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Anterior Gradient Protein 3 Is Associated with Less Aggressive Tumors and Better Outcome of Breast Cancer Patients

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Anterior Gradient Protein 3 Is Associated with Less Aggressive Tumors and Better Outcome of Breast Cancer Patients Journal name: OncoTargets and Therapy Article Designation: Original Research Year: 2015 Volume: 8 OncoTargets and Therapy Dovepress Running head verso: Obacz et al Running head recto: Significance of AGR3 in breast cancer open access to scientific and medical research DOI: http://dx.doi.org/10.2147/OTT.S82235 Open Access Full Text Article ORIGINAL RESEARCH Anterior gradient protein 3 is associated with less aggressive tumors and better outcome of breast cancer patients Joanna Obacz1 Abstract: Anterior gradient protein (AGR) 3 is a highly related homologue of pro-oncogenic Veronika Brychtova1 AGR2 and belongs to the family of protein disulfide isomerases. Although AGR3 was found Jan Podhorec1 in breast, ovary, prostate, and liver cancer, it remains of yet poorly defined function in tumo- Pavel Fabian2 rigenesis. This study aimed to determine AGR3 expression in a cohort of 129 primary breast Petr Dobes1 carcinomas and evaluate the clinical and prognostic significance of AGR3 in these tumors. Borivoj Vojtesek1 The immunohistochemical analysis revealed the presence of AGR3 staining to varying degrees in 80% of analyzed specimens. The percentage of AGR3-positive cells significantly Roman Hrstka1 correlated with estrogen receptor, progesterone receptor (both P,0.0001) as well as low 1 Regional Centre for Applied histological grade (P=0.003), and inversely correlated with the level of Ki-67 expression Molecular Oncology (RECAMO), 2Department of Pathology, Masaryk (P,0.0001). In the whole cohort, AGR3 expression was associated with longer progression- Memorial Cancer Institute, Brno, free survival (PFS), whereas AGR3-positive subgroup of low-histological grade tumors showed Czech Republic both significantly longer PFS and overall survival. In conclusion, AGR3 is associated with the level of differentiation, slowly proliferating tumors, and more favorable prognosis of breast cancer patients. Keywords: AGR3, patient survival, protein disulfide isomerase, ER-positive breast cancer, immuno histochemistry Introduction Breast cancer is the most common female malignancy and a leading cause of deaths among women worldwide. Only in 2012, in Europe, roughly 464,000 new incidences were registered, and 131,000 women died from breast cancer.1 Despite intensive research on various diagnostic and/or prognostic markers, thorough understanding of factors affecting breast cancer patients’ outcome remains of great importance. In recent years, an increasing number of reports have linked anterior gradient protein (AGR) 2 with many aspects of breast tumor biology. AGR2 is a human homologue of Xenopus laevis-secreted protein XAG-2 and belongs to an evolutionary broad family with prominent role in developmental processes and regeneration of body appendages.2,3 There are three subfamilies of AGRs: AGR1, AGR2, and AGR3, all showing the highest homology to non-secreted protein disulfide isomerase (PDI) of the TLP19 subfamily.3 PDIs are involved in proper folding and maturation of newly synthesized proteins and the regulation of endoplasmic reticulum homeostasis.4 Following the first characterization of AGR2 in the estrogen receptor (ER)- positive breast cancer cell line MCF-7,5 AGR2 has been frequently shown as an Correspondence: Roman Hrstka RECAMO, Masaryk Memorial Cancer estrogen-responsive gene/protein. It was demonstrated that AGR2 is upregulated Institute, Zluty kopec 7, 65653 Brno, in response to estradiol treatment both in vitro5 and in vivo,6 and its high expres- Czech Republic Email [email protected] sion correlates with ER status7 and predicts poor prognosis in ER-positive breast submit your manuscript | www.dovepress.com OncoTargets and Therapy 2015:8 1523–1532 1523 Dovepress © 2015 Obacz et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further http://dx.doi.org/10.2147/OTT.S82235 permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Obacz et al Dovepress cancers8,9 as well as resistance to tamoxifen.10 Moreover, Memorial Cancer Institute (MMCI) between 2003 and 2006. chromatin immunoprecipitation (ChIP) confirmed direct Patient age at the time of diagnosis ranged from 29 years AGR2 regulation by ER.10–12 In normal mammary gland, to 84 years (median 57 years). The clinical, histological, AGR2 induces cell proliferation and differentiation as and molecular characteristics of the analyzed set of tumors shown in the mouse models,13 whereas in breast tumors, are summarized in Table 1. Histological typing of tumors it promotes cell progression and survival through, among was carried out according to the criteria of World Health others, ER, cyclin D1, c-Myc, and survivin signaling Organization.23 Tumor stage was determined according to pathways.14 Furthermore, when introduced into benign rat the guidelines of the Union for International Cancer Control mammary epithelial cell line, AGR2 was found to contribute (UICC).24 Tumor grade was established according to Bloom to metastasis development.15 and Richardson in the modification of Elston and Ellis.25 ER, Closely related AGR2 homologue, AGR3,7 has also been progesterone receptor (PR), human epidermal growth factor identified in breast cancer cell lines using proteomics screen receptor 2 (Her2/neu), and Ki-67 statuses were extracted from as one of the membrane-associated proteins.16 Although both pathological records obtained from the MMCI database. For molecules share 71% sequence identity and lie adjacent to the evaluation of AGR3 prognostic relevance without regard one another at chromosomal position 7p21,7,17 AGR2, but to ER status, additional ER-negative group of 90 breast not AGR3, is a dominant factor identified in many OMICS screens. Thus, to date, only few reports describing AGR3 Clinicopathological characteristics of primary breast car­ expression in various tumors were published, and there are Table 1 cinomas limiting amount of data depicting AGR3 prognostic rele- Variablea Group Nb %c vance in these malignancies. It has been shown that AGR3 Histology Ductal 95 73.6 is strongly expressed in breast carcinomas when compared Lobular 18 14 16 to healthy tissues and that its expression correlates with ER Other 9 7 status in breast tumors.7 In another study, single ER-binding NA 7 5.4 site on AGR3 promoter has been found using ChIP-Seq Histological grade G1 27 20.9 G2 43 33.4 12 approach. Our group has recently demonstrated that intra- G3 56 43.4 hepatic cholangiocarcinomas (ICCs) express AGR3 protein, NA 3 2.3 while hepatocellular carcinomas are predominantly AGR3 Tumor size pT1 44 34.1 pT 65 50.3 negative. Furthermore, we postulated that together with acid 2 pT 6 4.7 mucopolysaccharides, AGR3 could serve as a diagnostic 3 pT4 9 7 marker of well-differentiated ICCs.18 It has also been shown NA 5 3.9 that AGR3 is overexpressed in different histological types Nodal status Negative 45 34.9 Positive 73 56.6 of ovarian cancers. In non-mucinous types (including serous NA 11 8.5 papillary, endometrioid, and clear cell), AGR3 expression ER status Negative 29 22.5 was found to be ER independent and uncoupled with AGR2 Positive 100 77.5 expression, whereas in mucinous ovarian cancers, both NA 0 0 PR status Negative 34 26.4 19 AGR2 and AGR3 showed cognate expression patterns. Positive 94 72.8 In serous type, AGR3 staining correlated with the level of dif- NA 1 0.8 ferentiation and was associated with longer patient survival.20 Her2/neu status Negative 92 71.3 Additionally, AGR3 was found to be androgen-regulated Positive 36 27.9 NA 1 0.8 21,22 gene, expression of which was highly elevated in human Ki­67d ,15% 55 42.6 21 prostate cancer. The aim of this study is to examine the $15% 61 47.3 significance between AGR3 expression, clinicopathologi- NA 13 10.1 cal characteristics, and patient outcome in primary breast AGR3 expression 1 25 19.4 2 25 19.4 carcinomas. 3 79 61.2 Notes: aDefined in the “Materials and methods” section. bNumber of patients. Materials and methods cPercentage of total patients, out of a total of 129. dCut­off for Ki­67 was used according to St Gallen Consensus in 2009. AGR3 expression: 1 – negative/border, Study group and tissue specimens 2 – weakly/moderately positive, and 3 – strongly positive. The study group consisted of 129 patients undergoing sur- Abbreviations: NA, not available; ER, estrogen receptor; PR, progesterone receptor; Her2/neu, human epidermal growth factor receptor 2; AGR3, anterior gradient gical procedure for primary breast cancer at the Masaryk protein 3. 1524 submit your manuscript | www.dovepress.com OncoTargets and Therapy 2015:8 Dovepress Dovepress Significance of AGR3 in breast cancer cancer patients treated at MMCI between 1995 and 2006 95°C, and then 40 cycles at 95°C for 15 seconds and at 60°C were included for survival analysis. Informed consent has for 1 minute. To obtain absolute quantification, dilution series been obtained from all patients involved in this study. The of plasmids pDEST12.2 with cloned respective sequences study was approved by ethical committee of MMCI, and the were used in range from 20 to 2 millions of copies to ge nerate data used were anonymized and were handled according to standard curves. For data normalization, 18S rRNA levels Czech Republic existing legislation. were determined using TaqMan assay for 18S rRNA (Thermo Fisher Scientific, Waltham, MA, USA). Immunohistochemistry Tumor samples were fixed in 10% neutral buffered formalin Statistical analysis for 24 hours and then embedded in paraffin wax. Immuno- All statistical analyses were performed using STATISTICA histochemical analysis was performed on 4 μm thick sections Version 12 (StatSoft, Inc., Tulsa, OK, USA) and IBM SPSS cut from formalin-fixed, paraffin-embedded archival tissue Statistics 20.0.
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