Article

Agr2 Mediates Paracrine Effects on Stromal Fibroblasts That Promote Invasion by Gastric Signet-Ring Carcinoma Cells

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Abstract

Agr2 is a member of the endoplasmic reticulum (ER) protein disulfide isomerase, which physiologically regulates protein folding and plays a pivotal role in resistance to ER stress. Agr2 is expressed primarily in adenocarcinomas of various organs, and Agr2 protein overexpression participates in neoplastic transformation and metastasis, therefore acts as a pro-oncogenic protein. Besides the normal ER-localization, extracellular Agr2 is present in serum and urine of cancer patients. However, the physiological significance of extracellular Agr2 is poorly understood. In this study, we demonstrated a novel function of extracellular Agr2 that activated stromal fibroblasts and promoted fibroblast-associated cancer invasion. Agr2 is highly expressed in gastric signet-ring cell carcinoma (SRCC). Agr2 secreted from SRCC cells was incorporated by the surrounding gastric fibroblasts and promoted invasion by these cells. In turn, activated fibroblasts promote the coordinated invasion by fibroblasts and cancer cells. Thus Agr2 plays pivotal roles in the progression of gastric SRCC by exerting paracrine effects on the surrounding fibroblasts. Furthermore, Agr2 increased the growth and resistance of SRCC cells to oxidative and hypoxic stress as cell autonomous effects. Our results indicate that Agr2 may be a suitable therapeutic target for gastric SRCC. Copyright © 2014, American Association for Cancer Research.

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... Stimulation of angiogenesis, VEGF expression; control of endothelial cells migration, fibroblast growth and collagen synthesis; recruitment of MSCs [61][62][63][64][65] constitutive presence in intact skin [66] eAGR2 induces lactate production, glucose uptake and HIF-1α expression; eAGR2 expression is upregulated by HIF-1α [15,67,68] VEGF Stimulation of angiogenesis, activation of endothelial cells, proteolytic enzymes secretion, releasing of MMPs for further proliferation and migration [69,70] lower levels [71] eAGR2 enhances VEGF homodimerization, thus controlling angiogenesis, endothelial cells and fibroblasts invasion [45,72] FGFs control cell proliferation, differentiation and migration; FGF 1, 2, 5, 7, 10 upregulation during cutaneous WH; in scarless WH FGF signaling downregulates; FGF2 expression increases under the hypoxia [51,64,65,73,74] constitutive presence in intact skin as morphogens; FGF7 and FGF10 downregulate in fetal scarless wounds; FGF2 expression decrease in both scarless and scarring fetal wounds [73,74] eAGR2 enhances FGF2 homodimerization, thus controlling angiogenesis, endothelial cells and fibroblasts invasion [45] MMPs Regulation of matrix stiffness, lysing surrounding tissues, control of ECM remodeling [70,75] no significant differences were found nAG activates MMP, increase collagen degradation [76,77] Collagen I, III collagen III (proliferation phase)-thin fibrils, part of the provisional matrix replaced by the collagen I, orientating along the epidermal surface; low ratio of type III (10%) to type I (20%) collagen [78,79] higher ratio of collagen III (60%) to collagen I (30%) [79] nAG suppresses collagen I and III synthesis, increases collagen degradation [76,77] Cell capabilities keratinocytes-proliferation, migration along the fibrin of the blood clot, closure of the wound surface [7,[80][81][82]; fibroblasts-recruitment to the wound bed, transformation to myofibroblast, secretion ECM [52,55,75,83]; M1-macrophages migration, secrete pro-inflammatory mediators, switch to M2 form [84]; M2-macrophages secrete TGF-β1, PDGF [85][86][87] lower number of M1, M2-macrophages, higher ratio of M2-macrophages to M1, reduced presentation time [88] eAGR2 accelerates fibroblasts and keratinocytes migration; eAGR2 promotes epithelial morphogenesis, disrupts cell-cell contact and basal laminin; iAGR2 prevents EMT induction [24,[89][90][91][92][93] In certain tumor systems, both TGF-β and TNF-α have been shown to inhibit eAGR2 expression ( Figure 3A) [56]. ...
... However, the relationship between AGR2 and TGF-β may be complex and context dependent, and further research is necessary to fully understand its implications for cancer progression and profibrotic changes. secrete pro-inflammatory mediators, switch to M2 form [84]; M2-macrophages secrete TGF-β1, PDGF [85][86][87] induction [24,[89][90][91][92][93] In certain tumor systems, both TGF-β and TNF-α have been shown to inhibit eAGR2 expression ( Figure 3A) [56]. Figure 3. ...
... Judging by studies conducted on human cancer cell lines, the AGR2 effect on EMT, migration and invasion abilities depends on the form of the protein, intracellular or extracellular ( Figure 3B) [89,90]. It was shown that extracellular AGR2 promotes epithelial morphogenesis and tumorigenesis by disrupting cell-cell contact, disrupting basal laminin and activating fibroblast-associated cancer invasion (Table 1) [91,92]. On the other hand, intracellular AGR2 protects the epithelial cellular phenotype by preventing EMT induction ( Figure 3A) [93]. ...
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The limited ability of mammals to regenerate has garnered significant attention, particularly in regard to skin wound healing (WH), which is a critical step for regeneration. In human adults, skin WH results in the formation of scars following injury or trauma, regardless of severity. This differs significantly from the scarless WH observed in the fetal skin of mammals or anamniotes. This review investigates the role of molecular players involved in scarless WH, which are lost or repressed in adult mammalian WH systems. Specifically, we analyze the physiological role of Anterior Gradient (AGR) family proteins at different stages of the WH regulatory network. AGR is activated in the regeneration of lower vertebrates at the stage of wound closure and, accordingly, is important for WH. Mammalian AGR2 is expressed during scarless WH in embryonic skin, while in adults, the activity of this gene is normally inhibited and is observed only in the mucous epithelium of the digestive tract, which is capable of full regeneration. The combination of AGR2 unique potencies in postnatal mammals makes it possible to consider it as a promising candidate for enhancing WH processes.
... by regulating the formation of focal adhesions. These AGR2 functions in wound healing involve FAK and JNK signaling pathways and require AGR2 adhesion domain (amino acids [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]. Our finding indicates that external AGR2 may represent an alternative strategy for the development of therapeutics for wounds with large exposure area or difficult-to-heal chronic wounds. ...
... and paracrine effect on the Fibro. to facilitate cell migration which is correlated with recent work published by Tsuji et al. [30]. It is also important to note that externally applied AGR2 accelerated early activation of Fibro. ...
... Tumor secreted AGR2 has cell-autocrine/paracrine effect which plays a pivotal role in tumor microenvironment [14]. Cancer-secreted AGR2 has been reported to promote the invasion and migration of Fibro., program cell death of stromal cells, and disrupt the polarity of epithelial cells [14,30,32,33]. Remarkably, several functional domains of AGR2 like PDI activity domain, dimerization site, and adhesion domain may be involved in the process of cell migration [24,26,34,35]. ...
Article
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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 as well as 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 and the recruitment of fibroblasts near the wounded area. External AGR2 also promoted the migration of keratinocytes and fibroblasts in vitro in a dose-dependent manner. The adhesion domain of AGR2 was required for the formation of focal adhesions in migrating fibroblasts, leading to the directional migration along AGR2 gradient. These results indicate that recombinant AGR2 accelerates skin wound healing through regulation of keratinocytes and fibroblasts migration, thus demonstrating its potential utility as an alternative strategy of the therapeutics to accelerate the healing of acute or chronic skin wounds. This article is protected by copyright. All rights reserved.
... A proteomic analysis comparing two gastric cancer cell lines with the differential potential of metastasis showed that AGR2 was the most differentially overexpressed protein, supporting the capacity of AGR2 to predict the risk of developing metastasis in gastric cancer [78]. In vitro analyses carried out on the gastric cancer cell line Tu-Kato III (TP53 deleted) showed that AGR2 favors cell growth and resistance to apoptosis under stress or hypoxia [79]. In a murine xenograft model of Tu-Kato-III treated with an AGR2 targeting miRNA, tumor growth was 8 fold lower and there were much less mice with peritoneal metastases than in the control group, suggesting AGR2 could contribute to gastric cancer aggressiveness and metastasis [79]. ...
... In vitro analyses carried out on the gastric cancer cell line Tu-Kato III (TP53 deleted) showed that AGR2 favors cell growth and resistance to apoptosis under stress or hypoxia [79]. In a murine xenograft model of Tu-Kato-III treated with an AGR2 targeting miRNA, tumor growth was 8 fold lower and there were much less mice with peritoneal metastases than in the control group, suggesting AGR2 could contribute to gastric cancer aggressiveness and metastasis [79]. Expression of AGR2 has a prognostic value in gastric adenocarcinoma, especially in the early stages of the disease. ...
Article
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Pancreatic cancer is one of the deadliest cancers, against which current immunotherapy strategies are not effective. Herein, we analyzed the immune cell composition of the tumor microenvironment of pancreatic cancer samples in The Cancer Genome Atlas and found that the presence of intratumoral natural killer (NK) cells correlates with survival. Subsequent analysis also indicated that NK cell exclusion from the microenvironment is found in a high percentage of clinical pancreatic cancers and in preclinical models of pancreatic cancer. Mechanistically, NK cell exclusion is regulated in part by complement C3a and its receptor signaling. Inhibition of the C3a receptor enhances NK cell infiltration in syngeneic mouse models of pancreatic cancer resulting in tumor growth delay. However, tumor growth inhibition mediated by NK cells is not sufficient alone for complete tumor regression, but is potentiated when combined with radiotherapy. Our findings indicate that although C3a inhibition is a promising approach to enhance NK cell–based immunotherapy against pancreatic cancer, its combination with radiotherapy holds greater therapeutic benefit. Significance Immunotherapeutic agents are not effective against pancreatic cancer. We show that the inhibition of complement C3a enhances NK cell infiltration in preclinical models of pancreatic cancer, resulting in tumor growth delay. This effect is further potentiated by radiotherapy, thereby leading to significant antitumor activity compared with either therapy alone.
... This motif allows AGR2 binding to KDEL receptors in Golgi to retrograde back to and reside in ER where it interacts with newly synthesized proteins to promote their maturation by facilitating proper folding [9,10]. Besides residing in lumens of ER and Golgi, AGR2 can also be secreted to extracellular compartments [11][12][13]. Extracellular AGR2 (eAGR2) can promote metastasis of human CRC cells via a non-classical Wnt/β-catenin pathway [8]. However, in contrast to eAGR2, intracellular AGR2's (iAGR2) functional roles and related molecular mechanisms in CRC metastasis remain largely unknown. ...
... As a member of the PDI family, AGR2 can reside in endoplasmic reticulum (ER) or be secreted into extracellular compartments [28]. An increasing number of studies reported that AGR2 overexpression or secreted AGR2 enhanced cell migration and metastasis in several cancers [3][4][5]7,8,12]. However, the functional roles of intracellular AGR2 in CRC remain largely unknown. ...
Article
Human anterior gradient homolog 2 (AGR2) reportedly acts as an oncogene in multiple types of cancers. As a secreted protein, the oncogenic roles of extracellular AGR2 have been the focus of the increasing number of studies. In contrast, the oncological functions of intracellular AGR2 (iAGR2) remain elusive. Here, we report that intracellular AGR2 (iAGR2) is sufficient to promote CRC metastasis. iAGR2 binds to KDEL receptors (KDELRs) via its KTEL motif to activate downstream Gs-PKA signaling. Activated PKA upregulates the expression of NF-κB subunit c-Rel (REL) and acetylates histone H3 at lysine 9 (H3K9ac) to promote the transcription of SNAIL and SLUG. AGR2 can be upregulated by prostaglandin E2 (PGE2) via EP4-PI3K-AKT pathway and is indispensable for PGE2-induced CRC metastasis. AGR2 knockdown enhances therapeutic effects of a COX-2 inhibitor, celecoxib, in CRC metastasis. Collectively, our study reveals a promoting role and molecular mechanisms of iAGR2 in CRC metastasis and uncovers a new tumor microenvironment signal regulating AGR2 expression, which may provide new targets for treating metastatic CRC.
... If the role of secreted PDIA6 and ERP44 must be the subject of future experimentations, the role of extracellular AGR2 (eAGR2) has been further studied in oncology. It is implicated in cancer invasion and tumour cell dissemination and is generally associated with worse clinical outcomes 41,56 . ...
... This eAGR2 is also known to interact with core components of the mTORC2 complex (RICTOR), known to be involved in cell proliferation and actin cytoskeleton organization in fibrosis57 . Finally, secreted AGR2 has been demonstrated to be involved in the fibroblasts recruitment and migration (eAGR2 can promote cutaneous wound healing by recruitment of fibroblasts in the wounded area but can also activate stromal fibroblasts and promote fibroblast-associated cancer invasion in gastric signet-ring cell carcinoma, for example)56,58,59 as well as in the murine fibroblasts elongation and proliferation60 . ...
Article
Background and aims: Intestinal fibrosis is a common complication of Crohn's disease (CD). It is characterised by an accumulation of fibroblasts differentiating into myofibroblasts secreting excessive extracellular matrix. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. Methods: We performed a pilot proteomic study comparing the proteome of surface epithelium, isolated by laser-capture microdissection, in normal and fibrotic zones of resected ileal CD strictures (13 zones collected in 5 patients). Proteins of interests were validated by immunohistochemistry (IHC) in ileal and colonic samples of stricturing CD (n=44), pure inflammatory CD (n=29) and control (n=40) subjects. The pro-fibrotic role of one selected epithelial protein was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. Results: Proteomic study revealed an endoplasmic reticulum (ER) stress proteins increase in the epithelium of CD ileal fibrotic strictures, including Anterior gradient protein 2 homolog (AGR2) and Binding-immunoglobulin protein (BiP). This was confirmed by IHC. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2 intracellular expression and its secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin, led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. By using recombinant protein and blocking agent for AGR2, we demonstrated that the secretion of this protein by epithelial cells can play a role in the myofibroblastic differentiation. Conclusions: The development of CD fibrotic strictures could involve epithelial ER stress and particularly the secretion of AGR2.
... 2,3 AGR2 levels are elevated in various tumors including prostate, breast, pancreas, and gastric carcinomas. [4][5][6][7] The expression of AGR2 is driven by the reaction to ER stress induced by an increase in unfolded proteins. AGR2 was initially identified as bound to nascent proteins from ER ribosomes. ...
... has been previously described that AGR2 expression is elevated in distinct tumors. [4][5][6][7]23 Our results demonstrate that the expression of AGR2 in the Caki-1 cells affects cellular pathways including the induction of inflammation, angiogenesis, the regulation of metabolism, and the response to oxygen deficiency. In an environment with a regular oxygen concentration, the destabilization of HIF-1α and HIF-2α through the ubiquitin-proteasome pathway is initiated by hydroxylation of HIF prolyl residues and subsequent ubiquitination by prolyl hydroxylases and the VHL ubiquitin ligase complex. ...
Article
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It has been previously established that hypoxia leads to tumor development, treatment resistance, and a poor prognosis. Under oxygen deprivation, hypoxia‐inducible factors (HIFs) are stimulated to activate the genes necessary for tumor development in a low‐oxygen environment. These genes encode regulators of angiogenesis, epithelial‐mesenchymal transition, and cellular metabolism. A disulfide isomerase, anterior gradient 2 (AGR2), has been shown to increase hypoxia‐inducible factor 1, alpha subunit (HIF‐1α) stability in breast cancer. Our goal was to determine if AGR2 affects the level of transcription factor hypoxia‐inducible factor 2, alpha subunit (HIF‐2α). As a model, we used the clear cell renal cell carcinoma (ccRCC) cell line Caki‐1. The cells were transduced with lentiviral vector (Tet‐On) encoding AGR2. After induction of AGR2 expression, cells were grown under either hypoxic (0.5% O2) or normoxic (21% O2) conditions. Our data showed that AGR2 upregulated both HIF‐1α and HIF‐2α expression in Caki‐1 cells increasing the expression of HIF‐activated genes (glucose transporter 1, phosphoglycerate kinase 1, vascular endothelial growth factor A, and transforming growth factor‐alpha) under the hypoxic conditions. Under the normoxic conditions, AGR2 strongly activated CCAAT‐enhancer binding protein beta (C/EBPβ). Upregulation of C/EBPβ correlated with increased expression and secretion of the interleukin‐6 and interleukin‐8, inducing angiogenesis and inflammation in Caki‐1 cells. In summary, our studies revealed that AGR2 has essential functions in ccRCC progression through upregulation of C/EBPβ and HIF‐2α expressions, which affects cell signaling and metabolism.
... Although AGR2 and AGR3 harbor an ER retention signal sequence (KTEL and QSEL, respectively), they were present in extracellular media such as gastrointestinal mucus, blood or urine (16)(17)(18)(19). Intriguingly, we and others have recently demonstrated an emerging role of AGR2 in the maintenance of the tumor microenvironment [including properties of cancer migration (20), invasion (21), chemoresistance (22) and epithelial-to-mesenchymal transition (EMT) (23)], which clearly indicates that extracellular AGR2 (eAGR2) protein assumes a gain-of-function role. AGR3 is a less characterized homologue of AGR2, with similar but apparently not identical function in the regulation JOANNA OBACZ 1-4 , LUCIA SOMMEROVA 3 , DARIA SICARI 1 , MICHAL DURECH 3 , TONY AVRIL 1,2 , FILIPPO IULIANO 4 , SILVIA PASTOREKOVA 3,4 , ROMAN HRSTKA 3 , ERIC CHEVET 1,2 , FREDERIC DELOM 5-7 and DELPHINE FESSART 1,2,5,6 of tumor-associated processes (11). ...
... In addition, AGR2 has been shown to be secreted by various cancer cell lines as well as in tumors tissues (17,18,37,38). Follow-up studies indicated that AGR2 can also act 'from the outside', as eAGR2, either through binding to membrane receptors or through incorporating within the cytoplasm of target cells (21,22). Despite high similarity between the two homologues-AGR2 and AGR3, there is no reported evidence of eAGR3 extracellular activity. ...
Article
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Human anterior gradient proteins AGR2 and AGR3 are overexpressed in a variety of adenocarcinomas and are often secreted in cancer patients' specimens, which suggests a role for AGR proteins in intra and extracellular compartments. Although these proteins exhibit high sequence homology, AGR2 is predominantly described as a pro-oncogene and a potential prognostic biomarker. However, little is known about the function of AGR3. Therefore, the aim of the present study was to investigate the role of AGR3 in breast cancer. The results demonstrated that breast cancer cells secrete AGR3. Furthermore, it was revealed that extracellular AGR3 (eAGR3) regulates tumor cell adhesion and migration. The current study indicated that the pharmacological and genetic perturbation of Src kinase signaling, through treatment with Dasatinib (protein kinase inhibitor) or investigating cells that express a dominant-negative form of Src, significantly abrogated eAGR3-mediated breast cancer cell migration. Therefore, the results indicated that eAGR3 may control tumor cell migration via activation of Src kinases. The results of the present study indicated that eAGR3 may serve as a microenvironmental signaling molecule in tumor-associated processes.
... Elevated AGR2 expression is associated with the poor survival of patients with breast and lung cancers [10][11][12]. In the tumor microenvironment, AGR2 plays a crucial role by engaging in a cross-talk with growth factors, thereby enhancing their function [13][14][15][16]. AGR2 interferes with cell survival and metastasis pathways by modulating survivin, cyclin D1, cathepsin B and D, CD147, and epidermal growth factor receptor (EGFR) levels. ...
... Next, we evaluated the effect of mAb18A4 on α-SMA level because AGR2 exerts a paracrine effect on fibroblasts [13]. Furthermore, we found that AGR2 expression levels were significantly reduced in the tumors isolated from the mAb18A4-treated mice (Supplementary Fig. S3B). ...
... Anterior gradient homolog 2 (AGR2) belongs to the protein disulfide isomerase family and serves as an ER retention protein [158]. It has been demonstrated that the extracellular AGR2 protein was shown to be up-regulated in SGC cells and was closely correlated with the capacity to prompt stromal fibroblasts and stimulate invasion, as well as the resistance to oxidative and hypoxic stresses [159]. ...
Article
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Gastric cancer (GC) is one of the most common cancers worldwide. Most patients are diagnosed at the progressive stage of GC, and progress in the development of effective anti-GC drugs has been insufficient. The tumor microenvironment (TME) regulates various functions of tumor cells, and interactions between the cellular and molecular components of the TME—e.g., inflammatory cells, fibroblasts, vasculature cells, and innate and adaptive immune cells—promote the aggressiveness of cancer cells and dissemination to distant organs. This review summarizes the roles of various TME cells and molecules in regulating the malignant progression and metastasis of GC. We also address the important roles of signaling pathways in mediating the interaction between cancer cells and the different components of the GC TME. Finally, we discuss the implications of these molecular mechanisms for developing novel and effective therapies targeting molecular and cellular components of the GC TME to control the malignant progression of GC.
... Matrix metalloproteinase 3 (MMP-3) was specifically abundant in CDH1-aberrant GSRCC [24]. Extracellular AGR2, which acts as a chaperon-like enhancer to vascular endothelial growth factor (VEGF) and FGF2, can activate stromal fibroblasts and promote fibroblast-associated cancer invasion by exerting paracrine effects on fibroblasts in the TME of GSRCC [25,26]. Therefore, inhibitors for MMP-3 or monoclonal antibody targeting AGR2 may also represent a promising choice to enhance the efficiency of ICB by remodeling immunosuppressive TME in GSRCC. ...
Article
Gastric signet-ring cell carcinoma (GSRCC) is a subtype of gastric cancer with distinct phenotype and high risk of peritoneal metastasis. Studies have shown that early GSRCC has a good prognosis, while advanced GSRCC is insensitive to radiotherapy, chemotherapy or immune checkpoint blockade therapy. With technological advancement of single-cell RNA sequencing analysis and cytometry by time of flight mass cytometry, more detailed atlas of tumor microenvironment (TME) in GSRCC and its association with prognosis could be investigated extensively. Recently, two single-cell RNA sequencing studies revealed that GSRCC harbored a unique TME, manifested as highly immunosuppressive, leading to high immune escape. The TME of advanced GSRCC was enriched for immunosuppressive factors, including the loss of CXCL13 +-cluster of differentiation 8+-Tex cells and declined clonal crosstalk among populations of T and B cells. In addition, GSRCC was mainly infiltrated by follicular B cells. The increased proportion of SRCC was accompanied by a decrease in mucosa-associated lymphoid tissue-derived B cells and a significant increase in follicular B cells, which may be one of the reasons for the poor prognosis of GSRCC. By understanding the relationship between immunosuppressive TME and poor prognosis in GSRCC and the underlying mechanism, more effective immunotherapy strategies and improved treatment outcomes of GSRCC can be anticipated.
... Through its protein disulfide isomerase (PDI)-like domain, it facilitates the proper folding and secretion of MUC2, and playing a vital role in maintaining the stability of the intestinal mucus barrier [16,17]. AGR2 synthesis is influenced by the cellular redox state, and recent research has found that oxidative stress caused by various diseases is a key factor in triggering AGR2 synthesis disorders [18,19]. During burns, trauma, and sepsis, cells are often under oxidative stress, where excessive production of reactive oxygen species and free radicals leads to the continuous depletion of reduced glutathione (GSH) and increased levels of oxidized glutathione (GSSG), resulting in GSH/GSSG imbalance [20]. ...
... Notably, immunotherapies targeting anti-eAGR2 antibodies have demonstrated potential applicability in solid tumors [19], with investigations hinting at the potential of eAGR2 targeting to impede cancer cell invasion [133]. A targeted reduction in eAGR2 expression may emerge as a therapeutic avenue for bolstering patient prognosis [134]. As previously highlighted, eAGR2 could impact the TME or engage with signaling molecules, ultimately promoting cancer invasion. ...
Article
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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.
... On the other hand, parasympathetic nerves bind to muscarinic acetylcholine receptors (mAChRs) on gastric cancer cells through the release of ACh, activating the transmission of various signaling pathways such as Wnt/betacatenin pathway and Hippo pathway, promoting high expression of oncogenes thereby in-creasing the aggressiveness of gastric cancer [57]. It has been demonstrated that high expression of the neuroregeneration-dependent anterior gradient homolog 2 (AGR2) in gastric cancer tissues activates the Hippo signaling pathway, promotes cancer-associated fibroblasts (CAF) invasiveness and induces peritoneal metastasis and proliferation of gastric cancer cells [58,59]. ...
Article
With the emergence of the scientific research field of tumor microenvironment, the idea that tumor growth and propagation cannot be separated from the tumor microenvironment has become common. The autonomic nervous system is involved in the whole process of growth and development of the organism, and it is undeniable that the tumor microenvironment is equally regulated by both the autonomic nervous system and the immune system. Our research focused on the cancer-nerve crosstalk process and revealed the regulatory mechanisms between the autonomic nervous system and prostate, gastric, pancreatic ductal and breast cancers, mainly elucidating that (1) the release of neurotransmitters and their receptors by autonomic nerves may be important for solid tumor progression, and (2) in combination with the latest targeted small molecule imaging technology, we summarized the biological pathways related to neurotransmitters as small molecule tracers to track solid tumor progression. This research focused on combining targeted small molecules and imaging techniques to observe sympathetic and parasympathetic processes that promote or inhibit cancer development, providing new potential therapeutic targets for prostate, gastric, pancreatic ductal and breast cancers. It also provided cutting-edge research evidence for the development of biological small molecule drugs and targeted tracers in cancer therapy.
... In recent years, the role of AGR2 in tumor development and progression has become more and more intensively studied [5,6]. The contribution of AGR2 to malignant transformation, drug resistance, and the development of metastasis has already been reported by various authors [7][8][9][10][11]. However, the mechanism of action, as well as the scope of AGR2 functions, remain unclear. ...
Article
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The TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-β signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-β effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated AGR2 knockout with and without TGF-β treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of COL4A1, COL4A2, FLNA, VAV3, VEGFA, and VINC mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-β treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-β have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both AGR2 knockout and TGF-β treatment and were validated by RT-qPCR of GPX2, PTGS2, and PLA2G4A. Since PGE2 is a product of arachidonic acid metabolism, its lowered concentration in media from AGR2-knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-β have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway.
... Several studies have shown that the downregulation of AGR2 decreases cell proliferation (15)(16)(17)30) and induces cell cycle arrest (31) or apoptosis (32,33) in some types of cancer. The present study demonstrated that AGR2 downregulation in TP53-wild-type ESCC cells suppressed cell proliferation and increased the proportion of G2/M-phase and apoptotic cells. ...
Article
Anterior gradient 2 (AGR2) reportedly promotes tumor growth and has an unfavorable impact on survival in several cancers. However, no comprehensive functional analysis of AGR2 in esophageal squamous cell carcinoma (ESCC) has been performed. In the present study, the function and clinical significance of AGR2 were examined using ESCC cell lines and clinical samples. AGR2 was upregulated in EC tissue and ESCC cell lines. The downregulation of AGR2 suppressed cell proliferation and increased the proportion of G2/M‑phase cells and phosphorylation of p53 in TP53‑wild‑type ESCC and osteosarcoma cells. However, these changes were not observed in TP53‑mutant ESCC cells. In addition, immunohistochemistry results demonstrated that high AGR2 and low p53 expression levels in ESCC tissues were correlated with a worse prognosis. These results suggested that although AGR2 enhanced cell proliferation by inhibiting p53 phosphorylation in TP53‑wild‑type ESCC, the same mechanism did not regulate cell functions in TP53‑mutant ESCC. Thus, AGR2 served an important role in ESCC progression and might be a useful prognostic marker in patients with TP53‑wild‑type ESCC.
... It has been reported that AGR2 secreted by Gastric signet-ring carcinoma cells mediates the paracrine effects on stromal fibroblasts that promote invasion (Tsuji et al., 2015). This ER-resident protein secreted in cancer microenvironment triggers novel signaling pathways that exhibit pro-oncogenic gain-of-functions in cancer (Ramachandran et al., 2008;Dumartin et al., 2011). ...
... Consistent with this observation, plates coated with recombinant AGR2 enhances the rate of adhesion of rat mammary epithelial cells, MCF-7, T47D eAGR2 promotes the IGF-1-induced EMT of estrogen receptor-positive breast cancer cells [31] MCF-7 eAGR2 is detected from cancer-cell spent media. Three times higher than prostate 22Rv1 cells [117] Lung Adenocarcinoma H23, HBEC-AGR2 eAGR2 is secreted in the medium and interacts with ECM components [74] Gastric signet-ring carcinoma Tu-katoIII, HSC-39 eAGR2 activates stromal fibroblasts and stimulates invasion by fibroblasts and cancer cells [118] Prostate PCa, PC3, PC3M-Luc, HUVECs, RWPE-1 eAGR2 directly interacts with VEGFA and promotes metastasis [61] LuCaP eAGR2 induce the formation of cellular protrusions in prostate stromal cells [37] PC3 eAGR2 promotes prostate cancer metastasis [77] 22Rv1 eAGR2 is detected from cancer-cell spent media [117] Colorectal HT-29, SW48 eAGR2 promotes invasion of colorectal cancer cell [76] Ovarian ES-2, A2780, SKOV3 eAGR2 promotes angiogenesis and invasion [75] Brain/Glioblastoma U87, LN18 eAGR2 promotes chemotaxis and tube formation in HUVECs suggesting a role of eAGR2 in cell adhesion [58]. Extracellular AGR2 can also induce the migration and tube formation of human umbilical vein endothelial cells (HUVEC) whereby AGR2 expression is controlled by hypoxia-induced factor-1 (HIF-1), signifying a possible role for extracellular AGR2 in promoting angiogenesis [71]. ...
Article
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Anterior gradient-2 (AGR2) protein mediates the formation, breakage and isomerization of disulphide bonds during protein maturation in the endoplasmic reticulum (ER) and contributes to the homoeostasis of the secretory pathway. AGR2 promotes tumour development and metastasis and its elevated expression is almost completely restricted to malignant tumours. Interestingly, this supposedly ER-resident protein can be localised to other compartments of cancer cells and can also be secreted into the extracellular milieu. There are emerging evidences that describe the gain-of-function activities of the extracellular AGR2, particularly in cancer development. Here, we reviewed studies detailing the expression, pathological and physiological roles associated with AGR2 and compared the duality of localization, intracellular and extracellular, with special emphasis on the later. We also discussed the possible mechanisms of AGR2 secretion as well as deliberating the functional impacts of AGR2 in cancer settings. Last, we deliberate the current therapeutic strategies and posit the potential use AGR2, as a prognosis and diagnosis marker in cancer.
... Lucia Sommerova et al. described that loss of intracellular AGR2 in cell lines caused a variation from epithelial to mesenchymal phenotype and establishment of intracellular AGR2 expression launched EMT 43 . However, other studies suggested that extracellular AGR2 could interrupt adherens junctions, disrupt basal laminin and activate broblastassociated cancer invasion, promoting epithelial morphogenesis and tumorigenesis 44 . To date, no researches about correlation between AGR2 and ribosome biogenesis have been reported. ...
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Background: Belonging to the protein disulfide isomerase (PDI) family, anterior gradient 2 (AGR2) is overexpressed in mucus-secreting cells and endocrine organs such as breast, lung, and ovarian, but its exact function and regulation remain unclear. We aimed to perform integrative analysis of AGR2 in breast cancer. Methods: In our study, a serious of bioinformatic online databases were used to analyze the expression, regulation, prognostic value, and function of AGR2 in breast cancer. Results: The results suggested that AGR2 mRNA was overexpressed in non-basal-like or non-triple-negative breast cancer, but underexpressed in basal-like/ triple-negative breast cancer. AGR2 mRNA expression was correlated with its protein expression. Moreover, the expression of AGR2 was lower in more malignant breast cancer. Furthermore, we also found that DNA methylation, rather than copy number variation, led to AGR2 mRNA overexpression. Prognosis analysis showed no significant correlation between AGR2 level and survival, but in subtype investigation, patients with higher AGR2 level had a significantly better outcome in patients with basal-like / triple-negative breast cancer. Enrichment analysis for co-expression genes of AGR2 revealed that gene sets enriched for chromosome segregation, DNA conformation change, chromosomal region, and GABA receptor activity may play important roles in breast cancer, and that the most significant pathway was “ribosome biogenesis in eukaryotes”, in which negative co-expression genes of AGR2 were enriched. Conclusions: Overexpression of AGR2 was found in less malignant breast cancer, for the first time, our results propose that DNA methylation rather than copy number variation leads to AGR2 overexpression, which can predict favorable prognosis in basal-like/ triple-negative breast cancer, and AGR2 could be a possible regulator of ribosome biogenesis in patients with breast cancer.
... Recently published research work shows that various molecules and factors expressed by cancer cells regulate the accumulation and transformation of normal fibroblasts (NFs) to cancerassociated fibroblasts (CAFs) which develop as most prominent stromal cell type [5][6][7]. Cancer cells secrete various molecules like transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1) and interleukin-6 [8][9][10][11][12]. These tumour niche secretome plays a pivotal role in cellular communications and thus regulates stromal fibroblasts to support tumour growth [13]. ...
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The 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.
... eAGR2 was also shown to affect adhesive and metastatic related molecules. In gastric signetring cell carcinoma cells, the eAGR2 protein was shown to be highly expressed extracellularly and was associated with the ability to activate stromal fibroblasts, promote cancer cells invasion and increase both the growth and the resistance to oxidative and hypoxic stresses [97]. In the metastatic cholangiocarcinoma KKU-213L5 cell line, the depletion of the AGR2vH isoform using siRNA resulted in the significant decrease in cell adhesion and in vimentin expression [98]. ...
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Effective diagnostic, prognostic and therapeutic biomarkers can help in tracking disease progress, predict patients’ survival, and considerably affect the drive for successful clinical management. The present review aims to determine how the metastatic-linked protein anterior gradient homologue 2 (AGR2) operates to affect cancer progression, and to identify associated potential diagnostic, prognostic and therapeutic biomarkers, particularly in central nervous system (CNS) tumors. Studies that show a high expression level of AGR2, and associate the protein expression with the resilience to chemotherapeutic treatments or with poor cancer survival, are reported. The primary protein structures of the seven variants of AGR2, including their functional domains, are summarized. Based on experiments in various biological models, this review shows an orchestra of multiple molecules that regulate AGR2 expression, including a feedback loop with p53. The AGR2-associated molecular functions and pathways including genomic integrity, proliferation, apoptosis, angiogenesis, adhesion, migration, stemness, and inflammation, are detailed. In addition, the mechanisms that can enable the rampant oncogenic effects of AGR2 are clarified. The different strategies used to therapeutically target AGR2-positive cancer cells are evaluated in light of the current evidence. Moreover, novel associated pathways and clinically relevant deregulated genes in AGR2 high CNS tumors are identified using a meta-analysis approach.
... Another PDI family member, anterior gradient 2 (Agr2), is highly expressed in gastric signet-ring cell carcinoma compared to in noncancerous tissue. Secreted Agr2 activates stromal fibroblasts and promotes fibroblast-associated cancer invasion in a paracrine manner, thereby remodeling the tumor microenvironment towards an invasive phenotype [64]. In addition, ER oxidoreductin-1-like (ERO1L), a PDI interaction partner and an important player in hypoxia-induced oxidative protein folding, is highly expressed in GC tissue, and its level is associated with adverse prognosis in patients with GC [65]. ...
Article
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The endoplasmic reticulum (ER) is the primary organelle responsible for the synthesis, modification, folding and secretion of proteins, especially in specialized secretory cells. It also contributes to the maintenance of cellular functions, such as Ca2+ storage, lipogenesis, gluconeogenesis, and organelle biogenesis. Cellular stress conditions, such as glucose deprivation, hypoxia and disturbance of Ca2+ homeostasis, may increase the risk of protein misfolding and perturb proteostasis. This activates ER stress and triggers the unfolded protein response (UPR), leading to either the restoration of homeostasis or cell death. ER stress and UPR have been shown to play crucial roles in the pathogenesis, progression and treatment response of various cancers. In gastric cancer (GC), one of the most aggressive cancer types, critical functions of ER stress signaling have also started to emerge. Herein, we summarize the current knowledge linking ER stress and UPR to GC; we also discuss the possible nodes of therapeutic intervention and propose directions of future research.
... Very recently, several studies showed that extracellular AGR2 promotes epithelial morphogenesis and tumorigenesis by interruption of adherens junctions, disruption of basal laminin and activation of fibroblast-associated cancer invasion [37,46]. That elevated levels of secreted AGR2 mediate more aggressive cellular phenotypes associated with loss of cellular polarity, remodeling of extracellular matrix and increased invasiveness may appear to be inconsistent with our results. ...
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Background During cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance. Methods The expression of AGR2 was analyzed in cancer cell lines exposed to TGF-β alone or to combined treatment with TGF-β and the Erk1/2 inhibitor PD98059 or the TGF-β receptor specific inhibitor SB431542. The impact of AGR2 silencing by specific siRNAs or CRISPR/Cas9 technology on EMT was investigated by western blot analysis, quantitative PCR, immunofluorescence analysis, real-time invasion assay and adhesion assay. ResultsInduction of EMT was associated with decreased AGR2 along with changes in cellular morphology, actin reorganization, inhibition of E-cadherin and induction of the mesenchymal markers vimentin and N-cadherin in various cancer cell lines. Conversely, induction of AGR2 caused reversion of the mesenchymal phenotype back to the epithelial phenotype and re-acquisition of epithelial markers. Activated Smad and Erk signaling cascades were identified as mutually complementary pathways responsible for TGF-β-mediated inhibition of AGR2. Conclusion Taken together our results highlight a crucial role for AGR2 in maintaining the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.
... Leys et al. suggested that ERp57 expression is down-regulated in GC and associated with tumor invasion depth, TNM stage, and patient survival [13]. Tsuji et al. showed that extracellular AGR2 can activate stromal fibroblasts and plays important roles in the progression of gastric signet-ring cell carcinoma (SRCC) [14]. Zhang et al. suggested that TXNDC5 was overexpressed in GC and could promote the growth and invasion of gastric cancer cells [15]. ...
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ERp29 is a novel endoplasmic reticulum (ER) protein that plays an important role in protein unfolding and secretion. Recently, it has been reported to be widely implicated in control of tumorigenesis in some tumors. However, the potential function of ERp29 in gastric cancer remains poorly understood. In this study, we found that the positive rate of ERp29 in gastric cancer tissues was significantly lower than that in adjacent non-tumor tissues. And tumor with high ERp29 expression had inclinations towards smaller tumor size and earlier TNM stage. The in vitro experiments indicated that over-expression of ERp29 in gastric cancer cells significantly suppressed the proliferation and migration of tumor cells, which is consistent with the result of the in vivo animal experiments. Furthermore, our mechanistic investigations revealed that ERp29 reversed EMT process in gastric carcinoma, and its effect was related to the inactivation of ERK1/2 and AKT phosphorylation. Thus, we conclude that ERp29 acts as a tumor suppressor gene in gastric cancer, and is expected to become a novel target of the treatment of GC.
... [15][16][17] We and others have also reported several extracellular AGR2 functions, including the promotion of angiogenesis and fibroblasts coordinated tumor cells invasion. [18][19][20] However, the exact mechanism of how secreted AGR2 performs its function is not understood, especially how AGR2 interacts with other extracellular signaling molecules, such as VEGF and FGF2, which are the major players in tumor angiogenesis. [21][22][23][24] Here, we report a mechanism for the tumor-promoting function of extracellular AGR2. ...
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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.
... Thus, AGR proteins cannot have a growth factor activity comparable with nAG in regeneration, and whether the loss of limb regeneration in higher vertebrates is due to the evolution of nAG toward a non-secreted protein remains to be elucidated. Nevertheless, AGR proteins are also expressed in human tumors (Tsuji et al., 2015;Zhang et al., 2016b), and whether their expression is related to nerve dependence in cancer should be investigated. ...
Article
Nerve dependence has long been described in animal regeneration, where the outgrowth of axons is necessary to the reconstitution of lost body parts and tissue remodeling in various species. Recent discoveries have demonstrated that denervation can suppress tumor growth and metastasis, pointing to nerve dependence in cancer. Regeneration and cancer share similarities in regard to the stimulatory role of nerves, and there are indications that the stem cell compartment is a preferred target of innervation. Thus, the neurobiology of cancer is an emerging discipline that opens new perspectives in oncology.
... Jako potenciální bio marker adenokarcinomu pankreatu je v poslední době uvažován protein AGR2. Tento protein se obvykle vyskytuje v endoplazmatickém retikulu, dále na povrchu buněk a často dochází k jeho sekreci ven z buněk [37]. Zvýšená exprese tohoto proteinu byla detekována v prekancerózních lézích, nádorových buňkách, buněčných liniích odvozených od karcinomu pankreatu a také v cirkulujících nádorových buňkách [38,39]. ...
Article
Background: Pancreatic ductal adenocarcinoma is a highly aggressive disease with 5-year overall survival not exceeding 5%. In the Czech Republic, the incidence of this tumor has been increasing; according to recent statistics, the Czech Republic is already number one worldwide in the occurrence of this malignancy. Delayed diagnosis due to asymptomatic course of the disease in the early stages is characteristic for this disease. Aim: The objective of this article is to give an overview of the most important factors, which according to current knowledge of pancreatic adenocarcinoma have a prognostic and predictive potential. This work describes both traditional prognostic factors, such as tumor resectability, its extent and localization, application of adjuvant chemotherapy, microscopically positive resection margin, presence of metastases in lymph nodes, histological grade, vascular and perineural invasion. Further, the paper lists a number of different biological markers that could contribute to early detection of cancer, better prognosis and optimization of treatment, for example hENTl (human equilibrative nucleoside transported), SPARC (secreted protein acidic and rich in cysteine), AGR2, Bcl-2, VEGF, Ki-67, COX-2 and more. Also, genetic mutations and significance of microRNA are discussed. Conclusion: Despite great efforts that have been devoted to the research of biological markers, so far the only clinically accepted and used marker is CA 19-9. Its use is primarily in patients already diagnosed with adenocarcinoma of the pancreas. A lot of attention has recently been focused on other potential biomarkers, their application in clinical practice would however still require further research.
... This protocol was adapted from the previously published studies, Satoyoshi et al. (2015a; 2015b) and Tsuji et al. (2015). This work was supported by JSPS KAKENHI (Grant Nos. ...
... AGR2 expression was up-regulated in a gastric cancer cell subline with high metastatic potential for invasion to lymph nodes [18]. Cancer-secreted AGR2 could activate stromal fibroblasts (cancer-associated stromal cells vs. normal tissue stromal cells) to promote fibroblast associated cancer invasion of gastric cancer cells [19]. AGR2 could activate ER stress response genes [11], to stimulate cell proliferation of AGR2-negative pancreatic tumor cells, and to enhance drug resistance [10]. ...
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Anterior Gradient 2 (AGR2) is a protein expressed in many solid tumor types including prostate, pancreatic, breast and lung. AGR2 functions as a protein disulfide isomerase in the endoplasmic reticulum. However, AGR2 is secreted by cancer cells that overexpress this molecule. Secretion of AGR2 was also found in salamander limb regeneration. Due to its ubiquity, tumor secretion of AGR2 must serve an important role in cancer, yet its molecular function is largely unknown. This study examined the effect of cancer-secreted AGR2 on normal cells. Prostate stromal cells were cultured, and tissue digestion media containing AGR2 prepared from prostate primary cancer 10-076 CP and adenocarcinoma LuCaP 70CR xenograft were added. The control were tissue digestion media containing no AGR2 prepared from benign prostate 10-076 NP and small cell carcinoma LuCaP 145.1 xenograft. In the presence of tumor-secreted AGR2, the stromal cells were found to undergo programmed cell death (PCD) characterized by formation of cellular blebs, cell shrinkage, and DNA fragmentation as seen when the stromal cells were UV irradiated or treated by a pro-apoptotic drug. PCD could be prevented with the addition of the monoclonal AGR2-neutralizing antibody P3A5. DNA microarray analysis of LuCaP 70CR media-treated vs. LuCaP 145.1 media-treated cells showed downregulation of the gene SAT1 as a major change in cells exposed to AGR2. RT-PCR analysis confirmed the array result. SAT1 encodes spermidine/spermine N1-acetyltransferase, which maintains intracellular polyamine levels. Abnormal polyamine metabolism as a result of altered SAT1 activity has an adverse effect on cells through the induction of PCD.
... AGR3 was firstly characterized in breast cancer cell membranes and was found to localize in secretory or endosome-like vesicles in both T47-D and MDA-MB-468 cells, 16 suggesting more prominent role of secreted form of AGR3. Indeed, recent works have depicted emerging role of extracellular AGR2 in the control of tumor aggressiveness through both autocrine and paracrine effects, 39,40 indicating that similar mechanism can also be valid for AGR3. Lastly, taking into account cognate expression pattern of AGR proteins in different carcinomas, 7,19 it can be speculated that there is a functional cross talk between these proteins. ...
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Anterior gradient protein (AGR) 3 is a highly related homologue of pro-oncogenic AGR2 and belongs to the family of protein disulfide isomerases. Although AGR3 was found in breast, ovary, prostate, and liver cancer, it remains of yet poorly defined function in tumorigenesis. This study aimed to determine AGR3 expression in a cohort of 129 primary breast carcinomas and evaluate the clinical and prognostic significance of AGR3 in these tumors. The immunohistochemical analysis revealed the presence of AGR3 staining to varying degrees in 80% of analyzed specimens. The percentage of AGR3-positive cells significantly correlated with estrogen receptor, progesterone receptor (both P<0.0001) as well as low histological grade (P=0.003), and inversely correlated with the level of Ki-67 expression (P<0.0001). In the whole cohort, AGR3 expression was associated with longer progression-free survival (PFS), whereas AGR3-positive subgroup of low-histological grade tumors showed both significantly longer PFS and overall survival. In conclusion, AGR3 is associated with the level of differentiation, slowly proliferating tumors, and more favorable prognosis of breast cancer patients.
Article
Gastric cancer is a leading cause of cancer-related deaths globally and chemotherapy is widely accepted as the standard treatment for gastric cancer. However, drug resistance in cancer cells poses a significant obstacle to the success of chemotherapy, limiting its effectiveness in treating gastric cancer. Although many studies have been conducted to unravel the mechanisms of acquired drug resistance, the existing studies were based on abnormalities of a single gene, that is, differential gene expression (DGE) analysis. Single gene-based analysis alone is insufficient to comprehensively understand the mechanisms of drug resistance in cancer cells, because the underlying processes of the mechanism involve perturbations of the molecular interactions. To uncover the mechanism of acquired gastric cancer drug resistance, we perform for identification of differentially regulated gene networks between drug-sensitive and drug-resistant cell lines. We develop a computational strategy for identifying phenotype-specific gene networks by extending the existing method, CIdrgn, that quantifies the dissimilarity of gene networks based on comprehensive information of network structure, that is, regulatory effect between genes, structure of edge, and expression levels of genes. To enhance the efficiency of identifying differentially regulated gene networks and improve the biological relevance of our findings, we integrate additional information and incorporate knowledge of network biology, such as hubness of genes and weighted adjacency matrices. The outstanding capabilities of the developed strategy are validated through Monte Carlo simulations. By using our strategy, we uncover gene regulatory networks that specifically capture the molecular interplays distinguishing drug-sensitive and drug-resistant profiles in gastric cancer. The reliability and significance of the identified drug-sensitive and resistance-specific gene networks, as well as their related markers, are verified through literature. Our analysis for differentially regulated gene network identification has the capacity to characterize the drug-sensitive and resistance-specific molecular interplays related to mechanisms of acquired drug resistance that cannot be revealed by analysis based solely on abnormalities of a single gene, for example, DGE analysis. Through our analysis and comprehensive examination of relevant literature, we suggest that targeting the suppressors of the identified drug-resistant markers, such as the Melanoma Antigen (MAGE) family, Trefoil Factor (TFF) family, and Ras-Associated Binding 25 (RAB25), while enhancing the expression of inducers of the drug sensitivity markers [e.g., Serum Amyloid A (SAA) family], could potentially reduce drug resistance and enhance the effectiveness of chemotherapy for gastric cancer. We expect that the developed strategy will serve as a useful tool for uncovering cancer-related phenotype-specific gene regulatory networks that provide essential clues for uncovering not only drug resistance mechanisms but also complex biological systems of cancer.
Article
Most of the organs of the digestive tract comprise secretory epithelia that require specialized molecular machines to achieve their functions. As such anterior gradient (AGR) proteins, which comprise AGR1, AGR2, and AGR3, belong to the protein disulfide isomerase family, and are involved in secretory and transmembrane protein biogenesis in the endoplasmic reticulum. They are generally expressed in epithelial cells with high levels in most of the digestive tract epithelia. To date, the vast majority of the reports concern AGR2, which has been shown to exhibit various subcellular localizations and exert pro-oncogenic functions. AGR2 overexpression has recently been associated with a poor prognosis in digestive cancers. AGR2 is also involved in epithelial homeostasis. Its deletion in mice results in severe diffuse gut inflammation, whereas in inflammatory bowel diseases, the secretion of AGR2 in the extracellular milieu participates in the reshaping of the cellular microenvironment. AGR2 thus plays a key role in inflammation and oncogenesis and may represent a therapeutic target of interest. In this review, we summarize the already known roles and mechanisms of action of the AGR family proteins in digestive diseases, their expression in the healthy digestive tract, and in digestive oncology. At last, we discuss the potential diagnostic and therapeutic implications underlying the biology of AGR proteins.
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Gastric cancer is globally the fourth leading cause of cancer-related deaths. Patients with diffuse-type gastric cancer (DGC) particularly have a poor prognosis that only marginally improved over the last decades, as conventional chemotherapies are frequently ineffective and specific therapies are unavailable. Early-stage DGC is characterized by intramucosal lesions of discohesive cells, which can be present for many years before the emergence of advanced DGC consisting of highly proliferative and invasive cells. The mechanisms underlying the key steps of DGC development and transition to aggressive tumors are starting to emerge. Novel mouse- and organoid models for DGC, together with multi-omic analyses of DGC tumors, revealed contributions of both tumor cell-intrinsic alterations and gradual changes in the tumor microenvironment to DGC progression. In this review, we will discuss how these recent findings are leading towards an understanding of the cellular and molecular mechanisms responsible for DGC initiation and malignancy, which may provide opportunities for targeted therapies.
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Warm-blooded vertebrates regenerate lost limbs and their parts in general much worse than fishes and amphibians. We previously hypothesized that this reduction in regenerative capability could be explained in part by the loss of some genes important for the regeneration in ancestors of warm-blooded vertebrates. One of such genes could be ag1, which encodes secreted protein disulfide isomerase of the Agr family. Ag1 is activated during limb and tail regeneration in the frog Xenopus laevis tadpoles and is absent in warm-blooded animals. The essential role of another agr family gene, agr2, in limb regeneration was demonstrated previously in newts. However, agr2, as well as the third member of agr family, agr3, are present in all vertebrates. Therefore, it is important to verify if the activity of ag1 lost by warm-blooded vertebrates is also essential for regeneration in amphibians, which could be a further argument in favor of our hypothesis. Here, we show that in the Xenopus laevis tadpoles in which the expression of ag1 or agr2 was artificially suppressed, regeneration of amputated tail tips was also significantly reduced. Importantly, overexpression of any of these agrs or treatment of tadpoles with any of their recombinant proteins resulted in the restoration of tail regeneration in the refractory period when these processes are severely inhibited in normal development. These findings demonstrate the critical roles of ag1 and agr2 in regeneration in frogs and present indirect evidence that the loss of ag1 in evolution could be one of the prerequisites for the reduction of regenerative ability in warm-blooded vertebrates.
Article
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.
Article
Tamoxifen treatment in breast cancer patients is associated with increased risk of endometrial malignancies. Significantly, higher AGR2 expression was found in endometrial cancers that developed in women previously treated with tamoxifen compared to those who had not been exposed to tamoxifen. An association of elevated AGR2 level with myometrial invasion occurrence and invasion depth was also found. In vitro analyses identified a stimulatory effect of AGR2 on cellular proliferation. Although adverse tamoxifen effects on endometrial cells remain elusive, our work identifies elevated AGR2 as a candidate tamoxifen-dependent mechanism of action responsible for increased incidence of endometrial cancer.
Article
Tamoxifen treatment in breast cancer patients is associated with increased risk of endometrial malignancies. Significantly, higher AGR2 expression was found in endometrial cancers that developed in women previously treated with tamoxifen compared to those who had not been exposed to tamoxifen. An association of elevated AGR2 level with myometrial invasion occurrence and invasion depth was also found. In vitro analyses identified a stimulatory effect of AGR2 on cellular proliferation. Although adverse tamoxifen effects on endometrial cells remain elusive, our work identifies elevated AGR2 as a candidate tamoxifen-dependent mechanism of action responsible for increased incidence of endometrial cancer.
Article
One recently discussed general mechanism affecting gene expression is 3’-untranslated region (3’UTR) length. Events such as shortening, translocation or loss of 3’UTRs are observed within oncogenes and are proposed to associate with increased expression. Thus, increased efforts are being made to understand constitutive and differential transcript 3’end formation. Investigation of AGR2 mRNA revealed a direct impact of its 3’UTR length on AGR2 expression. In silico analyses identified several regulatory sequences within the distal part of AGR2 mRNA that may regulate 3’UTR length and associated protein levels. Short 3’UTRs were observed in a panel of AGR2-positive cancer cell lines and in human breast cancer specimens, in which more extensive 3’UTR shortening correlated with increased AGR2 protein levels. AGR2 is an important member of PI3K/AKT signalling pathway, which along with the proposed involvement of mTOR in the regulation of alternative polyadenylation, prompted us to study the role of mTOR in relation to AGR2 mRNA 3’UTR shortening. A direct impact of mTOR signalling on AGR2 3’UTR shortening associated with increased protein synthesis was found, which led to the identification of a novel molecular mechanism involved in upregulation of AGR2 levels in mTOR-activated cells via modulating the 3’UTR length of AGR2 mRNA.
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Anterior Gradient 2 (AGR2) is a potential anti-tumor target and we previously reported a murine antibody 18A4 with specific binding to AGR2. However, humanization is a must to overcome immunogenicity before considering for clinical use and optimized vectors for mammalian expression are also necessary for following industrialized manufacture. Here, we describe an anti-tumor humanized antibody blocking secreted AGR2 activity. We employed the CDR grafting technique and deimmunization analysis to construct humanized antibody variants of 18A4, and 18A4Hu I was selected as the best humanization candidate, characterized by physical and chemical property comparison. Mouse xenograft study showed that 18A4Hu I could effectively inhibit the xenograft tumor growth, antibody blocking epitope analysis using AGR2 mutants indicated that the inhibition activity of 18A4Hu I is exerted probably through blocking the AGR2 functions which rely on the amino acid sites of E60-H76 and A86-E153. What's more, in this report, we also describe a pHAb-FAST vector system which is specifically designed for humanized antibody mammalian expression vector fast construction. With pHAb-FAST system, expression vector of 18A4Hu I could be quickly constructed only through twice overlapping PCR reactions. To our knowledge, AGR2-targeted 18A4Hu I is a promising humanized anti-tumor drug candidate, and pHAb-FAST system is a useful optimized mammalian expression vector construction tool. Our findings are supposed to accelerate the development of antibody-based cancer therapy.
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ERp19, a mammalian thioredoxin-like protein, plays a key role in defense against endoplasmic reticulum stress. It belongs to the protein disulfide isomerize (PDI) family, whose members have been implicated in development of breast, ovarian and gastrointestinal cancers. However, the role of ERp19 in gastric cancer (GC) remains undefined. Therefore, we sought to investigate the expression and prognostic value of ERp19 in GC patients, and to explore the role of ERp19 in tumorigenicity. Expression of ERp19 in gastric tissues was assessed by immunohistochemical staining and real-time PCR in clinical samples of GC patients. Statistical analysis of clinical cases revealed that the expression levels of ERp19 were higher in tumor tissues than non-tumor tissues. And the level of ERp19 expression was correlated with tumor size, lymph node involvement and poor clinical prognosis. Furthermore, ERp19 knockdown dramatically suppressed gastric cancer cell growth, inhibited cellular migration/invasion and down-regulated the phosphorylation of FAK and paxillin, whereas ERp19 over-expression reversed these changes. We conclude that ERp19 contributes to tumorigenicity and metastasis of GC by activating the FAK signaling pathway, and may function as an oncogene in GC. ERp19 may represent a new diagnostic and prognostic marker and a novel target for the treatment of GC.
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Scirrhous gastric cancer, which has the worst prognosis among the various types of gastric cancer, is highly invasive and associated with abundant stromal fibroblasts. Although cancer-associated fibroblasts (CAFs) have been proposed to generate a tumor-supportive extracellular matrix that promotes the expansion of this type of cancer, the molecular mechanisms by which CAFs assist cancer cells are not yet fully understood. Here, we show for the first time that Asporin, a small leucine-rich proteoglycan (SLRP), is predominantly expressed in CAFs, and has essential roles in promoting co-invasion of CAFs and cancer cells. CAFs of scirrhous gastric cancer possess high potential for invasion, and invasion by CAFs frequently proceeded invasion by cancer cells, both in vitro and in vivo. Expression of Asporin was induced in fibroblasts by exposure to gastric cancer cells. Asporin secreted from CAFs activates Rac1 via an interaction with CD44 and promotes invasion by CAFs themselves. Moreover, Asporin promoted invasion by neighboring cancer cells, via paracrine effects mediated by activation of the CD44-Rac1 pathway. These results suggest that Asporin is a unique SLRP that promotes progression of scirrhous gastric cancer and is required for coordinated invasion by CAFs and cancer cells. Therefore, Asporin may represent a new therapeutic target molecule for the development of drugs aimed at manipulating the cancer microenvironment.Oncogene advance online publication, 20 January 2014; doi:10.1038/onc.2013.584.
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Background Signet ring cell carcinoma (SRC) of the stomach is a histological type based on microscopic characteristics. Although the distinctive clinicopathological features of SRC have been reported, results are inconsistent and survival outcomes are uncertain. Methods We retrospectively studied 769 patients with gastric carcinoma who underwent gastrectomy in our institute from 1999 to 2009. Among them, 326 patients (42.4 %) had early gastric cancer (EGC) and 443 patients (57.6 %) had advanced gastric cancer (AGC). Sex, age, tumor location, macroscopic type, tumor size, microscopic invasion, and survival rate were compared between patients with SRC, differentiated-, and undifferentiated-type gastric carcinomas. Results Fifty-one patients (15.6 %) had SRC in EGC; there were significant differences in sex, age, location, macroscopic type, and size between SRC and the differentiated histological type. However, there was no difference between SRC and undifferentiated-type gastric carcinoma, except for the macroscopic type. Fifty-seven patients (12.9 %) had SRC in AGC. Sex, age, location, size, macroscopic type, perineural invasion, N stage, and hepatic metastasis were significantly different between SRC and the differentiated histological type. Undifferentiated-type gastric carcinoma differed in sex, macroscopic type, and hepatic metastasis. The overall survival rate differed between SRC and other cell types (P P = 0.041] and tumor, node, and metastasis (TNM) stage (HR 2.350, P P Conclusions Patients with SRC showed similar clinicopathological features with undifferentiated histology. The survival of patients with SRC reflected a better prognosis in patients with undifferentiated gastric carcinoma. However, when narrowing the patients to those with EGC only, survival in EGC patients exhibited no difference between histological types. Among AGC patients, SRC patients had a worse prognosis than other cell types.
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Anterior gradient-2 (AGR2) is a normal endoplasmic reticulum protein that has two important abnormal functions, amphibian limb regeneration and human cancer metastasis promotion. These normal intracellular and abnormal extracellular roles can be attributed to the multidomain structure of AGR2. The NMR structure shows that AGR2 consists of an unstructured N-terminus region followed by a thioredoxin fold. The protein exists in monomer-dimer equilibrium with a K(d) of 8.83 μM and intermolecular salt bridges involving E60 and K64 within the folded domain serve to stabilize the dimer. The unstructured region is primarily responsible for the ability of AGR2 to promote cell adhesion while dimerisation is less important for this activity. The structural data of AGR2 shows a separation between potential catalytic redox activity and adhesion function within the context of metastasis and development.
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Recent studies of epithelial tissues have revealed the presence of tissue-specific stem cells that are able to establish multiple cell lineages within an organ. The stem cells give rise to progenitors that replicate before differentiating into specific cell lineages. The mechanism by which homeostasis is established between proliferating stem or progenitor cells and terminally differentiated cells is unclear. This study demonstrates that Agr2 expression by mucous neck cells in the stomach promotes the differentiation of multiple cell lineages while also inhibiting the proliferation of stem or progenitor cells. When Agr2 expression is absent, gastric mucous neck cells increased in number as does the number of proliferating cells. Agr2 expression loss also resulted in the decline of terminally differentiated cells, which was supplanted by cells that exhibited nuclear SOX9 labeling. Sox9 expression has been associated with progenitor and stem cells. Similar effects of the Agr2 null on cell proliferation in the intestine were also observed. Agr2 consequently serves to maintain the balance between proliferating and differentiated epithelial cells.
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Clinical studies have defined the core 'genetic blueprint' of a cancer cell, but this information does not necessarily predict the cancer phenotype. Signalling hubs that mediate such phenotype have been identified largely using OMICS platforms that measure dynamic molecular changes within the cancer cell landscape. The pro-oncogenic protein anterior gradient 2 (AGR2) is a case in point; AGR2 has been shown using a range of expression platforms to be involved in asthma, inflammatory bowel disease, cell transformation, cancer drug resistance and metastatic growth. AGR2 protein is also highly overexpressed in a diverse range of human cancers and can be secreted and detected in extracellular fluids, thus representing a compelling pro-oncogenic signalling intermediate in human cancer. AGR2 belongs to the protein disulphide isomerase family with all the key features of an endoplasmic reticulum-resident protein-this gives clues into how it might function as an oncoprotein through the regulation of protein folding, maturation and secretion that can drive metastatic cell growth. In this review, we will describe the known aspects of AGR2 molecular biology, including gene structure and regulation, emerging protein interaction networks and how its subcellular localization mediates its biological functions. We will finally review the cases of AGR2 expression in human cancers, the pathophysiological consequences of AGR2 overexpression, its potential role as a tumour biomarker that predicts the response to therapy and how the AGR2 pathway might form the basis for drug discovery programmes aimed at targeting protein folding/maturation pathways that mediate secretion and metastasis.Oncogene advance online publication, 3 September 2012; doi:10.1038/onc.2012.346.
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The mechanisms controlling expression of the putative oncogene Anterior gradient 2 (AGR2) in pancreatic ductal adenocarcinoma (PDAC) are not well understood. We now show that AGR2 is a transforming growth factor-β (TGF-β)-responsive gene in human pancreatic cancer cells, whose downregulation is SMAD4 dependent. We also provide evidence supporting a role for AGR2 as an ER-chaperone for the cancer-associated mucin, MUC1. AGR2 is both sufficient and required for MUC1 expression in pancreatic cancer cells. Furthermore, AGR2 is coexpressed with MUC1 in mouse pancreatic intraepithelial neoplasia (mPanIN)-like lesions and in the cancer cells of four distinct genetically engineered mouse models of PDAC. We also show that Pdx1-Cre/LSL-Kras(G12D)/Smad4(lox/lox) mice heterozygous for Agr2 exhibit a delay in mPanIN initiation and progression to PDAC. It is proposed that loss of Smad4 may convert TGF-β from a tumor suppressor to a tumor promoter by causing the upregulation of AGR2, which then leads to increased MUC1 expression, at which point both AGR2 and MUC1 facilitate mPanIN initiation and progression to PDAC.Oncogene advance online publication, 3 September 2012; doi:10.1038/onc.2012.394.
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Gastric cancer remains one of the deadly diseases with poor prognosis. New classification of gastric cancers based on histologic features, genotypes and molecular phenotypes helps better understand the characteristics of each subtype, and improve early diagnosis, prevention and treatment. The objective of this article is to review the new classification of gastric cancers and the up-to-date guidance in the application of molecular testing.
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Background Poorly differentiated signet-ring cell carcinoma (SRCC) and non signet-ring cell carcinoma (NSRCC) are prevalent histological subtypes of gastric cancers with distinct morphological features. To date, however, the molecular basis of their growth, differentiation, and metastasis still remains unclear, because of the limitation of available cell lines. Methods In the present study, we established novel SRCC and NSRCC cell lines (designated GPM-2 and GPM-1) derived from the ascites of two individual gastric cancer patients with peritoneal metastasis. Results Immunohistochemical and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that GPM-2 cells showed both gastric and intestinal differentiation phenotypes (E-cadherin+/MUC5AC+/MUC6+/Villin+), and formed xenografted tumors with typical SRCC histology in nude mice. In contrast, GPM-1 cells only weakly expressed differentiation markers, showing a phenotype of E-cadherinlow+/MUC2-/MUC5AC-/Villinlow+. Characteristically, GPM-2 cells were found to highly express both membrane-bound mucin (MUC1/MUC4) and secreted mucin glycoproteins (MUC5AC/MUC6), whose expression is regulated by an epigenetic mechanism such as histone acetylation. GPM-2 cells also secreted a large amount of sTn antigen into the culture medium. These mucin profiles of GPM-2 cells are distinct from those of conventional SRCC cell lines (KATO III and HSC-39), which preferentially express intestinal MUC2/MUC4 as well as sLex and sLeA antigens. In addition, GPM-2 cells showed a slow growth rate, and a lower metastatic potential than GPM-1 cells. Conclusions These results indicate that the cells of the new SRCC line, GPM-2 cells, are more differentiated and less aggressive than NSRCC-type GPM-1 cells, and would thus offer an excellent model for understanding the molecular mechanism underlying the growth, differentiation, and mucin production of an SRCC gastric cancer cell line.
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The protein-disulfide isomerase (PDI) family member anterior gradient 2 (AGR2) is reportedly overexpressed in numerous cancers and plays a role in cancer development. However, to date the molecular functions of AGR2 remain to be characterized. Herein we have identified AGR2 as bound to newly synthesized cargo proteins using a proteomics analysis of endoplasmic reticulum (ER) membrane-bound ribosomes. Nascent protein chains that translocate into the ER associate with specific ER luminal proteins, which in turn ensures proper folding and posttranslational modifications. Using both imaging and biochemical approaches, we confirmed that AGR2 localizes to the lumen of the ER and indirectly associates with ER membrane-bound ribosomes through nascent protein chains. We showed that AGR2 expression is controlled by the unfolded protein response and is in turn is involved in the maintenance of ER homeostasis. Remarkably, we have demonstrated that siRNA-mediated knockdown of AGR2 significantly alters the expression of components of the ER-associated degradation machinery and reduces the ability of cells to cope with acute ER stress, properties that might be relevant to the role of AGR2 in cancer development.
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Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers largely due to disseminated disease at the time of presentation. Here, we investigated the role and mechanism of action of the metastasis-associated protein anterior gradient 2 (AGR2) in the pathogenesis of pancreatic cancer. AGR2 was induced in all sporadic and familial pancreatic intraepithelial precursor lesions (PanIN), PDACs, circulating tumor cells, and metastases studied. Confocal microscopy and flow cytometric analyses indicated that AGR2 localized to the endoplasmic reticulum (ER) and the external surface of tumor cells. Furthermore, induction of AGR2 in tumor cells regulated the expression of several ER chaperones (PDI, CALU, RCN1), proteins of the ubiquitin-proteasome degradation pathway (HIP2, PSMB2, PSMA3, PSMC3, and PSMB4), and lysosomal proteases [cathepsin B (CTSB) and cathepsin D (CTSD)], in addition to promoting the secretion of the precursor form pro-CTSD. Importantly, the invasiveness of pancreatic cancer cells was proportional to the level of AGR2 expression. Functional downstream targets of the proinvasive activity of AGR2 included CTSB and CTSD in vitro, and AGR2, CTSB, and CTSD were essential for the dissemination of pancreatic cancer cells in vivo. Taken together, the results suggest that AGR2 promotes dissemination of pancreatic cancer and that its cell surface targeting may permit new strategies for early detection as well as therapeutic management.
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In a search for proteins involved in cancer metastasis, we analyzed proteomes of the human gastric cancer cell OCUM-2M and its metastatic subline OCUM-2MLN. We observed that aspartate aminotransferase (AAT), D-site binding protein (DBP), and anterior gradient protein 2 (AGR2) are differentially expressed in metastatic OCUM-2MLN cells. Measurement of protein expression in clinical samples indicated that DBP and AAT are also down-regulated in metastatic adenocarcinoma. Additionally, urokinase-type tissue plasminogen activator is up-regulated in OCUM-2MLN cells and also in metastatic gastric cancer samples. Collectively, these results raise a possibility that AAT, DBP and AGR2 are functionally implicated in the invasiveness of gastric cancer cells.
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Ovarian cancer is a leading cause of death in women. Early detection of ovarian cancer is essential to decrease mortality. However, the early diagnosis of ovarian cancer is difficult due to a lack of clinical symptoms and suitable molecular diagnostic markers. Thus, identification of meaningful tumor biomarkers with potential clinical application is clearly needed. To search for a biomarker for the early detection of ovarian cancer, we identified human anterior gradient 2 (AGR2) from our systematic analysis of paired normal and ovarian tumor tissue cDNA microarray. We noted a marked overexpression of AGR2 mRNA and protein in early stage mucinous ovarian tumors compared to normal ovarian tissues and serous type ovarian tumors by Western blot analysis and immunohistochemistry. To further elucidate the role of AGR2 in ovarian tumorigenesis, stable 2774 human ovarian cancer cell lines overexpressing AGR2 were established. Forced expression of AGR2 in 2774 cells enhanced the growth and migration of ovarian cancer cells. AGR2 protein was detected in the serum of mucinous ovarian cancer patients by Western blot and ELISA analysis. Thus, AGR2 is a potential biomarker for the diagnosis of mucinous ovarian cancer and an ELISA assay may facilitate the early detection of mucinous ovarian cancer using patient serum.
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Transcriptomic screens in breast cancer cell lines have identified a protein named anterior gradient-2 (AGR2) as a potentially novel oncogene overexpressed in estrogen receptor (ER) positive tumours. As targeting the ER is responsible for major improvements in cure rates and prevention of breast cancers, we have evaluated the pro-oncogenic function of AGR2 in anti-hormone therapeutic responses. We show that AGR2 expression promotes cancer cell survival in clonogenic assays and increases cell proliferation and viability in a range of cancer cell lines. Chromatin immunoprecipitation and reporter assays indicate that AGR2 is transcriptionally activated by estrogen through ERalpha. However, we also found that AGR2 expression is elevated rather than inhibited in response to tamoxifen, thus identifying a novel mechanism to account for an agonistic effect of the drug on a specific pro-oncogenic pathway. Consistent with these data, clinical analysis indicates that AGR2 expression is related to treatment failure in ERalpha-positive breast cancers treated with tamoxifen. In contrast, AGR2 is one of the most highly suppressed genes in cancers of responding patients treated with the anti-hormonal drug letrozole. These data indicate that the AGR2 pathway represents a novel pro-oncogenic pathway for evaluation as anti-cancer drug developments, especially therapies that by-pass the agonist effects of tamoxifen.
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Anterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein. Its upregulation has been well documented in a number of cancers, particularly breast cancer, for which mixed data exist on the prognostic implications of AGR2 expression. Although emerging evidence indicates that AGR2 is associated with poor prognosis, its function and impact on cancer-relevant pathways have not been elucidated in breast cancer. To investigate the biologic role of AGR2 in breast cancer, AGR2 was transiently knocked down, by using siRNA, in T47 D and ZR-75-1 (estrogen receptor-alpha (ER)-positive) and MDA-MB-231 and SK-BR-3 (ER-negative) human breast cancer cell lines. The impact of silencing AGR2 was evaluated in both anchorage-dependent and anchorage-independent growth (soft agar, spheroid) assays. Cell-cycle profiles in ER-positive cell lines were determined with BrdU incorporation, and cell death was measured with Annexin V, JC-1, and F7-26 staining. After transiently silencing AGR2 or stimulating with recombinant AGR2, modulation of key regulators of growth and survival pathways was assessed with Western blot. Combination studies of AGR2 knockdown with the antiestrogens tamoxifen and fulvestrant were carried out and assessed at the level of anchorage-dependent growth inhibition and target modulation (cyclin D1, ER). AGR2 knockdown inhibited growth in anchorage-dependent and anchorage-independent assays, with a more-pronounced effect in ER-positive cell lines. Cyclin D1 levels and BrdU incorporation were reduced with AGR2 knockdown. Conversely, cyclin D1 was induced with recombinant AGR2. AGR2 knockdown induced cell death in ZR-75-1 and T47 D cells, and also downregulated survivin and c-Myc. Evidence of AGR2-ER crosstalk was demonstrated by a reduction of ER at the protein level after transiently silencing AGR2. AGR2 knockdown in combination with fulvestrant or tamoxifen did not preclude the efficacy of the antiestrogens, but enhanced it. In addition, p-Src, implicated in tamoxifen resistance, was downregulated with AGR2 knockdown. Transiently silencing AGR2 in ER-positive breast cancer cell lines inhibited cell growth and cell-cycle progression and induced cell death. Breast cancer drivers (ER and cyclin D1) as well as cancer-signaling nodes (pSrc, c-Myc, and survivin) were demonstrated to be downstream of AGR2. Collectively, the data presented support the utility of anti-AGR2 therapy in ER-positive breast cancers because of its impact on cancer-relevant pathways.
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Pancreatic cancer is a major oncological challenge due to its aggressive growth and metastasis. In the current study, we investigated the role of anterior gradient 2 (AGR2) in these processes. AGR2 mRNA, as assessed by quantitative real-time reverse transcription-PCR (Q-RT-PCR), was 14-fold higher in pancreatic cancer compared with normal and pancreatitis tissues. Immunohistochemistry revealed high expression of AGR2 in neoplastic cells with 98% (56 of 57) positivity on pancreatic cancer and minimal staining in normal and pancreatitis tissues. AGR2 was also expressed in early pancreatic intraepithelial neoplastic lesions. RT-PCR and Western blotting showed elevated AGR2 expression in seven of nine pancreatic cancer cell lines. AGR2, as detected in conditioned media from cancer cells, indicated that it was secreted. The influence of AGR2 on pancreatic cancer cells was evaluated by silencing with small interfering RNA and short hairpin RNA. Silencing of AGR2 significantly reduced cell proliferation (MTS assay) and invasion (Boyden chamber assay) and improved gemcitabine sensitivity (fluorescence-activated cell sorting analysis). Conditioned media from cells in which AGR2 was silenced had a reduced ability to stimulate proliferation of pancreatic cancer cells, suggesting that secreted AGR2 was active. In vivo, silencing of AGR2 in MPanc-96 cells led to a significant reduction of tumor growth and increased the effectiveness of gemcitabine treatments in orthotopic tumor models evaluated by noninvasive bioluminescence imaging. In summary, AGR2 is expressed and secreted during pancreatic cancer development and plays an important role in cancer cell growth and survival. These observations suggest that AGR2 may be a useful molecular target in pancreatic cancer.
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The DF3/MUC1 mucin-like, transmembrane glycoprotein is aberrantly overexpressed in most human carcinomas. The MUC1 cytoplasmic domain interacts with the c-Src tyrosine kinase and thereby increases binding of MUC1 and beta-catenin. In the present work, coimmunoprecipitation studies demonstrate that MUC1 associates constitutively with the epidermal growth factor receptor (EGF-R) in human ZR-75-1 breast carcinoma cells. Immunofluorescence studies show that EGF-R and MUC1 associate at the cell membrane. We also show that the activated EGF-R phosphorylates the MUC1 cytoplasmic tail on tyrosine at a YEKV motif that functions as a binding site for the c-Src SH2 domain. The results demonstrate that EGF-R-mediated phosphorylation of MUC1 induces binding of MUC1 to c-Src in cells. Moreover, in vitro and in vivo studies demonstrate that EGF-R increases binding of MUC1 and beta-catenin. These findings support a novel role for EGF-R in regulating interactions of MUC1 with c-Src and beta-catenin.
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The anterior gradient protein-2 (AGR2) is inducible by oestrogen and itself can induce metastasis in a rat model for breast cancer. Here, a rabbit antibody to recombinant human AGR2 was used to assess its prognostic significance in a retrospective cohort of 351 breast cancer patients treated by adjuvant hormonal therapy. The antibody stains 66% of breast carcinomas to varying degrees. The percentage of positive carcinoma cells in tumours directly correlates with the level of AGR2 mRNA (Spearman's rank correlation, P = 0.0007) and protein (linear regression analysis r2 = 0.95, P = 0.0002). There is a significant association of staining of carcinomas for AGR2 with oestrogen receptor alpha (ERalpha) staining and with low histological grade (both Fisher's Exact test P<0.0001). In the ERalpha-positive cases, but not the ERalpha-negative cases, when subdivided into the separate staining classes for AGR2, there is a significantly progressive decrease in patient survival with increased staining (log rank test, P = 0.006). The significant association of staining for AGR2 with patient death over a 10-year period (log rank test P = 0.007, hazard ratio = 3) only becomes significant at 6 years of follow-up. This may be due to the cessation of adjuvant hormonal therapy at an earlier time, resulting in adverse re-expression of the metastasis-inducing protein AGR2.
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The esophageal epithelium is subject to damage from bile acid reflux that promotes normal tissue injury resulting in the development of Barrett's epithelium. There is a selection pressure for mutating p53 in this preneoplastic epithelium, thus identifying a physiologically relevant model for discovering novel regulators of the p53 pathway. Proteomic technologies were used to identify such p53 regulatory factors by identifying proteins that were overexpressed in Barrett's epithelium. A very abundant polypeptide selectively expressed in Barrett's epithelium was identified as anterior gradient-2. Immunochemical methods confirmed that anterior gradient-2 is universally up-regulated in Barrett's epithelium, relative to normal squamous tissue derived from the same patient. Transfection of the anterior gradient-2 gene into cells enhances colony formation, similar to mutant oncogenic p53 encoded by the HIS175 allele, suggesting that anterior gradient-2 can function as a survival factor. Deletion of the C-terminal 10 amino acids of anterior gradient-2 neutralizes the colony enhancing activity of the gene, suggesting a key role for this domain in enhancing cell survival. Constitutive overexpression of anterior gradient-2 does not alter cell-cycle parameters in unstressed cells, suggesting that this gene is not directly modifying the cell cycle. However, cells overexpressing anterior gradient-2 attenuate p53 phosphorylation at both Ser(15) and Ser(392) and silence p53 transactivation function in ultraviolet (UV)-damaged cells. Deletion of the C-terminal 10 amino acids of anterior gradient-2 permits phosphorylation at Ser(15) in UV-damaged cells, suggesting that the C-terminal motif promoting colony survival also contributes to suppression of the Ser(15) kinase pathway. These data identify anterior gradient-2 as a novel survival factor whose study may shed light on cellular pathways that attenuate the tumor suppressor p53.
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AZD2171 is an oral, highly potent, and selective vascular endothelial growth factor signaling inhibitor that inhibits all vascular endothelial growth factor receptor tyrosine kinases. The purpose of this study was to investigate the activity of AZD2171 in gastric cancer. We examined the antitumor effect of AZD2171 on the eight gastric cancer cell lines in vitro and in vivo. AZD2171 directly inhibited the growth of two gastric cancer cell lines (KATO-III and OCUM2M), with an IC(50) of 0.15 and 0.37 micromol/L, respectively, more potently than the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib. Reverse transcription-PCR experiments and immunoblotting revealed that sensitive cell lines dominantly expressed COOH terminus-truncated fibroblast growth factor receptor 2 (FGFR2) splicing variants that were constitutively phosphorylated and spontaneously dimerized. AZD2171 completely inhibited the phosphorylation of FGFR2 and downstream signaling proteins (FRS2, AKT, and mitogen-activated protein kinase) in sensitive cell lines at a 10-fold lower concentration (0.1 micromol/L) than in the other cell lines. An in vitro kinase assay showed that AZD2171 inhibited kinase activity of immunoprecipitated FGFR2 with submicromolar K(i) values ( approximately 0.05 micromol/L). Finally, we assessed the antitumor activity of AZD2171 in human gastric tumor xenograft models in mice. Oral administration of AZD2171 (1.5 or 6 mg/kg/d) significantly and dose-dependently inhibited tumor growth in mice bearing KATO-III and OCUM2M tumor xenografts. AZD2171 exerted potent antitumor activity against gastric cancer xenografts overexpressing FGFR2. The results of these preclinical studies indicate that AZD2171 may provide clinical benefit in patients with certain types of gastric cancer.
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Context.—During the last 50 years, the incidence and mortality of gastric cancer has declined in many countries. This decline has primarily included the intestinal type (Lauren classification). However, there is an impression among pathologists that the diffuse type, especially the signet ring cell subtype, has become more prevalent. Objectives.—Using data from the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute, we analyzed the trends of the 2 primary types (intestinal and diffuse) of gastric carcinomas from 1973 through 2000. Design.—Trends in age-adjusted rates were determined for gastric carcinomas through the SEER statistical program (SEER*Stat), which is available on the Internet to the public. Results.—During the period studied, the intestinal type continued to decline in males, females, African Americans, and whites. The intestinal type was more common in males than in females and more common in African Americans than in whites. In contrast, a consistent increase in the rate of the diffuse type of gastric carcinoma was seen during this period. The rate increased from 0.3 cases per 100 000 persons in 1973 to 1.8 cases per 100 000 persons in 2000. This increase was seen in males, females, African Americans, and whites. The predominant increase occurred in the signet ring type. Conclusions.—The results indicate a progressive decrease in the incidence of the intestinal type of gastric cancer and an increase in the diffuse type of gastric carcinoma, especially the signet ring cell type. The clinical implications of the increase are considered.
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Anterior gradient 2 (AGR2) is a secreted, cancer-associated protein in many types of epithelial cancer cells. We developed a highly sensitive targeted mass spectrometric assay for quantification of AGR2 in urine and serum. Digested peptides from clinical samples were processed by PRISM (high pressure and high resolution separations coupled with intelligent selection and multiplexing), which incorporates high pH reversed-phase LC separations to fractionate and select target fractions for follow-on LC-SRM analyses. The PRISM-SRM assay for AGR2 showed a reproducibility of <10% CV and LOQ values of ~130 pg/mL in serum and ~10 pg per 100 μg total protein mass in urine, respectively. A good correlation (R2 = 0.91) was observed for the measurable AGR2 concentrations in urine between SRM and ELISA. Based on an initial cohort of 37 subjects, urinary AGR2/PSA concentration ratios showed a significant difference (P = 0.026) between non-cancer and cancer. Large clinical cohort studies are needed for the validation of AGR2 as a useful diagnostic biomarker for prostate cancer. Our work validated the approach of identifying candidate secreted protein biomarkers through genomics and measurement by targeted proteomics, especially for proteins where no immunoassays are available.
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Mucins are high-molecular-weight glycoproteins expressed throughout the gastrointestinal tract, with a key role in mucosal protection and function. In gastric cancer expression of MUC5AC and MUC1 is reduced and denovo expression of MUC2 occurs. With progressive loss of tumor differentiation and increased tumor stage, expression of MUC5AC and MUC1 is further reduced, and MUC2 decreases. Isolated MUC2 expression (the intestinal phenotype) correlates with metastatic spread and poor survival. There is emerging evidence that MUC1 acts as an oncoprotein when overexpressed. The cytoplasmic tail of MUC1 interacts with the H. pylori virulence factor cagA and is a major effector of the wnt-β catenin intracellular signalling cascade. Polymorphism in the MUC1 gene has been identified in gastric cancer patients and may have a prospective role in the stratification of high-risk subjects. The MUC1 gene also mediates resistance to the recombinant HER2/neu antibody trastuzumab. Future research efforts will examine targeting MUC1 for therapeutic purposes.
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The prevention of oral squamous cell carcinoma metastasis to improve overall patient survival has provided the rationale for biomarker development. We used the Transwell invasion assay to isolate a highly metastatic subpopulation, HSC-3-5 cells, with almost the same genetic background as HSC-3 cells but a higher metastatic capacity accompanied by cytoskeletal rearrangements and mesenchymal transformation. HSC-3-5 cells also showed tumorigenic and metastatic characteristics in vivo. In addition, Anterior gradient 2 (agr2), a pro-oncogenic signaling intermediate, was identified from gene expression profiling, and overexpression of AGR2 showed a positive correlation with oral tumor metastasis. Taken together, our data suggest that AGR2 may be a novel sensitive biomarker for metastatic oral cancer.
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Many regulatory proteins are homo-oligomeric and designing assays that measure self-assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein-interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer-associated oncoprotein AGR2. A two site-sandwich microtiter assay ((2S) MTA) was designed using a DyLight800-labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self-peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N-terminal region of AGR2 increase in trans oligomer stability as defined using the (2S) MTA assay. A DSS-crosslinking assay that traps the AGR2 dimer through K95-K95 adducts confirmed that Δ45-AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt-AGR2, Δ45-AGR2 (more stable dimer), and monomeric AGR2(E60A) revealed that Δ45-AGR2 was more active in binding to Reptin than either wt-AGR2 or the AGR2(E60A) mutant. Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its' biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its' oncogenic potential.
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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.
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Mucins are gel-forming proteins that are responsible for the characteristic viscoelastic properties of mucus. Mucin overproduction is a hallmark of asthma, but the cellular requirements for airway mucin production are poorly understood. The endoplasmic reticulum (ER) protein anterior gradient homolog 2 (AGR2) is required for production of the intestinal mucin MUC2, but its role in the production of the airway mucins MUC5AC and MUC5B is not established. Microarray data were analyzed to examine the relationship between AGR2 and MUC5AC expression in asthma. Immunofluorescence was used to localize AGR2 in airway cells. Coimmunoprecipitation was used to identify AGR2-immature MUC5AC complexes. Agr2(-/-) mice were used to determine the role of AGR2 in allergic airway disease. AGR2 localized to the ER of MUC5AC- and MUC5B-producing airway cells and formed a complex with immature MUC5AC. AGR2 expression increased together with MUC5AC expression in airway epithelium from "Th2-high" asthmatics. Allergen-challenged Agr2(-/-) mice had greater than 50% reductions in MUC5AC and MUC5B proteins compared with allergen-challenged wild-type mice. Impaired mucin production in Agr2(-/-) mice was accompanied by an increase in the proportion of mucins contained within the ER and by evidence of ER stress in airway epithelium. This study shows that AGR2 increases with mucin overproduction in individuals with asthma and in mouse models of allergic airway disease. AGR2 interacts with immature mucin in the ER and loss of AGR2 impairs allergen-induced MUC5AC and MUC5B overproduction.
Article
AGR2 has evolutionarily conserved roles in development and tissue regeneration and is linked with several human cancers. The exact functions and regulation of AGR2 are poorly understood, but current data identify AGR2 as a clinically relevant factor that modulates the behavior and response of hormone-dependent cancers (breast, prostate) and hormone-independent cancers (colorectal, pancreatic, esophageal and other common cancers). AGR2 protein expression induces metastasis, acts as a p53 tumor suppressor inhibitor and survival factor, participates directly in neoplastic transformation and is involved in drug resistance. Thus, AGR2 is an important tumor biomarker and negative prognostic factor potentially exploitable in clinical practice.
Article
Reports of clinicopathological features and prognosis in patients with signet ring cell carcinoma of the stomach (SRC) are conflicting. The aim was to describe the clinicopathological features and prognosis of patients with SRC in comparison with non-signet ring cell carcinoma of the stomach (NSRC). In this retrospective study, we reviewed the records of 1,439 consecutive patients diagnosed with gastric carcinoma who were resected surgically from 1993 to 2003. Among them, 218 patients (15.1%) with SRC were compared with 1,221 patients with NSRC. There were significant differences in tumor size, tumor location, macroscopic type, depth on invasion, lymph node metastasis, lymphatic invasion, tumor stage, chemotherapy, and curability between the patients with SRC histology and NSRC. The overall 5-year survival of patients with SRC was 44.9% as compared with 36.0% for patients with NSRC (P = 0.013). Multivariate analysis showed that lymph node metastasis and curative resection were significant factors affecting survival. A significant survival benefit for curative resection was observed, with a 5-year survival rate of 58.5% compared with non-curatively resected cases (8.4%). When stage matched, SRC patients had a similar survival to NSRC patients. Curative resection is recommended to improve the prognosis of patients with SRC.
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The secreted metastasis-inducing protein, human anterior gradient 2 (AGR2), has been independently reported to be associated with either a reduced or an increased survival of different groups of patients with breast cancer. We now aim to analyze the expression of AGR2 in a third completely independent group of patients using a specific AGR2 monoclonal antibody (mAb). Primary tumors from a group of 315 patients suffering from operable (stage I and II) breast cancer with 20-years follow-up were immunocytochemically stained with a specific mAb to AGR2 and associations with prognostic factors and patient survival were analyzed. The mAb specifically recognized AGR2 in Western blots, and positive staining for AGR2 was significantly associated with involved lymph nodes and staining for estrogen receptor alpha, progesterone receptor, and the metastasis-inducing proteins osteopontin, S100P, and S100A4. After 20 years of follow-up, only 26% of patients with AGR2-positive carcinomas survived compared with 96% of those with AGR2 negative carcinomas, with the highly significant difference in median survival times of 68 and >216 months, respectively (P < 0.0001). Cox's multivariate regression analysis showed that staining for AGR2 was one of the most significant independent prognostic indicators, with a corrected relative risk of 9.4. The presence of AGR2 in the primary tumor is therefore a possible prognostic indicator of poor patient outcome in breast cancer.
Article
Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals, but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells. Here, we show that AGR2 is present within the ER of intestinal secretory epithelial cells and is essential for in vivo production of the intestinal mucin MUC2, a large, cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, indicating a direct role for AGR2 in mucin processing. Mice lacking AGR2 were viable but were highly susceptible to colitis, indicating a critical role for AGR2 in protection from disease. We conclude that AGR2 is a unique member of the PDI family, with a specialized and nonredundant role in intestinal mucus production.
Article
Two unique human signet ring cell gastric carcinoma cell lines (designated HSC-39 and HSC-40A) were established in vitro from the ascites of a 54-year-old male patient. Both cell lines were biologically quite similar, grew in vitro in suspension with a population doubling time of 28-30 h, and had cytological features of mucinous epithelial tumor cells. They formed colonies in soft agar, with a cloning efficiency of 0.8-1.0%. Ultrastructurally, numerous granules were observed in the cytoplasm, suggesting secretory activity. The frequent presence of desmosome and the tight junction at the cell boundary certifies the epithelial origin of the lines. Immunocytochemistry and radioimmunoassay showed production of tumor marker antigens (carcinoembryonic antigen, CA 19-9, and sialyl-Lex-i) and gastrin in both lines. These lines were transplantable in athymic BALB/c nude mice. The histopathology of each line growing in athymic BALB/c nude mice was similar to that of the original tumor. The karyotype of the cells was highly aberrant with structural and numerical changes. The presence of numerous double minute chromosomes and loss of the 13 chromosome and Y-chromosome characterize these lines. In addition, the amplified c-myc oncogene (16-32-fold) was found in both cell lines and original ascitic tumor cells. Overexpression of the c-myc mRNA was noted. These cell lines may be a useful tool, providing both in vivo and in vitro systems for further studies of the biology and therapy of human signet ring cell (or Borrmann's type IV carcinoma) gastric carcinoma.
Article
In an attempt to elucidate various histological features of gastric cancers, seven human gastric adenocarcinomas were studied in vitro and in nude mice. Growth pattern of each cultured cell line in vitro corresponded well to the histological type of parent tumor. The cell lines, MKN7, MKN74, and MKN28 derived from differentiated carcinomas showed morphological characteristics of intestinal differentiation in cell polarity and microvilli with core-filaments in vitro as well as in nude mice. However, they gradually diminished the characteristics in course of time. The cell lines, MKN 45 and OKAJIMA, derived from undifferentiated carcinomas, had natures of not only ordinary gastric mucosa but also intestinal metaplastic mucosa. They seem to have multipotentiality for differentiation, and preserved well the natures for long periods of culture. The KWS-I cell line composed of undifferentiated cells in vitro displayed the potential for differentiation in nude mice. However, the differentiation of KATO-III cells derived from a signet-ring cell carcinoma was suppressed in nude mice. The common abnormality of chromosome was not found, and the growth rate in vitro was not dependent on the histological type of parent tumor.
Article
Gastric carcinomas had various pathological features. Based on patterns of growth and invasiveness, however, they fell into two types: expanding type and infiltrative type. These types were readily recognizable histologically: expanding carcinoma grew en masse and by expansion, resulting in the formation of discrete tumor nodules, whereas in infiltrative carcinoma tumor cells invaded individually. Both types showed varying degrees of cell maturation, but glands were much more common in expanding carcinoma. The difference in growth pattern was reflected partly by gross appearance of the tumors. These two types of carcinoma appeared to be different in their histogenetic origins. Intestinal metaplasia probably played a role in the development of expanding, but not infiltrative, carcinoma. There were differences also in the sex and age of the patients, survival rate, and epidemiological distribution. Thus, this classification provided a simple basis for evaluation of various aspects of gastric cancer.
Article
During the last 50 years, the incidence and mortality of gastric cancer has declined in many countries. This decline has primarily included the intestinal type (Lauren classification). However, there is an impression among pathologists that the diffuse type, especially the signet ring cell subtype, has become more prevalent. Using data from the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute, we analyzed the trends of the 2 primary types (intestinal and diffuse) of gastric carcinomas from 1973 through 2000. Trends in age-adjusted rates were determined for gastric carcinomas through the SEER statistical program (SEER*Stat), which is available on the Internet to the public. During the period studied, the intestinal type continued to decline in males, females, African Americans, and whites. The intestinal type was more common in males than in females and more common in African Americans than in whites. In contrast, a consistent increase in the rate of the diffuse type of gastric carcinoma was seen during this period. The rate increased from 0.3 cases per 100 000 persons in 1973 to 1.8 cases per 100 000 persons in 2000. This increase was seen in males, females, African Americans, and whites. The predominant increase occurred in the signet ring type. The results indicate a progressive decrease in the incidence of the intestinal type of gastric cancer and an increase in the diffuse type of gastric carcinoma, especially the signet ring cell type. The clinical implications of the increase are considered.
Article
AGR2, the human homologue of Xenopus anterior gradient 2 (XAG2), was identified by a suppression subtractive hybridization-based technique as an androgen-inducible gene. There are two AGR2 transcripts, which encode the same secretory protein of 175 amino acids. The androgen induction was time- and dose-dependent, with more than a 10-fold increase in the level of AGR2 mRNA after 48 hr of treatment with 10(-9) M R1881. Expression of AGR2 mRNA was specifically detected in limited human tissue rich in epithelial cells, including the prostate gland. Analysis of 46 microdissected primary prostate adenocarcinoma samples showed that AGR2 mRNA expression was markedly elevated in the majority of tumors as compared to matched adjacent benign tissues. Androgen-induced AGR2 protein expression was demonstrated in LNCaP cells by Western blot analysis with an anti-AGR2 antibody. Immunohistochemistry analysis indicated that AGR2 protein expression was highly restricted to the secretory epithelial cells in the prostate gland. In tissue sections from radical prostatectomy specimens, immunohistochemical staining of AGR2 showed markedly increased expression in high-grade prostatic intraepithelial neoplasia and Gleason pattern 3-4 prostatic adenocarcinoma. Therefore, the androgen-induced secretory protein AGR2 may serve as a potential therapeutic target and/or molecular marker for prostate cancer.
Article
The number of published studies on peritoneal dissemination of scirrhous gastric carcinoma is very small as a result of the unavailability of highly reproducible animal models. Orthotopic implantation of HSC-44PE and HSC-58 (scirrhous gastric carcinoma-derived cell lines) cells into nude mice led to dissemination of the tumor cells to the greater omentum, mesenterium, peritoneum and so on, and caused ascites in a small number of animals. Cycles of isolation of the ascitic tumor cells and orthotopic inoculation of these cells were repeated in turn to animals. This was to isolate highly metastatic cell lines with a strong capability of inducing the formation of ascites (44As3 from HSC-44PE; 58As1 and 58As9 from HSC-58). All three cell lines induced tumor formation at the site of orthotopic injection, and caused fatal cancerous peritonitis and bloody ascites in 90-100% of the animals approximately 3-5 weeks after the inoculation. When the parent cells were implanted, the animals became moribund in approximately 12-18 weeks, however, none of the animals developed ascites. Complementary DNA microarray and immunohistochemical analyses revealed differences in the expression levels of genes coding for the matrix proteinase, cell adhesion, motility, angiogenesis and proliferation between the highly metastatic- and parent-cell lines. The usefulness of this model for the evaluation of drugs was assessed by analyzing the stability of the metastatic potential of the cells and the reproducibility. Animals intravenously treated with CPT-11 and GEM showed suppressed tumor growth and significantly prolonged survival. The metastatic cell lines and the in vivo model established in the present study are expected to serve as a model of cancerous peritonitis developing from primary lesions, and as a useful means of clarifying the pathophysiology of peritoneal dissemination of scirrhous gastric carcinoma and the development of drugs for its treatment.
Article
The AGR2 gene encodes a secretory protein that is highly expressed in adenocarcinomas of the esophagus, pancreas, breast, and prostate. This study explores the effect of AGR2 expression with well-established in vitro and in vivo assays that screen for cellular transformation and tumor growth. AGR2 expression in SEG-1 esophageal adenocarcinoma cells was reduced with RNA interference. Cellular transformation was examined using NIH3T3 cells that express AGR2 after stable transfection. The cell lines were studied in vitro with assays for density-dependent and anchorage-independent growth, and in vivo as tumor xenografts in nude mice. SEG-1 cells with reduced AGR2 expression showed an 82% decrease in anchorage-independent colony growth and a 60% reduction in tumor xenograft size. In vitro assays of AGR2-expressing NIH3T3 cells displayed enhanced foci formation and anchorage-independent growth. In vivo, AGR2-expressing NIH3T3 cells established tumors in nude mice. Thus, AGR2 expression promotes tumor growth in esophageal adenocarcinoma cells and is able to transform NIH3T3 cells. Immunohistochemistry of the normal mouse intestine detected AGR2 expression in proliferating and differentiated intestinal cells of secretory lineage. AGR2 may be important for the growth and development of the intestine as well as esophageal adenocarcinomas.
AZD2171 shows potent antitumor activity against gastric cancer
  • M Takeda
  • T Arao
  • H Yokote
  • T Komatsu
  • K Yanagihara
  • H Sasaki
Takeda M, Arao T, Yokote H, Komatsu T, Yanagihara K, Sasaki H, et al. AZD2171 shows potent antitumor activity against gastric cancer
TNM classification of malignant tumors Japanese classification of gastric carcinoma: 3rd English edition
  • L Sobin
  • Witteknd
Sobin L, Witteknd C. TNM classification of malignant tumors. 6th ed. New York, NY: Wiley-Liss; 2002. 31. Japanese classification of gastric carcinoma: 3rd English edition. Gastric Cancer 2011;14:101–12.