Anterior Gradient 2 Research Articles

Abstract 5892: miR-217 sensitizes chronic myelogenous leukemia cells to tyrosine kinase inhibitors by targeting pro-oncogenic anterior gradient 2

Abstract BCR-ABL1 independent mechanisms had been indicated to mediate drug resistance to tyrosine kinase inhibitors (TKIs) in patients with chronic myelogenous leukemia (CML). In this study, we observed increased level of pro-oncogenic anterior gradient 2 (AGR2) in TKIs-resistant CML cells. Silencing of AGR2 in dasatinib-resistant K562 (K562DR) cells led to restored sensitivity to dasatinib both in vitro and in vivo. Exposure to dasatinib induced up-regulation of AGR2 in K562 cells, which indicated a probable treatment related drug resistance. We further investigated the potential interaction between microRNA (miRNA) and AGR2 in K562DR cells, and found that down-regulation of miR-217 was associated with over-expression of AGR2 in K562DR cells. Luciferase reporter assay identified that miR-217 negatively regulated expression of AGR2 through binding 3'-untranslated region (3'-UTR) of AGR2. Forced expression of miR-217 led to decreased expression of AGR2 as well as compromised TKIs-resistant potential of K562DR cells. Similarly, over-expression of miR-217 re-sensitized K562DR cells to dasatinib-treatment in a murine xenograft transplantation model. Taken together, TKIs-treatment induced drug-resistance is correlated with decrease of miR-217 and up-regulation of AGR2. miR-217/AGR2 interaction might be a potential therapeutic target in treating CML patients with TKIs-resistant. Citation Format: Bin Pan, Xiangmin Wang, Kailin Xu, Takayuki Ikezoe. miR-217 sensitizes chronic myelogenous leukemia cells to tyrosine kinase inhibitors by targeting pro-oncogenic anterior gradient 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5892.

Anterior gradient 2 as a supervisory marker for tumor vessel normalization induced by anti-angiogenic treatment.

Anti-angiogenic therapy provides transient tumor vascular normalization, which results in a window of opportunity for improvement of radio- or chemotherapy. Biomarkers indicating this window are required for rationalizing anti-angiogenesis. Anterior gradient 2 (AGR2), the majority of which is secreted from tumor cells, is an easily detected plasma protein. In the present study, it was demonstrated that AGR2 could be applied as a biomarker for the supervision of vascular normalization during anti-angiogenic treatment with gold nanoparticles (AuNPs). Nude mice inoculated with SW620 human colorectal cancer cells were treated with AuNPs. Vessel density, pericyte coverage, vessel permeability, tumor hypoxia, tumor growth and AGR2 secretion were detected following treatment with AuNPs at days 0, 4, 6, 9 and 14. Tumor volume and vessel density were reduced, whereas pericyte coverage was increased, and hypoxia and vessel permeability were improved between days 6–9; however, these improvements decreased by day 14, revealing a time frame for tumor vascular normalization, namely days 4–9, during treatment with AuNPs in mice. AGR2 levels in tumor tissues and plasma were significantly low at day 9, along with vascular normalization; therefore, AGR2 can be used as a potential marker for monitoring tumor vascular normalization during anti-angiogenic treatment.

Association of increased primary breast tumor AGR2 with decreased disease-specific survival.

ObjectiveTumor expression of Anterior Gradient 2 (AGR2), an endoplasmic reticulum protein disulfide isomerase, was associated with decreased breast cancer survival. We aimed to validate the association of tumor AGR2 mRNA expression with disease-specific survival (DSS) and identify differentially expressed signaling pathways between high and low AGR2 expression tumor groups.MethodsPrimary tumor mRNA expression data from the METABRIC study was used to evaluate AGR2 expression as a prognostic factor for DSS while adjusting for survival-determining confounders using Cox proportional-hazards regression. Differentially expressed genes and signaling pathway differences between high and low AGR2 groups were determined by modular enrichment analyses using DAVID and Ingenuity Pathway Analysis.ResultsIncreased tumor AGR2 mRNA expression was associated with decreased DSS among 1,341 women (per each standard deviation increase of AGR2 expression: HR 1.14, 95% CI: 1.01-1.29, P = 0.03). Pathway analyses supported prior experimental studies showing that estrogen receptor 1 (ESR1) regulated AGR2 expression. Canonical signaling pathways significantly differentially represented between high and low AGR2 groups included those involved in inflammation and immunity.ConclusionIncreased primary tumor AGR2 expression was associated with decreased DSS. Pathway analyses suggested that increased AGR2 was associated with endoplasmic reticular homeostasis, possibly allowing tumor cells to overcome hypoxic stress and meet the increased protein demand of tumorigenesis, thereby preventing unfolded protein response-mediated apoptosis.

The expression of anterior gradient-2 and vascular endothelial growth factor-C in prostate cancer and clinical data

The expression of anterior gradient-2 and vascular endothelial growth factor-C in prostate cancer and clinical data

Electrochemical sensing of interaction of anterior gradient-2 protein with peptides at a charged interface

Anterior gradient-2 protein (AGR2) is overexpressed in many human cancers, and this protein presents a novel promising cancer biomarker. We show significant progress in understanding of the electric field effects on AGR2-peptides complexes using constant current chronopotentiometric stripping (CPS) analysis. Surface-attached AGR2-peptide complexes can be disintegrated as a result of their exposure to negative potentials. By controlling the exposure time and temperature, peaks of weakly bound non-specific complexes can be discriminated from tightly bound specific complexes. Using CPS analysis we found that mutant E60A-AGR2 forms weaker complex with peptide aptamer in comparison to wild type AGR2. These data highlight the utility of this method for studying real-time dynamics of surface-attached protein-peptide complexes.

Secreted AGR2 promotes invasion of colorectal cancer cells via Wnt11-mediated non-canonical Wnt signaling

Human anterior gradient-2 (AGR2), a member of protein disulfide isomerase (PDI) family, is present in both intracellular and extracellular compartments. Although AGR2 is overexpressed in various human cancers and reported to promote aggressive tumor features, little is known regarding AGR2′s extracellular functions during tumorigenesis. Here, we demonstrate that secreted AGR2 promotes cell migration and metastasis of colorectal cancer (CRC) in vitro and in vivo. Mechanistically, secreted AGR2 elevated Wnt11 expression, triggering non-canonical Wnt signaling: the Ca2+/Calmodulin-dependent protein kinase II (CaMKII) and c-jun amino-terminal kinase (JNK) pathways. Knockdown of Wnt11 or pretreatment with CaMKII and JNK inhibitors reversed the secreted AGR2′s migration-promoting effect. Further studies revealed that AGR2 antagonized canonical Wnt/β-catenin signaling via activating CaMKII. Collectively, our study uncovers a critical role of Wnt11-mediated non-canonical Wnt signaling (CaMKII and JNK pathways) in secreted AGR2′s promoted migration of CRC cells. These results raise the possibility that secreted AGR2 may be a potential therapeutic target towards inhibiting CRC metastasis.

Pro-metastatic activity of AGR2 interrupts angiogenesis target bevacizumab efficiency via direct interaction with VEGFA and activation of NF-κB pathway

Anterior gradient 2 (AGR2), an endoplasmic reticulum (ER)-resident protein-disulfide isomerase (PDI), is associated with cancer development and malignant progression. Here, we show that high level of AGR2 promotes the aggressive phenotype of prostate cancer (PCa) mouse models developed by either patient-derived xenografts or surgical intra-prostate implantation of PCa cells, associated with enrichment of the blood vessel network in tumor tissues. Angiogenesis markers VEGFR2 and CD34, accompanied with the invasive marker Vimentin, were predominantly stained in metastatic liver tissues. Secreted AGR2 was defined to enhance VEGFR2 activity as evidenced by physical interaction of purified recombinant human AGR2 (rhAGR2) with rhVEGFA through the formation of a disulfide bond. Mutant or deleted thioredoxin motif in rhAGR2 was also unable to bind to rhVEGFA that led to the significant abolishment in the vessel formation, but partially affecting the aggressive process, implicating alternative mechanisms are required for AGR2-conferring metastasis. Cytosolic AGR2 contributed to cell metastasis ascribed to its stabilizing effect on p65 protein, which subsequently activated the NF-κB and facilitated epithelial to mesenchymal transition (EMT). Importantly, GSH and cabozantinib, but not bevacizumab, effectively blocked the pro-angiogenic effect of rhAGR2 in vitro and in vivo, providing evidence that secreted AGR2 acts as a predictive biomarker for selection of angiogenesis-targeting therapeutic drugs based on its levels in the circular system.

FOXM1 activates AGR2 and causes progression of lung adenomas into invasive mucinous adenocarcinomas.

Lung cancer remains one of the most prominent public health challenges, accounting for the highest incidence and mortality among all human cancers. While pulmonary invasive mucinous adenocarcinoma (PIMA) is one of the most aggressive types of non-small cell lung cancer, transcriptional drivers of PIMA remain poorly understood. In the present study, we found that Forkhead box M1 transcription factor (FOXM1) is highly expressed in human PIMAs and associated with increased extracellular mucin deposition and the loss of NKX2.1. To examine consequences of FOXM1 expression in tumor cells in vivo, we employed an inducible, transgenic mouse model to express an activated FOXM1 transcript in urethane-induced benign lung adenomas. FOXM1 accelerated tumor growth, induced progression from benign adenomas to invasive, metastatic adenocarcinomas, and induced SOX2, a marker of poorly differentiated tumor cells. Adenocarcinomas in FOXM1 transgenic mice expressed increased MUC5B and MUC5AC, and reduced NKX2.1, which are characteristics of mucinous adenocarcinomas. Expression of FOXM1 in KrasG12D transgenic mice increased the mucinous phenotype in KrasG12D-driven lung tumors. Anterior Gradient 2 (AGR2), an oncogene critical for intracellular processing and packaging of mucins, was increased in mouse and human PIMAs and was associated with FOXM1. FOXM1 directly bound to and transcriptionally activated human AGR2 gene promoter via the -257/-247 bp region. Finally, using orthotopic xenografts we demonstrated that inhibition of either FOXM1 or AGR2 in human PIMAs inhibited mucinous characteristics, and reduced tumor growth and invasion. Altogether, FOXM1 is necessary and sufficient to induce mucinous phenotypes in lung tumor cells in vivo.

The prognostic value of AGR2 expression in solid tumours: a systematic review and meta-analysis

The prognostic value of anterior gradient-2 (AGR2) in tumours remains inconclusive. Here, we systematically reviewed the literature evidence and assessed the association between AGR2 expression and prognosis in solid tumours. The primary outcomes were overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS)/recurrence-free survival (RFS)/progression-free survival (PFS). All analyses were performed by STATA 12.0, with the hazard ratio (HR) or odds ratios (OR), and 95% confidence interval (CI) as the effect size estimate. A total of 20 studies containing 3285 cases were included. Pooled analyses revealed that AGR2 overexpression had an unfavourable impact on OS (HR 1.93, 95% CI 1.32–2.81) and time to tumour progression (TTP) (DFS/RFS/PFS) (HR 1.60 95% CI 1.06–2.40) in solid tumour patients. Subgroup analyses indicated that AGR2 overexpression in breast cancer patients was significantly associated with poor OS (HR 3.02, 95% CI 1.03–8.81) and TTP (HR 1.93, 95% CI 1.17–3.20). Excluding breast cancer, AGR2 overexpression was also found to have a significant correlation with poor OS in the remaining solid tumour patients (HR 1.51, 95% CI 1.04–2.19). Overall, AGR2 might be a potential biomarker to predict prognosis in solid tumour patients.

Relationship of serum anterior gradient 2 level with clinicopathologic features and prognosis of patients with prostate cancer

Objective To investigate the relationship of preoperative level of serum anterior gradient 2 (AGR2) with clinicopathological features and prognosis of patients with prostate cancer. Methods The serum levels of AGR2 were detected by ELISA in 72 patients with prostate cancer, 30 patients with benign prostatic hyperplasia (BPH) and 20 healthy controls. The receiver operating characteristic (ROC) curve was drawn to determine the optimal cut-off value of clinical diagnosis, and to analyze the diagnostic value of serum AGR2 in prostate cancer. Kaplan-Meier method was used to plot the survival curve. Log-rank test was used to analyze the difference of survival time between the two groups. The effect of AGR2 on the prognosis of prostate cancer patients was analyzed by Cox regression. Results The level of serum AGR2 in prostate cancer group was obviously higher than those in BPH group and health control group (t= 4.441, t= 5.285, both P < 0.01). Serum AGR2 level was correlated with Gleason score, tumor stage, lymph node metastasis and preoperative PSA level (F= 11.343, F= 9.613, t= 3.882, t= 7.514, all P < 0.01). The area under the ROC curve (AUC) of AGR2 was 0.803 (95% CI 0.726-0.880, P= 0.000), when the cut-off value was 17.25 ng/ml, the sensitivity rate was 63.9%, specificity rate was 80.0%. Kaplan-Meier survival analysis showed that the survival rate in low AGR2 expression group was significantly higher than that in high AGR2 expression group (χ 2= 5.565, P= 0.018). Cox regression analysis showed that AGR2 was an independent risk factor for prostate cancer patients (HR= 5.412, 95% CI 1.143-25.624, P= 0.033). Conclusions The elevated level of serum AGR2 is related with tumor progression in prostate cancer. It may be a potential marker for predicting prognosis of patients with prostate cancer. Key words: Prostatic neoplasms; Anterior gradient 2; Enzyme-linked immunosorbent assay; Prognosis

Expression and clinical significance of serum anterior gradient-2 in prostate cancer patients

Objective To determine the level of serum anterior gradient-2 (AGR2) in prostate cancer, and investigate its clinical application value. Methods The serum levels of AGR2 were detected by enzyme-linked immuno sorbent assay (ELISA) in 46 patients with prostate cancer, 20 patients with benign prostatic hyperplasia (BPH) and 20 cases of healthy control. Results ①The levels of serum AGR2 in patients with prostate cancer were obviously higher than that in BPH group and health control group (P 0.05). ②The level of serum AGR2 after treatment was significantly lower than that before treatment (P<0.01). Before operation in patients with prostate cancer, the level of preoperative serum AGR2 was higher than that in post-operation (P<0.01). The level of serum AGR2 before endocrine therapy was higher than after endocrine therapy in patients with prostate cancer (P<0.01). ③The levels of serum AGR2 in prostate cancer were positively correlated with clincal stage and Gleason pathologic stage (P<0.05). The level of AGR2 was positively correlated with PSA (P<0.01). ④The area under the ROC curve (AUC) of AGR2 was 0.792, when the best threshold was 13.75 ng/mL, sensitivity rate was 71.7%, specificity rate was 75%. Conclusions The level of serum AGR2 in prostate cancer is higher, positively correlated with the clinical stage and Gleason pathologic stage. It may be play certain clinical significance in diagnosis, malignant degree and clinical curative effect of judgement for prostate cancer. Key words: Prostatic Neoplasms; Proteins

Anterior gradient 2 is induced in cutaneous wound and promotes wound healing through its adhesion domain.

Anterior gradient 2 (AGR2), a member of protein disulfide isomerase (PDI) family, is both located in cytoplasm and secreted into extracellular matrix. The orthologs of AGR2 have been linked to limb regeneration in newt and wound healing in zebrafish. In mammals, AGR2 influences multiple cell signaling pathways in tumor formation and in normal cell functions related to new tissue formation like angiogenesis. However, the function of AGR2 in mammalian wound healing remains unknown. This study aimed to investigate AGR2 expression and its function during skin wound healing and the possible application of external AGR2 in cutaneous wound to accelerate the healing process. Our results showed that AGR2 expression was induced in the migrating epidermal tongue and hyperplastic epidermis after skin excision. Topical application of recombinant AGR2 significantly accelerated wound-healing process by increasing the migration of keratinocytes (Kera.) and the recruitment of fibroblasts (Fibro.) near the wounded area. External AGR2 also promoted the migration of Kera. and Fibro. invitro in a dose-dependent manner. The adhesion domain of AGR2 was required for the formation of focal adhesions in migrating Fibro., leading to the directional migration along AGR2 gradient. These results indicate that recombinant AGR2 accelerates skin wound healing through regulation of Kera. and Fibro. migration, thus demonstrating its potential utility as an alternative strategy of the therapeutics to accelerate the healing of acute or chronic skin wounds.

Correlation between the level of serum anterior gradient 2 and clinical pathological characteristics of nasopharyngeal carcinoma patients

Objective To determine the levels of serum anterior gradient 2 (AGR2) before and after treatment in nasopharyngeal carcinoma (NPC) patients, and investigate the relationship of AGR2 and clinical pathological characteristics of NPC patients. Methods The serum levels of AGR2 were detected by enzyme linked immunosorbent assay (ELISA) in 55 NPC patients (NPC group) before and after treatment, 30 patients with nasopharyngeal inflammation (inflammation group) and 20 healthy controls (health control group). The correlations between serum AGR2 before and after treatment and clinical pathological characteristics of NPC were analyzed. The NPC patients received radiotherapy and were followed up for 6 months, and the therapeutic effect was evaluated. Results The serum AGR2 levels in NPC group before treatment, inflammation group and health control group were (22.92±5.24)μg/L, (9.50±4.15)μg/L and (8.75±2.18)μg/L respectively, and the difference was statistically significant (F=268.400, P=0.000). The level of serum AGR2 in NPC group was obviously higher than that in inflammation group (t=14.241, P=0.000) and health control group (t=15.254, P=0.000). The level of serum AGR2 in NPC group after treatment was significantly lower than that before treatment [(15.15±10.33)μg/L vs. (22.92±5.24)μg/L, t=12.774, P=0.000]. In NPC patients, serum AGR2 levels of clinical Ⅲ-Ⅳ stage group before and after treatment were higher than those of clinical Ⅰ-Ⅱ stage group (t=5.938, P=0.000; t=0.759, P=0.032). Serum AGR2 levels of lymph node metastasis group before and after treatment were higher than those of no lymph node metastasis group (t=6.879, P=0.000; t=2.700, P=0.009). Serum AGR2 levels of carotid sheath and skull base involvement groups before treatment were higher than those of non-involvement groups (t=8.342, P=0.000; t=8.255, P=0.009). Serum AGR2 levels of cranial nerve involvement group before and after treatment were higher than those of non-involvement group (t=7.743, P=0.000; t=3.021, P=0.004). The serum AGR2 level after treatment in complete response patients [(13.86±2.93)μg/L] was significantly lower than that in partial response patients [(15.85±3.24)μg/L, t=2.267, P=0.028] and invalid patients [(20.65±6.59)μg/L, t=4.935, P=0.000]. The serum AGR2 level in partial response patients was significantly lower than that in invalid patients (t=3.196, P=0.004). Conclusion The level of serum AGR2 in NPC patients increases obviously. AGR2 plays an important role in the genesis and development of NPC, and can be used as a new marker of NPC for judging the clinical therapeutic efficacy and prognosis. Key words: Nasopharyngeal neoplasms; Pathology, clinical; Anterior gradient 2

Tumor-secreted anterior gradient-2 binds to VEGF and FGF2 and enhances their activities by promoting their homodimerization.

The importance of the tumor microenvironment in targeted anticancer therapies has been well recognized. Various protein factors participate in the cross-talk between tumor cells and non-malignant cells. Anterior gradient-2 (AGR2) is overexpressed in diverse human adenocarcinomas and it exists in both intracellular and extracellular spaces. Although intracellular AGR2 has been intensively investigated, the function of secreted AGR2, especially its exact mechanism of action is still poorly understood. Here we report that the secreted AGR2 promotes the angiogenesis and the invasion of vascular endothelial cells and fibroblasts by enhancing the activities of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). Further study indicated that AGR2 directly binds to these extracellular signaling molecules, and enhances their homodimerization. The extracellular AGR2 activity can be blocked to reduce angiogenesis and inhibit tumor growth in vitro and in vivo by a monoclonal antibody targeting the AGR2 self-dimerization region, and combined treatment with bevacizumab produced maximum inhibition effect. In conclusion, our investigation reveals a mechanism that directly links the secreted AGR2 with extracellular signaling networks, and we propose that the secreted AGR2 is a blockable molecular target, which acts as a chaperon-like enhancer to VEGF and FGF2.

Chronopotentiometric sensing of anterior gradient 2 protein

The study of new proteins and particularly those involved in cancer development is still a focal point of interest. We studied the anterior gradient 2 (AGR2) oncoprotein, aberrantly expressed in a number of human cancers. For the first time the electrochemical behaviour of various variants of the AGR2 was described using constant current chronopotentiometric stripping (CPS) analysis at mercury electrodes. In the first part, we show that mutation of AGR2 protein at its sole cysteine significantly changed its CPS response compared to wild type AGR2, probably due to their different adsorption and some deviations in their structures, which were obtained by analysis of hydrogen − deuterium (H/D) exchange connected with high resolution mass spectrometry (MS). In the second part we studied the influence of His-tag modification, widely used in the purification of recombinant proteins, on CPS response and H/D exchange. Addition of a His-tag, containing positively charged and electroactive residues, affected the CPS peak H of His6-tagged AGR2 compared to non-tagged AGR2 protein due to different adsorption as well as variation in the structure at the negatively charged interface. H/D exchange MS analysis confirmed differences in the structure of these two variants even in solution. The uncovering of AGR2 behaviour at charged surfaces gives us the opportunity to study its interactions with other biomedically important proteins.

Targeted epigenetic editing of SPDEF reduces mucus production in lung epithelial cells.

Airway mucus hypersecretion contributes to the morbidity and mortality in patients with chronic inflammatory lung diseases. Reducing mucus production is crucial for improving patients' quality of life. The transcription factor SAM-pointed domain-containing Ets-like factor (SPDEF) plays a critical role in the regulation of mucus production and, therefore, represents a potential therapeutic target. This study aims to reduce lung epithelial mucus production by targeted silencing SPDEF using the novel strategy, epigenetic editing. Zinc fingers and CRISPR/dCas platforms were engineered to target repressors (KRAB, DNA methyltransferases, histone methyltransferases) to the SPDEF promoter. All constructs were able to effectively suppress both SPDEF mRNA and protein expression, which was accompanied by inhibition of downstream mucus-related genes [anterior gradient 2 (AGR2), mucin 5AC (MUC5AC)]. For the histone methyltransferase G9A, and not its mutant or other effectors, the obtained silencing was mitotically stable. These results indicate efficient SPDEF silencing and downregulation of mucus-related gene expression by epigenetic editing, in human lung epithelial cells. This opens avenues for epigenetic editing as a novel therapeutic strategy to induce long-lasting mucus inhibition.

Serum AGR2 as a useful biomarker for pituitary adenomas

ObjectiveThis study aims to evaluate whether the serum Anterior Gradient-2 (AGR2) can be used as a potential biomarker screening in the diagnosis of Pituitary adenomas(PAs). Patients and methodsThe serum AGR2 protein levels were preoperatively measured in 163 PA patients, 43 patients with other sellar lesions excluding PAs, 7 patients with prostate cancer as a positive control and 20 normal people(10 female and 10 male) using Enzyme-Linked ImmunoSorbent Assay (ELISA). Differences in the serum AGR2 level between different groups were analyzed for statistical significance with a Mann-Whitney U test. ResultsThe data showed that serum AGR2 level was significantly higher in the serum of PA patients (250.10±79.14ng/ml) than the patients with other sellar lesions (220.84±79.62ng/ml, P=0.017) and normal people (163.67±50.38ng/ml, P <0.001). Receiver operating characteristic (ROC) curve analysis was used. The detected area under the curve (AUC) was 0.835. The calculated optimal cut-off point for AGR2 level in serum samples was 158.63ng/ml (Youden index=0.564). The sensitivity was 91.4% and the specificity was 65.0%. Despite the variety of PA clinical features, the serum level of AGR2 are definite in PAs, although there may be a difference between male or female patients. ConclusionOur data suggests AGR2 as a potential biomarker for the diagnosis of PAs.

ER stress protein AGR2 precedes and is involved in the regulation of pancreatic cancer initiation

The mechanisms of initiation of pancreatic ductal adenocarcinoma (PDAC) are still largely unknown. In the present study, we analysed the role of anterior gradient-2 (AGR2) in the earliest stages of pancreatic neoplasia. Immunohistochemical analysis of chronic pancreatitis (CP) and peritumoral areas in PDAC tissues showed that AGR2 was present in tubular complexes (TC) and early pancreatic intraepithelial neoplasia (PanINs). Moreover, AGR2 was also found in discrete subpopulations of non-transformed cells neighbouring these pre-neoplastic lesions. In primary cells derived from human patient-derived xenograft (PDX) model, flow-cytometry revealed that AGR2 was overexpressed in pancreatic cancer stem cells (CSC) compared with non-stem cancer cells. In LSL-KrasG12D;Pdx1-Cre (KC) mouse model Agr2 induction preceded the formation of pre-neoplastic lesions and their development was largely inhibited by Agr2 deletion in engineered LSL-KrasG12D;Pdx1-Cre; Agr2−/− mice. In vitro, AGR2 expression was stimulated by tunicamycin-induced endoplasmic reticulum (ER) stress in both KRAS wild-type normal pancreas cells, as well as in KRAS mutated pancreatic cancer cells and was essential for ER homoeostasis. The unfolded protein response proteins GRP78, ATF6 and XBP1s were found expressed in CP and PDAC peritumoral tissues, but in contrast to AGR2, their expression was switched off during TC and PanIN formation. Real-time PCR and ELISA analyses showed that ER stress induced a pro-inflammatory phenotype in pancreatic normal, cancer and stellate cells. Moreover, AGR2 expression was inducible by paracrine transfer of ER stress and pro-inflammation between different pancreatic cell types. Our findings demonstrate that AGR2 induced in ER-stressed and inflammatory pre-neoplastic pancreas is a potential marker of cancer progenitor cells with an important functional role in PDAC initiation.

An efficient method for native protein purification in the selected range from prostate cancer tissue digests.

Prostate cancer (CP) cells differ from their normal counterpart in gene expression. Genes encoding secreted or extracellular proteins with increased expression in CP may serve as potential biomarkers. For their detection and quantification, assays based on monoclonal antibodies are best suited for development in the clinical setting. One approach to obtain antibodies is to use recombinant proteins as immunogen. However, the synthesis of recombinant protein for each identified candidate is time-consuming and expensive. It is also not practical to generate high quality antibodies to all identified candidates individually. Furthermore, non-native forms (e.g., recombinant) of proteins may not always lead to useful antibodies. Our approach was to purify a subset of proteins from CP tissue specimens for use as immunogen. In the present investigation, ten cancer specimens obtained from cases scored Gleason 3+3, 3+4 and 4+3 were digested by collagenase to single cells in serum-free tissue culture media. Cells were pelleted after collagenase digestion, and the cell-free supernatant from each specimen were pooled and used for isolation of proteins in the 10-30 kDa molecular weight range using a combination of sonication, dialysis and Amicon ultrafiltration. Western blotting and mass spectrometry (MS) proteomics were performed to identify the proteins in the selected size fraction. The presence of cancer-specific anterior gradient 2 (AGR2) and absence of prostate-specific antigen (PSA)/KLK3 were confirmed by Western blotting. Proteomics also detected AGR2 among many other proteins, some outside the selected molecular weight range, as well. Using this approach, the potentially harmful (to the mouse host) exogenously added collagenase was removed as well as other abundant prostatic proteins like ACPP/PAP and AZGP1 to preclude the generation of antibodies against these species. The paper presents an optimized scheme for convenient and rapid isolation of native proteins in any desired size range with minor modifications.

Anterior Gradient 2 (AGR2) Induced Epidermal Growth Factor Receptor (EGFR) Signaling Is Essential for Murine Pancreatitis-Associated Tissue Regeneration.

A recently published study identified Anterior Gradient 2 (AGR2) as a regulator of EGFR signaling by promoting receptor presentation from the endoplasmic reticulum to the cell surface. AGR2 also promotes tissue regeneration in amphibians and fish. Whether AGR2-induced EGFR signaling is essential for tissue regeneration in higher vertebrates was evaluated using a well-characterized murine model for pancreatitis. The impact of AGR2 expression and EGFR signaling on tissue regeneration was evaluated using the caerulein-induced pancreatitis mouse model. EGFR signaling and cell proliferation were examined in the context of the AGR2-/- null mouse or with the EGFR-specific tyrosine kinase inhibitor, AG1478. In addition, the Hippo signaling coactivator YAP1 was evaluated in the context of AGR2 expression during pancreatitis. Pancreatitis-induced AGR2 expression enabled EGFR translocation to the plasma membrane, the initiation of cell signaling, and cell proliferation. EGFR signaling and tissue regeneration were partially inhibited by the tyrosine kinase inhibitor AG1478, but absent in the AGR2-/- null mouse. AG1478-treated and AGR2-/- null mice with pancreatitis died whereas all wild-type controls recovered. YAP1 activation was also dependent on pancreatitis-induced AGR2 expression. AGR2-induced EGFR signaling was essential for tissue regeneration and recovery from pancreatitis. The results establish tissue regeneration as a major function of AGR2-induced EGFR signaling in adult higher vertebrates. Enhanced AGR2 expression and EGFR signaling are also universally present in human pancreatic cancer, which support a linkage between tissue injury, regeneration, and cancer pathogenesis.