Astrocyte elevated gene-1 is overexpressed in non-small-cell lung cancer and associated with increased tumour angiogenesis

Astrocyte elevated gene-1 is overexpressed in non-small-cell lung cancer and associated with... Abstract OBJECTIVES Astrocyte elevated gene-1 (AEG-1) functions to mediate angiogenesis, and its upregulation is responsible for tumour angiogenesis during cancer development. This study analysed AEG-1 expression in non-small-cell lung cancer (NSCLC) for association with NSCLC clinicopathological features and tumour angiogenesis. METHODS The expression of AEG-1, vascular endothelial growth factor and intratumoural microvessel density (assessed using the expression of CD105) was detected by immunohistochemistry in 88 paired tumour tissue and adjacent normal tissue specimens obtained from NSCLC patients. The Kaplan–Meier curves were used for survival analysis through an online tool (http://kmplot.com/analysis/). RESULTS AEG-1 was overexpressed in 61.3% of NSCLC tissues vs 6.8% (6/88) of normal tissues (P < 0.001). AEG-1 expression in NSCLC was significantly associated with advanced pTNM stage (P = 0.021), tumour dedifferentiation (P = 0.034), vascular invasion (P = 0.035), lymph node metastasis (P < 0.001) and poor overall survival (P = 0.024). Moreover, the expression of AEG-1 in NSCLC was associated with tumour angiogenesis; that is, vascular endothelial growth factor overexpression (P < 0.001) and intratumoural microvessel density (P < 0.001). CONCLUSIONS This study demonstrates that AEG-1 expression is associated with NSCLC development, angiogenesis, progression and poor prognosis, indicating that the adjuvant therapy with antiangiogenic agent be adopted for the early postoperative period before the start of conventional chemotherapy in patients with AEG-1 overexpressed NSCLC. Astrocyte elevated gene-1, Non-small-cell lung cancer, Intratumoural microvessel density, Angiogenesis, Immunohistochemistry INTRODUCTION Lung cancer is the most commonly diagnosed malignant tumour with a high incidence and mortality rate in the world [1]. It has been estimated that there were approximately 733 300 new lung cancer cases and 610 200 lung cancer-related deaths in China in 2015 [2]. Histologically, lung cancer can be divided into small-cell lung cancer and non-small-cell lung cancer (NSCLC), and the latter accounts for 80% of all primary cancer cases. The 5-year survival rate after surgery remains poor, despite recent advances in early diagnosis and treatment options such as targeted therapy, which could be due to the poor understanding of the molecular mechanism(s) underlying NSCLC pathogenesis. To date, it has been widely accepted that NSCLC is a multifactorial and multistep disease, where angiogenesis plays a major role, and that the unbalanced expression of pro- and antiangiogenic factors in the tumour microenvironment results in neovascularization, which conversely facilitates tumour growth, further loss of tumour cell differentiation and increased tumour cell invasiveness [3, 4]. Thus, the further search for novel biomarkers to target angiogenesis could lead to a promising therapeutic strategy for NSCLC. Astrocyte elevated gene (AEG-1), also named as metadherin and protein LYRIC, has been previously identified as a novel protein induced by human immunodeficiency virus 1 or tumour necrosis factor α (TNF-α) in human primary foetal astrocytes [5, 6]. However, subsequent studies have found that AEG-1 is a tumour-related factor that promotes cancer progression and metastasis [7–10]. For example, Song et al. [8] revealed that AEG-1 expression was associated with NSCLC development and progression, and its elevated expression was dramatically associated with tumour cell aggressiveness and poor patient survival. Lu et al. [10] demonstrated that AEG-1 expression was an independent prognostic factor for poor overall survival (OS) and disease-free survival in NSCLC patients. In addition, AEG-1 has been shown to be an essential mediator of tumour angiogenesis and has been thereby associated with poor prognosis of various cancers, including cervical cancer, oral squamous cell carcinoma (SCC) and breast cancer [11–13]. In this study, immunohistochemistry was performed to detect and compare the expression of AEG-1 protein in 88 paired NSCLC and normal tissue specimens obtained from patients and investigate the association of AEG-1 expression with clinicopathological features, vascular endothelial growth factor (VEGF) expression and intratumoural microvessel density (iMVD). We expect to provide further information on AEG-1 as a biomarker for detecting NSCLC progression and prognosis and evaluate AEG-1 as a potential target for NSCLC therapy. MATERIALS AND METHODS Patients and tissue specimens A total of 88 consecutive newly diagnosed primary NSCLC patients in Huzhou Centre Hospital between September 2013 and December 2015 were recruited, and their clinicopathological characteristics were retrieved (Table 1). All patients were histologically diagnosed with NSCLC according to the eighth edition of the TNM classification of lung cancer [14]. None of these patients received radiotherapy or chemotherapy before surgery. Paired tissue specimens were collected during surgery, including tumour and normal adjacent tissues. These specimens were formalin fixed and paraffin embedded for histological examination and immunohistochemical analysis. This study was approved by the ethics committee of Huzhou Central Hospital (Huzhou, Zhejiang, China) with authorization number HZCHEC-276. All patients provided an informed consent prior to participating into this study. Table 1: Association of AEG-1 expression with NSCLC clinicopathological features (n = 88) Parameters  n  AEG-1 expression   χ2  P-value  High  Low  n = 54  n = 34  Gender   Male  56  34  22  0.027  0.869   Female  32  20  12  Age (years)   ≤60  28  19  9  0.730  0.393   >60  60  35  25  Smoking   Yes  50  28  22  1.405  0.236   No  38  26  12  Tumour size (cm)   ≤3  28  18  10  0.148  0.701   >3  60  36  24  Histology   ADC  55  31  24  1.547  0.214   SCC  33  23  10  pTNM stage   I/II  54  28  26  5.334  0.021   III/IV  34  26  8  Differentiation   Well moderated  58  31  27  4.496  0.034   Poor  30  23  7  Vascular invasion   No  61  33  28  4.426  0.035   Yes  27  21  6  Location   Central  38  24  14  0.091  0.763   Peripheral  50  30  20  Lymph node metastasis   No  57  28  29  10.22  0.001   Yes  31  26  5  Parameters  n  AEG-1 expression   χ2  P-value  High  Low  n = 54  n = 34  Gender   Male  56  34  22  0.027  0.869   Female  32  20  12  Age (years)   ≤60  28  19  9  0.730  0.393   >60  60  35  25  Smoking   Yes  50  28  22  1.405  0.236   No  38  26  12  Tumour size (cm)   ≤3  28  18  10  0.148  0.701   >3  60  36  24  Histology   ADC  55  31  24  1.547  0.214   SCC  33  23  10  pTNM stage   I/II  54  28  26  5.334  0.021   III/IV  34  26  8  Differentiation   Well moderated  58  31  27  4.496  0.034   Poor  30  23  7  Vascular invasion   No  61  33  28  4.426  0.035   Yes  27  21  6  Location   Central  38  24  14  0.091  0.763   Peripheral  50  30  20  Lymph node metastasis   No  57  28  29  10.22  0.001   Yes  31  26  5  ADC: adenocarcinoma; AEG-1: astrocyte elevated gene-1; NSCLC: non-small-cell lung cancer; SCC: squamous cell carcinoma. Immunohistochemistry The formalin-fixed and paraffin-embedded tumour specimens were sectioned into 4-μm-thick tissue sections and mounted onto poly-l-lysine-coated glass slides. For immunohistochemistry, these sections were deparaffinized in xylene and rehydrated into water in a series of ethanol solution (100–0%). Then, these sections were cooked in a high-pressure cooker in 10 mmol/l of citrate buffer (pH 6.0) for 3 min to repair tissue antigens during formalin fixation and paraffin embedding. After cooling down to room temperature and tap water washing and rinsing, the sections were incubated in 3% hydrogen peroxide for 20 min to block potential endogenous peroxidase activity. Then, these sections were washed with phosphate-buffered saline for 3 times, incubated with normal serum for 30 min and further incubated with rabbit monoclonal anti-LYRIC (1:200 dilution; ab124789, Abcam, Cambridge, MA, USA), rabbit monoclonal anti-VEGF receptor (1:200 dilution; ab32152, Abcam) or mouse monoclonal anti-CD105 (1:100 dilution; ab114052, Abcam) at 4 °C overnight. On the next day, these sections were washed with phosphate-buffered saline 3 times and further incubated with the secondary antibody. For VEGF immunostaining, the sections were colour developed using the 3,3'-diaminobenzidine solution, whereas for AEG-1 and CD105 double labelling, the sections were separately colour developed using 3,3'-diaminobenzidine and Ap-Red with the polymer double-staining assay kit (no. DS-0006; Zhongshan Goldbridge, Beijing, China), according to manufacturer’s recommendations. Haematoxylin solution was used to counterstain cell nuclei. The primary antibody was replaced with phosphate-buffered saline for negative control sections. Evaluation of astrocyte elevated gene-1, vascular endothelial growth factor and intratumoural microvessel density immunostaining These immunostained sections were evaluated and scored by 2 independent pathologists who were blinded to the clinical data of these sections. AEG-1 immunostaining was scored using both the percentage of staining and the staining intensity. That is, the percentage of staining was scored as 0 (negative), 1 (≤ 10%), 2 (10%–50%) and 3 (> 50%); and staining intensity was calculated as 0 (no staining), 1 (weakly stained), 2 (moderately stained) and 3 (strongly stained). The staining index was established by multiplying these 2 scores. A total score of <4 was defined as lowly expressed, whereas a total score of ≥4 was considered as highly expressed. VEGF staining was assessed according to a previous study reported by Mattern et al. [15]. Briefly, the percentage score was defined as 0 (negative), 1 (≤ 25%), 2 (25%–50%) and 3 (>50%), whereas the intensity score was calculated similar to that performed for AEG-1 immunostaining. The staining index was established by summing these 2 scores. VEGF expression was considered to be low when the score was 0, 1, 2, 3 or 4; VEGF expression was considered to be high when the score was 5 or 6. For iMVD, the tissue sections were first immunostained with the anti-CD105 antibody to visualize the microvessels. Any red-stained endothelial cell or endothelial cell cluster that could clearly separate contiguous microvessels, tumour cells and connective tissue elements was considered a single, countable microvessel. Microvessels were assessed by 2 observers using a microscope at a magnification of 200×. The data were summarized from 5 of the most vascularized areas in each tissue section, and the mean values of these measurements were taken and considered as the final iMVD level. Online lung cancer tissue database and data retrieval The OS of NSCLC patients was analysed using the online messenger RNA expression database developed by Gyorffy B (http://kmplot.com) [16]. NSCLC data were retrieved from the Gene Expression Omnibus (GEO), and the OS information of NSCLC, adenocarcinoma, SCC related to the expression of AEG-1 was obtained. Statistical analysis Statistical analyses were conducted using SPSS 18.0 (SPCC Inc, Chicago, IL, USA). The Kolmogorov–Smirnov test was performed to determine the distribution of these samples, the Student’s t-test was applied for normally distributed data and the Mann–Whitney U-test was performed for asymmetrical distribution data. The χ2-test was employed to associate the expression of AEG-1 with the clinicopathological parameters obtained from patients, and the Spearman’s correlation coefficient test was used to determine the correlation of AEG-1 with the expression of VEGF. The Kaplan–Meier curves were used to analyse the OS of patients, which was stratified by AEG-1 expression using the online tool (http://kmplot.com/analysis/). A P-value <0.05 was considered statistically significant. RESULTS Association of astrocyte elevated gene-1 expression with clinicopathological parameters obtained from non-small-cell lung cancer patients In this study, AEG-1 expression was first assessed by immunohistochemistry. It was found that AEG-1 protein was primarily localized in the cytoplasm, nuclear membrane and/or cell junction (Fig. 1). Furthermore, high AEG-1 expression was mostly found in cancer cells or cancer nests (Fig. 1C–F), whereas no or low AEG-1 expression was found in normal epithelial cells (Fig. 1A and B). According to staining scores, it was found that AEG-1 was lowly expressed in 82 normal tissues and 34 NSCLC tissues and highly expressed in 54 NSCLC tissues (Table 2, P < 0.001). Table 2: Association of AEG-1 with VEGF expression in NSCLC tissue specimens (n = 88) Parameters  n  VEGF expression (n)   χ2/r  P-value  High  Low  Tissue types   NSCLC  88  50  38       Normal  88  3  85  59.639  <0.001  Lymph node metastasis   No  57  26  31  8.279  0.004   Yes  31  24  7  AEG-1 expression   Low  34  5  29  0.657  <0.001   High  54  45  9  Parameters  n  VEGF expression (n)   χ2/r  P-value  High  Low  Tissue types   NSCLC  88  50  38       Normal  88  3  85  59.639  <0.001  Lymph node metastasis   No  57  26  31  8.279  0.004   Yes  31  24  7  AEG-1 expression   Low  34  5  29  0.657  <0.001   High  54  45  9  AEG-1: astrocyte elevated gene-1; VEGF: vascular endothelial growth factor; NSCLC: non-small-cell lung cancer. Figure 1: View largeDownload slide Immunohistochemical staining of astrocyte elevated gene-1 (AEG-1) protein in non-small-cell lung cancer tissue specimens. (A) and (B) Negative and weak AEG-1 staining in normal alveolar pneumocytes. (C) and (D) Weak and strong AEG-1 staining in lung adenocarcinoma. (E) and (F) Weak and strong AEG-1 staining in lung squamous cell carcinomas (original magnification ×200). Figure 1: View largeDownload slide Immunohistochemical staining of astrocyte elevated gene-1 (AEG-1) protein in non-small-cell lung cancer tissue specimens. (A) and (B) Negative and weak AEG-1 staining in normal alveolar pneumocytes. (C) and (D) Weak and strong AEG-1 staining in lung adenocarcinoma. (E) and (F) Weak and strong AEG-1 staining in lung squamous cell carcinomas (original magnification ×200). Next, AEG-1 expression was associated with clinicopathological features obtained from patients (Table 1). It was found that AEG-1 expression was significantly associated with advanced pTNM stage (P = 0.021), tumour dedifferentiation (P = 0.034), vascular invasion (P = 0.035) and lymph node metastasis (P < 0.001). However, there was no significant difference between AEG-1 expression and gender, age, pathology classification, tumour size, location and history of tobacco smoking (P > 0.05). Association of astrocyte elevated gene-1 expression with vascular endothelial growth factor in non-small-cell lung cancer tissues VEGF protein was preferentially localized in the cytoplasm of tumour cells (data not known), which further confirms the data reported in a previous study [15]. In many cases, our data also revealed that VEGF immunostaining did occur in tumour stromal and vascular endothelial cells, although the staining intensity was weak. VEGF was overexpressed in 3 normal tissues and in 50 cases of NSCLC tissues (Table 2). Furthermore, VEGF expression was dramatically associated with NSCLC lymph node metastasis (χ2 = 8.279, P = 0.004; Table 2). However, VEGF expression was not significantly associated with other clinicopathological factors such as tobacco smoking, tumour status or tumour differentiation. In addition, Spearman’s correlation analysis revealed that AEG-1 expression in NSCLC tissues was moderately associated with NSCLC (r = 0.657, P < 0.001; Table 2). Association of astrocyte elevated gene-1 with intratumoural microvessel density in non-small-cell lung cancer patients Altered expression of AEG-1 has been previously shown to be associated with tumour angiogenesis. In this study, the double immunostaining of AEG-1 and CD105 proteins in tissue specimens was assessed. It was found that CD105 protein was mainly localized in the membrane or cytoplasm of microvascular endothelial cells, manifesting as red granules (Fig. 2A–C). Compared with the faint or weak CD105 immunostaining in normal lung epithelial cells (Fig. 2A), strong and intensive CD105 immunostaining was found in tumour stroma around neoplastic cells (Fig. 2B and C). In summary, the mean iMVD counted in NSCLC tissues was 47.70 ± 16.74, which was significantly higher than that in normal tissues (19.02 ± 5.01, P < 0.001). Figure 2: View largeDownload slide Coimmunolocalization of AEG-1 and CD105 proteins in non-small-cell lung cancer. (A) CD105 staining around normal alveolar pneumocytes. Weak CD105 staining is shown by the arrows. (B) Coimmunolocalization of AEG-1 and CD105 in the low iMVD area. (C) Coimmunolocalization of AEG-1 and CD105 in the high iMVD area (original magnification ×200). (D) iMVD levels in the high- and low-AEG-1 expression groups. AEG-1: astrocyte elevated gene-1; AEG-1-H: AEG-1 high-expression group; AEG-1-L: AEG-1 low-expression group; iMVD: intratumoural microvessel density. Figure 2: View largeDownload slide Coimmunolocalization of AEG-1 and CD105 proteins in non-small-cell lung cancer. (A) CD105 staining around normal alveolar pneumocytes. Weak CD105 staining is shown by the arrows. (B) Coimmunolocalization of AEG-1 and CD105 in the low iMVD area. (C) Coimmunolocalization of AEG-1 and CD105 in the high iMVD area (original magnification ×200). (D) iMVD levels in the high- and low-AEG-1 expression groups. AEG-1: astrocyte elevated gene-1; AEG-1-H: AEG-1 high-expression group; AEG-1-L: AEG-1 low-expression group; iMVD: intratumoural microvessel density. Figure 3: View largeDownload slide The Kaplan–Meier curves of the overall survival (OS) in non-small-cell lung cancer (NSCLC) patients stratified by astrocyte elevated gene-1 expression. (A) The Kaplan–Meier survival curve of OS in NSCLC patient. (B) The Kaplan–Meier survival curve of OS in adenocarcinoma patients. (C) The Kaplan–Meier survival curve of OS in squamous cell carcinoma patients. HR: hazard ratio. Figure 3: View largeDownload slide The Kaplan–Meier curves of the overall survival (OS) in non-small-cell lung cancer (NSCLC) patients stratified by astrocyte elevated gene-1 expression. (A) The Kaplan–Meier survival curve of OS in NSCLC patient. (B) The Kaplan–Meier survival curve of OS in adenocarcinoma patients. (C) The Kaplan–Meier survival curve of OS in squamous cell carcinoma patients. HR: hazard ratio. Furthermore, a significant increase in mean iMVD (53.02 ± 16.88) was found in NSCLC tissues with high AEG-1 expression, and a low iMVD (39.26 ± 12.72) was found in NSCLC tissues with low AEG-1 expression (P < 0.001, Fig. 2D). Association of astrocyte elevated gene-1 expression with poor overall survival in non-small-cell lung cancer patients To further extend our observations to a clinicopathologically relevant context, we performed the Kaplan–Meier survival analysis of AEG-1 on an online platform (http://kmplot.com/analysis/), with the autoselect best cut-off was chosen. The results showed that higher AEG-1 expression was associated with a worse OS of NSCLC patients (P = 0.024) and SCC patients (P = 0.001) but not of adenocarcinoma patients (P = 0.085). DISCUSSION Although AEG-1 was originally identified as an human immunodeficiency virus-inducible gene in primary human foetal astrocytes [5, 6], recent studies have revealed that AEG-1 expression was also upregulated in various human cancers, such as gastric [17], colorectal [18], cervical [11, 12], breast [13] and ovarian cancers [19], glioma [20] and NSCLC [8]. Subsequent investigations have indicated that AEG-1 is a tumour metastasis-associated protein and participates in tumour angiogenesis. For example, in hepatocellular carcinoma HepG3 cells, AEG-1 overexpression was able to significantly increase tumour cell proliferation, invasion and anchorage-independent growth in vitro, as well as tumourigenesis, angiogenesis and metastasis in nude mice [21]. The study conducted by Emdad et al. [7] demonstrated that abnormal increase in AEG-1 expression was associated with increased microvessel density and expression of angiogenesis molecular markers in tumour sections. The in vitro experiment in this study further illustrated that AEG-1 was able to promote tube formation in Matrigel and increased the invasion of human umbilical vein endothelial cells via the PI3K/Akt signalling pathway. All these studies suggest that aberrant AEG-1 expression possessed great effects on oncogenic transformation and angiogenesis. Consistent with this speculation, we first observed the AEG-1 expression pattern in NSCLC tissues and linked AEG-1 expression to angiogenesis. Our present data revealed that the level of AEG-1 expression was dramatically higher in NSCLC tissues than in adjacent normal lung tissues. AEG-1 expression was statistically associated with advanced pTNM stages, vascular invasion, dedifferentiation and lymph node metastasis. However, it was independent of age, gender, tumour size and tobacco smoking status. This study further supports the data reported on a previous study conducted by Song et al. [8], indicating that AEG-1 expression promoted NSCLC development and progression. Angiogenesis is a process in which neovascularization is formed from existing vascular networks, which is a fundamental event for tumour growth and metastasis [22, 23]. During this process, tumour cells would induce the expression of VEGF (a key angiogenic regulator during neovascularization), resulting in an increase in iMVD, a conventional event observed in different tumour tissues related to their normal tissues. For example, the study conducted by Lin et al. [24] demonstrated that upregulated VEGF expression frequently occurred in NSCLC tissue specimens and was associated with the formation of new blood vessels, iMVD and shorter OS in patients. CD105, also referred as Endoglin, is a Type I membrane glycoprotein localized on cell surfaces and is a part of the TGF-beta receptor complex [25]. CD105 plays a crucial role in angiogenesis. It is usually lowly expressed in resting endothelial cells and highly expressed in endothelial cells during neoangiogenesis in cancers, inflamed tissues or vascular injury [25, 26]. In contrast, CD34 or CD31 is a panendothelial marker, and its panendothelial antibody could react not only to ‘newly forming’ vessels but also to pre-existing normal vessels in tumour tissues. Since CD105 has a higher affinity to neovascularized, activated endothelial cells [27, 28], the CD105 molecule is a better endothelial marker than panendothelial markers (such as CD34 or CD31). Therefore, in this study, we utilized both VEGF and CD105 expression to evaluate and associate the relationship between AEG-1 expression and angiogenesis. We found that NSCLC tissues had a significant level of VEGF expression and association with AEG-1 expression (r = 0.657, P < 0.001). In addition, the iMVD assessed by CD105 immunostaining was also considerably higher in AEG-1-expressed NSCLC tissues compared with NSCLC tissues with lowly expressed AEG-1 (t = 4.341, P < 0.001). The results of this study were consistent with the findings reported by previous studies on gastric cancer [17], cervical cancer [12] and oral SCC [9]. Indeed, a recent in vivo study reported by Zhang et al. [29] demonstrated that AEG-1 shRNA was able to successfully inhibit the tumourigenic and angiogenic ability of H460 cells using the in vivo chick embryo chorioallantoic membrane model. However, for surgeons, it should be appreciated that surgery may accelerate the growth of residual disease not only by removal of antiangiogenic factors produced by the primary tumour but also by the production of proangiogenic factors [30]. Therefore, to benefit patients with AEG-1-overexpressed NSCLC in tissue sections, we strongly recommended that adjuvant therapy with antiangiogenic agent be adopted for the early postoperative period before the start of conventional chemotherapy to control or inhibit tumour recurrence or metastasis. In addition, this study revealed that the Kaplan–Meier curve analysis stratified AEG-1 expression to associate with the poor prognosis of NSCLC (P = 0.024). Histologically, higher AEG-1 expression was associated with a worse OS in patients with SCC (P = 0.001) but not in patients with adenocarcinoma (P = 0.085). Although such data have been reported in various cancer types such as cervical cancer, oral SCC, lung cancer and breast cancer [8–13], the underlying molecular mechanisms responsible for AEG-1 promotion in NSCLC poor prognosis remain to be determined. AEG-1 might participate in the multiplex process of angiogenesis, potentially by raising VEGF expression and resulting in neovascularization. However, NSCLC progression is also very complicated, and tumour cell aggressiveness would lead to resistance to different therapy selections. Thus, further studies on angiogenesis and NSCLC cell aggressiveness could lead to the efficient control of this presently deadly disease. CONCLUSION In conclusion, our study demonstrated that AEG-1 protein overexpression frequently occurred in NSCLC, which may contribute to oncogenic transformation and angiogenesis. The detection of AEG-1 expression may serve as a useful biomarker for the prediction of NSCLC progression and poor prognosis, although the present data need to be confirmed through other studies with larger sample sizes and multiple institutional patients. ACKNOWLEDGEMENTS We would like to thank the staff of Huzhou Cancer Biobank and the Department of Surgery, Huzhou Central Hospital, for their excellent assistance. Funding This work was supported in part by grants from the Zhejiang Provincial Natural Science Foundation of China (LQ14H160015) and the Huzhou General Science and Social Development Project Foundation (2013GY17). Conflict of interest: none declared. REFERENCES 1 Torre LA, Bray F, Siegel RL, Ferlay J, Lortet‐Tieulent J et al.   Global cancer statistics, 2012. CA Cancer J Clin  2015; 65: 87– 108. 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Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Interactive CardioVascular and Thoracic Surgery Oxford University Press

Astrocyte elevated gene-1 is overexpressed in non-small-cell lung cancer and associated with increased tumour angiogenesis

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Oxford University Press
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© The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.
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1569-9293
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1569-9285
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10.1093/icvts/ivx340
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Abstract

Abstract OBJECTIVES Astrocyte elevated gene-1 (AEG-1) functions to mediate angiogenesis, and its upregulation is responsible for tumour angiogenesis during cancer development. This study analysed AEG-1 expression in non-small-cell lung cancer (NSCLC) for association with NSCLC clinicopathological features and tumour angiogenesis. METHODS The expression of AEG-1, vascular endothelial growth factor and intratumoural microvessel density (assessed using the expression of CD105) was detected by immunohistochemistry in 88 paired tumour tissue and adjacent normal tissue specimens obtained from NSCLC patients. The Kaplan–Meier curves were used for survival analysis through an online tool (http://kmplot.com/analysis/). RESULTS AEG-1 was overexpressed in 61.3% of NSCLC tissues vs 6.8% (6/88) of normal tissues (P < 0.001). AEG-1 expression in NSCLC was significantly associated with advanced pTNM stage (P = 0.021), tumour dedifferentiation (P = 0.034), vascular invasion (P = 0.035), lymph node metastasis (P < 0.001) and poor overall survival (P = 0.024). Moreover, the expression of AEG-1 in NSCLC was associated with tumour angiogenesis; that is, vascular endothelial growth factor overexpression (P < 0.001) and intratumoural microvessel density (P < 0.001). CONCLUSIONS This study demonstrates that AEG-1 expression is associated with NSCLC development, angiogenesis, progression and poor prognosis, indicating that the adjuvant therapy with antiangiogenic agent be adopted for the early postoperative period before the start of conventional chemotherapy in patients with AEG-1 overexpressed NSCLC. Astrocyte elevated gene-1, Non-small-cell lung cancer, Intratumoural microvessel density, Angiogenesis, Immunohistochemistry INTRODUCTION Lung cancer is the most commonly diagnosed malignant tumour with a high incidence and mortality rate in the world [1]. It has been estimated that there were approximately 733 300 new lung cancer cases and 610 200 lung cancer-related deaths in China in 2015 [2]. Histologically, lung cancer can be divided into small-cell lung cancer and non-small-cell lung cancer (NSCLC), and the latter accounts for 80% of all primary cancer cases. The 5-year survival rate after surgery remains poor, despite recent advances in early diagnosis and treatment options such as targeted therapy, which could be due to the poor understanding of the molecular mechanism(s) underlying NSCLC pathogenesis. To date, it has been widely accepted that NSCLC is a multifactorial and multistep disease, where angiogenesis plays a major role, and that the unbalanced expression of pro- and antiangiogenic factors in the tumour microenvironment results in neovascularization, which conversely facilitates tumour growth, further loss of tumour cell differentiation and increased tumour cell invasiveness [3, 4]. Thus, the further search for novel biomarkers to target angiogenesis could lead to a promising therapeutic strategy for NSCLC. Astrocyte elevated gene (AEG-1), also named as metadherin and protein LYRIC, has been previously identified as a novel protein induced by human immunodeficiency virus 1 or tumour necrosis factor α (TNF-α) in human primary foetal astrocytes [5, 6]. However, subsequent studies have found that AEG-1 is a tumour-related factor that promotes cancer progression and metastasis [7–10]. For example, Song et al. [8] revealed that AEG-1 expression was associated with NSCLC development and progression, and its elevated expression was dramatically associated with tumour cell aggressiveness and poor patient survival. Lu et al. [10] demonstrated that AEG-1 expression was an independent prognostic factor for poor overall survival (OS) and disease-free survival in NSCLC patients. In addition, AEG-1 has been shown to be an essential mediator of tumour angiogenesis and has been thereby associated with poor prognosis of various cancers, including cervical cancer, oral squamous cell carcinoma (SCC) and breast cancer [11–13]. In this study, immunohistochemistry was performed to detect and compare the expression of AEG-1 protein in 88 paired NSCLC and normal tissue specimens obtained from patients and investigate the association of AEG-1 expression with clinicopathological features, vascular endothelial growth factor (VEGF) expression and intratumoural microvessel density (iMVD). We expect to provide further information on AEG-1 as a biomarker for detecting NSCLC progression and prognosis and evaluate AEG-1 as a potential target for NSCLC therapy. MATERIALS AND METHODS Patients and tissue specimens A total of 88 consecutive newly diagnosed primary NSCLC patients in Huzhou Centre Hospital between September 2013 and December 2015 were recruited, and their clinicopathological characteristics were retrieved (Table 1). All patients were histologically diagnosed with NSCLC according to the eighth edition of the TNM classification of lung cancer [14]. None of these patients received radiotherapy or chemotherapy before surgery. Paired tissue specimens were collected during surgery, including tumour and normal adjacent tissues. These specimens were formalin fixed and paraffin embedded for histological examination and immunohistochemical analysis. This study was approved by the ethics committee of Huzhou Central Hospital (Huzhou, Zhejiang, China) with authorization number HZCHEC-276. All patients provided an informed consent prior to participating into this study. Table 1: Association of AEG-1 expression with NSCLC clinicopathological features (n = 88) Parameters  n  AEG-1 expression   χ2  P-value  High  Low  n = 54  n = 34  Gender   Male  56  34  22  0.027  0.869   Female  32  20  12  Age (years)   ≤60  28  19  9  0.730  0.393   >60  60  35  25  Smoking   Yes  50  28  22  1.405  0.236   No  38  26  12  Tumour size (cm)   ≤3  28  18  10  0.148  0.701   >3  60  36  24  Histology   ADC  55  31  24  1.547  0.214   SCC  33  23  10  pTNM stage   I/II  54  28  26  5.334  0.021   III/IV  34  26  8  Differentiation   Well moderated  58  31  27  4.496  0.034   Poor  30  23  7  Vascular invasion   No  61  33  28  4.426  0.035   Yes  27  21  6  Location   Central  38  24  14  0.091  0.763   Peripheral  50  30  20  Lymph node metastasis   No  57  28  29  10.22  0.001   Yes  31  26  5  Parameters  n  AEG-1 expression   χ2  P-value  High  Low  n = 54  n = 34  Gender   Male  56  34  22  0.027  0.869   Female  32  20  12  Age (years)   ≤60  28  19  9  0.730  0.393   >60  60  35  25  Smoking   Yes  50  28  22  1.405  0.236   No  38  26  12  Tumour size (cm)   ≤3  28  18  10  0.148  0.701   >3  60  36  24  Histology   ADC  55  31  24  1.547  0.214   SCC  33  23  10  pTNM stage   I/II  54  28  26  5.334  0.021   III/IV  34  26  8  Differentiation   Well moderated  58  31  27  4.496  0.034   Poor  30  23  7  Vascular invasion   No  61  33  28  4.426  0.035   Yes  27  21  6  Location   Central  38  24  14  0.091  0.763   Peripheral  50  30  20  Lymph node metastasis   No  57  28  29  10.22  0.001   Yes  31  26  5  ADC: adenocarcinoma; AEG-1: astrocyte elevated gene-1; NSCLC: non-small-cell lung cancer; SCC: squamous cell carcinoma. Immunohistochemistry The formalin-fixed and paraffin-embedded tumour specimens were sectioned into 4-μm-thick tissue sections and mounted onto poly-l-lysine-coated glass slides. For immunohistochemistry, these sections were deparaffinized in xylene and rehydrated into water in a series of ethanol solution (100–0%). Then, these sections were cooked in a high-pressure cooker in 10 mmol/l of citrate buffer (pH 6.0) for 3 min to repair tissue antigens during formalin fixation and paraffin embedding. After cooling down to room temperature and tap water washing and rinsing, the sections were incubated in 3% hydrogen peroxide for 20 min to block potential endogenous peroxidase activity. Then, these sections were washed with phosphate-buffered saline for 3 times, incubated with normal serum for 30 min and further incubated with rabbit monoclonal anti-LYRIC (1:200 dilution; ab124789, Abcam, Cambridge, MA, USA), rabbit monoclonal anti-VEGF receptor (1:200 dilution; ab32152, Abcam) or mouse monoclonal anti-CD105 (1:100 dilution; ab114052, Abcam) at 4 °C overnight. On the next day, these sections were washed with phosphate-buffered saline 3 times and further incubated with the secondary antibody. For VEGF immunostaining, the sections were colour developed using the 3,3'-diaminobenzidine solution, whereas for AEG-1 and CD105 double labelling, the sections were separately colour developed using 3,3'-diaminobenzidine and Ap-Red with the polymer double-staining assay kit (no. DS-0006; Zhongshan Goldbridge, Beijing, China), according to manufacturer’s recommendations. Haematoxylin solution was used to counterstain cell nuclei. The primary antibody was replaced with phosphate-buffered saline for negative control sections. Evaluation of astrocyte elevated gene-1, vascular endothelial growth factor and intratumoural microvessel density immunostaining These immunostained sections were evaluated and scored by 2 independent pathologists who were blinded to the clinical data of these sections. AEG-1 immunostaining was scored using both the percentage of staining and the staining intensity. That is, the percentage of staining was scored as 0 (negative), 1 (≤ 10%), 2 (10%–50%) and 3 (> 50%); and staining intensity was calculated as 0 (no staining), 1 (weakly stained), 2 (moderately stained) and 3 (strongly stained). The staining index was established by multiplying these 2 scores. A total score of <4 was defined as lowly expressed, whereas a total score of ≥4 was considered as highly expressed. VEGF staining was assessed according to a previous study reported by Mattern et al. [15]. Briefly, the percentage score was defined as 0 (negative), 1 (≤ 25%), 2 (25%–50%) and 3 (>50%), whereas the intensity score was calculated similar to that performed for AEG-1 immunostaining. The staining index was established by summing these 2 scores. VEGF expression was considered to be low when the score was 0, 1, 2, 3 or 4; VEGF expression was considered to be high when the score was 5 or 6. For iMVD, the tissue sections were first immunostained with the anti-CD105 antibody to visualize the microvessels. Any red-stained endothelial cell or endothelial cell cluster that could clearly separate contiguous microvessels, tumour cells and connective tissue elements was considered a single, countable microvessel. Microvessels were assessed by 2 observers using a microscope at a magnification of 200×. The data were summarized from 5 of the most vascularized areas in each tissue section, and the mean values of these measurements were taken and considered as the final iMVD level. Online lung cancer tissue database and data retrieval The OS of NSCLC patients was analysed using the online messenger RNA expression database developed by Gyorffy B (http://kmplot.com) [16]. NSCLC data were retrieved from the Gene Expression Omnibus (GEO), and the OS information of NSCLC, adenocarcinoma, SCC related to the expression of AEG-1 was obtained. Statistical analysis Statistical analyses were conducted using SPSS 18.0 (SPCC Inc, Chicago, IL, USA). The Kolmogorov–Smirnov test was performed to determine the distribution of these samples, the Student’s t-test was applied for normally distributed data and the Mann–Whitney U-test was performed for asymmetrical distribution data. The χ2-test was employed to associate the expression of AEG-1 with the clinicopathological parameters obtained from patients, and the Spearman’s correlation coefficient test was used to determine the correlation of AEG-1 with the expression of VEGF. The Kaplan–Meier curves were used to analyse the OS of patients, which was stratified by AEG-1 expression using the online tool (http://kmplot.com/analysis/). A P-value <0.05 was considered statistically significant. RESULTS Association of astrocyte elevated gene-1 expression with clinicopathological parameters obtained from non-small-cell lung cancer patients In this study, AEG-1 expression was first assessed by immunohistochemistry. It was found that AEG-1 protein was primarily localized in the cytoplasm, nuclear membrane and/or cell junction (Fig. 1). Furthermore, high AEG-1 expression was mostly found in cancer cells or cancer nests (Fig. 1C–F), whereas no or low AEG-1 expression was found in normal epithelial cells (Fig. 1A and B). According to staining scores, it was found that AEG-1 was lowly expressed in 82 normal tissues and 34 NSCLC tissues and highly expressed in 54 NSCLC tissues (Table 2, P < 0.001). Table 2: Association of AEG-1 with VEGF expression in NSCLC tissue specimens (n = 88) Parameters  n  VEGF expression (n)   χ2/r  P-value  High  Low  Tissue types   NSCLC  88  50  38       Normal  88  3  85  59.639  <0.001  Lymph node metastasis   No  57  26  31  8.279  0.004   Yes  31  24  7  AEG-1 expression   Low  34  5  29  0.657  <0.001   High  54  45  9  Parameters  n  VEGF expression (n)   χ2/r  P-value  High  Low  Tissue types   NSCLC  88  50  38       Normal  88  3  85  59.639  <0.001  Lymph node metastasis   No  57  26  31  8.279  0.004   Yes  31  24  7  AEG-1 expression   Low  34  5  29  0.657  <0.001   High  54  45  9  AEG-1: astrocyte elevated gene-1; VEGF: vascular endothelial growth factor; NSCLC: non-small-cell lung cancer. Figure 1: View largeDownload slide Immunohistochemical staining of astrocyte elevated gene-1 (AEG-1) protein in non-small-cell lung cancer tissue specimens. (A) and (B) Negative and weak AEG-1 staining in normal alveolar pneumocytes. (C) and (D) Weak and strong AEG-1 staining in lung adenocarcinoma. (E) and (F) Weak and strong AEG-1 staining in lung squamous cell carcinomas (original magnification ×200). Figure 1: View largeDownload slide Immunohistochemical staining of astrocyte elevated gene-1 (AEG-1) protein in non-small-cell lung cancer tissue specimens. (A) and (B) Negative and weak AEG-1 staining in normal alveolar pneumocytes. (C) and (D) Weak and strong AEG-1 staining in lung adenocarcinoma. (E) and (F) Weak and strong AEG-1 staining in lung squamous cell carcinomas (original magnification ×200). Next, AEG-1 expression was associated with clinicopathological features obtained from patients (Table 1). It was found that AEG-1 expression was significantly associated with advanced pTNM stage (P = 0.021), tumour dedifferentiation (P = 0.034), vascular invasion (P = 0.035) and lymph node metastasis (P < 0.001). However, there was no significant difference between AEG-1 expression and gender, age, pathology classification, tumour size, location and history of tobacco smoking (P > 0.05). Association of astrocyte elevated gene-1 expression with vascular endothelial growth factor in non-small-cell lung cancer tissues VEGF protein was preferentially localized in the cytoplasm of tumour cells (data not known), which further confirms the data reported in a previous study [15]. In many cases, our data also revealed that VEGF immunostaining did occur in tumour stromal and vascular endothelial cells, although the staining intensity was weak. VEGF was overexpressed in 3 normal tissues and in 50 cases of NSCLC tissues (Table 2). Furthermore, VEGF expression was dramatically associated with NSCLC lymph node metastasis (χ2 = 8.279, P = 0.004; Table 2). However, VEGF expression was not significantly associated with other clinicopathological factors such as tobacco smoking, tumour status or tumour differentiation. In addition, Spearman’s correlation analysis revealed that AEG-1 expression in NSCLC tissues was moderately associated with NSCLC (r = 0.657, P < 0.001; Table 2). Association of astrocyte elevated gene-1 with intratumoural microvessel density in non-small-cell lung cancer patients Altered expression of AEG-1 has been previously shown to be associated with tumour angiogenesis. In this study, the double immunostaining of AEG-1 and CD105 proteins in tissue specimens was assessed. It was found that CD105 protein was mainly localized in the membrane or cytoplasm of microvascular endothelial cells, manifesting as red granules (Fig. 2A–C). Compared with the faint or weak CD105 immunostaining in normal lung epithelial cells (Fig. 2A), strong and intensive CD105 immunostaining was found in tumour stroma around neoplastic cells (Fig. 2B and C). In summary, the mean iMVD counted in NSCLC tissues was 47.70 ± 16.74, which was significantly higher than that in normal tissues (19.02 ± 5.01, P < 0.001). Figure 2: View largeDownload slide Coimmunolocalization of AEG-1 and CD105 proteins in non-small-cell lung cancer. (A) CD105 staining around normal alveolar pneumocytes. Weak CD105 staining is shown by the arrows. (B) Coimmunolocalization of AEG-1 and CD105 in the low iMVD area. (C) Coimmunolocalization of AEG-1 and CD105 in the high iMVD area (original magnification ×200). (D) iMVD levels in the high- and low-AEG-1 expression groups. AEG-1: astrocyte elevated gene-1; AEG-1-H: AEG-1 high-expression group; AEG-1-L: AEG-1 low-expression group; iMVD: intratumoural microvessel density. Figure 2: View largeDownload slide Coimmunolocalization of AEG-1 and CD105 proteins in non-small-cell lung cancer. (A) CD105 staining around normal alveolar pneumocytes. Weak CD105 staining is shown by the arrows. (B) Coimmunolocalization of AEG-1 and CD105 in the low iMVD area. (C) Coimmunolocalization of AEG-1 and CD105 in the high iMVD area (original magnification ×200). (D) iMVD levels in the high- and low-AEG-1 expression groups. AEG-1: astrocyte elevated gene-1; AEG-1-H: AEG-1 high-expression group; AEG-1-L: AEG-1 low-expression group; iMVD: intratumoural microvessel density. Figure 3: View largeDownload slide The Kaplan–Meier curves of the overall survival (OS) in non-small-cell lung cancer (NSCLC) patients stratified by astrocyte elevated gene-1 expression. (A) The Kaplan–Meier survival curve of OS in NSCLC patient. (B) The Kaplan–Meier survival curve of OS in adenocarcinoma patients. (C) The Kaplan–Meier survival curve of OS in squamous cell carcinoma patients. HR: hazard ratio. Figure 3: View largeDownload slide The Kaplan–Meier curves of the overall survival (OS) in non-small-cell lung cancer (NSCLC) patients stratified by astrocyte elevated gene-1 expression. (A) The Kaplan–Meier survival curve of OS in NSCLC patient. (B) The Kaplan–Meier survival curve of OS in adenocarcinoma patients. (C) The Kaplan–Meier survival curve of OS in squamous cell carcinoma patients. HR: hazard ratio. Furthermore, a significant increase in mean iMVD (53.02 ± 16.88) was found in NSCLC tissues with high AEG-1 expression, and a low iMVD (39.26 ± 12.72) was found in NSCLC tissues with low AEG-1 expression (P < 0.001, Fig. 2D). Association of astrocyte elevated gene-1 expression with poor overall survival in non-small-cell lung cancer patients To further extend our observations to a clinicopathologically relevant context, we performed the Kaplan–Meier survival analysis of AEG-1 on an online platform (http://kmplot.com/analysis/), with the autoselect best cut-off was chosen. The results showed that higher AEG-1 expression was associated with a worse OS of NSCLC patients (P = 0.024) and SCC patients (P = 0.001) but not of adenocarcinoma patients (P = 0.085). DISCUSSION Although AEG-1 was originally identified as an human immunodeficiency virus-inducible gene in primary human foetal astrocytes [5, 6], recent studies have revealed that AEG-1 expression was also upregulated in various human cancers, such as gastric [17], colorectal [18], cervical [11, 12], breast [13] and ovarian cancers [19], glioma [20] and NSCLC [8]. Subsequent investigations have indicated that AEG-1 is a tumour metastasis-associated protein and participates in tumour angiogenesis. For example, in hepatocellular carcinoma HepG3 cells, AEG-1 overexpression was able to significantly increase tumour cell proliferation, invasion and anchorage-independent growth in vitro, as well as tumourigenesis, angiogenesis and metastasis in nude mice [21]. The study conducted by Emdad et al. [7] demonstrated that abnormal increase in AEG-1 expression was associated with increased microvessel density and expression of angiogenesis molecular markers in tumour sections. The in vitro experiment in this study further illustrated that AEG-1 was able to promote tube formation in Matrigel and increased the invasion of human umbilical vein endothelial cells via the PI3K/Akt signalling pathway. All these studies suggest that aberrant AEG-1 expression possessed great effects on oncogenic transformation and angiogenesis. Consistent with this speculation, we first observed the AEG-1 expression pattern in NSCLC tissues and linked AEG-1 expression to angiogenesis. Our present data revealed that the level of AEG-1 expression was dramatically higher in NSCLC tissues than in adjacent normal lung tissues. AEG-1 expression was statistically associated with advanced pTNM stages, vascular invasion, dedifferentiation and lymph node metastasis. However, it was independent of age, gender, tumour size and tobacco smoking status. This study further supports the data reported on a previous study conducted by Song et al. [8], indicating that AEG-1 expression promoted NSCLC development and progression. Angiogenesis is a process in which neovascularization is formed from existing vascular networks, which is a fundamental event for tumour growth and metastasis [22, 23]. During this process, tumour cells would induce the expression of VEGF (a key angiogenic regulator during neovascularization), resulting in an increase in iMVD, a conventional event observed in different tumour tissues related to their normal tissues. For example, the study conducted by Lin et al. [24] demonstrated that upregulated VEGF expression frequently occurred in NSCLC tissue specimens and was associated with the formation of new blood vessels, iMVD and shorter OS in patients. CD105, also referred as Endoglin, is a Type I membrane glycoprotein localized on cell surfaces and is a part of the TGF-beta receptor complex [25]. CD105 plays a crucial role in angiogenesis. It is usually lowly expressed in resting endothelial cells and highly expressed in endothelial cells during neoangiogenesis in cancers, inflamed tissues or vascular injury [25, 26]. In contrast, CD34 or CD31 is a panendothelial marker, and its panendothelial antibody could react not only to ‘newly forming’ vessels but also to pre-existing normal vessels in tumour tissues. Since CD105 has a higher affinity to neovascularized, activated endothelial cells [27, 28], the CD105 molecule is a better endothelial marker than panendothelial markers (such as CD34 or CD31). Therefore, in this study, we utilized both VEGF and CD105 expression to evaluate and associate the relationship between AEG-1 expression and angiogenesis. We found that NSCLC tissues had a significant level of VEGF expression and association with AEG-1 expression (r = 0.657, P < 0.001). In addition, the iMVD assessed by CD105 immunostaining was also considerably higher in AEG-1-expressed NSCLC tissues compared with NSCLC tissues with lowly expressed AEG-1 (t = 4.341, P < 0.001). The results of this study were consistent with the findings reported by previous studies on gastric cancer [17], cervical cancer [12] and oral SCC [9]. Indeed, a recent in vivo study reported by Zhang et al. [29] demonstrated that AEG-1 shRNA was able to successfully inhibit the tumourigenic and angiogenic ability of H460 cells using the in vivo chick embryo chorioallantoic membrane model. However, for surgeons, it should be appreciated that surgery may accelerate the growth of residual disease not only by removal of antiangiogenic factors produced by the primary tumour but also by the production of proangiogenic factors [30]. Therefore, to benefit patients with AEG-1-overexpressed NSCLC in tissue sections, we strongly recommended that adjuvant therapy with antiangiogenic agent be adopted for the early postoperative period before the start of conventional chemotherapy to control or inhibit tumour recurrence or metastasis. In addition, this study revealed that the Kaplan–Meier curve analysis stratified AEG-1 expression to associate with the poor prognosis of NSCLC (P = 0.024). Histologically, higher AEG-1 expression was associated with a worse OS in patients with SCC (P = 0.001) but not in patients with adenocarcinoma (P = 0.085). Although such data have been reported in various cancer types such as cervical cancer, oral SCC, lung cancer and breast cancer [8–13], the underlying molecular mechanisms responsible for AEG-1 promotion in NSCLC poor prognosis remain to be determined. AEG-1 might participate in the multiplex process of angiogenesis, potentially by raising VEGF expression and resulting in neovascularization. However, NSCLC progression is also very complicated, and tumour cell aggressiveness would lead to resistance to different therapy selections. Thus, further studies on angiogenesis and NSCLC cell aggressiveness could lead to the efficient control of this presently deadly disease. CONCLUSION In conclusion, our study demonstrated that AEG-1 protein overexpression frequently occurred in NSCLC, which may contribute to oncogenic transformation and angiogenesis. The detection of AEG-1 expression may serve as a useful biomarker for the prediction of NSCLC progression and poor prognosis, although the present data need to be confirmed through other studies with larger sample sizes and multiple institutional patients. ACKNOWLEDGEMENTS We would like to thank the staff of Huzhou Cancer Biobank and the Department of Surgery, Huzhou Central Hospital, for their excellent assistance. Funding This work was supported in part by grants from the Zhejiang Provincial Natural Science Foundation of China (LQ14H160015) and the Huzhou General Science and Social Development Project Foundation (2013GY17). Conflict of interest: none declared. REFERENCES 1 Torre LA, Bray F, Siegel RL, Ferlay J, Lortet‐Tieulent J et al.   Global cancer statistics, 2012. CA Cancer J Clin  2015; 65: 87– 108. 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Journal

Interactive CardioVascular and Thoracic SurgeryOxford University Press

Published: Mar 1, 2018

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