Anterior Gradient Protein 2 Research Articles

7274 Assessing The Interactome Of Hexose-6-Phosphate Dehydrogenase In Breast Cancer Cells And Identifying Anterior Gradient Protein 2 As Interacting Partner By Using A BioID Method

Abstract Disclosure: G. Sakalauskaite: None. M. Weingartner: None. T. Bock: None. J. Birk: None. M. Tsachaki: None. A. Odermatt: None. Hexose-6-phosphate dehydrogenase (H6PD) catalyzes the first two steps of the pentose-phosphate pathway in the endoplasmic reticulum (ER). It is so far the only enzyme shown to generate NADPH in the ER. H6PD is involved in physiological processes such as glucose and steroid metabolism, redox regulation, and response to ER-stress. So far, only one H6PD interacting partner, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), has been reported. This enzyme uses NADPH for its well-reported role in glucocorticoid activation. However, H6PD has 11β-HSD1-independent functions, such as the recently described modulation of breast cancer cell properties. Here, we adapted the BioID approach to identify novel H6PD interacting partners in the ER. First, we generated a triple negative breast cancer cell line MDA-MB 231 stably expressing a H6PD-biotin ligase fusion protein. Then, we validated the method to confirm the known interaction between H6PD and 11β-HSD1. Next, we analyzed enriched biotinylated proteins by mass spectrometry and further assessed potential candidates by co-immunoprecipitation and functional assays. The resulting interactome revealed a potential hit cluster of protein disulfide isomerase (PDI) family members, different ER-resident chaperones and luminal calcium binding proteins. Due to its association with breast cancer, we further examined the PDI Anterior gradient protein 2 (AGR2) as H6PD interacting partner. Physical interaction between AGR2 and H6PD was confirmed by co-immunoprecipitation assay in MCF7 cells. Downregulation of AGR2 by siRNA resulted in elevated H6PD protein levels but decreased activity. Furthermore, increased H6PD activity was observed when this protein was co-expressed with AGR2 in HEK-293 cells. In conclusion, the results validated the use of the BioID approach to identify protein interactions within the ER and they revealed that AGR2 physically interacts with H6PD, thereby controlling its expression and enzyme activity. Presentation: 6/3/2024

AGR2: The Covert Driver and New Dawn of Hepatobiliary and Pancreatic Cancer Treatment.

The anterior gradient protein 2 (AGR2) plays a crucial role in facilitating the formation of protein disulfide bonds within the endoplasmic reticulum (ER). Research suggests that AGR2 can function as an oncogene, with its heightened expression linked to the advancement of hepatobiliary and pancreatic cancers through invasion and metastasis. Notably, AGR2 not only serves as a pro-oncogenic agent but also as a downstream targeting protein, indirectly fostering cancer progression. This comprehensive review delves into the established functions and expression patterns of AGR2, emphasizing its pivotal role in cancer progression, particularly in hepatobiliary and pancreatic malignancies. Furthermore, AGR2 emerges as a potential cancer prognostic marker and a promising target for immunotherapy, offering novel avenues for the treatment of hepatobiliary and pancreatic cancers and enhancing patient outcomes.

Modeling of new markers for the diagnosis and prognosis of pancreatic cancer based on the transition from inflammation to cancer.

Pancreatic adenocarcinoma (PAAD) is a lethal disease with a poor prognosis. Genes involved in acute pancreatitis (AP) or chronic pancreatitis (CP) might be important for PAAD development. This study sought to identify potential PAAD diagnosis markers and to establish a PAAD prognosis prediction model based on AP- and CP-related genes. The significantly differentially expressed genes in both AP or CP and PAAD were obtained by a bioinformatics analysis. A risk-score model for predicting survival was constructed based on The Cancer Genome Atlas (TCGA) data and validated using an International Cancer Genome Consortium (ICGC) cohort. Protein expression and the effects of the genes in the risk models were validated by immunohistochemistry, or Cell Counting Kit-8 (CCK-8) and transwell assays. The study sample data included six AP tissue samples and five normal pancreatic tissue samples, six CP tissue samples and six normal pancreatic tissue samples from the Gene Expression Omnibus (GEO) expression profiling microarrays GSE109227 and GSE41418 data sets, respectively, and fragments per kilobase per million mapped fragments (FPKM) data from four normal controls and 150 PAAD cases from TCGA database, and 182 cancer patient samples with complete survival prognostic data from the ICGC database. In total, 508 significantly differentially expressed genes were found in both AP or CP and PAAD. Trefoil factor 2 (TFF2), tubulointerstitial nephritis antigen (TINAG), trefoil factor 1 (TFF1), aquaporin 5 (AQP5), SAM pointed domain containing ETS transcription factor (SPDEF), anterior gradient protein 2 (AGR2), apolipoprotein B messenger RNA editing enzyme catalytic subunit 1 (APOBEC1), kallikrein-related peptidase 6 (KLK6), dopa decarboxylase (DDC), mucin 13 (MUC13), claudin 18 (CLDN18), annexin A10 (ANXA10), and tetraspanin 1 (TSPAN1) were found to be present in PAAD and had the largest fold change. A risk-score model, comprising 19 genes, was constructed for prognostic prediction. A high-risk score indicated a poor prognosis. TINAG, DDC, SPDEF, and APOBEC1 proteins were increased in PAAD, while TINAG and DDC were correlated with the pathologic grade. Decreased TINAG, APOBEC1, transmembrane protein 94 (TMEM94), and kelch like family member 36 (KLHL36) expression inhibited PAAD cell proliferation, while decreased SPDEF, TMEM94, and KLHL36 expression significantly inhibited PAAD cell migration. The AP and CP co-related genes were significantly correlated with PAAD. TINAG, DDC, SPDEF, and APOBEC1 could serve as new PAAD predictors. The risk model developed in this study could be used to predict the prognosis of PAAD patients.

The relationship between AGR2 levels and intestinal barrier function in high-fat diet animal models.

This study aimed to observe the effect of anterior gradient protein 2 (AGR2) levels on intestinal barrier function in HFD animal models. For this purpose, thirty healthy male clean-grade C57BL/6 mice were randomly separated into a normal control group and a high-fat group. The normal control group was fed a normal diet, while the high-fat group was fed an HFD for a total of 8 weeks. It collected body weight changes before and after modeling of two groups of rats and serum samples and detected fasting blood glucose, total cholesterol, triglycerides, AGR2, and diamine oxidase (DAO) concentrations. It collected the expression levels of AGR2 in the colon of rats after modeling, evaluated the permeability of the colon and small intestine barrier by Ussing chamber and Evan's blue (EB) methods, and analyzed the correlation between AGR2 levels and intestinal barrier function using Pearson correlation. Results showed that when the two groups of mice were fed for 8 weeks, their body weight, fasting blood glucose, total cholesterol, and triglycerides all met the characteristics of an HFD mouse model, and the model was successfully established. When the two groups of mice were fed for 8 weeks, the serum AGR2 concentration, relative expression of AGR2 in colon tissue, Gt, and EB content of the high-fat group mice were higher than those of the normal control group, and the difference was significant (P<0.05); Meanwhile, the serum DAO concentration and Isc of the high-fat group mice were lower than those of the normal control group, with statistically significant differences (P<0.05); The relative expression levels of serum AFR2 and colon AGR2 were negatively correlated with Isc (r=-0.503, -0.623, P<0.05), and positively correlated with Gt (r=0.461, 0.560, P<0.05). There was a homogeneous distribution characteristic between the relative expression levels of serum AFR2 and serum DAO, colon AGR2, and Isc variables. It was concluded that HFD could upregulate the expression of AGR2 in mice, downregulate the level of DAO, and damage the intestinal barrier function of mice. Both serum AGR2 concentration and colonic AGR2 relative expression can participate in the regulation of colonic intestinal barrier function and can serve as potential indicators for evaluating intestinal barrier damage.

Pancreatic ductal adenocarcinoma with acinar-to-ductal metaplasia-like cancer cells shows increased cellular proliferation

Background/objectivesAcinar-to-ductal metaplasia (ADM) has been shown to contribute to the development of pancreatic ductal adenocarcinoma (PDAC) in genetically engineered mouse models, but little is known about whether acinar cell plasticity contributes to carcinogenesis in human PDAC. We aimed to assess whether cancer cells that stain positive for amylase and CK19 (ADM-like cancer cells) are present in human resected PDAC and to investigate their role in tumor progression. MethodsWe immunohistochemically investigated the presence of ADM-like cancer cells, and compared the clinical and histological parameters of PDAC patients with and without ADM-like cancer cells. ResultsADM-like cancer cells were detected in 16 of 60 (26.7%) PDAC specimens. Positive staining for anterior gradient protein 2 (AGR2) was observed in 14 of 16 (87.5%) PDAC specimens with ADM-like cancer cells. On the other hand, the intensity of AGR2 expression (negative, low/moderate or high) was lower in PDAC with ADM-like cancer cells (9/7) than in PDAC without these cells (11/33) (P = 0.032). The presence of ADM-like cancer cells was significantly correlated with increased cell proliferation (P = 0.012) and tended to be associated with MUC1 expression (P = 0.067). ConclusionsThese results indicated that acinar cells may act as the origin of human PDAC, and that their presence may be useful for the stratification of human PDAC to predict prognosis.

Anterior gradient protein 2 is a marker of tumor aggressiveness in breast cancer and favors chemotherapy‑induced senescence escape.

Among the different chemotherapies available, genotoxic drugs are widely used. In response to these drugs, particularly doxorubicin, tumor cells can enter into senescence. Chemotherapy-induced senescence (CIS) is a complex response. Long described as a definitive arrest of cell proliferation, the present authors and various groups have shown that this state may not be complete and could allow certain cells to reproliferate. The mechanism could be due to the activation of new signaling pathways. In the laboratory, the proteins involved in these pathways and triggering cell proliferation were studied. The present study determined a new role for anterior gradient protein 2 (AGR2) in vivo in patients and in vitro in a senescence escape model. AGR2's implication in breast cancer patients and proliferation of senescent cells was assessed based on a SWATH-MS proteomic study of patients' samples and RNA interference technology on cell lines. First, AGR2 was identified and it was found that its concentration is higher in the serum of patients with breast cancer and that this high concentration is associated with metastasis occurrence. An inverse correlation between intratumoral AGR2 expression and the senescence marker p16 was also observed. This observation led to the study of the role of AGR2 in the CIS escape model. In this model, it was found that AGR2 is overexpressed in cells during senescence escape and that its loss considerably reduces this phenomenon. Furthermore, it was shown that the extracellular form of AGR2 stimulated the reproliferation of senescent cells. The power of proteomic analysis based on the SWATH-MS approach allowed the present study to highlight the mammalian target of rapamycin (mTOR)/AKT signaling pathway in the senescence escape mechanism mediated by AGR2. Analysis of the two signaling pathways revealed that AGR2 modulated RICTOR and AKT phosphorylation. All these results showed that AGR2 expression in sera and tumors of breast cancer patients is a marker of tumor progression and metastasis occurrence. They also showed that its overexpression regulates CIS escape via activation of the mTOR/AKT signaling pathway.

Serum Level of Tumor-Overexpressed AGR2 Is Significantly Associated with Unfavorable Prognosis of Canine Malignant Mammary Tumors.

Simple SummaryCanine malignant mammary tumor (MMT) is a prevalent malignancy in intact female dogs. A current lack of easily accessible tumor biomarkers hinders a timely assessment of the disease outcome. This study reveals that anterior gradient protein 2 (AGR2) is overexpressed in canine MMT tissues, and elevated levels of extracellular AGR2 in sera of MMT dogs are significantly associated with progression and remote metastasis of MMT and an unfavorable overall survival of the patients. Hence, serum eAGR2 level is significantly associated with an adverse outcome of MMT dogs and holds a predictive potential in MMT prognosis.Canine malignant mammary tumors (MMTs) are prevalent malignancy in intact female dogs with a high incidence of metastasis and recurrence. A current lack of easily accessible tumor biomarkers hinders a timely assessment of the disease outcome. We previously identified anterior gradient protein 2 (AGR2) with higher protein abundance in canine MMT tissues compared with normal counterparts. AGR2 is an endoplasmic reticulum-resident protein disulfide isomerase involved in the regulation of protein processing and also exists extracellularly via secretion to exert pro-oncogenic functions. In the present study, we validated overexpression of AGR2 in canine MMT tissues from 45 dogs using immunohistochemistry and immunoblotting, and assessed serum AGR2 levels in 81 dogs with MMTs and 21 benign cases using a competitive enzyme-linked immunosorbent assay (ELISA). Our data revealed that serum eAGR2 levels are significantly correlated with MMT progression (p = 0.0007) and remote tumor metastasis (p = 0.002). Moreover, elevated levels of serum eAGR2 are associated with an unfavorable overall survival of MMT dogs in later stage (p = 0.0158). Area under the time-dependent ROC curve (AUC) of serum eAGR2 level as a prognostic indicator was 0.839. Collectively, this study uncovered that serum eAGR2 level is significantly associated with an adverse outcome of MMT dogs and holds a predictive potential in MMT prognosis.

Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2).

Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.

Anterior Gradient Protein 2 Promotes Mucosal Repair in Pediatric Ulcerative Colitis.

Mucosal healing comprises a key goal of ulcerative colitis (UC) treatment. Anterior gradient protein 2 (AGR2) plays an important role in maintaining intestinal homeostasis in UC. However, the role of AGR2 in the repair of mucosal injury is not yet clear. This study is aimed at investigating the expression of AGR2 in the intestinal tissues of children with UC and its role in repairing mucosal injury. Forty UC patients who were hospitalized in the Pediatric Gastroenterology Ward of Shengjing Hospital affiliated with China Medical University between July 1, 2013, and May 31, 2020, and 20 children who had normal colonoscopy results during the same period (control group) made up the study sample. The disease activity of UC was evaluated based on the pediatric ulcerative colitis activity index, and the ulcerative colitis endoscopic index was evaluated according to the Rachmilewitz score. Immunohistochemical staining was employed to examine the differences in AGR2 expression in the intestinal mucosa between groups. The protective effect of AGR2 in a model of tumor necrosis factor-alpha- (TNF-α-) induced intestinal mucosal barrier injury and the underlying molecular mechanism were explored through in vitro experiments. The results showed that compared with the normal control group, UC patients in the remission or active period had significantly higher expression of AGR2 in the intestine. AGR2 expression was positively correlated with Ki67, an intestinal epithelial cell proliferation marker, but negatively correlated with the degree of endoscopic mucosal injury. In an in vitro model, AGR2 overexpression promoted cell proliferation and migration and inhibited TNF-α-induced intestinal epithelial barrier damage by activating yes-associated protein (YAP). Collectively, our study suggests that AGR2 might serve as a valuable biomarker to help assess the condition and mucosal healing status of UC patients. In vitro, AGR2 promoted the repair of intestinal mucosal barrier injury by activating YAP.

ZEB1/miR-200c/AGR2: A New Regulatory Loop Modulating the Epithelial-Mesenchymal Transition in Lung Adenocarcinomas.

Epithelial-mesenchymal transition (EMT) is a process involved not only in morphogenesis and embryonic development, but also in cancer progression, whereby tumor cells obtain a more aggressive metastatic phenotype. Anterior gradient protein 2 (AGR2) maintains the epithelial phenotype and blocks the induction of EMT, thus playing an undeniable role in tumor progression. However, the mechanism through which AGR2 expression is regulated, not only during EMT, but also in the early stages of cancer development, remains to be elucidated. In the present study, we show an inverse correlation of AGR2 with ZEB1 (zinc finger enhancer binding protein, δEF1) that was verified by analysis of several independent clinical data sets of lung adenocarcinomas. We also identified the ZEB1 binding site within the AGR2 promoter region and confirmed AGR2 as a novel molecular target of ZEB1. The overexpression of ZEB1 decreased the promoter activity of the AGR2 gene, which resulted in reduced AGR2 protein level and the acquisition of a more invasive phenotype of these lung cancer cells. Conversely, silencing of ZEB1 led not only to increased levels of AGR2 protein, but also attenuated the invasiveness of tumor cells. The AGR2 knockout, vice versa, increased ZEB1 expression, indicating that the ZEB1/AGR2 regulatory axis may function in a double negative feedback loop. In conclusion, we revealed for the first time that ZEB1 regulates AGR2 at the transcriptional level, while AGR2 presence contributes to ZEB1 mRNA degradation. Thus, our data identify a new regulatory mechanism between AGR2 and ZEB1, two rivals in the EMT process, tightly associated with the development of metastasis.

Crystal structure of human anterior gradient protein 3.

Oxidative protein folding in the endoplasmic reticulum is catalyzed by the protein disulfide isomerase family of proteins. Of the 20 recognized human family members, the structures of eight have been deposited in the PDB along with domains from six more. Three members of this family, ERp18, anterior gradient protein 2 (AGR2) and anterior gradient protein 3 (AGR3), are single-domain proteins which share sequence similarity. While ERp18 has a canonical active-site motif and is involved in native disulfide-bond formation, AGR2 and AGR3 lack elements of the active-site motif found in other family members and may both interact with mucins. In order to better define its function, the structure of AGR3 is required. Here, the recombinant expression, purification, crystallization and crystal structure of human AGR3 are described.

Establishment and characterization of a novel human cholangiocarcinoma cell line with high metastatic activity.

Cholangiocarcinoma (CCA) is a highly metastatic tumor, and the lung is a common site of metastasis. A greater understanding of the biology of metastases is needed to improve treatment outcomes. Herein, a highly metastatic human CCA subline, KKU-213L5 from an original cell line, KKU-213 that has marginally metastatic ability, was established and characterized. KKU-213L5 was selected invivo through the fifth serial passage of pulmonary metastasized tissues via tail-vein injection in NOD/scid/Jak3 mice. The metastatic abilities of the KKU-213L5 cells were compared with the parental line invitro and invivo. The expression profile of this metastatic cell line was determined using real-time PCR. KKU-213L5 cells were found to possess higher metastatic phenotypes, i.e., growth rates, stem cell surface markers (CD133), migration and invasion characteristics when compared with the parental cells. Compared to the KKU-213 cells, KKU-213L5 cells formed larger tumors in subcutaneous xenografted mice and had a >10-fold increase in lung metastases in the tail-vein injected metastatic mouse model. Mice injected intravenously with KKU-213L5 cells had a significantly shorter survival. Analysis of the expressed genes related to progression of cancer revealed significant upregulation of anterior gradient protein-2 (AGR2) and suppression of KiSS-1 in the KKU-213L5 cells. The association of these two genes with metastasis was affirmed in CCA patient tissues since increased AGR2 expression and decreased KiSS-1 expression were found in higher stage patient tumors. In conclusion, a highly metastatic human CCA cell line was established and characterized. It is plausible that the differential expression between the parental KKU-213 and highly metastatic KKU-213L5 cells may be beneficial to classify novel genes associated with metastasis. The KKU-213L5 cell line should serve as a valued device for discovering the molecular mechanisms of CCA metastasis and enabling the search for an effective therapy for the unmet clinical need in CCA.

AGR2 is associated with gastric cancer progression and poor survival.

Anterior gradient protein 2 (AGR2) has been reported as a novel biomarker with a potential oncogenic role. However, its association with the prognosis and survival rate of gastric cancer (GC) has not yet been determined. Therefore, the present study aimed to examine the expression and prognostic significance of AGR2 in patients with GC. Immunohistochemistry was used to analyze AGR2 and cathepsin D (CTSD) protein expression in 436 clinicopathologically characterized GC cases and 92 noncancerous tissue samples. AGR2 and CTSD expression were both elevated in GC lesions compared with noncancerous tissues. In 204/436 (46.8%) GC patients, high expression of AGR2 was positively correlated with the expression of CTSD (r=0.577, P<0.01). Furthermore, several clinicopathological parameters were significantly associated with AGR2 expression level, including tumor size, depth of invasion and TNM stage (P<0.05). Using Kaplan-Meier survival analysis, it was determined that the mean survival time of patients with low levels of AGR2 expression was significantly longer than those with high ARG2 expression (in stages I, II and III; P<0.05). For stage IV disease, no significant difference in survival time was identified. Multivariate survival analysis demonstrated that AGR2 was an independent prognostic factor and was associated in the progression of GC. The findings of the present study indicate that AGR2 expression is significantly associated with location and size of GC, depth of invasion, TNM stage, lymphatic metastasis, vessel invasion, distant metastasis, Lauren's classification, high CTSD expression and poor prognosis. Thus, AGR2 may be a novel GC marker and may present a potential therapeutic target for GC.

Anterior gradient protein 2 expression in high grade head and neck squamous cell carcinoma correlated with cancer stem cell and epithelial mesenchymal transition.

Anterior gradient protein 2 (AGR2) is a novel biomarker with potential oncogenic role. We sought to investigate the diagnostic and prognostic role of AGR2 on head and neck squamous cell carcinoma (HNSCC) with an emphasis on its correlation of cancer stemloid cells (CSC) and epithelial mesenchymal transition (EMT). We found that AGR2 protein levels were higher in HNSCC than in normal oral mucosa. High levels of AGR2 were associated with the T category, pathological grade and lymph node metastasis of HNSCC. Expression of AGR2 increased in recurring HNSCC after radiotherapy and in post cisplatin-based chemotherapeutic tissues. In HNSCC cell lines, knock-down of AGR2 induced apoptosis, reduced sphere formation, and down-regulated Survivin, Cyclin D1, Bcl2, Bcl2l1, Slug, Snail, Nanog and Oct4. In addition, over-expressed AGR2 in transgenic mice with spontaneous HNSCC was associated with lost function of Tgfbr1 and/ or lost function of Pten. In vitro knockdown TGFBR1 in HNSCC cell lines increased AGR2 expression. These results suggest that AGR2 is involved in EMT and self-renewal of CSC and may present a potential therapeutic target (oncotarget) for HNSCC.

Anterior gradient protein 2 promotes survival, migration and invasion of papillary thyroid carcinoma cells.

BackgroundThrough a transcriptome microarray analysis, we have isolated Anterior gradient protein 2 (AGR2) as a gene up-regulated in papillary thyroid carcinoma (PTC). AGR2 is a disulfide isomerase over-expressed in several human carcinomas and recently linked to endoplasmic reticulum (ER) stress. Here, we analyzed the expression of AGR2 in PTC and its functional role.MethodsExpression of AGR2 was studied by immunohistochemistry and real time PCR in normal thyroids and in PTC samples. The function of AGR2 was studied by knockdown in PTC cells and by ectopic expression in non-transformed thyroid cells. The role of AGR2 in the ER stress was analyzed upon treatment of cells, expressing or not AGR2, with Bortezomib and analyzing by Western blot the expression levels of GADD153.ResultsPTC over-expressed AGR2 at mRNA and protein levels. Knockdown of AGR2 in PTC cells induced apoptosis and decreased migration and invasion. Ectopic expression of AGR2 in non-transformed human thyroid cells increased migration and invasion and protected cells from ER stress induced by Bortezomib.ConclusionsAGR2 is a novel marker of PTC and plays a role in thyroid cancer cell survival, migration, invasion and protection from ER stress.

Anterior gradient protein-2 is a regulator of cellular adhesion in prostate cancer.

Anterior Gradient Protein (AGR-2) is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s) has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis.

Hilar cholangiocarcinoma is pathologically similar to pancreatic duct adenocarcinoma: suggestions of similar background and development

Routine experiences suggest that cholangiocarcinomas (CCAs) show different clinicopathological behaviors along the biliary tree, and hilar CCA apparently resembles pancreatic duct adenocarcinoma (PDAC). Herein, the backgrounds for these similarities were reviewed. While all cases of PDAC, hilar CCA, intrahepatic CCA (ICCA) and CCA components of combined hepatocellular-cholangiocarcinoma (cHC-CCA) were adenocarcinomas, micropapillary patterns and columnar carcinoma cells were common in PDAC and hilar CCA, and trabecular components and cuboidal carcinoma cells were common in ICCA and CCA components of cHC-CCA. Anterior gradient protein-2 and S100P were frequently expressed in perihilar CCA and PDAC, while neural cell adhesion molecule and luminal epithelial membrane antigen were common in CCA components of c-HC-CCA. Pdx1 and Hes1 were frequently and markedly expressed aberrantly in PDAC and perihilar CCA, although their expression was rare and mild in CCA components in cHC-CCA and ICCA. Hilar CCA showed a similar postoperative prognosis to PDAC but differed from ICCA and cHC-CCA. Taken together, hilar CCA may differ from ICCA and CCA components of cHC-CCA but have a similar development to PDAC. These similarities may be explained by the unique anatomical, embryological and reactive nature of the pancreatobiliary tract. Further studies of these intractable malignancies are warranted.

AGR2 Expression is Regulated by HIF-1 and Contributes to Growth and Angiogenesis of Glioblastoma

Glioblastoma multiforme (GBM) tumors are the most common type of brain tumors characterized by extensive angiogenesis that is mostly orchestrated by tumor hypoxia. The hypoxia induced factor-1 (HIF-1) transcriptional complex is the "master control switch" for hypoxia. Dysregulation of anterior gradient protein 2 (AGR2) expression is associated with tumor growth and metastasis. Whether AGR2 is a hypoxia-responsive factor and affects tumor progression via angiogenesis remains unknown. Here, we show that GBM cell lines, U87 and LN18, exhibited enhanced hypoxic responses compared with control normal human astrocytes, and a corresponding HIF-1-dependent increase in AGR2 mRNA and protein. Recombinant AGR2 and conditioned medium from GBM cells induced human umbilical vein endothelial cell (HUVEC) migration and tube formation, which were abrogated by anti-AGR2 neutralizing antibodies. Expression of the HIF-1α oxygen-dependent degradation domain mutant in cells resulted in elevated AGR2 levels and an increased ability to induce HUVEC migration and tube formation in vitro and enhanced growth and vascularity of tumor xenografts in vivo, which were prevented by AGR2 knockdown. Taken together, these results indicate that AGR2 expression is regulated by HIF-1 and plays an important role in control of glioblastoma growth and vascularity. Our findings suggest that inhibiting AGR2 may represent a new therapeutic target for anti-angiogenic cancer treatment.

Hilar cholangiocarcinoma and pancreatic ductal adenocarcinoma share similar histopathologies, immunophenotypes, and development-related molecules

Embryologically, intrahepatic small bile ducts arise from hepatic progenitor cells via ductal plates, whereas the pancreato-extrahepatic biliary progenitor cells expressing the transcription factors PDX1 and HES1 are reportedly involved in the development of the extrahepatic biliary tract and ventral pancreas. The expression of cellular markers characteristic of the different anatomical levels of the biliary tree and pancreas, as well as PDX1 and HES1, was examined in cholangiocarcinoma components of combined hepatocellular cholangiocarcinoma (12 cases), intrahepatic cholangiocarcinoma (21 cases), hilar cholangiocarcinoma (25 cases), and pancreatic ductal adenocarcinoma (18 cases). Anterior gradient protein-2 and S100P were frequently expressed in hilar cholangiocarcinoma and pancreatic ductal adenocarcinoma, whereas neural cell adhesion molecule and luminal expression of epithelial membrane antigen were common in cholangiocarcinoma components of combined hepatocellular cholangiocarcinoma. PDX1 and HES1 were frequently and markedly expressed in pancreatic ductal adenocarcinoma and, to a lesser degree, in hilar cholangiocarcinoma, although their expression was rare and mild in cholangiocarcinoma components in combined hepatocellular cholangiocarcinoma. The expression patterns of these molecules in intrahepatic cholangiocarcinoma were intermediate between those in hilar cholangiocarcinoma and cholangiocarcinoma components of combined hepatocellular cholangiocarcinoma. Pancreatic ductal adenocarcinoma and hilar cholangiocarcinoma had a similar expression of mucin, immunophenotypes, as well as transcription factors. Pancreatic ductal adenocarcinoma and hilar cholangiocarcinoma showed similar postoperative prognosis. In conclusion, the similar expression of phenotypes related to pancreatobiliary anatomy and embryology may in part explain why these 2 types of carcinoma present similar clinicopathologic features. Further studies on the carcinogenesis of these carcinomas based on their similarities are warranted.

Anterior gradient protein 2 (AGR2) is an independent prognostic factor in ovarian high-grade serous carcinoma

Ovarian high-grade serous carcinoma (HGSC, type 2 ovarian carcinoma) is a poor prognosis cancer with limited therapeutic options. We aimed to investigate the expression pattern and prognostic potential of the metastasis-promoting protein anterior gradient 2 (AGR2) in primary HGSC. Immunohistochemistry was applied to a cohort of 124 primary HGSCs using tissue microarrays. Additionally, in 48 type 1 carcinomas (low-grade serous (LGSC), endometrioid (EC), clear cell (CCC), and mucinous carcinoma (MC)), AGR2 expression was investigated in an exploratory approach. A strong expression of AGR2 was seen in 15 HGSCs (12.1%) and was significantly linked to shortened overall survival (OS, p = 0.011) and also for progression-free survival (PFS, p = 0.001) in the setting of adjuvant platinum-based chemotherapy (CTX). Multivariate survival analysis including age, stage, and residual tumor after surgery revealed that AGR2 expression was an independent prognostic marker for OS (p = 0.001) and PFS (p = 0.001) in HGSC. In type 1 carcinomas, AGR2 was significantly increased as compared to HGSC (p = 0.001) and was seen in subsets of all histological types, low-grade serous LGSC, EC, CCC, and MC. In particular, strong diffuse staining was seen in LGSC and MC. There was no association between AGR2 and estrogen receptor expression in ovarian type 1 or type 2 carcinomas. AGR2 expression identifies highly aggressive HGSC with a compromised prognosis for which novel therapeutic options are needed. Our data strongly support the further evaluation of AGR2 as a therapeutic target and a potential marker for response to platinum-based CTX in this tumor entity.