|Year : 2015 | Volume
| Issue : 5 | Page : 638-643
Correlation of astrocyte elevated gene-1, basic-fibroblast growth factor, beta-catenin, Ki-67, tumor necrosis factor-alfa with prognostic parameters in ductal carcinomas and ductal intraepithelial neoplasms
H Erdem1, B Gundogdu2, H Ankarali3, M Yasar4, E Sener5, M Oktay1, A Bahadir6, AK Uzunlar1, N Gursan2, I Ozaydin4, C Sahiner1
1 Department of Pathology, Duzce University of Medical Faculty, Duzce, Turkey
2 Department of Pathology, Ataturk University of Medical Faculty, Erzurum, Turkey
3 Department of Biostatistics, Duzce University of Medical Faculty, Duzce, Turkey
4 General Surgery, Duzce University of Medical Faculty, Duzce, Turkey
5 Department of Urology, Erzurum Region Education and Research Hospital, Erzurum, Turkey
6 Department of Biophysics, Medical Faculty, Duzce, Turkey
|Date of Acceptance||03-Feb-2015|
|Date of Web Publication||22-Jun-2015|
Department of Pathology, Duzce University Medical Faculty, Duzce, 81000
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Breast cancer is the second most frequent cancer in the world. Although it is widely accepted that the etiology of breast cancer includes both genetic and environmental factors, the molecular mechanism of its development and progression remains poorly understood, and thus far, no specific signature of breast cancer gene expression has been reported to allow for patient-tailored therapy strategies. Hence, it is of great clinical value to further understand the molecular mechanisms underlying the progression of breast cancer and to identify effective early markers for the diagnosis and prognosis of the disease as well as novel therapeutic targets.
Materials and Methods: This study was conducted on a total of 90 paraffin-embedded breast tumor samples. Immunohistochemical stains for astrocyte elevated gene-1 (AEG-1), basic-fibroblast growth factor (b-FGF), beta-catenin, Ki-67, tumor necrosis factor-α (TNF-α) were performed on tissue microarray using standard procedures. Each patient age, grade, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) status, size, lymphovascular invasion, metastasis of lymph node (LNM), nipple and ductal hyperplasia areas were assessed.
Results: We observed significant relationship between the age and LNM or FGF (P = 0.018, 0.035, respectively). The relationship between histological and nuclear grade, LNM, ER, PR, HER-2 and prognostic parameters was evaluated in cases of ductal carcinomas (DC). There was a significant positive correlation between TNF-α, size, LNM (P ≤ 0.0001, 0.002, 0.005). We found that significant relationship between AEG-1 and TNF-α. There was a significant positive correlation between FGF and Ki-67 and negative correlation AEG-1. Although, FGF, TNF-α, AEG-1 staining in DC were observed higher than ductal intraepithelial neoplasms, this observation could not statistically (P ≥ 0.05).
Conclusions: The present work aims to investigate the relationship between the expression of AEG-1, b-FGF, beta-catenin, Ki-67, TNF-α other prognostic parameters in DC and ductal intraepithelial neoplasm. We found a relationship between these factors.
Keywords: Astrocyte elevated gene-1, beta-catenin, basic-fibroblast growth factor, ductal carcinomas, prognostic parameters, tumor necrosis factor-α
|How to cite this article:|
Erdem H, Gundogdu B, Ankarali H, Yasar M, Sener E, Oktay M, Bahadir A, Uzunlar A K, Gursan N, Ozaydin I, Sahiner C. Correlation of astrocyte elevated gene-1, basic-fibroblast growth factor, beta-catenin, Ki-67, tumor necrosis factor-alfa with prognostic parameters in ductal carcinomas and ductal intraepithelial neoplasms. Niger J Clin Pract 2015;18:638-43
|How to cite this URL:|
Erdem H, Gundogdu B, Ankarali H, Yasar M, Sener E, Oktay M, Bahadir A, Uzunlar A K, Gursan N, Ozaydin I, Sahiner C. Correlation of astrocyte elevated gene-1, basic-fibroblast growth factor, beta-catenin, Ki-67, tumor necrosis factor-alfa with prognostic parameters in ductal carcinomas and ductal intraepithelial neoplasms. Niger J Clin Pract [serial online] 2015 [cited 2019 Sep 21];18:638-43. Available from: http://www.njcponline.com/text.asp?2015/18/5/638/158961
| Introduction|| |
Breast cancer is the second most common malignancy in the world to date.  Classification of this cancer is based on a number of aspects such as pathology and tumor progression, estrogen receptor (ER), progesterone receptor (PR) status and human epidermal growth factor receptor 2 (HER-2) status. Prognosis is related to a variety of clinical, pathologic and molecular features, which include classical prognostic factors (such as histologic type, grade, tumor size and metastasis of lymph node [LNM]). All of these clinical parameters dictate the most suitable patient treatment. 
However, continued improvements in treatment will undoubtedly depend on our ability to identify novel targets contributing to this disease. Fibroblast growth factor receptors (FGFRs) as well as a number of fibroblast growth factor (FGF) ligands, play a critical role in regulating normal mammary gland development and tissue homeostasis. Such target, which has been implicated in the etiology of breast cancer, is the FGFR. 
In addition, high-throughput sequencing studies have shown activating FGF mutations to be highly common in multiple forms of human cancer, including breast cancer. There is also increasing evidence for the importance of FGF signaling in human cancers, including breast cancer. 
Almost 25% of cancers are reported to occur through chronic inflammation related processes.  The pro-inflammatory regulators such as tumor necrosis factor-α (TNF-α) and other cytokines and their receptor networks seem to play crucial functions in tumorigenesis. 
Beta-catenin is considered to be involved in the carcinogenesis of many tumors and is also thought to be effective in the development of breast cancer.  It is one of the proteins that E-cadherin, which is an adhesion molecule, has to interact with in order to carry out its mission, and it is also involved in intracellular signal transduction pathways. 
Recent studies have showed that astrocyte elevated gene-1 (AEG-1) increases the invasiveness of malignant cells, favoring tumor genesis, neovascularization, metastasis and that up-regulation in epithelial cells inhibits apoptosis. 
The possible involvement of AEG-1, basic-FGF (b-FGF), beta-catenin, TNF-α in tumorigenesis, angiogenesis and metastasis led us to examine the relationship between AEG-1, b-FGF, beta-catenin, Ki-67, TNF-α and prognostic parameters in 90 primary breast carcinomas samples.
| Materials and Methods|| |
This study was conducted on a total of 90 paraffin-embedded breast tumor (ductal carcinomas [DC]) samples, which were histopathologically diagnosed at the Department of Pathology between 2005 and 2012. All the diagnoses were made by two pathologists, following the pathology of World Health Organization (WHO) Classification of Tumors. The samples consisted of 90 (all cases) DC. Age distribution of the cases ranged from the 31 to 83 (56.4 ± 13.4). The tumor size distribution was as follows: Less than 2 cm in diameter in 9 (10%) patients, from 2 to 5 cm in 70 (77.8%) patients and more than 5 cm in 11 (12.2%) patients. Lymph node metastasis number distribution of the cases ranged from the 0 to 16 (3.32 ± 2.37). In addition, 47 (52.2%) patients in LNM.
In all cases, the histological diagnosis and prognostic parameters were confirmed by corresponding paraffin-embedded materials, and when necessary, immunohistochemical study panels were carried out according to the most recent WHO classification.
The tumor grade of invasive carcinoma was classified according to the Scarff-Bloom-Richardson system. Based on the frequency of cell mitosis, tubule formation, and nuclear pleomorphism, invasive carcinoma was graded as grade 1 (low), 2 (moderate) or 3 (high). The presence of lymph node metastases was reviewed for each patient. Prognostic parameters were compared with the results of AEG-1, b-FGF, beta-catenin, TNF-α, Ki-67 stains.
Among the Hematoxylin-Eosin stained slides, one suitable paraffin block was chosen. For AEG-1, Genetex brand 2F11C3 clone was used, and it was diluted at a ratio of 1:200. The degree of immunostaining was reviewed. They were scored independently by two observers, based on both the proportion of positively-stained tumor cells and the intensity of staining. The proportion of tumor cells was scored as follows: 0 (no positive tumor cells), 1 (<10% positive tumor cells), 2 (10-50% positive tumor cells), and 3 (>50% positive tumor cells). The intensity of staining was graded 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 was calculated from the staining intensity score and proportion of positive tumor cells. Using this method of assessment, we evaluated the expression of AEG-1 [Figure 1] in normal tubular epithelia and malignant lesions by determining the staining index, which was scored as 0, 1, 2, 3, 4, 6, and 9. The staining index score of ≥4 was used to define tumors as having high AEG-1 expression and ≤3 as having low expression of AEG-1. 
|Figure 1: Astrocyte elevated gene-1 expression seen in malign glands (AEG-1 ×100)|
Click here to view
The paraffin-embedded tumor sections were deparaffinized for immunohistochemical detection of cells positively-stained for TNF-α. Endogenous peroxidase activity was blocked using 3% H 2 O 2 for 10 min, followed by incubation with nonspecific staining blocking reagent (DakoCytomation) for 10 min. The sections were incubated with the optimal dilutions of anti-TNF-α (15 μg/mL), antibodies overnight at 4°C followed by 1 h with horse radish peroxidase-labeled secondary antibodies. Immune complexes were visualized with using the streptavidin-peroxidase method with 3,3'-diaminobenzidine (DAB) as chromogen. (Vector Laboratories Inc., Burlingame, California, USA).
Immunostaining was performed by the streptavidin-biotin-peroxidase method. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide. No antigen retrieval was used for b-FGF antibody. The sections were incubated at room temperature for 1 h reactive (b-FGF [1-24] [Polyclonal] concentration 0.1 ml [1:100], code: F3393, Sigma Aldrich). After washing in phosphate-buffered saline with Tween-20, the tissues were incubated with a biotin-conjugated secondary antibody and then with a biotin-streptavidin complex for 30 min at room temperature. Reactions were visualized with 3,3-diaminobenzidine tetrahydrochloride. Sections were counterstained with hematoxylin, rinsed, and mounted.
The avidin biotin complex immunoperoxidase method with primer antibody (catenin, beta [17C2] C.Lyo. 1 ml (1:100-200, code: B-CAT, Leica/Novocastra) and Ki-67 (BioGenex: Species mouse, clone Ki-88, isotype IgG1/kappa) were applied to the cases included in the study group [Figure 2]. Normal breast tissue surrounding the tumor was used as a positive control.
|Figure 2: Beta-catenin expression seen in malign glands and intraductal areas (lower ×100) ki-67 staining of malign glands (upper x400)|
Click here to view
Immunoreactivity for b-FGF, beta-catenin, Ki-67, TNF-α was scored using a semi-quantitative scale for intensity of staining: 0 negative, no staining; 1 + weak positive; 2 + moderately positive; 3 + strongly positive [Figure 1] [Figure 2] [Figure 3] [Figure 4].
|Figure 3: Tumor necrosis factor alpha expression seen in ductal carcinomas and intraductal areas (TNF alpha x100)|
Click here to view
|Figure 4: Fibroblast growth factor expression seen in ductal carcinomas (FGF x100)|
Click here to view
Descriptive statistics were computed as count and percent frequency. Spearman rank correlation analyze was used for determination of the relations between prognostic factors and the degree of staining, and the relation of staining results to each other. The level of significance was determined to be 0.05 (P = 0.05). Analyses were performed using the statistical PASW SPSS version 18 (IBM, Chicago, USA).
| Results|| |
We observed significant relationship between the age and LNM or FGF (r = 0.250 and P = 0.018; r = 0.224 and 0.035, respectively). The positive relationship between histological grade and nuclear grade, tumor size, LNM, HER-2 (P ≤ 0.0001, 0.010, 0.044, <0.0001, 0.002, 0.037, respectively). However, the negative significant correlation were found between histological grade and ER, PR (P ≤ 0.0001, 0.002). There was a significant positive correlation between TNF-α, size, LNM (P ≤ 0.0001, 0.002, 0.005). We found that a significant relationship between AEG-1 and TNF-α [Table 1]. There was a significant positive correlation between FGF and Ki-67 and negative correlation AEG-1. No relationship was found beta-catenin and FGF, TNF-α and prognostic parameters [Table 1].
In addition to, FGF and TNF-α staining in DC were observed higher than ductal intraepithelial neoplasms [Figure 3] and [Figure 4]. However, this observation could not statistically (P ≥ 0.05).
| Discussion|| |
Recently, anti-cancer research continues to rapidly. In this study, it was found a significant relationship between FGF, TNF-α, AEG-, Ki-67 and tumor prognosis.
Fibroblast growth factor is a potent angiogenic molecule that has been shown to promote tumor cell mitosis. Dow et al. found that FGF has been implicated in the differentiation of stromal and epithelial cells from a dormant to an invasive phenotype. 
Basic-fibroblast growth factor has been the focus of a plethora of studies into human tumor biology and has important implications for cancer therapies and clinical outcomes. Presta et al. showed that interact with various vascular endothelial growth factors and cell surface receptors that are known to play a role in tumor growth and angiogenesis. 
The extent of metastatic disease and the correlation between angiogenesis has been widely demonstrated in a large and diverse range of human cancers. ,
Abnormally high concentrations of b-FGF have been shown to correlate significantly with the extent of disease, clinical status and risk of future mortality. They have been found in the serum of patients with active metastatic cancers. 
In this study, FGF was found a significant relationship between the age and LNM, also, FGF ekspression was higher metastatic tumors than nonmetastatic tumors.
Tumor necrosis factor-α is highly expressed in tumors where it has been considered initially as a potent tumor cell killer and an anti-vascular cytokine at high doses. 
However, low chronic doses of the cytokine are thought to be pro-angiogenic.  In order to test the involvement of TNF-α activation/endothelial dysfunction of the breast tumor vasculature. Perrot-Applanat et al. further explored if the correlations identified in their ex vivo cellular model (endothelial cells) between the expression of TNF-α mRNA and the expression of putative TNF-α-inducible nuclear factor-kB (NF-kB) target genes were also observed ex vivo in human breast tumors. 
Two additional members of the TNF/TNF receptor superfamily involved in the immune system.  Such induction occurs through TNFR1 and TNFR2 and via NF-kB activation.
Tumor necrosis factor-α and its related NF-kB transcription factor are described as critical components of tumor progression.  NF-kB is deregulated in breast cancer patients and in breast cancer cells. 
Several studies have shown that tumor associated neovascularization is a precondition of rapid growth and metastasis. 
Different markers and many essential interactions have to be yet determined though great strides have been made in the elucidation of molecular mechanisms of tumor vasculature. ,,,
In this study, TNF-α was found a significant relationship between the poor prognostic parameters (such as size and LNM). It was correlation between angiogenesis, also, TNF-α ekspression was metastatic tumors higher than nonmetastatic tumors, similarly FGF.
This study, beta-catenin staining in ductal intraepithelial neoplasms (DIN) was observed higher than DC [Figure 3], but this observation could not statistically (P ≥ 0.05). For this reason, it could be small of DIN areas in slides.
A possible interaction partner of AEG-1 was found by Emdad et al. They reported that AEG-1 promotes anchorage-independent growth and invasion through IkBα degradation, NF-kB binding, and nuclear translocation.  Furthermore, several studies demonstrated a functional role of AEG-1 in several aspects of cancer development including angiogenesis, invasion, metastasis, apoptosis and chemo-resistance. ,,
In this study, was found a significant relationship between AEG-1 and TNF-α. This correlation could be due to NF-kB signaling pathway.
Cell-cell and cell-matrix interactions play a role in every stage of metastasis. Adhesion molecules enable these interactions. 
Interaction with catenins is essential for adhesive functions of E-cadherin, which is a conventional cadherin type involved in Ca+2 mediated cell adhesion in epithelial cells and this interaction largely depends on beta-catenin expression and function. The cadherin family should occur changes in cell-cell adhesions mediated for a cancer cell to break free from the primary tumor.  Many studies regarding the expression of adhesion molecules in breast carcinomas have been conducted to date. 
Beta-catenin expression was demonstrated to be decreased or completely lost in cultured human breast cancer cell lines in studies. ,,
Jonsson et al. have revealed that beta-catenin signals also play a role in human breast cancers by showing increased levels of cytosolic beta-catenin in 13% of primary breast tumors. 
In this study, no relationship was found beta-catenin and FGF, TNF-α and prognostic parameters.
| Conclusions|| |
The relationship between TNF and FGF, AEG-1 protocols may be useful new treatment.
Difference stain between DIN and DC in terms of beta-catenin FGF, TNF-α, AEG-1 expressions in our study shows that any change in beta-catenin FGF, TNF-α, AEG-1 expressions develop in early carcinogenesis. In addition to, in this study was found a significant relationship between AEG-1 and TNF-α. However, more comprehensive studies should be performed in order to demonstrate at what stage the change in beta-catenin FGF, TNF-α, AEG-1 expression occurs.
| Acknowledgment|| |
This project is supported by Dόzce University Research. We thank Dr. Hilal Balta for help this study.
| References|| |
Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893-917.
Rampaul RS, Pinder SE, Elaston CW, Ellis IO. Prognostic and predictive factors in primary breast cancer and their role in patient management; the Nottingham breast team. Eur J Surg Oncol 2001;27:229-38.
Grose R, Dickson C. Fibroblast growth factor signaling in tumorigenesis. Cytokine Growth Factor Rev 2005;16:179-86.
Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, Bignell G, et al.
Patterns of somatic mutation in human cancer genomes. Nature 2007;446:153-8.
Balkwill F, Coussens LM. Cancer: An inflammatory link. Nature 2004;431:405-6.
Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:860-7.
Bukholm IK, Nesland JM, Kåresen R, Jacobsen U, Børresen-Dale AL. E-cadherin and alpha-, beta-, and gamma-catenin protein expression in relation to metastasis in human breast carcinoma. J Pathol 1998;185:262-6.
Hülsken J, Birchmeier W, Behrens J. E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton. J Cell Biol 1994;127:2061-9.
Hu G, Wei Y, Kang Y. The multifaceted role of MTDH/AEG-1 in cancer progression. Clin Cancer Res 2009;15:5615-20.
Dow JK, deVere White RW. Fibroblast growth factor 2: Its structure and property, paracrine function, tumor angiogenesis, and prostate-related mitogenic and oncogenic functions. Urology 2000;55:800-6.
Presta M, Dell'Era P, Mitola S, Moroni E, Ronca R, Rusnati M. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev 2005;16:159-78.
Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol 1993;143:401-9.
Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis - Correlation in invasive breast carcinoma. N Engl J Med 1991;324:1-8.
Nguyen M, Watanabe H, Budson AE, Richie JP, Hayes DF, Folkman J. Elevated levels of an angiogenic peptide, basic fibroblast growth factor, in the urine of patients with a wide spectrum of cancers. J Natl Cancer Inst 1994;86:356-61.
ten Hagen TL, Seynhaeve AL, Eggermont AM. Tumor necrosis factor-mediated interactions between inflammatory response and tumor vascular bed. Immunol Rev 2008;222:299-315.
Mocellin S, Rossi CR, Pilati P, Nitti D. Tumor necrosis factor, cancer and anticancer therapy. Cytokine Growth Factor Rev 2005;16:35-53.
Perrot-Applanat M, Vacher S, Toullec A, Pelaez I, Velasco G, Cormier F, et al.
Similar NF-kB gene signatures in TNF-a treated human endothelial cells and breast tumor biopsies. PLoS One 2011;6:e21589.
Karin M, Cao Y, Greten FR, Li ZW. NF-kappaB in cancer: From innocent bystander to major culprit. Nat Rev Cancer 2002;2:301-10.
Cogswell PC, Guttridge DC, Funkhouser WK, Baldwin AS Jr. Selective activation of NF-kappa B subunits in human breast cancer: Potential roles for NF-kappa B2/p52 and for Bcl-3. Oncogene 2000;19:1123-31.
Kim DW, Sovak MA, Zanieski G, Nonet G, Romieu-Mourez R, Lau AW, et al.
Activation of NF-kappaB/Rel occurs early during neoplastic transformation of mammary cells. Carcinogenesis 2000;21:871-9.
Cao Y, Karin M. NF-kappaB in mammary gland development and breast cancer. J Mammary Gland Biol Neoplasia 2003;8:215-23.
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;66:1509-16.
Yoo BK, Emdad L, Su ZZ, Villanueva A, Chiang DY, Mukhopadhyay ND, et al.
Astrocyte elevated gene-1 regulates hepatocellular carcinoma development and progression. J Clin Invest 2009;119:465-77.
Lee SG, Su ZZ, Emdad L, Sarkar D, Franke TF, Fisher PB. Astrocyte elevated gene-1 activates cell survival pathways through PI3K-Akt signaling. Oncogene 2008;27:1114-21.
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;26:7647-55.
Gillett CE, Miles DW, Ryder K, Skilton D, Liebman RD, Springall RJ, et al.
Retention of the expression of E-cadherin and catenins is associated with shorter survival in grade III ductal carcinoma of the breast. J Pathol 2001;193:433-41.
Karayiannakis AJ, Nakopoulou L, Gakiopoulou H, Keramopoulos A, Davaris PS, Pignatelli M. Expression patterns of beta-catenin in in situ
and invasive breast cancer. Eur J Surg Oncol 2001;27:31-6.
Gonzalez MA, Pinder SE, Wencyk PM, Bell JA, Elston CW, Nicholson RI, et al.
An immunohistochemical examination of the expression of E-cadherin, alpha-and beta/gamma-catenins, and alpha2-and beta1-integrins in invasive breast cancer. J Pathol 1999;187:523-9.
Karabacak T, Eðılmez R, Arpaci RB, Pfeiffer ES. B-catenin expression in in situ
and infiltrative ductal carcinomas of the breast. Turk J Pathol 2011;27:185-8.
Jonsson M, Borg A, Nilbert M, Andersson T. Involvement of adenomatous poliposis coli (APC)/β-catenin signalling in human breast cancer. Eur J Cancer 2000;36:242-8.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]