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The Pro-Metastatic Protein Anterior Gradient-2 Predicts Poor Prognosis in Tamoxifen-Treated Breast Cancers

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The Pro-Metastatic Protein Anterior Gradient-2 Predicts Poor Prognosis in Tamoxifen-Treated Breast Cancers Oncogene (2010) 29, 4838–4847 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE The pro-metastatic protein anterior gradient-2 predicts poor prognosis in tamoxifen-treated breast cancers R Hrstka1, R Nenutil1, A Fourtouna2, MM Maslon2, C Naughton2, S Langdon2, E Murray2, A Larionov3, K Petrakova1, P Muller1, MJ Dixon3, TR Hupp2 and B Vojtesek1 1Department of Oncological and Experimental Pathology, Masaryk Memorial Cancer Institute, Brno, Czech Republic; 2University of Edinburgh, Institute of Genetics and Molecular Medicine and Cancer Research UK p53 Signal Transduction Laboratories, Edinburgh, UK and 3The Edinburgh Breast Unit, Western General Hospital, Edinburgh, UK Transcriptomic screens in breast cancer cell lines have the past. In line with this, suppression subtractive identified a protein named anterior gradient-2 (AGR2) as hybridization has allowed the identification of anterior a potentially novel oncogene overexpressed in estrogen gradient-2 (AGR2) gene, the human homologue of the receptor (ER) positive tumours. As targeting the ER is Xenopus laevis cement gland gene XAG-2, as being responsible for major improvements in cure rates and coexpressed with estrogen receptors (ERs) in breast prevention of breast cancers, we have evaluated the pro- cancer cell lines (Thompson and Weigel, 1998). More oncogenic function of AGR2 in anti-hormone therapeutic recently, gene expression profiling techniques identified responses. We show that AGR2 expression promotes AGR2 as highly expressed in human cancers (Iacobuzio- cancer cell survival in clonogenic assays and increases cell Donahue et al., 2003; Kondrakhin et al., 2008; proliferation and viability in a range of cancer cell lines. Barraclough et al., 2010). A proteomic analysis to Chromatin immunoprecipitation and reporter assays indicate discover cell membrane-associated proteins with poten- that AGR2 is transcriptionally activated by estrogen through tial clinical relevance to breast cancer also identified ERa. However, we also found that AGR2 expression is AGR2 (Adam et al., 2003). elevated rather than inhibited in response to tamoxifen, thus AGR2 was originally identified as a potential identifying a novel mechanism to account for an agonistic secretory protein that is highly expressed in frog eggs effect of the drug on a specific pro-oncogenic pathway. (Aberger et al., 1998). The human AGR2-coding Consistent with these data, clinical analysis indicates that sequence is located on chromosome 7p21, well known AGR2 expression is related to treatment failure in ERa- as a locus of frequent genetic alterations (Petek et al., positive breast cancers treated with tamoxifen. In contrast, 2000). Subsequent studies have shown a significant AGR2 is one of the most highly suppressed genes in cancers function for AGR2 in a range of biological pathways of responding patients treated with the anti-hormonal drug including regulation of p53 (Pohler et al., 2004), cell letrozole. These data indicate that the AGR2 pathway migration and cellular transformation (Wang et al., represents a novel pro-oncogenic pathway for evaluation 2008). In a rat somatic model for breast carcinoma, as anti-cancer drug developments, especially therapies that although the overexpression of AGR2 in grafted cells by-pass the agonist effects of tamoxifen. did not enhance tumour initiation, the resulting tumours Oncogene (2010) 29, 4838–4847; doi:10.1038/onc.2010.228; exhibited considerably greater propensity to form lung published online 7 June 2010 metastases compared with AGR2-negative counterparts (Liu et al., 2005). Importantly, the evidence suggested Keywords: AGR2; tamoxifen; letrozole; estrogen receptor; that AGR2 does not promote cell migration but instead breast cancer controls the cell adhesion rates of detached cells, which may correlate with the ability of metastatic cells to colonize distal sites. Strikingly, AGR2 was identified as the first protein able to promote cell migration and Introduction regeneration in severed nerves, which normally provide signal to stimulate regeneration (Kumar et al., 2007). Genomic and proteomic technologies provide the Clinical studies have also implicated the protein in opportunity to discover clinically relevant and novel inflammatory bowel disease (Zheng et al., 2006), oncogenic pathways that would have been overlooked hormone-dependent breast cancers (Thompson and by genetic and model organism cancer gene screens of Weigel, 1998; Fletcher et al., 2003; Innes et al., 2006), and a range of non-hormone cancers (Lee et al., 2006; Correspondence: Dr B Vojtesek, Department of Oncological and Zhu et al., 2007). Furthermore, AGR2 predicts poor Experimental Pathology, Masaryk Memorial Cancer Institute, Zluty prognosis in prostate cancers (Zhang et al., 2007). kopec 7, Brno 656 53, Czech Republic. Finally, expression of genes including AGR2 and others E-mail: [email protected] Received 25 November 2009; revised 29 January 2010; accepted 9 May could detect circulating tumour cells in peripheral blood 2010; published online 7 June 2010 of advanced cancer patients (Smirnov et al., 2005). Association of AGR2 expression with tamoxifen agonist effect R Hrstka et al 4839 The clinical effects of AGR2 expression in breast contrasting suppression of AGR2 by letrozole suggests a cancers are unclear as AGR2 has been associated with strategy for optimizing treatment of cancer based on the either increased or decreased survival (Fritzsche et al., AGR2 pathway. 2006; Innes et al., 2006), although the former study included ER-positive and ER-negative tumours, which may complicate the analysis due to the overwhelming impact of ER for prognosis after treatment. A more Results recent study using patients that have only undergone AGR2 expression promotes cell survival operative breast cancer surgery indicated that high AGR2 expression is linked to poor survival We evaluated whether transfection of the AGR2 gene (Barraclough et al., 2009). Therefore, the precise function into ER-negative and AGR2-negative cell lines H1299 of these proteins and the mechanisms of their hormonal (lung cancer, p53-null) and MDA-MB-231 (breast regulation in breast cancers remain to be elucidated. cancer, mutant p53) can alter cell survival in a As AGR2 expression is presumed to occur in clonogenic assay in vitro. Transient transfection of hormone-responsive tumours, development of a breast vectors encoding AGR2 gene indicated that AGR2 cancer model to study the function of novel proteins increases colony-forming ability in both cell lines such as AGR2 is required. Recent research has still (Figures 1a and b). To investigate whether AGR2 failed to translate a broader clinical utilization of expression increases survival after chemotherapy in a range of breast cancer biomarkers because only two breast cancer, we used the MTT assay in MDA-MB-231 markers, ER and ErbB2 (Her2), are presently accepted cells transfected with AGR2 and demonstrated an worldwide for breast cancer diagnosis. These proteins increase in cell viability after drug treatment (Figure 1c). have important functions in breast cancer development Together, these results clearly show that AGR2 promotes and provide effective treatments for many breast cellular viability, indicating that AGR2 can mediate tumours. As breast cancer is a classical hormone- survival under conditions of stress. These effects are dependent tumour, estrogen is well known to have a independent of p53 and ER status. In the light of these major function in the development and progression of facts, we evaluated AGR2 expression in the more the disease. Therefore, the determination of the ER common breast cancer cell systems that naturally express status is an essential part of the diagnostic procedure in both ER and AGR2. We tested four miRNAs, denoted as all breast cancer patients. The presence of ER and 261, 522, 575 and 654, for silencing AGR2 expression progesterone receptors is indicative of response to (Supplementary Figure 1). Two miRNAs, 261 and 575, endocrine therapy and improved disease-free survival the most potent AGR2 inhibitors, were chosen for further (Robertson et al., 1996; Katzenellenbogen and Frasor, experiments. MCF-7 cells (breast cancer, ER-positive, 2004). ER expression is generally found in only 6–10% wild-type p53) with silenced AGR2 expression showed of normal breast epithelial cells, whereas B60–70% of twofold to fourfold reduced clonogenic efficiency in primary breast cancers are ER-positive (Jacquemier response to tamoxifen treatment compared with control et al., 1990; Normanno et al., 2005). cells (Figures 1d and e). ER-positive/Her2-negative breast cancers typically respond well to endocrine therapy, which suppresses AGR2 gene is expressed in response to tamoxifen in vitro estrogen signalling in cancer cells and halts their and represents agonist responsive locus proliferation and/or induces apoptosis. Although endo- These data validating the pro-survival activity of AGR2 crine therapy is proven effective, its clinical benefit is directed us to analyse whether the expression of AGR2 seriously limited by drug resistance. Approximately 40% is associated with response to tamoxifen, which is the of ER-positive early stage breast cancers, which initially endocrine treatment of choice for all stages of ER- respond to anti-hormonal therapy, become resistant positive breast cancer for the past 30 years. Treatment of during the course of therapy (acquired resistance) MCF-7 cells with dialyzed serum resulted in a loss of and
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