Anterior Gradient Homolog Research Articles

Potential toxicity of graphene (oxide) quantum dots via directly covering the active site of anterior gradient homolog 2 protein

Graphene quantum dots (GQDs) have attracted significant attention in biomedicine, while extensive investigations have revealed a reverse regarding the potential biotoxicity of GQDs. In order to supplementing the understanding of the toxicity profile of GQDs, this study employs a molecular dynamics (MD) simulation approach to systematically investigate the potential toxicity of both GQDs and Graphene Oxide Quantum Dots (GOQDs) on the Anterior Gradient Homolog 2 (AGR2) protein, a key protein capable of protecting the intestine. We construct two typical simulation systems, in which an AGR2 protein is encircled by either GQDs or GOQDs. The MD results demonstrate that both GQDs and GOQDs can directly make contact with and even cover the active site (specifically, the Cys81 amino acid) of the AGR2 protein. This suggests that GQDs and GOQDs have the capability to inhibit or interfere with the normal biological interaction of the AGR2 active site with its target protein. Thus, GQDs and GOQDs exhibit potential detrimental effects on the AGR2 protein. Detailed analyses reveal that GQDs adhere to the Cys81 residue due to van der Waals (vdW) interaction forces, whereas GOQDs attach to the Cys81 residue through a combination of vdW (primary) and Coulomb (secondary) interactions. Furthermore, GQDs aggregation typically adsorb onto the AGR2 active site, while GOQDs adsorb to the active site of AGR2 one by one. Consequently, these findings shed new light on the potential adverse impact of GQDs and GOQDs on the AGR2 protein via directly covering the active site of AGR2, providing valuable molecular insights for the toxicity profile of GQD nanomaterials.

Intracellular AGR2 transduces PGE2 stimuli to promote epithelial–mesenchymal transition and metastasis of colorectal cancer

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.

Cloning, Expression, Purification, and Characterization of Soluble Bioactive Recombinant Human Anterior Gradient Homolog 2 - DsRed Monomer Protein in Escherichia coli

Cloning, Expression, Purification, and Characterization of Soluble Bioactive Recombinant Human Anterior Gradient Homolog 2 - DsRed Monomer Protein in Escherichia coli

Dietary Soy Protein Isolate Attenuates Intestinal Immunoglobulin and Mucin Expression in Young Mice Compared with Casein.

Dietary protein sources have profound effects on children and young animals, and are important for the gut barrier function and immune resilience. Milk and soy are the main sources of protein for children and young animals after weaning. The objective of this study was to compare the effects of dairy and soy proteins on the intestinal barrier in early development. Weanling C57BL/6 mice were fed AIN-93G diets prepared with casein or soy protein isolate (SPI) for 21 days. Compared with those fed with the casein diet, mice fed with the SPI diet did not change their body weight and organ coefficients, but increased their feed intake and ratio of feed to gain. SPI lowered the level of luminal secretory immunoglobulin A (SIgA) and downregulated the levels of IL-4, IL-13, polymeric immunoglobulin receptor (Pigr), Janus kinase 1 (Jak1), signal transducer and activator of transcription 6 (Stat6), and transforming growth factor-β (Tgfb) in the mouse ileum. Western blotting of ileal proteins confirmed that SPI suppressed the activation of the JAK1/STAT6 signaling pathway. Furthermore, SPI attenuated intestinal mucin production, as demonstrated by the decreased numbers of intestinal goblet cells and the reduced relative expression levels of mucin 1 (Muc1), mucin 2 (Muc2), trefoil factor 3 (Tff3), glucose-regulated protein 94 (Grp94), and anterior gradient homolog 2 (Agr2). The results indicated that the SPI diet could attenuate mouse intestinal immunity, as demonstrated by decreased SIgA and mucin production in the intestine. Therefore, we suggest that our findings should be of consideration when SPI or casein are used as dietary protein sources.

Long noncoding RNA LINC00460 conduces to tumor growth and metastasis of hepatocellular carcinoma through miR-342-3p-dependent AGR2 up-regulation.

Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor in the world. It ranks third among cancer-induced deaths worldwide and has the characteristics of high metastasis and high recurrence rate. Long non-coding RNA (LncRNA) LINC00460 is significantly up-regulated in multiple types of cancers and is closely related to the progression of tumors. However, effects of LINC00460 and corresponding regulatory path in HCC are still poorly investigated.In our study, we found that expression of LINC00460 was up-regulated in HCC tissues and cell lines compared with the control. Then we revealed that knockdown of LINC00460 suppressed cell proliferation and cell mobility and induced cell apoptosis in HCC cells. Further study demonstrated that knockdown of LINC00460 suppressed the progression of HCC by elevating the expression of microRNA (miRNA, miR)-342-3p. Besides that, metastasis marker, Anterior gradient homolog 2 (AGR2) was found to be a target of miR-342-3p and overexpression of AGR2 promoted the progression of HCC. Finally, the in vivo experiments further verified the anti-tumor effects of LINC00460 / miR-342-3p / AGR2 axis in HCC.The LINC00460 / miR-342-3p / AGR2 axis exerts anti-tumor effect in HCC in vitro and in vivo, consolidating and expanding the research about targeted gene therapy for early diagnosis and treatment of HCC.

Identification, characterization and application of a new peptide against anterior gradient homolog 2 (AGR2)

The cancer-associated protein Anterior Gradient 2 (AGR2) has been described, predominantly in adenocarcinomas. Increased levels of extracellular AGR2 (eAGR2) have been correlated with poor prognosis in cancer patients, making it a potential biomarker. Additionally, neutralizing AGR2 antibodies showed preclinical effectiveness in murine cancer models suggesting eAGR2 may be a therapeutic target.We set out to identify a peptide by mRNA display that would serve as a theranostic tool targeting AGR2. This method enables the selection of peptides from a complex (>1011) library and incorporates a protease incubation step that filters the selection for serum stable peptides. We performed six successive rounds of enrichment using a 10-amino acid mRNA display library and identified several AGR2 binding peptides. One of these peptides (H10), demonstrated high affinity binding to AGR2 with a binding constant (KD) of 6.4 nM. We developed an AGR2 ELISA with the H10 peptide as the capture reagent. Our H10-based ELISA detected eAGR2 from cancer cell spent media with a detection limit of (20-50 ng/ml). Furthermore, we investigated the therapeutic utility of H10 and discovered that it inhibited cell viability at IC50 (9-12 μmoles/L) in cancer cell lines. We also determined that 10 μg/ml of H10 was sufficient to inhibit cancer cell migration in breast and prostate cancer cell lines. A control peptide did not show any appreciable activity in these cells. The H10 peptide showed promise as both a novel diagnostic and a potential therapeutic peptide.

Hybridization chain reaction coupled with the fluorescence quenching of gold nanoparticles for sensitive cancer protein detection

An ultrasensitive and enzyme-free sensing platform was designed for sensitive detection of anterior gradient homolog 2 (AGR2) based on the fluorescence quenching of gold nanoparticles (AuNPs) coupled with hybridization chain reaction (HCR) amplification. To construct the sensor, two fluorophore labeled hairpin probes (HP1 and HP2) that contained the sticky tails were designed, and AGR2 aptamer was used as an initiator for the happen of HCR between HP1 and HP2 to form a long nicked dsDNA duplex. Such DNA duplex could not adsorb onto the surface of AuNPs, and a strong fluorescence signal appeared due to the label of HP1 and HP2 with fluorophore. In the presence of AGR2, AGR2 aptamer would specifically recognize with it and accordingly could not trigger the happen of HCR. As a result, the sticky tails of HP1 and HP2 would adsorb onto the surface of AuNPs to bring the fluorophore into the close proximity of the AuNPs, and lead to the quenching of the fluorescence signal. By using such method, AGR2 could be sensitively detected in the range of 5.0pM–1.0nM based on monitoring the decrease of the fluorescence signal. Importantly, the assay could realize the detection of AGR2 effectively in diluted human serum samples.

Epidermal Growth Factor Receptor (EGFR) Signaling Requires a Specific Endoplasmic Reticulum Thioredoxin for the Post-translational Control of Receptor Presentation to the Cell Surface

The epidermal growth factor receptor (EGFR) is a well characterized receptor-tyrosine kinase that functions in development and serves a vital role in many human cancers. Understanding EGFR regulatory mechanisms, and hence approaches for clinical intervention, has focused on ligand-receptor interactions and tyrosine kinase activity. Here, we show using the NCI-H460 lung and A431 epidermoid human cancer cell lines that EGFR binding to anterior gradient homolog 2 (AGR2) in the endoplasmic reticulum is required for receptor delivery to the plasma membrane and thus EGFR signaling. Reduced AGR2 protein levels or mutation of an essential cysteine in the active site result in decreased cell surface EGFR and a concomitant decrease in signaling as reflected by AREG, EGR1, and FOS expression. Similar to previously described EGFR nulls, an AGR2 null also resulted in embryonic lethality. Consistent with its role in regulating EGFR-mediated signaling, AGR2 expression is also enhanced in many human cancers and promotes the transformed phenotype. Furthermore, EGFR-mediated signaling in NCI-H460 cells, which are resistant to the tyrosine kinase inhibitor AG1478, is also disrupted with reduced AGR2 expression. The results provide insights into why cancer prognosis or response to therapy often does not correlate with EGFR protein or RNA levels because they do not reflect delivery to the cell surface where signaling is initiated. AGR2, therefore, represents a novel post-translational regulator of EGFR-mediated signaling and a promising target for treating human cancers.

The sandwich-type aptasensor based on gold nanoparticles/DNA/magnetic beads for detection of cancer biomarker protein AGR2

A simple and stable method for the detection of cancer biomarker anterior gradient homolog 2 (AGR2) using UV–vis spectrometry is described. The sensing system is constructed easily with magnetic beads, aptamer, its complementary probe, and regular-shaped gold nanoparticles. The designed aptasensor exhibits excellent efficiency, selectivity and sensitivity toward AGR2 protein detection, and shows a picomolar range of detection limit. This extraordinary performance can be attributed to the effective separation of magnetic beads, the highly selective Gquadruplex formation of AGR2 proteins recognized by aptamers, the unique optical properties of gold nanoparticles and the special sandwich structure of the aptasensor.

AGR2-mediated lung adenocarcinoma metastasis novel mechanism network through repression with interferon coupling cytoskeleton to steroid metabolism-dependent humoral immune response

7 anterior gradient homolog 2 (AGR2)-inhibited different molecular mutual positive correlation network was constructed in lung adenocarcinoma compared with human normal adjacent tissues by 17 overlapping molecules of 358 GRNInfer and 19 Pearson (AGR2 CC⩽−0.25). Based on GO, KEGG, GenMAPP, BioCarta and disease databases, we determined AGR2-mediated lung adenocarcinoma metastasis through repression with cytoskeleton of MAST1; steroid metabolism of SOAT2; humoral immune response of POU2AF1; interferon alpha-inducible of IFI6; immunoglobulin of IGKC_3, CTA_246H3.1. Thus we proposed AGR2-mediated lung adenocarcinoma metastasis novel mechanism network through repression with interferon coupling cytoskeleton to steroid metabolism-dependent humoral immune response.

Validation of four candidate pancreatic cancer serological biomarkers that improve the performance of CA19.9

BackgroundThe identification of new serum biomarkers with high sensitivity and specificity is an important priority in pancreatic cancer research. Through an extensive proteomics analysis of pancreatic cancer cell lines and pancreatic juice, we previously generated a list of candidate pancreatic cancer biomarkers. The present study details further validation of four of our previously identified candidates: regenerating islet-derived 1 beta (REG1B), syncollin (SYCN), anterior gradient homolog 2 protein (AGR2), and lysyl oxidase-like 2 (LOXL2).MethodsThe candidate biomarkers were validated using enzyme-linked immunosorbent assays in two sample sets of serum/plasma comprising a total of 432 samples (Sample Set A: pancreatic ductal adenocarcinoma (PDAC, n = 100), healthy (n = 92); Sample Set B: PDAC (n = 82), benign (n = 41), disease-free (n = 47), other cancers (n = 70)). Biomarker performance in distinguishing PDAC from each control group was assessed individually in the two sample sets. Subsequently, multiparametric modeling was applied to assess the ability of all possible two and three marker panels in distinguishing PDAC from disease-free controls. The models were generated using sample set B, and then validated in Sample Set A.ResultsIndividually, all markers were significantly elevated in PDAC compared to healthy controls in at least one sample set (p ≤ 0.01). SYCN, REG1B and AGR2 were also significantly elevated in PDAC compared to benign controls (p ≤ 0.01), and AGR2 was significantly elevated in PDAC compared to other cancers (p < 0.01). CA19.9 was also assessed. Individually, CA19.9 showed the greatest area under the curve (AUC) in receiver operating characteristic (ROC) analysis when compared to the tested candidates; however when analyzed in combination, three panels (CA19.9 + REG1B (AUC of 0.88), CA19.9 + SYCN + REG1B (AUC of 0.87) and CA19.9 + AGR2 + REG1B (AUC of 0.87)) showed an AUC that was significantly greater (p < 0.05) than that of CA19.9 alone (AUC of 0.82). In a comparison of early-stage (Stage I-II) PDAC to disease free controls, the combination of SYCN + REG1B + CA19.9 showed the greatest AUC in both sample sets, (AUC of 0.87 and 0.92 in Sets A and B, respectively).ConclusionsAdditional serum biomarkers, particularly SYCN and REG1B, when combined with CA19.9, show promise as improved diagnostic indicators of pancreatic cancer, which therefore warrants further validation.

15-Deoxy-Δ12,14-prostaglandin J2 induces growth inhibition, cell cycle arrest and apoptosis in human endometrial cancer cell lines

15-Deoxy-∆(12,14)-prostaglandin J(2) (15d-PGJ(2)), a peroxisome proliferator-activated receptor γ ligand, has been reported to have antiproliferative activity in certain types of cancer. The purpose of this study was to elucidate the effect of 15d-PGJ(2) on endometrial cancer cells, as well as the mechanism of action. Endometrial cancer-derived cells (HHUA, Ishikawa and HEC-59) were treated with various concentrations of 15d-PGJ(2), and its effects on cell growth, the cell cycle and apoptosis were investigated in vitro. Using cDNA microarrays, some potential targets of this drug were identified. All endometrial cancer cell lines were sensitive to the growth-inhibitory effect of 15d-PGJ(2). Cell cycle arrest at the G2/M phase of the cell cycle and induction of apoptosis were observed. Concerning the gene expression changes induced by 15d-PGJ(2) treatment, the upregulation of aldo-keto reductase family 1 member C3 (AKR1C3) and the downregulation of anterior gradient homolog 3 (AGR3) and nitric oxide synthase 2A (NOS2A) were confirmed using western blot analysis in all the cell lines examined. These results suggest that 15d-PGJ(2) may be a novel therapeutic option for the treatment of endometrial cancer.

The ER stress transducer IRE1β is required for airway epithelial mucin production.

Inflammation of human bronchial epithelia (HBE) activates the endoplasmic reticulum (ER) stress transducer inositol-requiring enzyme 1 (IRE1)α, resulting in IRE1α-mediated cytokine production. Previous studies demonstrated ubiquitous expression of IRE1α and gut-restricted expression of IRE1β. We found that IRE1β is also expressed in HBE, is absent in human alveolar cells, and is upregulated in cystic fibrosis and asthmatic HBE. Studies with Ire1β(-/-) mice and Calu-3 airway epithelia exhibiting IRE1β knockdown or overexpression revealed that IRE1β is expressed in airway mucous cells, is functionally required for airway mucin production, and this function is specific for IRE1β vs. IRE1α. IRE1β-dependent mucin production is mediated, at least in part, by activation of the transcription factor X-box binding protein-1 (XBP-1) and the resulting XBP-1-dependent transcription of anterior gradient homolog 2, a gene implicated in airway and intestinal epithelial mucin production. These novel findings suggest that IRE1β is a potential mucous cell-specific therapeutic target for airway diseases characterized by mucin overproduction.

Identification, Characterization and Application of a G-Quadruplex Structured DNA Aptamer against Cancer Biomarker Protein Anterior Gradient Homolog 2

BackgroundAnterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism.Methodology/Principal FindingsThrough a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with Kd values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2.Conclusion/SignificanceIn this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.

An accurate and rapid detection of lymph node metastasis in non-small cell lung cancer patients based on one-step nucleic acid amplification assay

A sublobar resection is currently recognized as an option for early small-sized non-small cell lung cancer (NSCLC), and intraoperative rapid and accurate lymph node assessment is required for a complete resection. To solve this issue, we investigated the clinical utility of one-step nucleic acid amplification (OSNA) assay, an automated rapid molecular diagnostic method and its optimal mRNA marker for detection of lymph node metastasis in lung cancer. We extracted 16 target candidate mRNA markers with high expression in lung cancer from a genetic database, and then quantified their expression levels by quantitative RT-PCR using surgically dissected lymph nodes with or without metastasis. Cytokeratin 19 (CK19), cytokeratin 7 (CK7), stratifin (SFN), and anterior gradient homolog 2 (AGR2) showed significant differences for mRNA expression between metastasis-negative and -positive lymph nodes in quantitative-RT-PCR screening. CK19 and CK7 were finally selected as potential target markers and were quantified using OSNA assay findings of 165 dissected lymph nodes obtained from 49 lung cancer patients. The OSNA assay with CK19 and CK7 were completed within 40min and their positive predictive value, negative predictive value, and accuracy comparing to pathological diagnosis with hematoxylin–eosin staining and immunohistochemistry were shown to be 95.0%, 99.3%, and 98.8%, and 85.0%, 97.9%, and 96.4%, respectively, using a cut-off value of 250 copies/μL. Among the 165 lymph nodes tested, 1 false negative result was due to massive necrosis of cancer cells and 1 false positive was caused by the allocation bias of cancer cells in the sampling in patient with pleural dissemination. The best performance was observed when CK19 was used as a marker, while the addition of CK7 mRNA as a marker did not increase sensitivity or specificity. In conclusion, an OSNA assay using CK19 could be effective for molecular diagnosis of lymph node metastasis in lung cancer. This is the first report suggesting the potential clinical utility of OSNA assay for intraoperative rapid diagnosis of nodal status in lung cancer.

Immunohistochemical panel for distinguishing esophageal adenocarcinoma from squamous cell carcinoma: a combination of p63, cytokeratin 5/6, MUC5AC, and anterior gradient homolog 2 allows optimal subtyping

Distinguishing adenocarcinoma and squamous cell carcinoma of the esophagus is often based on morphological criteria and can be difficult in small biopsies. We analyzed commonly used immunohistochemical markers (p63, cytokeratin 5/6, cytokeratin 7, CDX2, MUC2, and MUC5AC) and 2 new markers, anterior gradient homolog 2 and SOX2, in esophageal carcinomas to establish the best panel to distinguish these tumors. Tissue microarrays with 69 esophageal adenocarcinomas and 41 whole sections of esophageal squamous cell carcinomas were stained for these markers and semiquantitatively scored. Sensitivities and specificities were calculated for individual markers and select combinations using the morphological diagnosis as a gold standard. All squamous cell carcinomas expressed p63 with 38 of 41 demonstrating reactivity in more than 75% of tumor cells. Cytokeratin 5/6 expression was seen in 40 of 41 squamous cell carcinomas with 39 of 41 demonstrating reactivity in more than 75% of tumor cells. SOX2 expression was present in 35 of 41 of squamous cell carcinomas but also in 24 of 69 of adenocarcinomas, frequently demonstrating extensive reactivity in adenocarcinomas. Anterior gradient homolog 2 was highly sensitive for adenocarcinoma and present in 68 of 69 of cases, but anterior gradient homolog 2 reactivity was also identified in 15 of 41 of squamous cell carcinomas, typically demonstrating focal reactivity in squamous cell carcinoma. MUC5AC expression was seen almost exclusively in adenocarcinomas with only a single squamous cell carcinoma demonstrating focal MUC5AC staining. Overall, the dual expression of both p63 and cytokeratin 5/6 was 99% specific and 98% sensitive for squamous cell carcinoma. In addition, anterior gradient homolog 2 and MUC5AC are useful positive markers of adenocarcinoma in the setting of absent or diminished p63 and cytokeratin 5/6 staining.

Abstract 3380: Comparison proteome analysis of scirrhous gastric carcinoma stem cell-like SP and hypoxia-resistant cell lines

Abstract Intro Cancer stem cells (CSCs) are considered to be responsible for cancer metastasis, and hypoxia is supposed to be an important regulator of CSCs differentiation. Furthermore, tumor hypoxia was reported to be associated with more aggressive tumor phenotypes such as high metastatic ability and resistance to various anti-cancer therapies which may lead to a poorer prognosis. This raises the question of whether there might be proteins representing similar alterations which are responsible for the correlation between hypoxic and CSCs phenotypes. We have established a diffuse-type of gastric carcinoma cell line (OCUM-12), and a hypoxia-resistant cancer cell line (OCUM-12/Hypo) cloned from parent OCUM-12 cells by continuous exposure to 1% oxygen. The side population (SP), as evaluated by a flow cytometric analysis using Hoechst 33342, has been known as CSC-rich population. To investigate and compare the proteomes of the hypoxic (OCUM-12/Hypo), stem cell (OCUM-12SP) and parent OCUM-12 diffuse-type gastric carcinoma cell lines, protein lysates from those cell lines were analyzed using QSTAR Elite LC MS/MS. Method Triplicate pooled samples (10 ug protein each) from stomach cancer cell lines were prepared and labeled with iTRAQ reagents. MS/MS data were searched against the Swiss Protein database (HUMAN) using ProteinPilot™ 2.0 software (AB Sciex). The Ingenuity Pathway analysis program was utilized, to assign biological significance, and to identify networks of interacting differentially expressed proteins, functional groups and pathways. Result In both OCUM-12SP and OCUM-12/Hypo cells, significant overexpression of Wnt signaling pathway was obvious. Furthermore, elevation of MTHFD1, a protein controlling histidine and purine nucleotide metabolism, cytokeratins 18 (CK18), and 19 (CK19) regulating cell cycle, apoptosis and angiogenesis, transcriptional regulator ANP32A, and cellular chaperones heat shock proteins A9 and STIP was observed as compared to the parent OCUM-12 cell line. Furthermore, anterior gradient homolog 2 (AGR2)was down-regulated in both cell lines. Conclusion Wnt signal, MTHFD1, CK18, CK19, ANP32A, HSPA9 and STIP, AGR2 might be responsible for the stem-like phenotype, hypoxia resistance and higher invasiveness of gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3380. doi:1538-7445.AM2012-3380

Evaluation of the Adenocarcinoma-Associated Gene AGR2 and the Intestinal Stem Cell Marker LGR5 as Biomarkers in Colorectal Cancer

We aim to estimate the diagnostic performances of anterior gradient homolog-2 (AGR2) and Leucine-rich repeat-containing-G-protein-coupled receptor 5 (LGR5) in peripheral blood (PB) as mRNA biomarkers in colorectal cancer (CRC) and to explore their prognostic significance. Real-time PCR was used to analyze AGR2 and LGR5 in 54 stages I-IV CRC patients and 19 controls. Both mRNAs were significantly increased in PB from CRC patients compared to controls. The area under the receiver-operating characteristic curves were 0.722 (p = 0.006), 0.376 (p = 0.123) and 0.767 (p = 0.001) for AGR2, LGR5 and combined AGR2/LGR5, respectively. The AGR2/LGR5 assay resulted in 67.4% sensitivity and 94.7% specificity. AGR2 correlated with pT3–pT4 and high-grade tumors. LGR5 correlated with metastasis, R2 resections and high-grade. The progression-free survival (PFS) of patients with high AGR2 was reduced (p = 0.037; HR, 2.32), also in the stage I-III subgroup (p = 0.046). LGR5 indicated a poor prognosis regarding both PFS (p = 0.007; HR, 1.013) and overall survival (p = 0.045; HR, 1.01). High AGR2/LGR5 was associated with poor PFS (p = 0.014; HR, 2.8) by multivariate analysis. Our findings indicate that the assessment of AGR2 and LGR5 in PB might reflect the presence of circulating tumor cells (CTC) and stem cell like CTC in CRC. Increased AGR2 and LGR5 are associated with poor outcomes.

AGR2 is induced in asthma and promotes allergen-induced mucin overproduction.

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.

Genome-Wide Transcriptomic Analysis of Intestinal Tissue to Assess the Impact of Nutrition and a Secondary Nematode Challenge in Lactating Rats

BackgroundGastrointestinal nematode infection is a major challenge to the health and welfare of mammals. Although mammals eventually acquire immunity to nematodes, this breaks down around parturition, which renders periparturient mammals susceptible to re-infection and an infection source for their offspring. Nutrient supplementation reduces the extent of periparturient parasitism, but the underlying mechanisms remain unclear. Here, we use a genome wide approach to assess the effects of protein supplementation on gene expression in the small intestine of periparturient rats following nematode re-infection.Methodology/Principal FindingsThe use of a rat whole genome expression microarray (Affymetrix Gene 1.0ST) showed significant differential regulation of 91 genes in the small intestine of lactating rats, re-infected with Nippostrongylus brasiliensis compared to controls; affected functions included immune cell trafficking, cell-mediated responses and antigen presentation. Genes with a previously described role in immune response to nematodes, such as mast cell proteases, and intelectin, and others newly associated with nematode expulsion, such as anterior gradient homolog 2 were identified. Protein supplementation resulted in significant differential regulation of 64 genes; affected functions included protein synthesis, cellular function and maintenance. It increased cell metabolism, evident from the high number of non-coding RNA and the increased synthesis of ribosomal proteins. It regulated immune responses, through T-cell activation and proliferation. The up-regulation of transcription factor forkhead box P1 in unsupplemented, parasitised hosts may be indicative of a delayed immune response in these animals.Conclusions/SignificanceThis study provides the first evidence for nutritional regulation of genes related to immunity to nematodes at the site of parasitism, during expulsion. Additionally it reveals genes induced following secondary parasite challenge in lactating mammals, not previously associated with parasite expulsion. This work is a first step towards defining disease predisposition, identifying markers for nutritional imbalance and developing sustainable measures for parasite control in domestic mammals.