Anterior Gradient 2 Research Articles

Physiological stress induces the metastasis marker AGR2 in breast cancer cells

As an approach to understanding the factors that activate expression of tumor progression genes, the role of physiological stress in the activation of a panel of tumor cell markers was investigated. These studies identify the developmental gene product, anterior gradient 2 (AGR2) as a cancer cell marker specifically up-regulated in response to depletion of serum and oxygen. AGR2 has been identified as a tumor marker in primary and secondary cancer lesions, and as a marker for detection of circulating tumor cells (CTCs). Elevated levels of AGR2 are known to increase the metastatic potential of cancer cells, but conditions leading to increased expression of AGR2 are not well understood. The present results identify novel physiological parameters likely to contribute to AGR2 induction in situ.

Expression of AGR2 in non small cell lung cancer.

We aimed to evaluate immunohistochemically the expression of the human Anterior Gradient-2 (AGR2), a gene which has recently been proposed as an oncogene for lung carcinoma development, in non small cell lung cancer and to correlate the findings to clinico-pathological data including patient survival. 95 cases of NSCLC were immunostained using a polyclonal AGR2 antibody and statistical analyses were applied to test for prognostic and diagnostic associations. AGR2 was expressed in 66.3% of cases, preferentially adenocarcinomas. There were no relevant associations with clinico-pathological parameters. A prognostic value of AGR2 could not be demonstrated neither in multivariate nor in univariate analyses. Interestingly, this is the first study to demonstrate AGR2 expression in squamous cell carcinomas. Although a prognostic value of AGR2 seems unlikely further studies are warranted to investigate the biological role of AGR2 in NSCLC and its differential expression according to histology.

Increased expression of anterior gradient-2 is significantly associated with poor survival of prostate cancer patients

Anterior gradient-2 (AGR2) expression was examined in a series of prostate cell lines and in an archival set of prostate tissues. The relative levels of AGR2 expression in the malignant cell lines PC-3 and PC-3M were, respectively, 5.3+/-0.1 and 3.8+/-0.2 times that detected in the benign cell line PNT-2. Immunohistochemical staining in 106 cases showed that amongst seven normal cases, one (14.3%) was unstained, five (71.4%) stained weakly positive and one (14.3%) stained moderately positive. Amongst 34 benign prostate hyperplastic (BPH) cases, 12 (35.3%) were unstained, 18 (52.9%) stained weakly positive and four (11.8%) stained moderately positive. Amongst 65 carcinomas, three (4.6%) were unstained, 14 (21.5%) stained weakly positive, 19 (29.2%) stained moderately positive and 29 (44.9%) stained strongly positive. AGR2 expression in carcinomas was significantly higher than that in BPH (chi(2)-test, P<0.001). Kaplan-Meier survival analysis showed that increased AGR2 expression was significantly (log rank test, P=0.007) associated with reduced patient-survival time. Increased joint Gleason score (GS) was significantly (log rank test, P=0.001) associated with poor patient survival. However, neither prostate specific antigen (PSA) level, nor androgen receptor (AR) index, was significantly associated with patient-survival time. Increased AGR2 expression was significantly correlated with high GS (two-sided Fisher's exact test, P<0.001) and PSA levels (Mann-Whitney U-test, P=0.047), but not significantly related to the level of AR (Mann-Whitney U-test, P=0.286). These results suggest that increased AGR2 expression is a valuable prognostic factor to predict the clinical outcome of the prostate cancer patients.

Expression of AGR-2 in Chicken Oviduct during Laying Period

The chicken oviduct is a dynamic organ that produces secretory proteins such as ovalbumin during the laying period. In this study, we identified oviduct-specific proteins in hens during the egg-laying period by proteomic analysis. Proteins extracted from the magnum of hens of different ages (5, 35, and 65 weeks) were analyzed by two-dimensional gel electrophoresis to compare the intensity of proteins among samples. Approximately 300 spots were detected on each gel. Based on the comparison of image gels, we found that the intensity of eight spots in 35-week magnums was increased at least by 2-fold compared with the others. Five of the eight spots were identified as calumenin, acidic ribosomal phosphoproteins (ARP), prohibitin, heart fatty acid-binding protein, and anterior gradient-2 (AGR-2). In particular, ARP and AGR-2 were highly expressed in 35- week magnums compared with 5- and 65-week magnums. In addition, the level of these proteins was consistent with their RNA levels. Expression of AGR-2 mRNA was detected in the mature magnum, whereas no signal was observed in premature tissue. Among various tissues, expression of AGR-2 mRNA was highest in the magnum, high in the isthmus, and five fold lower in muscle. It was undetectable in the liver and in other tissues (heart and kidney). However, the mRNA levels of other proteins were ubiquitous among tissues. In transcriptional activity of AGR-2, a 3.0 kb fragment of promoter region containing potential estrogen receptor binding sites had enhanced its activity strongly. In conclusion, these results suggest that AGR-2 has functional regulatory roles in the chicken oviduct during the egglaying period.

Transplacental exposure to inorganic arsenic at a hepatocarcinogenic dose induces fetal gene expression changes in mice indicative of aberrant estrogen signaling and disrupted steroid metabolism.

Exposure to inorganic arsenic in utero in C3H mice produces hepatocellular carcinoma in male offspring when they reach adulthood. To help define the molecular events associated with the fetal onset of arsenic hepatocarcinogenesis, pregnant C3H mice were given drinking water containing 0 (control) or 85 ppm arsenic from day 8 to 18 of gestation. At the end of the arsenic exposure period, male fetal livers were removed and RNA isolated for microarray analysis using 22K oligo chips. Arsenic exposure in utero produced significant (p<0.001) alterations in expression of 187 genes, with approximately 25% of aberrantly expressed genes related to either estrogen signaling or steroid metabolism. Real-time RT-PCR on selected genes confirmed these changes. Various genes controlled by estrogen, including X-inactive-specific transcript, anterior gradient-2, trefoil factor-1, CRP-ductin, ghrelin, and small proline-rich protein-2A, were dramatically over-expressed. Estrogen-regulated genes including cytokeratin 1-19 and Cyp2a4 were over-expressed, although Cyp3a25 was suppressed. Several genes involved with steroid metabolism also showed remarkable expression changes, including increased expression of 17beta-hydroxysteroid dehydrogenase-7 (HSD17beta7; involved in estradiol production) and decreased expression of HSD17beta5 (involved in testosterone production). The expression of key genes important in methionine metabolism, such as methionine adenosyltransferase-1a, betaine-homocysteine methyltransferase and thioether S-methyltransferase, were suppressed. Thus, exposure of mouse fetus to inorganic arsenic during a critical period in development significantly alters the expression of various genes encoding estrogen signaling and steroid or methionine metabolism. These alterations could disrupt genetic programming at the very early life stage, which could impact tumor formation much later in adulthood.

P227: Anterior gradient 2 and TFF1 coexpression in breast cancer lymph node metastases from estrogen receptor-negative (ER-) primary tumors contributes to the metastatic phenotype

A more complete understanding of the critical genes regulating the phenotypic characteristics associated with the development of metastatic breast cancer could help impact on the effectiveness of therapy for this disease. In order to identify target genes involved in breast cancer metastasis, we performed a microarray analysis of three metastatic lymph nodes. Thirty-five genes that were most highly expressed as compared to negative control cervical lymph nodes were identifyed. After cytokeratin 19, the second most highly expressed gene was anterior gradient 2 (AGR2), a gene known to be upregulated in estrogen receptor positive (ER+) tumors.

Fetal onset of aberrant gene expression relevant to pulmonary carcinogenesis in lung adenocarcinoma development induced by in utero arsenic exposure.

Arsenic is a human pulmonary carcinogen. Our work indicates that in utero arsenic exposure in mice can induce or initiate lung cancer in female offspring. To define early molecular changes, pregnant C3H mice were given 85 ppm arsenic in drinking water from days 8 to 18 of gestation and expression of selected genes in the fetal lung or in lung tumors developing in adults was examined. Transplacental arsenic exposure increased estrogen receptor-alpha (ER-alpha) transcript and protein levels in the female fetal lung. An overexpression of various estrogen-regulated genes also occurred, including trefoil factor-3, anterior gradient-2, and the steroid metabolism genes 17-beta-hydroxysteroid dehydrogenase type 5 and aromatase. The insulin growth factor system, which can be influenced by ER and has been implicated in the pulmonary oncogenic process, was activated in fetal lung after gestational arsenic exposure. In utero arsenic exposure also induced overexpression of alpha-fetoprotein, epidermal growth factor receptor, L-myc, and metallothionein-1 in fetal lung, all of which are associated with lung cancer. Lung adenoma and adenocarcinoma from adult female mice exposed to arsenic in utero showed widespread, intense nuclear ER-alpha expression. In contrast, normal adult lung and diethylnitrosamine-induced lung adenocarcinoma showed little evidence of ER-alpha expression. Thus, transplacental arsenic exposure at a carcinogenic dose produced aberrant estrogen-linked pulmonary gene expression. ER-alpha activation was specifically associated with arsenic-induced lung adenocarcinoma and adenoma but not with nitrosamine-induced lung tumors. These data provide evidence that arsenic-induced aberrant ER signaling could disrupt early life stage genetic programing in the lung leading eventually to lung tumor formation much later in adulthood.

Characterization of peptide aptamers to the Anterior Gradient 2: a novel inhibitor of the tumour suppressor protein p53

Anterior Gradient-2 was identified using proteomic technologies as a protein overexpressed in human cancers. We show here properties of AG2 suggesting it has proto-oncogenic properties:(i) the AG2 protein and gene are unregulated in a tamoxifen resistance panel of breast cancer cells lines, (ii) cell lines overproducing AG2 have an elevated clonogenic activity in vitro and also increase cell growth in a xenograft model, (iii) AG2 protein inhibits the tumour suppressor protein p53, and (iv) AG2 localizes to the endoplasmic reticulum, suggesting a proto-onocogenic signalling pathway exists from endoplasmic reticulum to the nucleus to inhibit p53. To validate the AG2-mediated endoplasmic reticulum pathway as a possible drug target, we developed peptide aptamers to AG2 protein in order to determine whether the oncogenic properties of the protein can be altered by the peptide ligand. These studies hold promise for developing new types of drugs that can release and reactivate the tumour suppressor p53 in breast cancers.

Prognostic Relevance of AGR2 Expression in Breast Cancer

We aimed to evaluate the expression of the human anterior gradient-2 (AGR2) in breast cancer on RNA and protein level and to correlate it with clinicopathologic data, including patient survival. AGR2 mRNA expression was assessed by reverse transcription-PCR in 25 breast cancer samples and normal tissues. A polyclonal rabbit AGR antiserum was used for immunohistochemistry on 155 clinicopathologically characterized cases. Statistical analyses were applied to test for prognostic and diagnostic associations. Immunohistochemical detection of AGR2 was statistically significantly associated with positive estrogen receptor status and lower tumor grade. AGR2-positive tumors showed significantly longer overall survival times in univariate analyses. For the subgroup of nodal-negative tumors, an independent prognostic value of AGR2 was found. The expression of AGR2 in breast cancer is strongly associated with markers of tumor differentiation (estrogen receptor positivity, lower tumor grade). A prognostic effect of AGR2 for overall survival could be shown, which became independently significant for the group of nodal-negative tumors.

Transcriptome profiling the gills of amoebic gill disease (AGD)-affected Atlantic salmon (Salmo salarL.): a role for tumor suppressor p53 in AGD pathogenesis?

Neoparamoeba spp. are amphizoic amoebae with the capacity to colonize the gills of some marine fish, causing AGD. Here, the gill tissue transcriptome response of Atlantic salmon (Salmo salar L.) to AGD is described. Tanks housing Atlantic salmon were inoculated with Neoparamoeba spp. and fish sampled at time points up to 8 days postinoculation (pi.). Gill tissues were taken from AGD-affected fish, and a DNA microarray was used to compare global gene expression against tissues from AGD-unaffected fish. A total of 206 genes, representing 190 unique transcripts, were reproducibly identified as up- or downregulated in response to Neoparamoeba spp. infection. Informative transcripts having GO biological process identifiers were grouped according to function. Although a number of genes were placed into each category, no distinct patterns were observed. One Atlantic salmon cDNA that was upregulated in infected gill relative to noninfected gill at 114 and 189 h pi. showed significant identity with the Xenopus, mouse, and human anterior gradient-2 (AG-2) homologs. Two Atlantic salmon AG-2 mRNA transcripts, designated asAG-2/1 and asAG-2/2, were cloned, sequenced, and shown to be predominantly expressed in the gill, intestine, and brain of a healthy fish. In AGD-affected fish, differential asAG-2 expression was confirmed in samples used for microarray analyses as well as in AGD-affected gill tissue taken from fish in an independent experiment. The asAG-2 upregulation was restricted to AGD lesions relative to unaffected tissue from the same gill arch, while p53 tumor suppressor protein mRNA was concurrently downregulated in AGD lesions. Differential expression of p53-regulated transcripts, proliferating cell nuclear antigen and growth arrest and DNA damage-inducible gene-45beta (GADD45beta) in AGD lesions, suggests a role for p53 in AGD pathogenesis. Thus AGD may represent a novel model for comparative analysis of p53 and p53-regulated pathways.

ANTERIOR GRADIENT-2 IS OVER-EXPRESSED IN PANCREATIC CANCER AND ITS SILENCING REDUCED CELL GROWTH, MIGRATION, INVASION AND SURVIVAL

Background: Pancreatic cancer is a major oncological challenge, due to its aggressive growth and extensive metastasis. Our microarray data showed over-expression of the human homologue of Anterior Gradient-2 (hAG-2) in pancreatic cancer when compared with normal pancreas or samples of chronic pancreatitis. hAG-2, a gene of differentiation, is associated with ER positive breast tumors. To date, nothing is known about the biological significance of its expression in pancreatic cancer. In the current study, we sought to determine the role of this molecule in pancreatic cancer. Methods: RT-PCR, Western blotting and IHC were used to assess hAG-2 expression in tissue samples and cell lines. hAG-2 was silenced in MPanc96 cells by shRNA technique. Growth was assessed by MTS method, migration by Corning migration chamber and invasion by Biocoat Matrigel Invasion assay. The influences of hAG-2 on survival were studied after serum starvation. Results: hAG-2 mRNA was expressed 14 fold higher in pancreatic cancer compared to normal pancreas and pancreatitis, with elevated expression in 4/7 pancreatic cancer cell lines. RT-PCR confirmed the microarray data and IHC revealed expression of hAG-2 specifically in neoplastic cells. Western blot analysis of conditioned media from cell lines indicated that hAG-2 is a secretory protein. Stable transfection with an shRNA for hAG-2 reduced the levels in Mpanc96 cells as indicated by Western blotting. Silencing of hAG-2 reduced the survival of Mpanc96 cells by 50% and the rate of cell growth to 62% of control. Cell migration and invasion were also reduced by 77% and 80%, respectively, in hAG-2 silenced cells. Conclusion: hAG-2 is highly expressed in pancreatic cancer, influences cell growth, survival, motility and invasion and thus might contribute to pancreatic cancer aggressiveness.

Expression of morphogenic genes in mature ovarian and testicular tissues: Potential stem‐cell niche markers and patterning factors

Morphogens are developmental regulators that modulate different tissue patterning, proliferation, differentiation, or remodeling processes in embryonic and adult tissues. Morphogens may also evoke specific regulatory programs in stem cells. Some of the morphogens involved in these processes have been characterized, while others remain unidentified. A microarray containing 3,557 salmonid cDNAs was used to compare the transcriptomes of rainbow trout precocious ovary at three different stages during second year (June, August, and October) with a reference (June normal ovary) transcriptome. During this study, we detected morphogen transcript hybridizations to salmonid elements and the study was enlarged to investigate these activities in various developmental stages of both ovary and testis. Genes from diverse development regulator families such as Anterior gradient-2, BMP, Epimorphin, Flightless, Frizzled, Notch, Tiarin, Twisted gastrulation, and Wnt were demonstrated to be expressed in the adult trout gonads. In mice or rats, expression of mammalian bmp-4, epimorphin, flightless, twisted gastrulation, and GW112 transcripts were localized to cell types isolated from the developed ovary and testis. Comparisons of salmonid and mammalian morphogens at the amino acid residue level show high similarities, suggesting functional conservation. This report provides evidence for local regulation by various morphogens and their potential to control distinct programs of gene expression in the gametes and their accessory cells during gametogenesis.

Human Homologue of Cement Gland Protein, a Novel Metastasis Inducer Associated with Breast Carcinomas

A suppression subtractive cDNA library representing mRNAs expressed at a higher level in the malignant human breast cancer cell line, MCF-7, relative to a benign breast tumor-derived cell line, Huma 123, contained a cDNA, M36, which was expressed in estrogen receptor alpha (ERalpha)-positive breast carcinoma cell lines but not in cell lines from normal/benign/ERalpha-negative malignant breast lesions. M36 cDNA had an identical coding sequence to anterior gradient 2 (AGR2), the human homologue of the cement gland-specific gene (Xenopus laevis). Screening of breast tumor specimens using reverse transcription-PCR and immunocytochemistry with affinity-purified anti-AGR2 antibodies showed that the presence of AGR2 mRNA and protein were both statistically significantly associated with ERalpha-positive carcinomas (P = 0.007, Fisher's exact test) and with malignancy (P < or = 0.025). When an expression vector for AGR2 cDNA was introduced into benign nonmetastatic rat mammary tumor cells, and three separate clones and two pools of cells were transferred to the mammary glands of syngeneic hosts, there were no consistent differences in the mean latent periods of tumor formation. However, metastases occurred in the lungs of animals receiving the AGR2 transfectants in 77% to 92% of animals with primary tumors (P = 0.0001) compared with no metastases in the control groups. The AGR2 transfectants exhibited enhanced rates of adhesion to a plastic substratum and extracellular AGR2 enhanced the rate of attachment of AGR2-negative but not AGR2-positive cells. These experiments are the first to link mechanistically the developmental gene product, AGR2, with metastasis in vivo.

The Barrett’s Antigen Anterior Gradient-2 Silences the p53 Transcriptional Response to DNA Damage

The esophageal epithelium is subject to damage from bile acid reflux that promotes normal tissue injury resulting in the development of Barrett's epithelium. There is a selection pressure for mutating p53 in this preneoplastic epithelium, thus identifying a physiologically relevant model for discovering novel regulators of the p53 pathway. Proteomic technologies were used to identify such p53 regulatory factors by identifying proteins that were overexpressed in Barrett's epithelium. A very abundant polypeptide selectively expressed in Barrett's epithelium was identified as anterior gradient-2. Immunochemical methods confirmed that anterior gradient-2 is universally up-regulated in Barrett's epithelium, relative to normal squamous tissue derived from the same patient. Transfection of the anterior gradient-2 gene into cells enhances colony formation, similar to mutant oncogenic p53 encoded by the HIS175 allele, suggesting that anterior gradient-2 can function as a survival factor. Deletion of the C-terminal 10 amino acids of anterior gradient-2 neutralizes the colony enhancing activity of the gene, suggesting a key role for this domain in enhancing cell survival. Constitutive overexpression of anterior gradient-2 does not alter cell-cycle parameters in unstressed cells, suggesting that this gene is not directly modifying the cell cycle. However, cells overexpressing anterior gradient-2 attenuate p53 phosphorylation at both Ser(15) and Ser(392) and silence p53 transactivation function in ultraviolet (UV)-damaged cells. Deletion of the C-terminal 10 amino acids of anterior gradient-2 permits phosphorylation at Ser(15) in UV-damaged cells, suggesting that the C-terminal motif promoting colony survival also contributes to suppression of the Ser(15) kinase pathway. These data identify anterior gradient-2 as a novel survival factor whose study may shed light on cellular pathways that attenuate the tumor suppressor p53.