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Anterior gradient-2 (AGR2) is highly expressed in several tumors and plays an important role in tumor development. However, the biological function of AGR2 in teratomas has not yet been thoroughly studied. In this study, AGR2 was found to be upregulated in teratoma tissues and in human testicular teratoma cell lines by Western blotting and qRT-PCR assays. A DNA Methylation-Specific PCR assay demonstrated that AGR2 upregulation resulted from hypomethylated AGR2 in teratoma cells. NCC-IT and NT2-D1 cells were transfected with pcDNA-AGR2 or sh-AGR2 to obtain AGR2-overexpressed or -silenced cells, and cell proliferation, invasion and glycolysis were determined using CCK-8, 5-ethynyl-2′-deoxyuridine (EdU), Transwell assays, and commercial kits. The results revealed that overexpression of AGR2 promoted teratoma cell proliferation and invasion and elevated glycolysis levels evidencing by the increase in lactate secretion, glucose consumption, ATP levels and the expression of glycolysis-related proteins, while knockdown of AGR2 showed the opposite results. The interactions between AGR2 and annexin A2 (AnXA2), as well as between AnXA2 and epidermal growth factor receptor (EGFR) were verified by co-immunoprecipitation assay. Mechanistic studies revealed that AGR2 interacts with AnXA2 and increases the level of AnXA2 to recruit more AnXA2 to EGFR, there by promoting EGFR expression. A series of rescue experiments showed that knockdown of AnXA2 or EGFR weakened the promotional effects of AGR2 overexpression on the proliferation, invasion, and glycolysis of teratoma cells. Finally, tumorigenicity assays were performed using NT2-D1 cells stably transfected with either LV-NC-shRNA or LV-shAGR2. The results showed that AGR2 knockdown significantly inhibited teratoma tumor growth in vivo. In conclusion, our data suggested that AGR2 facilitates glycolysis in teratomas through promoting EGFR expression by interacting with AnXA2, thereby promoting teratoma cells proliferation and invasion. [ABSTRACT FROM AUTHOR]

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

Proteins targeted to secretory pathway enter the endoplasmic reticulum where they undergo post-translational modification and subsequent quality control executed by exquisite catalysts of protein folding, protein disulphide isomerases (PDIs). These enzymes can often provide strict conformational protein folding solutions to highly cysteine-rich cargo as they facilitate disulphide rearrangement in the endoplasmic reticulum. Under conditions when PDI substrates are not isomerised properly, secreted proteins can accumulate in the endoplasmic reticulum leading to endoplasmic reticulum stress initiation with implications for human disease development. Anterior Gradient-2 (AGR2) is an endoplasmic reticulum-resident PDI superfamily member that has emerged as a dominant effector of basic biological properties in vertebrates including blastoderm formation and limb regeneration. AGR2 perturbation in mammals influences disease processes including cancer progression and drug resistance, asthma, and inflammatory bowel disease. This review will focus on the molecular characteristics, function, and regulation of AGR2, views on its emerging biological functions and misappropriation in disease, and prospects for therapeutic intervention into endoplasmic reticulum-resident protein folding pathways for improving the treatment of human disease. [ABSTRACT FROM AUTHOR]

Abstract: 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-terminal region followed by a thioredoxin fold. The protein exists in monomer–dimer equilibrium with a K d of 8.83μM, 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 dimerization is less important for this activity. The structural data of AGR2 show a separation between potential catalytic redox activity and adhesion function within the context of metastasis and development. [Copyright &y& Elsevier]

Abstract: Anterior Gradient 2 (AGR2), an ER stress-inducible protein, has been reported to be localized in endoplasmic reticulum (ER) and its level is elevated in numerous metastatic cancers. Recently, it has been demonstrated that AGR2 is involved in the control of ER homeostasis. However, the molecular mechanism how AGR2 regulates ER stress response remains unclear. Herein we show that AGR2 homo-dimerizes through an intermolecular disulfide bond. Moreover, dimerization of AGR2 attenuates ER stress-induced cell death through the association with BiP/GRP78. Thus, these results suggest that dimerization of AGR2 is crucial in mediating the ER stress signaling pathway. [Copyright &y& Elsevier]

Abstract: Agr2 is a putative protein disulfide isomerase (PDI) initially identified as an estrogen-responsive gene in breast cancer cell lines. While Agr2 expression in breast cancer is positively correlated with estrogen receptor (ER) expression, it is upregulated in both hormone dependent and independent carcinomas. Several in vitro and xenograft studies have implicated Agr2 in different oncogenic features of breast cancer; however, the physiological role of Agr2 in normal mammary gland development remains to be defined. Agr2 expression is developmentally regulated in the mammary gland, with maximum expression during late pregnancy and lactation. Using a mammary gland specific knockout mouse model, we show that Agr2 facilitates normal lobuloalveolar development by regulating mammary epithelial cell proliferation; we found no effects on apoptosis in Agr2 -/- mammary epithelial cells. Consequently, mammary glands of Agr2 -/- females exhibit reduced expression of milk proteins, and by two weeks post-partum their pups are smaller in size. Utilizing a conditional mouse model, we show that Agr2 constitutive expression drives precocious lobuloalveolar development and increased milk protein expression in the virgin mammary gland. In vitro studies using knock down and overexpression strategies in estrogen receptor positive and negative mammary epithelial cell lines demonstrate a role for Agr2 in estradiol-induced cell proliferation. In conclusion, the estrogen-responsive Agr2, a candidate breast cancer oncogene, regulates epithelial cell proliferation and lobuloalveolar development in the mammary gland. The pro-proliferative effects of Agr2 may explain its actions in early tumorigenesis. [Copyright &y& Elsevier]

Abstract: Anterior gradient-2 (AGR2) functions in a range of biological systems, including goblet cell formation, limb regeneration, inhibition of p53, and metastasis. There are no well-validated binding proteins for AGR2 protein despite the wealth of data implicating an important cellular function in vertebrates. The yeast two-hybrid system was used to isolate the ATP binding protein Reptin as an AGR2-interacting protein. AGR2 formed a stable complex in human cell lysates with Reptin, thus validating Reptin as an AGR2 binding protein in cells. Reptin was also shown to be overproduced in a panel of primary breast cancer biopsy specimens, relative to normal adjacent tissue from the same patient, suggesting a role in cancer growth in vivo. Mutations were made at the two ATP binding motifs in Reptin to evaluate the effects of ATP on Reptin–AGR2 complex stability. Loss-of-ATP binding mutations at the Walker A motif (K83A) or gain-of-ATP binding mutations at the Walker B motif (D299N) resulted in Reptin mutants with altered oligomerization, thermostability, and AGR2 binding properties. These data indicate that the two ATP binding motifs of Reptin play a role in regulating the stability of the AGR2–Reptin complex. The minimal region of AGR2 interacting with Reptin was localized using overlapping peptide libraries derived from the AGR2 protein sequence. The Reptin docking site was mapped to a divergent octapeptide loop in the AGR2 superfamily between amino acids 104 and 111. Mutations at codon Y104 or F111 in full-length AGR2 destabilized the binding of Reptin. These data highlight the existence of a protein docking motif on AGR2 and an ATP-regulated peptide-binding activity for Reptin. This knowledge has implications for isolating other AGR2-interacting proteins, for developing assays to isolate small molecules that target the Reptin ATP binding site, and for measuring the effects of the Reptin–AGR2 complex in cancer cell growth. [Copyright &y& Elsevier]

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