Anterior Gradient 2 Expression Research Articles

AGR2 is controlled by DNMT3a-centered signaling module and mediates tumor resistance to 5-Aza in colorectal cancer

Human anterior gradient-2 (AGR2), a member of protein disulfide isomerase (PDI) family, is upregulated in various human cancers and reportedly has oncogenic activities. However, the functional roles of AGR2 and its regulation in colorectal cancer (CRC) remain unclear. Here, we showed that AGR2 promoted CRC tumorigenesis and progression in vitro and in vivo and acted as an independent prognostic factor of poor outcome. AGR2 was negatively regulated by DNA methyltransferase 3a (DNMT3a) through directly methylating AGR2 promoter and by a DNMT3a–SPRY2–miR-194 axis. Moreover, AGR2 mediated the resistance to 5-Aza-2′-deoxycytidine (5-Aza) treatment. Knockdown of AGR2 improved the therapeutic effect of 5-Aza in human CRC xenograft tumor model. Thus, our work supports AGR2's oncogenic role in CRC, reveals DNMT3a-mediated epigenetic modulation on AGR2 promoter, and uncovers a new DNMT3a signaling module controlling expression of AGR2. Upregulated AGR2 offset 5-Aza mediated epigenetic therapy. This work might provide potential targets for clinical anti-cancer therapy.

SECRETED PROTEINS AS POTENTIAL BIOMARKERS IN ADENOID CYSTIC CARCINOMA OF THE SALIVARY GLANDS

Secreted proteins are involved in several physiological mechanisms. In solid tumors, it can be used as diagnostic and prognostic tools. Human anterior gradient 2 (AGR2) and CD10 are proteins secreted in body fluids in prostate cancer. The main of this study was to evaluate expression of the secreted proteins AGR2 and CD10 in adenoid cystic carcinoma (ACC) of the salivary gland. A total of 20 cases of ACC of the salivary glands were examined by immunohisto-chemistry method. Female was more affected (70%) with age varying from 10 to 79. Tumor sizes ranged from 1.3 to 9 (mean 2.7 cm), located mostly in minor salivary glands and submandibular gland. Eleven cases showed neural invasion. AGR2 was typically citoplasmatic and focally expressed in 75% of cases. Its expression was observed in all solid subtype, followed by tubular (88.8%) and cribiform (55.5%). Interestingly, half of ACC exhibited AGR2 expression in the extracellular space or inside ductiform structures. Clinically, neural invasion, nodal involvement, and systemic metastasis were associated to AGR2 expression. The surface protein CD10 was also focally expressed mainly in ductal structures in solid and tubular subtypes. Interestingly, its expression was restricted only to stroma in cribriform subtype. This secreted protein was also found freely inside ductiform structures. Furthermore, peripheral nerves involved in the tumor significantly expressed CD10 (p = 0.029) in the tubular subtype. Conclusion Neural invasion may involve participation of CD10 in ACC of the salivary gland. Further studies should confirm if extracellular AGR2 and CD10 represent potential biomarkers for salivary detection as diagnostic and prognostic tools in the clinic.

AGR2 is a target of canonical Wnt/β-catenin signaling and is important for stemness maintenance in colorectal cancer stem cells

Colorectal cancer is one of the leading causes of cancer-related deaths. Due to relapse after current therapy regimens, cancer stem cells (CSCs) are being studied to target this small tumor-initiating population. Anterior gradient 2 (AGR2), a disulfide isomerase protein, is a well-known pro-oncogenic/metastatic oncogene overexpressed in various tumor tissues, including colon cancer. We found that AGR2 was a novel stem cell marker that was regulated by the canonical Wnt/β-catenin pathway in colon CSCs. AGR2 was highly co-expressed with surface stem cell markers in spheroidal culture. Silencing of AGR2 resulted in decreased sphere-forming ability and down-regulated expression of stem cell markers, whereas the opposite effects were seen with AGR2 overexpression. Moreover, patients with high β-catenin and AGR2 expression showed lower overall survival than those with low expression. In conclusion, our study describes a novel role for AGR2 as a stem cell marker that is highly regulated by canonical Wnt/β-catenin signaling in colorectal cancer.

The role of the XBP-1/AGR2 signaling pathway in the regulation of airway Mucin5ac hypersecretion under hypoxia

Oversecretion of Mucin5ac (MUC5AC), which is primarily synthesized by goblet cells and is the major gel-forming mucin, is a hallmark of various pulmonary inflammatory diseases. Hypoxia is considered a common pathophysiologic feature in various pulmonary inflammatory diseases. It has been suggested that hypoxia-inducible factor 1α (HIF-1α) acts as a key factor in hypoxia-induced MUC5AC hypersecretion; however, the exact mechanisms that maintain the stability of HIF-1α and support oversecretion by airway epithelial cells under hypoxia are still unclear. With immunohistochemistry, we found overexpression of anterior gradient 2 (AGR2) in the bronchial epithelial cells of hypoxia-treated mice. With specific shRNA transduction, AGR2 was demonstrated to be a key factor in MUC5AC hypersecretion in vitro. Additionally, co-immunoprecipitation, cell immunochemistry and confocal microscopy experiments were performed to explore the interaction between HIF-1α and AGR2 during hypoxia-induced MUC5AC hypersecretion in vitro. The results indicated increased binding and intracytoplasmic colocation of HIF-1α and AGR2. Our findings suggest that AGR2 acts as a key regulator in hypoxia-induced airway MUC5AC hypersecretion by increasing the stability of HIF-1α. Additionally, the elevated expression of AGR2 induced by hypoxia in bronchial epithelial cells likely depends on an XBP-1-associated pathway.

MiR-135b-5p enhances doxorubicin-sensitivity of breast cancer cells through targeting anterior gradient 2

BackgroundThe pro-oncogenic anterior gradient 2 (AGR2) is involved in tumor growth and drug resistance of breast cancer. Mechanisms that regulate expression of AGR2 still need to be elucidated.MethodsIn this study, expression levels of AGR2 and miR-135b-5p were analyzed in different breast cancer cell lines as well as in clinical breast cancer tissues. The in vitro and in vivo functional effect of AGR2 and miR-135b-5p were also investigated. A luciferase reporter assay was applied to confirm the interaction between miR-135b-5p and AGR2 mRNA.ResultsWe identified AGR2 as a target of miR-135b-5p. Expression of AGR2 was up-regulated in doxorubicin-resistant breast cancer cells. AGR2 mediated doxorubicin-sensitivity of breast cancer cells both in vitro and in vivo. miR-135b-5p negatively regulated AGR2-expression of breast cancer cells increasing doxorubicin-sensitivity. However, miR-135b-5p was down-regulated in doxorubicin-resistant breast cancer cells as well as during treatment with doxorubicin, which might be a probable reason for over-expression of AGR2. Up-regulation of miR-135b-5p increased doxorubicin-sensitivity of breast cancer cells in vivo. In addition, levels of AGR2 negatively correlated with levels of miR-135b-5p in clinical breast cancer tissue samples.ConclusionOur results highlight the potential of miR-135b-5p as a target for treating AGR2-expressing breast cancer with doxorubicin-resistance.

Proteasome inhibition boosts autophagic degradation of ubiquitinated-AGR2 and enhances the antitumor efficiency of bevacizumab

Anterior gradient 2 (AGR2), a protein belonging to the protein disulfide isomerase (PDI) family, is overexpressed in multiple cancers and promotes angiogenesis to drive cancer progression. The mechanisms controlling AGR2 abundance in cancer remain largely unknown. Here, we observed that AGR2 expression is significantly suppressed by proteasome inhibitor MG132/bortezomib at mRNA and protein levels in lung cancer cells. MG132-mediated repression of AGR2 transcription was independent of ROS generation and ER stress induction, but partially resulted from the downregulated E2F1. Further investigation revealed that MG132 facilitated polyubiquitinated AGR2 degradation through activation of autophagy, as evidenced by predominant restoration of AGR2 level in cells genetic depletion of Atg5 and Atg7, or by autophagy inhibitors. Activation of autophagy by rapamycin noticeably reduced the AGR2 protein in cells and in the mouse tissue samples administrated with bortezomib. We also provided evidence identifying the K48-linked polyubiquitin chains conjugating onto K89 of AGR2 by an E3 ligase UBR5. In addition, an autophagy receptor NBR1 was demonstrated to be important in polyubiquitinated AGR2 clearance in response to MG132 or bortezomib. Importantly, downregulation of AGR2 by proteasome inhibition significantly enhanced antitumor activity of bevacizumab, highlighting the importance of AGR2 as a predictive marker for selection of subgroup patients in chemotherapy.

MiR-217 sensitizes chronic myelogenous leukemia cells to tyrosine kinase inhibitors by targeting pro-oncogenic anterior gradient 2

BCR-ABL1-independent mechanisms had been thought to mediate drug resistance to tyrosine kinase inhibitors (TKIs) in patients with chronic myelogenous leukemia (CML). The pro-oncogenic anterior gradient 2 (AGR2) mediates drug resistance of cancer cells. In this study, we observed an increased level of AGR2 in TKI-resistant CML cells. Silence of AGR2 in dasatinib-resistant K562 (K562DR) cells led to restored sensitivity to dasatinib both in vitro and in vivo. Exposure to dasatinib induced upregulation 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 downregulation of miR-217 was associated with overexpression of AGR2 in K562DR cells. Luciferase reporter assay identified that miR-217 negatively regulated expression of AGR2 through binding the 3'-untranslated region of AGR2. Hypermethylation of the CpG island on the promoter region of the MIR217 gene is a probable reason for the downregulation of miR-217 in dasatinib-treated K562 cells. Forced expression of miR-217 led to decreased expression of AGR2 as well as compromised TKI-resistant potential of K562DR cells. Similarly, overexpression of miR-217 resensitized K562DR cells to dasatinib treatment in a murine xenograft transplantation model. TKI treatment-induced drug resistance is correlated with a decrease of miR-217 and upregulation of AGR2. The miR-217/AGR2 interaction might be a potential therapeutic target in treating CML patients with TKI resistance.

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.

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 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

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.

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.

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.

MicroRNA-1291 targets the FOXA2-AGR2 pathway to suppress pancreatic cancer cell proliferation and tumorigenesis.

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Better understanding of pancreatic cancer biology may help identify new oncotargets towards more effective therapies. This study investigated the mechanistic actions of microRNA-1291 (miR-1291) in the suppression of pancreatic tumorigenesis. Our data showed that miR-1291 was downregulated in a set of clinical pancreatic carcinoma specimens and human pancreatic cancer cell lines. Restoration of miR-1291 expression inhibited pancreatic cancer cell proliferation, which was associated with cell cycle arrest and enhanced apoptosis. Furthermore, miR-1291 sharply suppressed the tumorigenicity of PANC-1 cells in mouse models. A proteomic profiling study revealed 32 proteins altered over 2-fold in miR-1291-expressing PANC-1 cells that could be assembled into multiple critical pathways for cancer. Among them anterior gradient 2 (AGR2) was reduced to the greatest degree. Through computational and experimental studies we further identified that forkhead box protein A2 (FOXA2), a transcription factor governing AGR2 expression, was a direct target of miR-1291. These results connect miR-1291 to the FOXA2-AGR2 regulatory pathway in the suppression of pancreatic cancer cell proliferation and tumorigenesis, providing new insight into the development of miRNA-based therapy to combat pancreatic cancer.

Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2

Anterior gradient 2 (AGR2) is a cancer-associated secreted protein found predominantly in adenocarcinomas. Given its ubiquity in solid tumors, cancer-secreted AGR2 could be a useful biomarker in urine or blood for early detection. However, normal organs express and might also secrete AGR2, which would impact its utility as a cancer biomarker. Uniform AGR2 expression is found in the normal bladder urothelium. Little AGR2 is secreted by the urothelial cells as no measurable amounts could be detected in urine. The urinary proteomes of healthy people contain no listing for AGR2. Likewise, the blood proteomes of healthy people also contain no significant peptide counts for AGR2 suggesting little urothelial secretion into capillaries of the lamina propria. Expression of AGR2 is lost in urothelial carcinoma, with only 25% of primary tumors observed to retain AGR2 expression in a cohort of lymph node-positive cases. AGR2 is secreted by the urothelial carcinoma cells as urinary AGR2 was measured in the voided urine of 25% of the cases analyzed in a cohort of cancer vs. non-cancer patients. The fraction of AGR2-positive urine samples was consistent with the fraction of urothelial carcinoma that stained positive for AGR2. Since cancer cells secrete AGR2 while normal cells do not, its measurement in body fluids could be used to indicate tumor presence. Furthermore, AGR2 has also been found on the cell surface of cancer cells. Taken together, secretion and cell surface localization of AGR2 are characteristic of cancer, while expression of AGR2 by itself is not.

The involvement of anterior gradient 2 in the stromal cell-derived factor 1-induced epithelial-mesenchymal transition of glioblastoma.

In recent years, it has been widely identified that the stromal cell-derived factor 1 (SDF-1) and anterior gradient 2 (AGR2) were implicated in the development of epithelial-mesenchymal transition (EMT) in a variety of cancers. However, the involvement of SDF-1-AGR2 pathway in the EMT of glioblastoma has not been investigated. In the present study, the in vitro assays were used to investigate the role of AGR2 in cell cycle, migration, and invasion. We found that the expressions of AGR2 and chemokine (C-X-C motif) receptor 4 (CXCR4) were obviously upregulated in glioblastoma cells T98G, A172, U87, and U251 than those in normal human astrocytes (NHA) (all p < 0.01), among which both U87 and U251 cells presented the highest expression (p > 0.05). Western blot revealed that SDF-1 induced the expression of p-AKT, AGR2, and EMT markers (N-cadherin, matrix metalloproteinase-2 (MMP2), and Slug) in a dose-dependent manner in U87 and U251 cells. However, the depletion of AGR2 reversed SDF-1-induced upregulation of EMT markers rather than p-AKT. Furthermore, functional analysis identified that knockdown of AGR2 induced cell cycle arrest in G0/G1 phase and suppressed the migration and invasion of U87 and U251 cells. Taken together, SDF-1-CXCR4 pathway induced the expression of AGR2 to control the progression of EMT likely via AKT pathway in the development of glioblastoma. Our findings lay a promising foundation for the SDF-1-AGR2 axis-targeting therapy in patients with glioblastoma.

High expression of AGR2 in lung cancer is predictive of poor survival.

BackgroundAnterior gradient 2 (AGR2) is a protein disulfide isomerase-like protein widely expressed in many normal tissues as well as cancers. In our study, non-neoplastic bronchial epithelial cells as well as non-small cell lung cancer (NSCLC) cells express AGR2 protein.MethodsAGR2 expression was analyzed on lung tissue microarrays. Tumor staining was correlated with clinical outcomes.ResultsOn a lung cancer tissue microarray using immunohistochemistry, expression levels in cancer showed generally decreasing intensities in order from adenocarcinomas with mucinous components, other adenocarcinomas, squamous carcinomas, to large cell carcinomas. The study cohort was comprised of 400 cases. As a group, there was a slight trend of lower expression with increasing tumor grade. AGR2 expression level was a significant predictor of overall survival in younger patients only. Patients under 65 with lower levels showed a significantly better survival for both men and women. Patients over 65, in contrast, showed no such trend.ConclusionsNearly all NSCLC tumors show AGR2 expression. Lung cancer expression of AGR2 has prognostic value for younger patients.

Expression of AGR2 in pituitary adenomas and its association with tumor aggressiveness.

The aim of the present study was to study the expression of anterior gradient 2 (AGR2) to determine its clinical significance in pituitary adenoma (PA), and to evaluate the potential effect of AGR2 on the diagnosis, therapy or prognosis of PA. Immunohistochemistry was performed to detect the expression of AGR2 in 117 PA tissue samples. Western blotting was performed to confirm the expression profile of AGR2 in different subtypes of PAs. The data showed that in 117 different histological subtypes of PA, 51.3% exhibited AGR2-positive expression. Although AGR2 expression occurred more frequently in PAs secreting growth hormone (GH; 62.5%), adrenocorticotropic hormone (ACTH; 80.0%) and follicle-stimulating hormone (FSH; 75.0%),the positive expression rate of AGR2 showed no statistically significant differences between PA subtypes (P>0.05). The association between AGR2 expression and clinical parameters was analyzed using a χ2 test, or Fisher's exact probability test when appropriate. The result showed that the aggressiveness of PA was significantly associated with AGR2 expression, and that in the majority of aggressive PAs, AGR2 was negative (P<0.001). In conclusion, the current study detected the expression of AGR2 in different subtypes of PAs for the first time. The data indicated that GH-, ACTH- and FSH-secreting PAs present marginally more frequently with AGR2 expression, howeverm the association was not significant (P>0.05). AGR2 may be a target for the study of PA aggressiveness, and a potential index for the diagnosis or prognosis of PAs.

Anterior gradient 2 is a binding stabilizer of hypoxia inducible factor-1α that enhances CoCl2 -induced doxorubicin resistance in breast cancer cells.

Hypoxia inducible factor-1α (HIF-1α) is associated with human breast cancer chemoresistance. Various reports have suggested that multiple pathways are involved in HIF-1α induction and that the molecular mechanisms regulating HIF-1α-induced chemoresistance are still not fully understood. Here, we report that anterior gradient 2 (AGR2), a proposed breast cancer biomarker, is an essential regulator in hypoxia-induced doxorubicin resistance through the binding and stabilization of HIF-1α. Our results show that knockdown of AGR2 in MCF-7 cells leads to the suppression of HIF-1α-induced doxorubicin resistance, whereas elevated levels of AGR2 in MDA-MB-231 cells enhance HIF-1α-induced doxorubicin resistance. AGR2 expression, in turn, is upregulated by the hypoxic induction of HIF-1α at both translational and transcriptional levels via a hypoxia-responsive region from −937 to −912 bp on the AGR2 promoter sequence. By specific binding to HIF-1α, the increased level of intracellular AGR2 stabilizes HIF-1α and delays its proteasomal degradation. Finally, we found that AGR2-stabilized HIF-1α escalates multiple drug resistance protein 1 (MDR1) mRNA levels and limits doxorubicin intake of MCF-7 cells, whereas MCF-7/ADR, a doxorubicin resistant cell line with deficient AGR2 and HIF-1α, acquires wild-type MDR1 overexpression. Our findings, for the first time, describe AGR2 as an important regulator in chemical hypoxia-induced doxorubicin resistance in breast cancer cells, providing a possible explanation for the variable levels of chemoresistance in breast cancers and further validating AGR2 as a potential anti-breast cancer therapeutic target.