Extracellular Anterior Gradient-2 Research Articles

Extracellular AGR2 activates neighboring fibroblasts through endocytosis and direct binding to β-catenin that requires AGR2 dimerization and adhesion domains

Anterior gradient 2 (AGR2) is often overexpressed in several types of cancer. AGR2 is cytoplasmic or secreted as an extracellular signal. Intracellular AGR2 properties and role in cancer have been well studied, but its extracellular function is largely unclear. It has been shown that extracellular AGR2 activates endothelial cells and fibroblasts in culture, but the mechanism of AGR2 signaling is not well elucidated. Here, we report that tumor secreted or externally added AGR2 translocates into cytoplasm by endocytosis, binds to β-catenin and further co-translocates to the nucleus in NIH3T3 fibroblasts. Externally added AGR2 also increased β-catenin expression, stability, and accumulation in the nucleus in both fibroblasts and cancer cells. External AGR2 rescued the expression of β-catenin, which was suppressed by EGFR inhibitor AG1478 indicating an alternative pathway to regulate β-catenin independent of EGFR signal. These effects were abolished when a monoclonal antibody against AGR2 was added to the experiments, confirming the effects are caused by AGR2 only. Putting together, our results show that extracellular AGR2 signaling pathway involves endocytosis mediated cellular translocation, direct binding and regulating β-catenin nuclear accumulation. It is also a target against tumor initiated AGR2 signaling to form and maintain tumor microenvironment.

Extracellular AGR2 triggers lung tumour cell proliferation through repression of p21CIP1

The human Anterior GRadient 2 (AGR2) protein is an Endoplasmic Reticulum (ER)-resident protein which belongs to the Protein-Disulfide Isomerase (PDI) superfamily and is involved to productive protein folding in the ER. As such AGR2, often found overexpressed in adenocarcinomas, contributes to tumour development by enhancing ER proteostasis. We previously demonstrated that AGR2 is secreted (extracellular AGR2 (eAGR2)) in the tumour microenvironment and plays extracellular roles independent of its ER functions. Herein, we show that eAGR2 triggers cell proliferation and characterize the underlying molecular mechanisms. We demonstrate that eAGR2 enhances tumour cell growth by repressing the tumour suppressor p21CIP1. Our findings shed light on a novel mechanism through which eAGR2 behaves as a growth factor in the tumour microenvironment, independently of its ER function, thus promoting tumour cell growth through repression of p21CIP1. Our results provide a rationale for targeting eAGR2/p21CIP1-based signalling as a potential therapeutic target to impede tumour growth.

The anterior gradient-2 interactome.

The anterior gradient-2 (AGR2) is an endoplasmic reticulum (ER)-resident protein belonging to the protein disulfide isomerase family that mediates the formation of disulfide bonds and assists the protein quality control in the ER. In addition to its role in proteostasis, extracellular AGR2 is responsible for various cellular effects in many types of cancer, including cell proliferation, survival, and metastasis. Various OMICs approaches have been used to identify AGR2 binding partners and to investigate the functions of AGR2 in the ER and outside the cell. Emerging data showed that AGR2 exists not only as monomer, but it can also form homodimeric structure and thus interact with different partners, yielding different biological outcomes. In this review, we summarize the AGR2 "interactome" and discuss the pathological and physiological role of such AGR2 interactions.

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.

Paracrine signalling of AGR2 stimulates RhoA function in fibroblasts and modulates cell elongation and migration

ABSTRACTThe most prominent cancer-associated fibroblasts (CAFs) in tumor stroma is known to form a protective structure to support tumor growth. Anterior gradient-2 (AGR2), a tumor secretory protein is believed to play a pivotal role during tumor microenvironment (TME) development. Here, we report that extracellular AGR2 enhances fibroblasts elongation and migration significantly. The early stimulation of RhoA showed the association of AGR2 by upregulation of G1-S phase-regulatory protein cyclin D1 and FAK phosphorylation through fibroblasts growth factor receptor (FGFR) and vascular endothelial growth factor receptor (VEGFR). Our finding indicates that secretory AGR2 alters fibroblasts elongation, migration, and organization suggesting the secretory AGR2 as a potential molecular target that might be responsible to alter fibroblasts infiltration to support tumor growth.

Loss of ER retention motif of AGR2 can impact mTORC signaling and promote cancer metastasis

Anterior gradient 2 (AGR2) is a member of the protein disulfide isomerase (PDI) family, which plays a role in the regulation of protein homeostasis and the unfolded protein response pathway (UPR). AGR2 has also been characterized as a proto-oncogene and a potential cancer biomarker. Cellular localization of AGR2 is emerging as a key component for understanding the role of AGR2 as a proto-oncogene. Here, we provide evidence that extracellular AGR2 (eAGR2) promotes tumor metastasis in various in vivo models. To further characterize the role of the intracellular-resident versus extracellular protein, we performed a comprehensive protein-protein interaction screen. Based on these results, we identify AGR2 as an interacting partner of the mTORC2 pathway. Importantly, our data indicates that eAGR2 promotes increased phosphorylation of RICTOR (T1135), while intracellular AGR2 (iAGR2) antagonizes its levels and phosphorylation. Localization of AGR2 also has opposing effects on the Hippo pathway, spheroid formation, and response to chemotherapy in vitro. Collectively, our results identify disparate phenotypes predicated on AGR2 localization. Our findings also provide credence for screening of eAGR2 to guide therapeutic decisions.

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.

Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties.

The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis.

Binding of anterior gradient 2 and estrogen receptor-α: Dual critical roles in enhancing fulvestrant resistance and IGF-1-induced tumorigenesis of breast cancer

Anterior gradient 2 (AGR2), an essential cancer biomarker, has been widely reported to be associated with estrogen receptor (ER) positive breast cancer development. Here, we uncovered the role of cytoplasmic and exogenous AGR2, through interaction with ER-α, in enhancing fulvestrant resistance and IGF-1-induced carcinogenesis respectively. Our present study revealed that the endogenous AGR2 level positively correlates with fulvestrant resistance in MCF-7 and T47D cells. AGR2-knockdown in MCF-7 cells strongly enhances the fulvestrant-induced G1 phase arrest and accelerates the fulvestrant-induced ER-α degradation. Furthermore, intracellular AGR2 exhibits a functional interaction with ER-α. On the other hand, extracellular AGR2 remarkably promotes the IGF-1-induced cell proliferation, migration, cell cycle progression and epithelial–mesenchymal transition. Extracellular AGR2 also enhances IGF-1 downstream signaling. We also showed that ER-α specifically interacts with both extracellular AGR2 and IGF-1 receptor as a potential intermediator. Finally, we revealed that the adjuvant therapy of AGR2 monoclonal antibody enhances the inhibitory effects of fulvestrant and linsitinib toward breast cancer development. Our findings, for the first time, point out the different functions of intra- and extra-cellular AGR2, providing new insights into the development of anti-tumor therapies targeting AGR2.

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

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