotide exchange factors (GEFs), which catalyzes guanine nucleotide exchange on the small GTP-binding protein, such as RhoA and Cdc42 [22]. In previous studies, knockdown of ECT2 with small
123
inhibitory RNA (siRNA) transfection resulted in growth inhibition of various tumor cells, such as NSCLC, glioma, indicating ECT2-derived RhoGTPases were indispensible for the proliferation [23, 24]. In the present study, ECT2 expression was significantly increased in tumor tissue compared with the adjacent tissue in patients with gastric carcinoma. ECT2 expression in tumor tissue was positively correlated with that in serum.
Clin Transl Oncol
Correlation between the ECT2 expression levels in tissues and the clinicopathological parameters of tumors revealed that the ECT2 expression was correlated with the histologic differentiation, TNM stage, and lymph node metastasis. The expression of ECT2 was reinforced significantly when the differentiation degree decreased, indicating that ECT2 may affect cell differentiation in early stage of gastric carcinoma. The expression of ECT2 was increased in gastric carcinoma with advanced stage and lymph node metastasis, indicating that ECT2 plays an important role in the proliferation and metastasis of gastric carcinoma. The proliferation and metastasis of tumor cells is important biological behavior of tumor and is related with prognosis closely; thus, ECT2 may be a useful predictor to the patients of gastric carcinoma. Furthermore, serum ECT2 in patients with gastric carcinoma was significantly increased compared with normal control. Serum ECT2 could discriminate gastric carcinoma patients from the healthy population with significant sensitivity and specificity. Serum ECT2 level in gastric carcinoma patients was related with some clinical parameters including TNM stage and lymph node metastasis. To the best of our knowledge, this is the first study to show the increased ECT2 expression in human gastric cancer serum, and identify the usefulness of serum ECT2 as a potential diagnostic biomarker for gastric carcinoma. We investigated whether ECT2 expression in gastric carcinoma tissue and serum differed from that of the adjacent non-tumorous tissue or in individuals without cancer. We successfully demonstrated that ECT2 expression in tumor tissue was up-regulated compared with adjacent non-tumorous tissue. A similar result was reported in a study using human oral cancer tissue [25]. This suggests that ECT2 expression might be up-regulated during gastric carcinoma tumorigenesis. However, to determine the precise role of ECT2 during gastric carcinoma carcinogenesis, further experiments are clearly required. In this study, ECT2 was detectable in human serum, and its level was increased in gastric carcinoma patients compared with healthy population. The exact mechanism how ECT2 is detectable in serum is not yet clear. However, influx of intracellular ECT2 to the bloodstream after cell death could be a possible explanation. Increased serum ECT2 level in gastric carcinoma patients might be attributable to increased tissue expression or to excessive turnover rate of cancer cells in the tumor and resultant high influx of ECT2 protein into circulation. This hypothesis might be supported by our demonstration of a positive correlation between tumor ECT2 expression and that in serum. ECT2 expression levels in tissue and serum were both significantly correlated with TNM stage and lymph node metastasis. Development of gastric cancer is a complex
multistep process that is still not fully understood. However, accumulating evidence firmly indicates that RhoGTPases and its Signaling pathways play an important role in various human cancers [26]. In fact, ECT2 is highly expressed in some human tumors including lung, bladder, esophageal, pancreatic, and ovarian tumors, and it is important for cell proliferation, migration, invasion, and tumorigenicity of human cancer cells [14–17]. Although it is not easy to demonstrate how ECT2 contributed to carcinogenesis, our results suggest that ECT2 overexpression might be a common phenomenon occurred in tumorigenesis of gastric cancer. Our data indicate the possibility of serum ECT2 as a potential diagnostic biomarker for gastric carcinoma. Standard biomarkers used for gastric cancer diagnosis include carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA19-9); however, they are not used in clinical practice because of their low diagnostic sensitivity [27]. In this study, ECT2 showed considerable sensitivity and specificity for the diagnosis for gastric carcinoma. Emerging candidate biomarkers including circulating tumor cells, microRNA, DNA methylation products, and autoantibodies have been widely studied and many promising results have been reported. However, several challenges hamper their use in clinical settings: the scarce quantity of any given biomarker, complexity of measurements, high cost of measurements, and lack of a wellverified assay technique [28]. This indicates that ECT2 may be promising because of its considerable discriminating performance and facility of measurement using a widely used assay technique. In conclusion, we verified the overexpression of ECT2 in tumor tissue and serum in patients with gastric carcinoma, and were correlated with specific clinicopathological features of gastric carcinoma. The data indicate that ECT2 has potential as a diagnostic marker and therapeutic molecular target for management of gastric carcinoma. Acknowledgments We thank for our colleagues in Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical University for helpful suggestions and their excellent technical assistance. Compliance with ethical standards Informed consent The study was approved by the local ethical committee and all patients gave informed consent. Conflict of interest
The authors declare no conflict of interest.
References 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90. 2. Cuschieri SA, Hanna GB. Meta-analysis of D1 versus D2 gastrectomy for gastric adenocarcinoma: let us move on to another era. Ann Surg. 2014;259:e90.
123
Clin Transl Oncol 3. Grabsch HI, Tan P. Gastric cancer pathology and underlying molecular mechanisms. Dig Surg. 2013;30:150–8. 4. Miki T, Smith CL, Long JE, Eva A, Fleming TP. Oncogene ect2 is related to regulators of small GTP-binding proteins. Nature. 1993;362:462–5. 5. Matthews HK, Delabre U, Rohn JL, Guck J, Kunda P, Baum B. Changes in Ect2 localization couple actomyosin-dependent cell shape changes to mitotic progression. Dev Cell. 2012;23:371–83. 6. Yuce O, Piekny A, Glotzer M. An ECT2-centralspindlin complex regulates the localization and function of RhoA. J Cell Biol. 2005;170:571–82. 7. Morita K, Hirono K, Han M. The Caenorhabditis elegans ect-2 RhoGEF gene regulates cytokinesis and migration of epidermal P cells. EMBO Rep. 2005;6:1163–8. 8. Kim JE, Billadeau DD, Chen J. The tandem BRCT domains of Ect2 are required for both negative and positive regulation of Ect2 in cytokinesis. J Biol Chem. 2005;280:5733–9. 9. Greer ER, Chao AT, Bejsovec A. Pebble/ECT2 RhoGEF negatively regulates the Wingless/Wnt signaling pathway. Development. 2013;140:4937–46. 10. Duquette PM, Lamarche-Vane N. Rho GTPases in embryonic development. Small GTPases. 2014;5:8. 11. Zegers MM, Friedl P. Rho GTPases in collective cell migration. Small GTPases. 2014;5:e28997. 12. Justilien V, Jameison L, Der CJ, Rossman KL, Fields AP. Oncogenic activity of Ect2 is regulated through protein kinase C iota-mediated phosphorylation. J Biol Chem. 2011;286:8149–57. 13. Fields AP, Justilien V. The guanine nucleotide exchange factor (GEF) Ect2 is an oncogene in human cancer. Adv Enzyme Regul. 2010;50:190–200. 14. Murata Y, Minami Y, Iwakawa R, Yokota J, Usui S, Tsuta K, et al. ECT2 amplification and overexpression as a new prognostic biomarker for early-stage lung adenocarcinoma. Cancer Sci. 2014;105:490–7. 15. Sano M, Genkai N, Yajima N, Tsuchiya N, Homma J, Tanaka R, et al. Expression level of ECT2 proto-oncogene correlates with prognosis in glioma patients. Oncol Rep. 2006;16:1093–8. 16. Chen J, Xia H, Zhang X, Karthik S, Pratap SV, Ooi LL, et al. ECT2 regulates the Rho/ERK signalling axis to promote early recurrence in human hepatocellular carcinoma. J Hepatol. 2015;62:1287–95.
123
17. Zhang ML, Lu S, Zhou L, Zheng SS. Correlation between ECT2 gene expression and methylation change of ECT2 promoter region in pancreatic cancer. Hepatobiliary Pancreat Dis Int. 2008;7:533–8. 18. Wang YY, Ye ZY, Zhao ZS, Tao HQ, Li SG. Systems biology approach to identification of biomarkers for metastatic progression in gastric cancer. J Cancer Res Clin Oncol. 2010;136:135–41. 19. Wang WF, Li J, Du LT, Wang LL, Yang YM, Liu YM, et al. Kruppel-like factor 8 overexpression is correlated with angiogenesis and poor prognosis in gastric cancer. World J Gastroenterol. 2013;19:4309–15. 20. Sinha S, Yang W. Cellular signaling for activation of Rho GTPase Cdc42. Cell Signal. 2008;20:1927–34. 21. Gomez del Pulgar T, Benitah SA, Valeron PF, Espina C, Lacal JC. Rho GTPase expression in tumourigenesis: evidence for a significant link. Bioessays. 2005;27:602–13. 22. Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol. 2005;6:167–80. 23. Hirata D, Yamabuki T, Miki D, Ito T, Tsuchiya E, Fujita M, et al. Involvement of epithelial cell transforming sequence-2 oncoantigen in lung and esophageal cancer progression. Clin Cancer Res. 2009;15:256–66. 24. Salhia B, Tran NL, Chan A, Wolf A, Nakada M, Rutka F, et al. The guanine nucleotide exchange factors trio, Ect2, and Vav3 mediate the invasive behavior of glioblastoma. Am J Pathol. 2008;173:1828–38. 25. Iyoda M, Kasamatsu A, Ishigami T, Nakashima D, Endo-Sakamoto Y, Ogawara K, et al. Epithelial cell transforming sequence 2 in human oral cancer. PLoS ONE. 2010;5:e14082. 26. Spuul P, Ciufici P, Veillat V, Leclercq A, Daubon T, Kramer I, et al. Importance of RhoGTPases in formation, characteristics, and functions of invadosomes. Small GTPases. 2014;5:e28195. 27. Pectasides D, Mylonakis A, Kostopoulou M, Papadopoulou M, Triantafillis D, Varthalitis J, et al. CEA, CA 19-9, and CA-50 in monitoring gastric carcinoma. Am J Clin Oncol. 1997;20:348–53. 28. Watari J, Moriichi K, Tanabe H, Kashima S, Nomura Y, Fujiya M, et al. Biomarkers predicting development of metachronous gastric cancer after endoscopic resection: an analysis of molecular pathology of Helicobacter pylori eradication. Int J Cancer. 2012;130:2349–58.
CLINICAL GUIDES IN ONCOLOGY
Clinical significance of ECT2 expression in tissue and serum of gastric cancer patients H.-B. Wang1 • H.-C. Yan2 • Y. Liu3
Received: 15 May 2015 / Accepted: 9 October 2015 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2015
Abstract The ECT2 (epithelial cell transforming sequence 2) oncogene acted as a guanine nucleotide exchange factor for RhoGTPases, and regulates cytokinesis; thus, it may play a role in the pathogenesis of gastric cancer. In this study, we investigated the expression ECT2 gene in tissues and serum of gastric cancer patients to explore its clinical significance. ECT2 mRNA expression levels in tissues and serum were examined by RT-PCR, and ECT2 protein expression in tissue was evaluated by Western blot, and was further validated by immunohistochemistry and enzyme-linked immunosorbent assay at serum level. ECT2 level was significantly increased in the GC tissues and serum compared to normal control. ECT2 expression was positively correlated with the histologic differentiation, stages of TNM, and lymph node metastasis in GC (P \ 0.05). Our results suggest that ECT2 plays an important role during GC progression and it may become a new diagnostic marker and therapeutic molecular target for management of GC. Keywords Gastric cancer Epithelial cell transforming sequence 2 Guanine nucleotide exchange factors
H.-B. Wang and H.-C. Yan have contributed equally to this work. & H.-B. Wang [email protected] 1
Department of Oncology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, China
2
Department of Oncology, The Second People’s Hospital of Lianyungang, Lianyungang, Jiangsu, China
3
Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical University, Xuzhou, Jiangsu, China
Introduction Gastric cancer (GC) is an aggressive neoplasm associated with a very poor prognosis. Worldwide, GC is the fourth most prevalent malignancy and the second leading cancer cause of death [1]. Although the early diagnosis and treatment of gastric cancer has improved in recent years, GC still remains among the leading causes of death from cancer in China [2]. The development and progression of gastric cancer involves a number of genetic alterations of tumor suppressor and tumor-related genes [3]. In recent years, a series of GC related gene have been found and been studied in depth, but obviously this theory cannot cover the entire process of carcinogenesis of GC. So it is critically important to understand the molecular mechanism of GC. ECT2 (epithelial cell transforming sequence 2) was found by Miki et al., in epithelial cells, and is located on chromosome 3q26. Human ECT2 encodes a polypeptide of 883 amino acids, with a predicted molecular weight of approximately 104 kDa [4]. ECT2 is a guanine nucleotide exchange factor (GEF) for Rho family GTPases related to cytokinesis [5–8]. The C-terminal protein of ECT2 catalyzes guanine nucleotide exchange on the Rho family GTPases, while the N-terminal contains a domain related to cell cycle regulator/checkpoint control proteins GEFs catalyze the exchange of GDP for GTP, thereby activating the RhoGTPases in signal transduction [9]. The RhoGTPases have been implicated in the malignant phenotype of human cancers as a result of their participation in aberrant signaling in tumor cells [10–12]. To date, ECT2 is considered as an oncogene in human cancer [13], and it has been showed to up-regulate in a variety of human tumors including lung cancer [14], glioma [15], hepatocellular carcinoma [16], pancreatic
123
Clin Transl Oncol
cancer [17], etc. However, few studies have analyzed the role of ECT2 expression in GC. In the current study, the expression of ECT2 in gastric carcinoma tissues and serum was analyzed and the correlation between ECT2 levels and the clinicopathological characteristics of gastric carcinoma was investigated, to determine the possible use of ECT2 as a diagnostic marker and therapeutic molecular target for management of GC.
Patients and methods Patients and sample collection 50 cases of primary gastric carcinoma patients, who had undergone a gastrectomy at the Affiliated Hospital of Xuzhou Medical University between April 2014 and September 2014, were involved in our study. 34 cases were male and 16 cases were female, with a mean age of 60 years. None of the patients had received chemotherapy or radiotherapy prior to the surgery and no additional malignancies were evident. All patients were informed of the nature of the study and provided written informed consent, and this study was approved by the ethics committee of Xuzhou Medical University. Peripheral venous blood (10 ml) was collected from each patient prior to the surgery and stored at -80 °C for the RT-PCR and enzyme-linked immunosorbent assay (ELISA). Tumor tissue samples were obtained immediately from the resected specimens and the adjacent tissue was collected from a distance of [5 cm from the tumor. For immunohistochemistry, the tissue samples were fixed with 10 % formalin, dehydrated with gradient alcohol, cleared with xylene and embedded in paraffin, and cut into serial sections at a thickness of 4 lm. In addition, peripheral serum from 30 healthy individuals was randomly selected to serve as controls. Classification of the tumors was conducted according to the tumor-node-metastasis (TNM) staging system issued by the Union Internationale Contrele Cancer (UICC) in 2010. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of ECT2 expression in tissues and serum Total RNA was extracted using Trizol (for tissues) (Invitrogen Inc, USA) or RNA extraction kit (Bioteke Biotech Co., LTD, Beijing, China) followed by complementary DNA synthesis using the reverse kit (Tiangen Biotech Co., LTD, Beijing, China). PCR was performed using Taq polymerase (Tiangen Biotech Co., LTD, Beijing, China) under the following conditions: 94 °C for 3 min; 30 cycles of 94 °C for 30 s, 55 °C (for b-actin) or 58 °C (for ECT2)
123
for 30 s and 72 °C for 60 s; followed by 72 °C for 5 min. b-actin served as an internal control. The PCR product lengths for ECT2 and b-actin were 326 and 285 bp, respectively, and were sequenced by Sangon Biotechnology Co., LTD (Shanghai, China). The primers for the PCR were as follows: Sense, 50 -GCCTTGCTTGTGAGGCCA CCAA-30 and anti-sense, 50 -TCCACTGAGAGCCGTG GGATGTCA-30 for ECT2; sense, 50 -GCCTTGCTTGT GAGGCCACCAA-30 , and anti-sense, 50 -TCCACTGA GCCGTGGGATGTCA-30 for b-actin. Each PCR product was examined by agarose gel electrophoresis and images were captured using a digital camera (PowerShot A2400; Canon Inc., Tokyo, Japan). The protein band intensities were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). Western blot of ECT2 Total proteins were extracted using RIPA lysis buffer from Beyotime Biotechnology (Nanjing, China), and the protein concentration was quantified using the BCA Protein Assay Kit (Beyotime Biotechnology, Nanjing, China). Total proteins were separated on 10 % SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5 % skimmed-milk powder in Tris-buffered saline with tween-20 (TBS-T), incubated with ECT2 antibody (1:400; Bioss Biotech Co., LTD, Beijing, China), and then incubated with anti-rabbit IgG secondary antibody (1:10,000; Sigma, St. Louis, MO, USA). Then they were subjected to quantitative immunoblot using the Odyssey imaging system (Li-Cor, USA). Immunohistochemical staining of ECT2 Routine hematoxylin and eosin (H&E) staining was conducted prior to immunohistochemical staining for serial sections. Paraffin-embedded tissues were dewaxed in xylene, rehydrated by serial concentrations of ethanol, and then rinsed in phosphate buffer solution (PBS) followed by treatment with 3 % H2O2 to refrain endogenous peroxidase. After being heated in a microwave for 15 min to retrieve the tissue antigen, the sections were incubated with 10 % normal goat serum at room temperature for 10 min to block non-specific reactions. This was followed by a PBS wash and incubation with rabbit polyclonal antibody against human ECT2 (1:400, Bioss Biotech Co., LTD, Beijing, China) for 12 h at 4 °C, and biotinylated goat antirabbit serum IgG was used as secondary antibody. After a PBS wash, the sections were developed in diaminobenzidine (DAB) substrate. The sections were then counterstained in hematoxylin for 2 min and then dehydrated in ethanol and xylene before being mounted. Sections were re-prepared by immunohistochemical staining. The
Clin Transl Oncol
staining results of colon cancer tissue sections which were already known to be ECT2 positive were regarded as positive control and PBS instead of primary antibodies was as negative control. ELISA of ECT2 The concentration of the ECT2 protein in the peripheral serum was measured using a double-antibody sandwich ELISA method (Shanghai Westang Biological Technology, Ltd, Shanghai, China) according to the manufacturer’s instructions. The optical density value was measured at a wavelength of 450 nm and a standard curve was determined to calculate the concentration of ECT2 protein. Statistical analysis Statistical analysis was performed using SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA). Student’s t test was applied for data analysis between the two groups, v2 was applied for numeration data, and a Pearson correlation coefficient was used to analyze the correlation between gene expression and the protein level. P \ 0.05 was considered to indicate a statistically significant difference.
Fig. 1 ECT2 expression in tumor tissue and adjacent tissue in gastric cancer patients. a RT-PCR showed stronger band intensity in tumor tissue (T) compared with adjacent tissue (NT). b Densitometric analysis result shows that the expression of ECT2 in tumor tissue is significantly increased comparing with the adjacent tissue (*P \ 0.05)
Results ECT2 expression levels in tissue samples Semi-quantitative RT-PCR (Fig. 1a) identified ECT2 expression in 70.0 % (35/50) of the gastric carcinoma tissues, 40.0 % (20/50) of the adjacent tissues. The mean level of ECT2 mRNA in the gastric carcinoma tissues (1.475 ± 0.047) was significantly higher than in the adjacent tissues (0.467 ± 0.034; P \ 0.05; Fig. 1b). Western blot showed that ECT2 level in gastric carcinoma tissues was higher than those in the adjacent normal tissues and the mean of ECT2/b-actin (gray scale value) was (1.275 ± 0.034) versus (0.414 ± 0.026), and there was significant difference (P \ 0.01; Fig. 2). H&E (Fig. 3) and immunohistochemical (Fig. 4) staining for ECT2 were observed in the majority of gastric carcinoma tissues. The expression rate of ECT2 was 76.0 % (38/50) in the gastric carcinoma tissues and 36.0 % (18/50) in the adjacent tissues. ECT2 expression level in preoperative serum Semi-quantitative RT-PCR (Fig. 5) identified ECT2 mRNA expression in 54.0 % (27/50) of the gastric carcinoma serum, 0 % (0/30) of the healthy volunteers.
Fig. 2 Western blot show ECT2 expression in tumor tissue (T) and adjacent tissue (NT) in gastric cancer patients (**P \ 0.01)
The mean preoperative serum ECT2 protein level in gastric carcinoma patients (1096.243 ± 214.332 lg/ml) was significantly higher than those of the control group (789.204 ± 79.709 lg/ml; P \ 0.05). Correlation between tissue ECT2 level and clinicopathological features of gastric carcinoma ECT2 mRNA expression was significantly associated with histological differentiation (P = 0.004), TNM stage (P = 0.001), and lymph node metastasis (P = 0.001), and there were no correlations between ECT2 mRNA expression and gender (P = 0.064), age (P = 0.355) (Table 1).
123
Clin Transl Oncol
Fig. 3 Hematoxylin and eosin staining of ECT2 in tumor tissue (T) and adjacent tissue (NT) in gastric cancer patients (magnification, 9100)
Fig. 4 Immunohistochemical staining of ECT2 in tumor tissue (T) and adjacent tissue (NT) in gastric cancer patients (magnification, 9400)
Fig. 5 ECT2 expression in gastric carcinoma and healthy volunteers
123
The correlation between ECT2 protein expression and clinicopathological features was also analyzed. The ECT2 protein level in patients with low differentiation (1.324 ± 0.036), an advanced TNM stage (1.474 ± 0.016), and lymph node metastasis (1.371 ± 0.032) was significantly higher than that in the patients at high differentiation (1.135 ± 0.072), an early TNM stage (1.042 ± 0.028), and exhibiting no lymph node metastasis (1.003 ± 0.037), respectively (P \ 0.05). However, there was no significant correlation between the ECT2 protein level and other clinicopathological characteristics, such as the gender and age (Table 1).
Clin Transl Oncol Table 1 Correlation between tissue ECT2 levels and clinicopathological features
Feature
Cases (n)
Positive expression of ECT2 mRNA (n (%))
P value
0.064
ECT2 protein (mean ± SE)
P value
1.259 ± 0.043
0.495
Gender Male
34
21 (61.8)
Female
16
14 (87.5)
1.310 ± 0.055
Age (year) B60
25
16 (64.0)
[60
25
19 (76.0)
0.355
Well
13
5 (38.5)
Poor
37
30 (81.1)
23 27
10 (43.5) 25 (92.6)
0.001**
0.001**
1.255 ± 0.050
0.554
1.296 ± 0.047
Differentiation 0.004**
1.135 ± 0.072
0.014*
1.324 ± 0.036
TNM stage I–II III–IV
1.042 ± 0.028 1.474 ± 0.016
0.001**
1.003 ± 0.037
0.001**
Lymphatic metastasis No
13
4 (30.8)
Yes
37
31 (83.8)
1.371 ± 0.032
* P \ 0.05; ** P \ 0.01
Correlation between preoperative serum ECT2 level and clinicopathological features of gastric carcinoma The expression of ECT2 mRNA in preoperative serum of gastric carcinoma was associated with TNM stage and lymph node metastasis (all P \ 0.01), and there were no correlations between ECT2 mRNA expression and gender, age, and histological differentiation (all P [ 0.05), (Table 2). The serum concentration of ECT2 protein level in patients with an advanced TNM stage (1256.316 ± 137.420) and lymph node metastasis (1170.813 ± 190.704) was significantly higher than that in the patients at an early TNM stage (908.332 ± 107.834) and exhibiting no lymph node metastasis (884.007 ± 111.953), respectively (P \ 0.05). However, there was no significant correlation between the ECT2 protein level and other clinicopathological characteristics, such as the gender, age, and histological differentiation (Table 2). Correlation between ECT2 expression level in gastric carcinoma tissues and preoperative serum ECT2 level The correlation between ECT2 mRNA level in gastric carcinoma tissues and the preoperative serum was identified to be statistically significant (r = 0.594; P = 0.000). The correlation between ECT2 protein level in gastric carcinoma tissues and the preoperative serum was identified to be statistically significant (r = 0.810; P = 0.000).
Diagnostic value of serum ECT2 level for gastric carcinoma To explore the diagnostic performance and the optimal cutoff value of serum ECT2 for gastric carcinoma, we carried out ROC curve analysis. With the healthy population as reference, the optimal discrimination of gastric carcinoma was defined at a cut-off value of 924.618 ng/l with a sensitivity of 68.0 % and specificity of 93.4 %. The area under the curve (AUC) value was 0.915 (Fig. 6).
Discussion As we known, most clinically patients with gastric cancer are in advanced, at this time, treatment options are limited [18]. Despite the advance in diagnosis and treatment, the prognosis for advanced gastric cancer is poor, the overall 5-year survival rate is low at 40 % [19], and it is predominantly attributed to their capacity to invade and metastasize. With the development of molecular biology techniques, gene therapy of gastric cancer has made great progress and is expected to become new avenues. Therefore, it is crucial to develop more target of molecular targeted therapy of gastric cancer. The small RhoGTPases is known to play important roles in essential cellular processes such as the regulation of actin cytoskeleton, gene transcription, cell motility, cell adhesion, and cytokinesis [20]. The RhoGTPases cycle between a GTP bound active state and a GDP bound
123
Clin Transl Oncol Table 2 Correlation between serum ECT2 levels and clinicopathological features
Feature
Cases (n)
Positive expression of ECT2 mRNA (n (%))
P value
0.151
ECT2 concentration, (mean ± SD (ng/l))
P value
1069.632 ± 213.320
0.204
Gender Male
34
16 (47.1)
Female
16
11 (68.8)
1152.792 ± 211.983
Age (year) B60
25
14 (56.0)
[60
25
13 (52.0)
0.777
Well
13
5 (38.5)
Poor
37
22 (59.4)
23 27
4 (17.4) 23 (85.2)
0.001**
0.001**
1125.006 ± 207.543
0.328
1065.084 ± 221.586
Differentiation 0.199
997.152 ± 573.766
0.052
1131.014 ± 208.494
TNM stage I–II III–IV
908.332 ± 107.834 1256.316 ± 137.420
0.001**
884.007 ± 111.953
0.001**
Lymphatic metastasis No
13
2 (15.4)
Yes
37
25 (67.6)
1170.813 ± 190.704
** P \ 0.01
Fig. 6 Receiver operating characteristic (ROC) curve analysis of serum ECT2 levels for the diagnosis of gastric carcinoma
inactive state, and have been implicated in the malignant phenotype of many human cancers as a result of their participation in aberrant signaling in tumor cells and overexpression in human tumors [21]. ECT2 is a member of the Dbl family of guanine nucleotide exchange factors (GEFs), which catalyzes guanine nucleotide exchange on the small GTP-binding protein, such as RhoA and Cdc42 [22]. In previous studies, knockdown of ECT2 with small
123
inhibitory RNA (siRNA) transfection resulted in growth inhibition of various tumor cells, such as NSCLC, glioma, indicating ECT2-derived RhoGTPases were indispensible for the proliferation [23, 24]. In the present study, ECT2 expression was significantly increased in tumor tissue compared with the adjacent tissue in patients with gastric carcinoma. ECT2 expression in tumor tissue was positively correlated with that in serum.
Clin Transl Oncol
Correlation between the ECT2 expression levels in tissues and the clinicopathological parameters of tumors revealed that the ECT2 expression was correlated with the histologic differentiation, TNM stage, and lymph node metastasis. The expression of ECT2 was reinforced significantly when the differentiation degree decreased, indicating that ECT2 may affect cell differentiation in early stage of gastric carcinoma. The expression of ECT2 was increased in gastric carcinoma with advanced stage and lymph node metastasis, indicating that ECT2 plays an important role in the proliferation and metastasis of gastric carcinoma. The proliferation and metastasis of tumor cells is important biological behavior of tumor and is related with prognosis closely; thus, ECT2 may be a useful predictor to the patients of gastric carcinoma. Furthermore, serum ECT2 in patients with gastric carcinoma was significantly increased compared with normal control. Serum ECT2 could discriminate gastric carcinoma patients from the healthy population with significant sensitivity and specificity. Serum ECT2 level in gastric carcinoma patients was related with some clinical parameters including TNM stage and lymph node metastasis. To the best of our knowledge, this is the first study to show the increased ECT2 expression in human gastric cancer serum, and identify the usefulness of serum ECT2 as a potential diagnostic biomarker for gastric carcinoma. We investigated whether ECT2 expression in gastric carcinoma tissue and serum differed from that of the adjacent non-tumorous tissue or in individuals without cancer. We successfully demonstrated that ECT2 expression in tumor tissue was up-regulated compared with adjacent non-tumorous tissue. A similar result was reported in a study using human oral cancer tissue [25]. This suggests that ECT2 expression might be up-regulated during gastric carcinoma tumorigenesis. However, to determine the precise role of ECT2 during gastric carcinoma carcinogenesis, further experiments are clearly required. In this study, ECT2 was detectable in human serum, and its level was increased in gastric carcinoma patients compared with healthy population. The exact mechanism how ECT2 is detectable in serum is not yet clear. However, influx of intracellular ECT2 to the bloodstream after cell death could be a possible explanation. Increased serum ECT2 level in gastric carcinoma patients might be attributable to increased tissue expression or to excessive turnover rate of cancer cells in the tumor and resultant high influx of ECT2 protein into circulation. This hypothesis might be supported by our demonstration of a positive correlation between tumor ECT2 expression and that in serum. ECT2 expression levels in tissue and serum were both significantly correlated with TNM stage and lymph node metastasis. Development of gastric cancer is a complex
multistep process that is still not fully understood. However, accumulating evidence firmly indicates that RhoGTPases and its Signaling pathways play an important role in various human cancers [26]. In fact, ECT2 is highly expressed in some human tumors including lung, bladder, esophageal, pancreatic, and ovarian tumors, and it is important for cell proliferation, migration, invasion, and tumorigenicity of human cancer cells [14–17]. Although it is not easy to demonstrate how ECT2 contributed to carcinogenesis, our results suggest that ECT2 overexpression might be a common phenomenon occurred in tumorigenesis of gastric cancer. Our data indicate the possibility of serum ECT2 as a potential diagnostic biomarker for gastric carcinoma. Standard biomarkers used for gastric cancer diagnosis include carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA19-9); however, they are not used in clinical practice because of their low diagnostic sensitivity [27]. In this study, ECT2 showed considerable sensitivity and specificity for the diagnosis for gastric carcinoma. Emerging candidate biomarkers including circulating tumor cells, microRNA, DNA methylation products, and autoantibodies have been widely studied and many promising results have been reported. However, several challenges hamper their use in clinical settings: the scarce quantity of any given biomarker, complexity of measurements, high cost of measurements, and lack of a wellverified assay technique [28]. This indicates that ECT2 may be promising because of its considerable discriminating performance and facility of measurement using a widely used assay technique. In conclusion, we verified the overexpression of ECT2 in tumor tissue and serum in patients with gastric carcinoma, and were correlated with specific clinicopathological features of gastric carcinoma. The data indicate that ECT2 has potential as a diagnostic marker and therapeutic molecular target for management of gastric carcinoma. Acknowledgments We thank for our colleagues in Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical University for helpful suggestions and their excellent technical assistance. Compliance with ethical standards Informed consent The study was approved by the local ethical committee and all patients gave informed consent. Conflict of interest
The authors declare no conflict of interest.
References 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90. 2. Cuschieri SA, Hanna GB. Meta-analysis of D1 versus D2 gastrectomy for gastric adenocarcinoma: let us move on to another era. Ann Surg. 2014;259:e90.
123
Clin Transl Oncol 3. Grabsch HI, Tan P. Gastric cancer pathology and underlying molecular mechanisms. Dig Surg. 2013;30:150–8. 4. Miki T, Smith CL, Long JE, Eva A, Fleming TP. Oncogene ect2 is related to regulators of small GTP-binding proteins. Nature. 1993;362:462–5. 5. Matthews HK, Delabre U, Rohn JL, Guck J, Kunda P, Baum B. Changes in Ect2 localization couple actomyosin-dependent cell shape changes to mitotic progression. Dev Cell. 2012;23:371–83. 6. Yuce O, Piekny A, Glotzer M. An ECT2-centralspindlin complex regulates the localization and function of RhoA. J Cell Biol. 2005;170:571–82. 7. Morita K, Hirono K, Han M. The Caenorhabditis elegans ect-2 RhoGEF gene regulates cytokinesis and migration of epidermal P cells. EMBO Rep. 2005;6:1163–8. 8. Kim JE, Billadeau DD, Chen J. The tandem BRCT domains of Ect2 are required for both negative and positive regulation of Ect2 in cytokinesis. J Biol Chem. 2005;280:5733–9. 9. Greer ER, Chao AT, Bejsovec A. Pebble/ECT2 RhoGEF negatively regulates the Wingless/Wnt signaling pathway. Development. 2013;140:4937–46. 10. Duquette PM, Lamarche-Vane N. Rho GTPases in embryonic development. Small GTPases. 2014;5:8. 11. Zegers MM, Friedl P. Rho GTPases in collective cell migration. Small GTPases. 2014;5:e28997. 12. Justilien V, Jameison L, Der CJ, Rossman KL, Fields AP. Oncogenic activity of Ect2 is regulated through protein kinase C iota-mediated phosphorylation. J Biol Chem. 2011;286:8149–57. 13. Fields AP, Justilien V. The guanine nucleotide exchange factor (GEF) Ect2 is an oncogene in human cancer. Adv Enzyme Regul. 2010;50:190–200. 14. Murata Y, Minami Y, Iwakawa R, Yokota J, Usui S, Tsuta K, et al. ECT2 amplification and overexpression as a new prognostic biomarker for early-stage lung adenocarcinoma. Cancer Sci. 2014;105:490–7. 15. Sano M, Genkai N, Yajima N, Tsuchiya N, Homma J, Tanaka R, et al. Expression level of ECT2 proto-oncogene correlates with prognosis in glioma patients. Oncol Rep. 2006;16:1093–8. 16. Chen J, Xia H, Zhang X, Karthik S, Pratap SV, Ooi LL, et al. ECT2 regulates the Rho/ERK signalling axis to promote early recurrence in human hepatocellular carcinoma. J Hepatol. 2015;62:1287–95.
123
17. Zhang ML, Lu S, Zhou L, Zheng SS. Correlation between ECT2 gene expression and methylation change of ECT2 promoter region in pancreatic cancer. Hepatobiliary Pancreat Dis Int. 2008;7:533–8. 18. Wang YY, Ye ZY, Zhao ZS, Tao HQ, Li SG. Systems biology approach to identification of biomarkers for metastatic progression in gastric cancer. J Cancer Res Clin Oncol. 2010;136:135–41. 19. Wang WF, Li J, Du LT, Wang LL, Yang YM, Liu YM, et al. Kruppel-like factor 8 overexpression is correlated with angiogenesis and poor prognosis in gastric cancer. World J Gastroenterol. 2013;19:4309–15. 20. Sinha S, Yang W. Cellular signaling for activation of Rho GTPase Cdc42. Cell Signal. 2008;20:1927–34. 21. Gomez del Pulgar T, Benitah SA, Valeron PF, Espina C, Lacal JC. Rho GTPase expression in tumourigenesis: evidence for a significant link. Bioessays. 2005;27:602–13. 22. Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol. 2005;6:167–80. 23. Hirata D, Yamabuki T, Miki D, Ito T, Tsuchiya E, Fujita M, et al. Involvement of epithelial cell transforming sequence-2 oncoantigen in lung and esophageal cancer progression. Clin Cancer Res. 2009;15:256–66. 24. Salhia B, Tran NL, Chan A, Wolf A, Nakada M, Rutka F, et al. The guanine nucleotide exchange factors trio, Ect2, and Vav3 mediate the invasive behavior of glioblastoma. Am J Pathol. 2008;173:1828–38. 25. Iyoda M, Kasamatsu A, Ishigami T, Nakashima D, Endo-Sakamoto Y, Ogawara K, et al. Epithelial cell transforming sequence 2 in human oral cancer. PLoS ONE. 2010;5:e14082. 26. Spuul P, Ciufici P, Veillat V, Leclercq A, Daubon T, Kramer I, et al. Importance of RhoGTPases in formation, characteristics, and functions of invadosomes. Small GTPases. 2014;5:e28195. 27. Pectasides D, Mylonakis A, Kostopoulou M, Papadopoulou M, Triantafillis D, Varthalitis J, et al. CEA, CA 19-9, and CA-50 in monitoring gastric carcinoma. Am J Clin Oncol. 1997;20:348–53. 28. Watari J, Moriichi K, Tanabe H, Kashima S, Nomura Y, Fujiya M, et al. Biomarkers predicting development of metachronous gastric cancer after endoscopic resection: an analysis of molecular pathology of Helicobacter pylori eradication. Int J Cancer. 2012;130:2349–58.
