Med Oncol (2014) 31:986 DOI 10.1007/s12032-014-0986-4
ORIGINAL PAPER
AEG-1 is associated with tumor progression in nonmuscleinvasive bladder cancer Guoliang Yang • Lianhua Zhang • Shilong Lin • Linfeng Li • Mengyao Liu Haige Chen • Ming Cao • Dongming Liu • Yi-ran Huang • Juanjie Bo
•
Received: 1 April 2014 / Accepted: 27 April 2014 / Published online: 15 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Astrocyte elevated gene-1 (AEG-1), a novel oncoprotein, has been implicated in oncogenesis and cancer progression in various types of human cancers. Here, immunohistochemistry was used to detect AEG-1 expression in nonmuscle-invasive bladder cancer (NMIBC), and these data were examined for correlation with clinicopathological parameters, and prognosis. Immunohistochemical analysis revealed that AEG-1 expression was significantly higher in bladder cancer tissues than that in normal tissues. High expression of AEG-1 was found in 45 % of bladder cancers and significantly associated with tumor grade (P = 0.002) and progression (P = 0.028). The Kaplan–Meier survival analysis demonstrated that AEG-1 expression was significantly associated with shorter progression-free survival (P = 0.0011). Multivariate analysis further demonstrated that AEG-1 was an independent prognostic factor for patients with BC. AEG-1 protein may contribute to the malignant progression of bladder cancer, and present as a novel marker to predict the progression of NMIBC. Keywords AEG-1 Bladder cancer Prognosis Progression
Guoliang Yang and Lianhua Zhang have contributed equally to this work. G. Yang L. Zhang S. Lin H. Chen M. Cao D. Liu Y. Huang J. Bo (&) Department of Urology, Ren Ji Hospital, Shanghai Jiao Tong University, Shanghai 200001, China e-mail: [email protected] L. Li M. Liu Clinical Stem Cell Research Center, Ren Ji Hospital, Shanghai Jiao Tong University, Shanghai 200001, China
Introduction It has been estimated that nearly 73, 510 new cases of bladder cancer were diagnosed in the USA in 2012, with approximately 14,880 people dying of the disease [1]. At initial diagnosis, approximately 75 % of patients have nonmuscle-invasive bladder cancer (NMIBC), which is generally treated with transurethral resection of the bladder (TURB) with or without intravesical therapy [2]. Recurrence rates for NMIBC range from 50 to 70 %, and approximately 10–15 % of tumors progress to muscleinvasive disease over a 5-year period [3, 4]. The remaining 25 % of patients are initially diagnosed with muscleinvasive bladder cancer (MIBC), and half of these patients die within 5 years [5]. Molecular markers hold considerable promise to predict clinical outcome of NMIBC. Nevertheless, the value of molecular markers over clinicopathologic indexes is still being questioned, and their clinical use is limited. Therefore, specific molecular markers that could serve as standard prognostic factors are required. Astrocyte elevated gene-1 (AEG-1) was originally characterized as a human immunodeficiency virus (HIV)-1inducible gene in primary human fetal astrocyte [6, 7]. As a downstream target of Ha-Ras, AEG-1 has an essential role in regulating tumorigenesis, invasion, metastasis and angiogenesis [8]. Intriguingly, expression analysis revealed that AEG-1 was significantly elevated in many types of human cancers including breast cancer, glioma, melanoma, esophageal squamous cell carcinoma, gastric cancer, salivary gland carcinoma, prostate cancer, hepatocellular carcinoma and colorectal cancer [9–18]. Studies in vivo and in vitro have shown that AEG-1 can mediate breast cancer lung metastasis through tumor cells adhesion to lung vasculature [19, 20]. These studies suggest that overexpression
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986 Page 2 of 6
of AEG-1 protein may lead to development and progression of diverse cancers. In bladder cancer, AEG-1 was identified as involved in a model significantly correlated to poor prognosis [21]. Here, we investigated the prognostic value of AEG-1 at the protein level in NMIBC.
Med Oncol (2014) 31:986 Table 1 Correlation between AEG-1 protein expression and clinicopathologic features of the patients with BC Parameters
Total
AEG-1 expression Low expression
High expression
Gender
Patients and methods
0.61
Female
35
18
17
Male
67
38
29
B65
40
20
20
[65
62
36
26
B3
63
36
27
[3
39
20
19
Unifocal
49
28
21
Mutlifocal
53
28
25
Age (yeas)
Patients and tissue specimens For immunohistochemical assay, a total of 102 paraffinembedded samples of transitional cell bladder cancer and 23 specimens of normal bladder tissue were collected from our hospital between January 2003 and December 2005. The criteria for enrollment were histopathological diagnosis of transitional cell carcinoma of the bladder, newly diagnosed and untreated, no history of other tumors and the potential to follow up. The carcinoma in situ is excluded from our study. The clinical materials were only for research purposes; prior patient’s consent and other ethical issues were approved by the Ethics Committee of our hospital. Clinical information about the samples is described in detail in Table 1. The patients included 67 males and 35 females from 41 to 88 years (mean age, 66.1 years). All patients underwent transurethral resection of bladder tumor (TURBT). All patients with NMIBC received intravesical mitomycin C(MMC) or pirarubicin (THP) instillations once weekly for the first 8 weeks and then monthly up to 1 year. Cystoscopy and urine cytology were performed at 3-month intervals during the first 2-year and 6-month intervals after 2 years. The median follow-up time for NMIBC patients was 33 months for patients at the time of analysis and ranged from 6 to 103 months. We defined recurrence as the recurrence of primary NMIBC with a lower or the same pathologic stage and progression as confirmed histologic muscle invasion (pathologic stage T2 or higher disease) or detectable distant metastases. The histopathological grade and clinical stage of bladder cancer in this study were defined according to the criteria of the World Health Organization (WHO, 2004) and the 6th edition of the pTNM classification of the International Union Against Cancer (UICC, 2002). All tumors were reevaluated for histological type and grade by two senior pathologists (Qiang Liu and Zhao Liang Wang) at the our Hospital. Immunohistochemistry Formalin-fixed and paraffin-embedded tissue sections (5-lm) were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3 % hydrogen
123
0.424
Tumor size (cm)
0.563
Tumor number
0.662
Grade Low grade High grade
P value
0.002 59 43
40 16
19 27
Ta
52
31
21
T1
50
25
25
T stage
0.329
Recurrence
0.642
Positive
44
23
21
Negative
58
33
25
Positive
23
8
15
Negative
79
48
31
Progression
0.028
peroxide for 10 min. Slides were submerged into a buffer [1 mmol/L EDTA/PBS (pH 9.0)] and microwaved for antigenic retrieval. Slides were incubated overnight at 4 °C in a humidified chamber with rabbit polyclonal anti-AEG1(Proteintech Group, Inc., USA) at the dilution of 1:100. Biotinylated anti-rabbit link was used as secondary antibody (30 min). Slides were then incubated with a streptavidin–horseradish peroxidase complex. Diaminobenzidine (DAB) was used as chromogen, and the sections were counterstained with hematoxylin. Samples incubated with normal serum instead of primary antibodies were used as negative controls. Evaluation of immunohistochemistry Immunohistochemical stainings were reviewed and scored independently by two observers, based on both the proportion of positively stained tumor cells and the intensity of staining. The proportion of positively stained tumor cells was scored as follows: 0 (no positive tumor cells), 1 (\10 %
Med Oncol (2014) 31:986
Page 3 of 6 986
Fig. 1 Immunohistochemical staining of AEG-1 protein in bladder
positive tumor cells), 2 (10–50 % positive tumor cells) and 3 ([50 % positive tumor cells). Staining intensity was classified according to the following criteria: 0 (no staining); 1(weak staining = light yellow), 2 (moderate staining = yellow brown) and 3 (strong staining = brown). The staining index (SI) was calculated as staining intensity score 9 proportion of positive tumor cells. Using this method of assessment, we evaluated the expression of AEG-1 in normal bladder epithelium and malignant lesions by determining the SI, which scores as 0, 1, 2, 3, 4, 6 and 9. An intensity score of 2 with at least 10 % of malignant cells positive for AEG-1 staining was to classify tumors as high expression, and \10 % of malignant cells with nuclear staining of intensity score of\2 was considered as low expression. Statistical analysis The significance of the relationships between AEG-1 protein expression and clinicopathological parameters was evaluated using v2 tests. Recurrence-free survival and progression-free survival curves were calculated using the Kaplan–Meier method and compared by log-rank test. Multivariate analysis was used to identify independent prognostic factors for recurrence-free survival and progression-free survival using the Cox proportional hazards model. SPSS 11.0 software was used for statistical analysis. A value of P \ 0.05 was considered statistically significant.
Results AEG-1 is overexpressed in NMIBC tissues To investigate the expression of AEG-1 protein in bladder tissues, immunohistochemistry was initially performed in 102 paraffin-embedded NMIBC samples. AEG-1 protein
was high expression in 45 % (46/102) of the NMIBC samples (Table 1) and mainly localized in the cytoplasm of primary cancer cells. However, AEG-1 staining was weaker in normal tissues than bladder cancer tissues (Fig. 1). Correlation between AEG-1 protein expression and clinicopathological features The association between AEG-1 protein expression and clinicopathological features of BC was examined as shown in Table 1. High levels of AEG-1 protein expression were significantly correlated with tumor grade and progression (P = 0.002 and P = 0.028, respectively). However, AEG1 protein expression was not associated with other clinicopathological features such as age, gender, tumor size, tumor number and recurrence. Prognostic value of AEG-1 protein in NMIBC We evaluated the ability of AEG-1 staining to predict tumor recurrence or progression in NMIBC. In NMIBC specimens, AEG-1 expression was higher in patients who experienced progression to muscle invasion, compared with those with no progression (P = 0.028). The results of univariate and multivariate analyses of recurrence-free survival and progression-free survival of bladder cancer patients are shown in Table 2. By Kaplan–Meier analysis, we observed that the expression of AEG-1 in bladder cancer was significantly correlated progression-free survival (P = 0.0011, Table 2). The log-rank test further demonstrated that the survival time was significantly different between groups with high and low expression of AEG-1 protein, indicating that high level of AEG-1 was tightly correlated with a shorter survival (Fig. 2). Multivariate analysis was also performed with the Cox proportional hazards model including gender, age, tumor size,
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986 Page 4 of 6 Table 2 Univariate analysis of recurrence-free and progressionfree survivals (months, mean ± SE) in patients with nonmuscle-invasive bladder cancer
Med Oncol (2014) 31:986
Variables
Case
Recurrence-free survival Mean ± SE (months)
95 % CI
Gender
P value
Mean ± SE (months)
95 % CI
84 ± 7
(70–96)
78 ± 5
(68–88)
77 ± 7
(64–90)
82 ± 5
(72–92)
0.667
Female
34
67 ± 7
(52–81)
Male
68
62 ± 5
(52–73)
Age (yeas) 40
62 ± 7
(48–75)
[65
62
66 ± 5
(55–76)
0.607
0.969
0.777
63
64 ± 5
(54–75)
81 ± 5
(71–91)
39
64 ± 7
(50–78)
78 ± 7
(65–92)
Unifocal
49
71 ± 6
(60–83)
90 ± 5
(80–100)
Mutlifocal
53
58 ± 6
(46–69)
71 ± 6
(60–83)
Low
59
77 ± 5
(67–87)
89 ± 4
(80–97)
High
43
44 ± 6
(32–57)
60 ± 8
(43–76)
[3 Tumor number
0.157
0.0268
\0.001
Grade
T stage
0.0017
0.0015
0.0002
Ta
52
77 ± 6
(66–87)
93 ± 4
(86–101)
T1
50
50 ± 6
(39–62)
64 ± 7
(51–77)
Low expression
56
71 ± 5
(60–81)
91 ± 4
(82–98)
High expression
46
47 ± 5
(37–58)
55 ± 6
(44–66)
AEG-1
P value 0.648
0.673
B65 Tumor size (cm) B3
Progression-free survival
0.059
0.0011
Fig. 2 Kaplan–Meier survival analysis of recurrence-free survival and progression-free survival
tumor number, grade, stage and AEG-1 expression. The results showed that AEG-1 protein expression had a significant correlation with bladder cancer progression and was found to be an independent prognostic factor of progression-free survival (P = 0.005) (Table 3).
Discussion The results of this study demonstrated that AEG-1 was overexpressed in bladder cancer tissues, compared with normal tissues. These results are consistent to previous
123
studies in other types of human cancers [9–18]. The overexpression of AEG-1 was also correlated with tumor grade and progression; statistical analysis showed that overexpression of AEG-1 protein in NMIBC was also shown to be significantly associated with poor prognosis. Taken together, our results imply that overexpression of AEG-1 could play a crucial role in the progression of bladder cancer and may represent an independent prognostic factor for NMIBC patient outcome. Many studies have documented that AEG-1 is associated with the initiation and progression of cancer. Upregulation of AEG-1 in HeLa cells significantly enhances
Med Oncol (2014) 31:986 Table 3 Multivariate Cox model analysis of recurrencefree survival and progressionfree survival
Page 5 of 6 986
Variables
Recurrence-free survival Hazard ratio (95 % CI)
Progression-free survival P value
Hazard ratio (95 % CI)
P value
1.179 (0.562–2.471)
0.663
2.029 (0.703–5.857)
0.191
0.942 (0.511–1.739)
0.849
0.988 (0.412–2.370)
0.979
1.9029 (0.517–2.048)
0.935
1.569 (0.548–4.486)
0.401
0.753 (0.398–1.423)
0.382
0.928 (0.379–2.274)
0.870
2.400 (1.194–4.826)
0.014
1.851 (0.690–4.964)
0.221
2.154 (1.026–4.521)
0.043
4.550 (1.354–15.292)
0.014
1.433 (0.745–2.756)
0.281
3.994 (1.533–10.411)
0.005
Gender Female vs [male Age (years) B65 vs [65 Tumor number unifocal vs mutlifocal Tumor size (cm) B3 vs [3 Grade Low vs high T stage Ta vs T1 AEG-1 expression Low vs high
the invasive ability of the cells, whereas AEG-1 knockdown reduces cell viability and promotes apoptosis in prostate cancer cells [8, 16]. It has been shown that the upregulation of AEG-1 increases and that the downregulation of AEG-1 decreases breast cancer cell metastasis in the lung [19]. In addition, the overexpression of AEG-1 enhances the migration and invasion of glioma cells, whereas the knockdown of AEG-1 in glioma cells significantly inhibits their migration and invasion [22]. The overexpression of AEG-1 protein in NMIBC was also shown to be significantly correlated with poor prognosis. Furthermore, a multivariate analysis showed that high level of AEG-1 protein expression was an independent prognostic factor for tumor progression (P = 0.005). A previous study on liver [10], esophagus [11], breast [12] and neuroblastoma [23] has revealed that AEG-1 represents a novel and useful prognostic marker for cancer progression, and there is inverse correlation between its expression and overall patient survival. Based on these results, the detection of AEG-1 in tissue samples after TURBT could be used as a prognostic marker for determining the risk of future progression in patients with NMIBC. In summary, the present study suggests the possibility of using AEG-1 as both a clinically relevant indicator of disease progression and a prognostic biomarker for survival in the patients with NMIBC. Thus, more studies should be conducted to draw definitive conclusions. However, our findings are worthwhile considering that this work is the first of its kind to explore the prognostic value of AEG-1 protein in NMIBC. Acknowledgments This work was supported by Grants from the National Key Clinical Specialty Project of China’s Ministry of Health and the Science and Technology Committee of Shanghai Municipal (No. 12ZR1417700).
Conflict of interest
There is no conflict of interest.
References 1. Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer J Clin. 2012;62(1):10–29. 2. Fernandez-Gomez J, Madero R, Solsona E, Unda M, MartinezPineiro L, Gonzalez M, et al. Predicting nonmuscle invasive bladder cancer recurrence and progression in patients treated with bacillus Calmette-Guerin: the CUETO scoring model. J Urol. 2009;182(5):2195–203. 3. Babjuk M, Oosterlinck W, Sylvester R, Kaasinen E, Bohle A, Palou-Redorta J, et al. EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder, the 2011 update. Eur Urol. 2011;59(6):997–1008. 4. Sylvester RJ, van der Meijden AP, Oosterlinck W, Witjes JA, Bouffioux C, Denis L, et al. Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables: a combined analysis of 2596 patients from seven EORTC trials. Eur Urol. 2006;49(3):466–5 (discussion 75-7). 5. Kaufman DS, Shipley WU, Feldman AS. Bladder cancer. Lancet. 2009;374(9685):239–49. 6. Su ZZ, Kang DC, Chen Y, Pekarskaya O, Chao W, Volsky DJ, et al. Identification and cloning of human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1 envelope glycoprotein by rapid subtraction hybridization, RaSH. Oncogene. 2002;21(22):3592–602. 7. Kang DC, Su ZZ, Sarkar D, Emdad L, Volsky DJ, Fisher PB. Cloning and characterization of HIV-1-inducible astrocyte elevated gene-1, AEG-1. Gene. 2005;353(1):8–15. 8. Lee SG, Su ZZ, Emdad L, Sarkar D, Fisher PB. Astrocyte elevated gene-1 (AEG-1) is a target gene of oncogenic Ha-ras requiring phosphatidylinositol 3-kinase and c-Myc. Proc Natl Acad Sci USA. 2006;103(46):17390–5. 9. Liu L, Wu J, Ying Z, Chen B, Han A, Liang Y, et al. Astrocyte elevated gene-1 upregulates matrix metalloproteinase-9 and induces human glioma invasion. Cancer Res. 2010;70(9):3750–9. 10. Yoo BK, Emdad L, Su ZZ, Villanueva A, Chiang DY, Mukhopadhyay ND, et al. Astrocyte elevated gene-1 regulates
123
986 Page 6 of 6
11.
12.
13.
14.
15.
16.
17.
hepatocellular carcinoma development and progression. J Clin Invest. 2009;119(3):465–77. Yu C, Chen K, Zheng H, Guo X, Jia W, Li M, et al. Overexpression of astrocyte elevated gene-1 (AEG-1) is associated with esophageal squamous cell carcinoma (ESCC) progression and pathogenesis. Carcinogenesis. 2009;30(5):894–901. Li J, Zhang N, Song LB, Liao WT, Jiang LL, Gong LY, et al. Astrocyte elevated gene-1 is a novel prognostic marker for breast cancer progression and overall patient survival. Clin Cancer Res. 2008;14(11):3319–26. Jian-bo X, Hui W, Yu-long H, Chang-hua Z, Long-juan Z, Shirong C, et al. Astrocyte-elevated gene-1 overexpression is associated with poor prognosis in gastric cancer. Med Oncol. 2011;28(2):455–62. Liao WT, Guo L, Zhong Y, Wu YH, Li J, Song LB. Astrocyte elevated gene-1 (AEG-1) is a marker for aggressive salivary gland carcinoma. J Transl Med. 2011;9:205. Emdad L, Sarkar D, Lee SG, Su ZZ, Yoo BK, Dash R, et al. Astrocyte elevated gene-1: a novel target for human glioma therapy. Mol Cancer Ther. 2010;9(1):79–88. Kikuno N, Shiina H, Urakami S, Kawamoto K, Hirata H, Tanaka Y, et al. Knockdown of astrocyte-elevated gene-1 inhibits prostate cancer progression through upregulation of FOXO3a activity. Oncogene. 2007;26(55):7647–55. Gnosa S, Shen YM, Wang CJ, Zhang H, Stratmann J, Arbman G, et al. Expression of AEG-1 mRNA and protein in colorectal
123
Med Oncol (2014) 31:986
18.
19.
20.
21.
22.
23.
cancer patients and colon cancer cell lines. J Transl Med. 2012;10:109. Emdad L, Sarkar D, Su ZZ, Randolph A, Boukerche H, Valerie K, et al. Activation of the nuclear factor kappaB pathway by astrocyte elevated gene-1: implications for tumor progression and metastasis. Cancer Res. 2006;66(3):1509–16. Brown DM, Ruoslahti E. Metadherin, a cell surface protein in breast tumors that mediates lung metastasis. Cancer Cell. 2004;5(4):365–74. Hu G, Chong RA, Yang Q, Wei Y, Blanco MA, Li F, et al. MTDH activation by 8q22 genomic gain promotes chemoresistance and metastasis of poor-prognosis breast cancer. Cancer Cell. 2009;15(1):9–20. Zhou J, Li J, Wang Z, Yin C, Zhang W. Metadherin is a novel prognostic marker for bladder cancer progression and overall patient survival. Asia Pac J Clin Oncol. 2012;8(3):e42–8. Emdad L, Sarkar D, Su ZZ, Lee SG, Kang DC, Bruce JN, et al. Astrocyte elevated gene-1: recent insights into a novel gene involved in tumor progression, metastasis and neurodegeneration. Pharmacol Ther. 2007;114(2):155–70. Liu H, Song X, Liu C, Xie L, Wei L, Sun R. Knockdown of astrocyte elevated gene-1 inhibits proliferation and enhancing chemo-sensitivity to cisplatin or doxorubicin in neuroblastoma cells. J Exp Clin Cancer Res. 2009;28:19.
ORIGINAL PAPER
AEG-1 is associated with tumor progression in nonmuscleinvasive bladder cancer Guoliang Yang • Lianhua Zhang • Shilong Lin • Linfeng Li • Mengyao Liu Haige Chen • Ming Cao • Dongming Liu • Yi-ran Huang • Juanjie Bo
•
Received: 1 April 2014 / Accepted: 27 April 2014 / Published online: 15 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Astrocyte elevated gene-1 (AEG-1), a novel oncoprotein, has been implicated in oncogenesis and cancer progression in various types of human cancers. Here, immunohistochemistry was used to detect AEG-1 expression in nonmuscle-invasive bladder cancer (NMIBC), and these data were examined for correlation with clinicopathological parameters, and prognosis. Immunohistochemical analysis revealed that AEG-1 expression was significantly higher in bladder cancer tissues than that in normal tissues. High expression of AEG-1 was found in 45 % of bladder cancers and significantly associated with tumor grade (P = 0.002) and progression (P = 0.028). The Kaplan–Meier survival analysis demonstrated that AEG-1 expression was significantly associated with shorter progression-free survival (P = 0.0011). Multivariate analysis further demonstrated that AEG-1 was an independent prognostic factor for patients with BC. AEG-1 protein may contribute to the malignant progression of bladder cancer, and present as a novel marker to predict the progression of NMIBC. Keywords AEG-1 Bladder cancer Prognosis Progression
Guoliang Yang and Lianhua Zhang have contributed equally to this work. G. Yang L. Zhang S. Lin H. Chen M. Cao D. Liu Y. Huang J. Bo (&) Department of Urology, Ren Ji Hospital, Shanghai Jiao Tong University, Shanghai 200001, China e-mail: [email protected] L. Li M. Liu Clinical Stem Cell Research Center, Ren Ji Hospital, Shanghai Jiao Tong University, Shanghai 200001, China
Introduction It has been estimated that nearly 73, 510 new cases of bladder cancer were diagnosed in the USA in 2012, with approximately 14,880 people dying of the disease [1]. At initial diagnosis, approximately 75 % of patients have nonmuscle-invasive bladder cancer (NMIBC), which is generally treated with transurethral resection of the bladder (TURB) with or without intravesical therapy [2]. Recurrence rates for NMIBC range from 50 to 70 %, and approximately 10–15 % of tumors progress to muscleinvasive disease over a 5-year period [3, 4]. The remaining 25 % of patients are initially diagnosed with muscleinvasive bladder cancer (MIBC), and half of these patients die within 5 years [5]. Molecular markers hold considerable promise to predict clinical outcome of NMIBC. Nevertheless, the value of molecular markers over clinicopathologic indexes is still being questioned, and their clinical use is limited. Therefore, specific molecular markers that could serve as standard prognostic factors are required. Astrocyte elevated gene-1 (AEG-1) was originally characterized as a human immunodeficiency virus (HIV)-1inducible gene in primary human fetal astrocyte [6, 7]. As a downstream target of Ha-Ras, AEG-1 has an essential role in regulating tumorigenesis, invasion, metastasis and angiogenesis [8]. Intriguingly, expression analysis revealed that AEG-1 was significantly elevated in many types of human cancers including breast cancer, glioma, melanoma, esophageal squamous cell carcinoma, gastric cancer, salivary gland carcinoma, prostate cancer, hepatocellular carcinoma and colorectal cancer [9–18]. Studies in vivo and in vitro have shown that AEG-1 can mediate breast cancer lung metastasis through tumor cells adhesion to lung vasculature [19, 20]. These studies suggest that overexpression
123
986 Page 2 of 6
of AEG-1 protein may lead to development and progression of diverse cancers. In bladder cancer, AEG-1 was identified as involved in a model significantly correlated to poor prognosis [21]. Here, we investigated the prognostic value of AEG-1 at the protein level in NMIBC.
Med Oncol (2014) 31:986 Table 1 Correlation between AEG-1 protein expression and clinicopathologic features of the patients with BC Parameters
Total
AEG-1 expression Low expression
High expression
Gender
Patients and methods
0.61
Female
35
18
17
Male
67
38
29
B65
40
20
20
[65
62
36
26
B3
63
36
27
[3
39
20
19
Unifocal
49
28
21
Mutlifocal
53
28
25
Age (yeas)
Patients and tissue specimens For immunohistochemical assay, a total of 102 paraffinembedded samples of transitional cell bladder cancer and 23 specimens of normal bladder tissue were collected from our hospital between January 2003 and December 2005. The criteria for enrollment were histopathological diagnosis of transitional cell carcinoma of the bladder, newly diagnosed and untreated, no history of other tumors and the potential to follow up. The carcinoma in situ is excluded from our study. The clinical materials were only for research purposes; prior patient’s consent and other ethical issues were approved by the Ethics Committee of our hospital. Clinical information about the samples is described in detail in Table 1. The patients included 67 males and 35 females from 41 to 88 years (mean age, 66.1 years). All patients underwent transurethral resection of bladder tumor (TURBT). All patients with NMIBC received intravesical mitomycin C(MMC) or pirarubicin (THP) instillations once weekly for the first 8 weeks and then monthly up to 1 year. Cystoscopy and urine cytology were performed at 3-month intervals during the first 2-year and 6-month intervals after 2 years. The median follow-up time for NMIBC patients was 33 months for patients at the time of analysis and ranged from 6 to 103 months. We defined recurrence as the recurrence of primary NMIBC with a lower or the same pathologic stage and progression as confirmed histologic muscle invasion (pathologic stage T2 or higher disease) or detectable distant metastases. The histopathological grade and clinical stage of bladder cancer in this study were defined according to the criteria of the World Health Organization (WHO, 2004) and the 6th edition of the pTNM classification of the International Union Against Cancer (UICC, 2002). All tumors were reevaluated for histological type and grade by two senior pathologists (Qiang Liu and Zhao Liang Wang) at the our Hospital. Immunohistochemistry Formalin-fixed and paraffin-embedded tissue sections (5-lm) were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3 % hydrogen
123
0.424
Tumor size (cm)
0.563
Tumor number
0.662
Grade Low grade High grade
P value
0.002 59 43
40 16
19 27
Ta
52
31
21
T1
50
25
25
T stage
0.329
Recurrence
0.642
Positive
44
23
21
Negative
58
33
25
Positive
23
8
15
Negative
79
48
31
Progression
0.028
peroxide for 10 min. Slides were submerged into a buffer [1 mmol/L EDTA/PBS (pH 9.0)] and microwaved for antigenic retrieval. Slides were incubated overnight at 4 °C in a humidified chamber with rabbit polyclonal anti-AEG1(Proteintech Group, Inc., USA) at the dilution of 1:100. Biotinylated anti-rabbit link was used as secondary antibody (30 min). Slides were then incubated with a streptavidin–horseradish peroxidase complex. Diaminobenzidine (DAB) was used as chromogen, and the sections were counterstained with hematoxylin. Samples incubated with normal serum instead of primary antibodies were used as negative controls. Evaluation of immunohistochemistry Immunohistochemical stainings were reviewed and scored independently by two observers, based on both the proportion of positively stained tumor cells and the intensity of staining. The proportion of positively stained tumor cells was scored as follows: 0 (no positive tumor cells), 1 (\10 %
Med Oncol (2014) 31:986
Page 3 of 6 986
Fig. 1 Immunohistochemical staining of AEG-1 protein in bladder
positive tumor cells), 2 (10–50 % positive tumor cells) and 3 ([50 % positive tumor cells). Staining intensity was classified according to the following criteria: 0 (no staining); 1(weak staining = light yellow), 2 (moderate staining = yellow brown) and 3 (strong staining = brown). The staining index (SI) was calculated as staining intensity score 9 proportion of positive tumor cells. Using this method of assessment, we evaluated the expression of AEG-1 in normal bladder epithelium and malignant lesions by determining the SI, which scores as 0, 1, 2, 3, 4, 6 and 9. An intensity score of 2 with at least 10 % of malignant cells positive for AEG-1 staining was to classify tumors as high expression, and \10 % of malignant cells with nuclear staining of intensity score of\2 was considered as low expression. Statistical analysis The significance of the relationships between AEG-1 protein expression and clinicopathological parameters was evaluated using v2 tests. Recurrence-free survival and progression-free survival curves were calculated using the Kaplan–Meier method and compared by log-rank test. Multivariate analysis was used to identify independent prognostic factors for recurrence-free survival and progression-free survival using the Cox proportional hazards model. SPSS 11.0 software was used for statistical analysis. A value of P \ 0.05 was considered statistically significant.
Results AEG-1 is overexpressed in NMIBC tissues To investigate the expression of AEG-1 protein in bladder tissues, immunohistochemistry was initially performed in 102 paraffin-embedded NMIBC samples. AEG-1 protein
was high expression in 45 % (46/102) of the NMIBC samples (Table 1) and mainly localized in the cytoplasm of primary cancer cells. However, AEG-1 staining was weaker in normal tissues than bladder cancer tissues (Fig. 1). Correlation between AEG-1 protein expression and clinicopathological features The association between AEG-1 protein expression and clinicopathological features of BC was examined as shown in Table 1. High levels of AEG-1 protein expression were significantly correlated with tumor grade and progression (P = 0.002 and P = 0.028, respectively). However, AEG1 protein expression was not associated with other clinicopathological features such as age, gender, tumor size, tumor number and recurrence. Prognostic value of AEG-1 protein in NMIBC We evaluated the ability of AEG-1 staining to predict tumor recurrence or progression in NMIBC. In NMIBC specimens, AEG-1 expression was higher in patients who experienced progression to muscle invasion, compared with those with no progression (P = 0.028). The results of univariate and multivariate analyses of recurrence-free survival and progression-free survival of bladder cancer patients are shown in Table 2. By Kaplan–Meier analysis, we observed that the expression of AEG-1 in bladder cancer was significantly correlated progression-free survival (P = 0.0011, Table 2). The log-rank test further demonstrated that the survival time was significantly different between groups with high and low expression of AEG-1 protein, indicating that high level of AEG-1 was tightly correlated with a shorter survival (Fig. 2). Multivariate analysis was also performed with the Cox proportional hazards model including gender, age, tumor size,
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986 Page 4 of 6 Table 2 Univariate analysis of recurrence-free and progressionfree survivals (months, mean ± SE) in patients with nonmuscle-invasive bladder cancer
Med Oncol (2014) 31:986
Variables
Case
Recurrence-free survival Mean ± SE (months)
95 % CI
Gender
P value
Mean ± SE (months)
95 % CI
84 ± 7
(70–96)
78 ± 5
(68–88)
77 ± 7
(64–90)
82 ± 5
(72–92)
0.667
Female
34
67 ± 7
(52–81)
Male
68
62 ± 5
(52–73)
Age (yeas) 40
62 ± 7
(48–75)
[65
62
66 ± 5
(55–76)
0.607
0.969
0.777
63
64 ± 5
(54–75)
81 ± 5
(71–91)
39
64 ± 7
(50–78)
78 ± 7
(65–92)
Unifocal
49
71 ± 6
(60–83)
90 ± 5
(80–100)
Mutlifocal
53
58 ± 6
(46–69)
71 ± 6
(60–83)
Low
59
77 ± 5
(67–87)
89 ± 4
(80–97)
High
43
44 ± 6
(32–57)
60 ± 8
(43–76)
[3 Tumor number
0.157
0.0268
\0.001
Grade
T stage
0.0017
0.0015
0.0002
Ta
52
77 ± 6
(66–87)
93 ± 4
(86–101)
T1
50
50 ± 6
(39–62)
64 ± 7
(51–77)
Low expression
56
71 ± 5
(60–81)
91 ± 4
(82–98)
High expression
46
47 ± 5
(37–58)
55 ± 6
(44–66)
AEG-1
P value 0.648
0.673
B65 Tumor size (cm) B3
Progression-free survival
0.059
0.0011
Fig. 2 Kaplan–Meier survival analysis of recurrence-free survival and progression-free survival
tumor number, grade, stage and AEG-1 expression. The results showed that AEG-1 protein expression had a significant correlation with bladder cancer progression and was found to be an independent prognostic factor of progression-free survival (P = 0.005) (Table 3).
Discussion The results of this study demonstrated that AEG-1 was overexpressed in bladder cancer tissues, compared with normal tissues. These results are consistent to previous
123
studies in other types of human cancers [9–18]. The overexpression of AEG-1 was also correlated with tumor grade and progression; statistical analysis showed that overexpression of AEG-1 protein in NMIBC was also shown to be significantly associated with poor prognosis. Taken together, our results imply that overexpression of AEG-1 could play a crucial role in the progression of bladder cancer and may represent an independent prognostic factor for NMIBC patient outcome. Many studies have documented that AEG-1 is associated with the initiation and progression of cancer. Upregulation of AEG-1 in HeLa cells significantly enhances
Med Oncol (2014) 31:986 Table 3 Multivariate Cox model analysis of recurrencefree survival and progressionfree survival
Page 5 of 6 986
Variables
Recurrence-free survival Hazard ratio (95 % CI)
Progression-free survival P value
Hazard ratio (95 % CI)
P value
1.179 (0.562–2.471)
0.663
2.029 (0.703–5.857)
0.191
0.942 (0.511–1.739)
0.849
0.988 (0.412–2.370)
0.979
1.9029 (0.517–2.048)
0.935
1.569 (0.548–4.486)
0.401
0.753 (0.398–1.423)
0.382
0.928 (0.379–2.274)
0.870
2.400 (1.194–4.826)
0.014
1.851 (0.690–4.964)
0.221
2.154 (1.026–4.521)
0.043
4.550 (1.354–15.292)
0.014
1.433 (0.745–2.756)
0.281
3.994 (1.533–10.411)
0.005
Gender Female vs [male Age (years) B65 vs [65 Tumor number unifocal vs mutlifocal Tumor size (cm) B3 vs [3 Grade Low vs high T stage Ta vs T1 AEG-1 expression Low vs high
the invasive ability of the cells, whereas AEG-1 knockdown reduces cell viability and promotes apoptosis in prostate cancer cells [8, 16]. It has been shown that the upregulation of AEG-1 increases and that the downregulation of AEG-1 decreases breast cancer cell metastasis in the lung [19]. In addition, the overexpression of AEG-1 enhances the migration and invasion of glioma cells, whereas the knockdown of AEG-1 in glioma cells significantly inhibits their migration and invasion [22]. The overexpression of AEG-1 protein in NMIBC was also shown to be significantly correlated with poor prognosis. Furthermore, a multivariate analysis showed that high level of AEG-1 protein expression was an independent prognostic factor for tumor progression (P = 0.005). A previous study on liver [10], esophagus [11], breast [12] and neuroblastoma [23] has revealed that AEG-1 represents a novel and useful prognostic marker for cancer progression, and there is inverse correlation between its expression and overall patient survival. Based on these results, the detection of AEG-1 in tissue samples after TURBT could be used as a prognostic marker for determining the risk of future progression in patients with NMIBC. In summary, the present study suggests the possibility of using AEG-1 as both a clinically relevant indicator of disease progression and a prognostic biomarker for survival in the patients with NMIBC. Thus, more studies should be conducted to draw definitive conclusions. However, our findings are worthwhile considering that this work is the first of its kind to explore the prognostic value of AEG-1 protein in NMIBC. Acknowledgments This work was supported by Grants from the National Key Clinical Specialty Project of China’s Ministry of Health and the Science and Technology Committee of Shanghai Municipal (No. 12ZR1417700).
Conflict of interest
There is no conflict of interest.
References 1. Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer J Clin. 2012;62(1):10–29. 2. Fernandez-Gomez J, Madero R, Solsona E, Unda M, MartinezPineiro L, Gonzalez M, et al. Predicting nonmuscle invasive bladder cancer recurrence and progression in patients treated with bacillus Calmette-Guerin: the CUETO scoring model. J Urol. 2009;182(5):2195–203. 3. Babjuk M, Oosterlinck W, Sylvester R, Kaasinen E, Bohle A, Palou-Redorta J, et al. EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder, the 2011 update. Eur Urol. 2011;59(6):997–1008. 4. Sylvester RJ, van der Meijden AP, Oosterlinck W, Witjes JA, Bouffioux C, Denis L, et al. Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables: a combined analysis of 2596 patients from seven EORTC trials. Eur Urol. 2006;49(3):466–5 (discussion 75-7). 5. Kaufman DS, Shipley WU, Feldman AS. Bladder cancer. Lancet. 2009;374(9685):239–49. 6. Su ZZ, Kang DC, Chen Y, Pekarskaya O, Chao W, Volsky DJ, et al. Identification and cloning of human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1 envelope glycoprotein by rapid subtraction hybridization, RaSH. Oncogene. 2002;21(22):3592–602. 7. Kang DC, Su ZZ, Sarkar D, Emdad L, Volsky DJ, Fisher PB. Cloning and characterization of HIV-1-inducible astrocyte elevated gene-1, AEG-1. Gene. 2005;353(1):8–15. 8. Lee SG, Su ZZ, Emdad L, Sarkar D, Fisher PB. Astrocyte elevated gene-1 (AEG-1) is a target gene of oncogenic Ha-ras requiring phosphatidylinositol 3-kinase and c-Myc. Proc Natl Acad Sci USA. 2006;103(46):17390–5. 9. Liu L, Wu J, Ying Z, Chen B, Han A, Liang Y, et al. Astrocyte elevated gene-1 upregulates matrix metalloproteinase-9 and induces human glioma invasion. Cancer Res. 2010;70(9):3750–9. 10. Yoo BK, Emdad L, Su ZZ, Villanueva A, Chiang DY, Mukhopadhyay ND, et al. Astrocyte elevated gene-1 regulates
123
986 Page 6 of 6
11.
12.
13.
14.
15.
16.
17.
hepatocellular carcinoma development and progression. J Clin Invest. 2009;119(3):465–77. Yu C, Chen K, Zheng H, Guo X, Jia W, Li M, et al. Overexpression of astrocyte elevated gene-1 (AEG-1) is associated with esophageal squamous cell carcinoma (ESCC) progression and pathogenesis. Carcinogenesis. 2009;30(5):894–901. Li J, Zhang N, Song LB, Liao WT, Jiang LL, Gong LY, et al. Astrocyte elevated gene-1 is a novel prognostic marker for breast cancer progression and overall patient survival. Clin Cancer Res. 2008;14(11):3319–26. Jian-bo X, Hui W, Yu-long H, Chang-hua Z, Long-juan Z, Shirong C, et al. Astrocyte-elevated gene-1 overexpression is associated with poor prognosis in gastric cancer. Med Oncol. 2011;28(2):455–62. Liao WT, Guo L, Zhong Y, Wu YH, Li J, Song LB. Astrocyte elevated gene-1 (AEG-1) is a marker for aggressive salivary gland carcinoma. J Transl Med. 2011;9:205. Emdad L, Sarkar D, Lee SG, Su ZZ, Yoo BK, Dash R, et al. Astrocyte elevated gene-1: a novel target for human glioma therapy. Mol Cancer Ther. 2010;9(1):79–88. Kikuno N, Shiina H, Urakami S, Kawamoto K, Hirata H, Tanaka Y, et al. Knockdown of astrocyte-elevated gene-1 inhibits prostate cancer progression through upregulation of FOXO3a activity. Oncogene. 2007;26(55):7647–55. Gnosa S, Shen YM, Wang CJ, Zhang H, Stratmann J, Arbman G, et al. Expression of AEG-1 mRNA and protein in colorectal
123
Med Oncol (2014) 31:986
18.
19.
20.
21.
22.
23.
cancer patients and colon cancer cell lines. J Transl Med. 2012;10:109. Emdad L, Sarkar D, Su ZZ, Randolph A, Boukerche H, Valerie K, et al. Activation of the nuclear factor kappaB pathway by astrocyte elevated gene-1: implications for tumor progression and metastasis. Cancer Res. 2006;66(3):1509–16. Brown DM, Ruoslahti E. Metadherin, a cell surface protein in breast tumors that mediates lung metastasis. Cancer Cell. 2004;5(4):365–74. Hu G, Chong RA, Yang Q, Wei Y, Blanco MA, Li F, et al. MTDH activation by 8q22 genomic gain promotes chemoresistance and metastasis of poor-prognosis breast cancer. Cancer Cell. 2009;15(1):9–20. Zhou J, Li J, Wang Z, Yin C, Zhang W. Metadherin is a novel prognostic marker for bladder cancer progression and overall patient survival. Asia Pac J Clin Oncol. 2012;8(3):e42–8. Emdad L, Sarkar D, Su ZZ, Lee SG, Kang DC, Bruce JN, et al. Astrocyte elevated gene-1: recent insights into a novel gene involved in tumor progression, metastasis and neurodegeneration. Pharmacol Ther. 2007;114(2):155–70. Liu H, Song X, Liu C, Xie L, Wei L, Sun R. Knockdown of astrocyte elevated gene-1 inhibits proliferation and enhancing chemo-sensitivity to cisplatin or doxorubicin in neuroblastoma cells. J Exp Clin Cancer Res. 2009;28:19.
