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Background: Anterior gradient-2 (AGR2) is a proto-oncogene involved in tumorigenesis and cancer progression. AGR2, predominantly localized in the endoplasmic reticulum (ER), is also a secreted protein detected in the extracellular compartment in multiple cancers. However, the biological functions of intracellular and extracellular AGR2 remain to be elucidated.Methods: Based on the biochemical structure of AGR2 protein, PANC-1 pancreatic cancer cells stably expressing ER-resident or secreted AGR2 were generated by a lentivirus-mediated stable overexpression system. The capacities of cell proliferation, migration, invasion and survival were assessed in PANC-1 stable cells. Moreover, EGFR expression and activation were determined to explore the possible mechanism of AGR2 roles in pancreatic cancer tumorigenesis.Results: It was discovered that secreted AGR2, but not ER-resident AGR2, promotes cell proliferation, migration and invasion of PANC-1 cells. Moreover, the data indicated that both the ER-resident and the secreted AGR2 enhance the survival capacity of PANC-1 cells after tunicamycin-induced ER stress and gemcitabine treatment. However, EGFR expression and activation were not found to be involved in AGR2-dependent oncogenic phenotypes in PANC-1 cells.Conclusions: Secreted AGR2 is predominantly involved in cell proliferation, migration and invasion in PANC-1 pancreatic cancer cells. Both secreted and ER-resident AGR2 contribute to the survival of PANC-1 cells under the challenging conditions. These findings provide insight into how different localizations of AGR2 have contributed to pancreatic cancer growth, metastasis, and drug sensitivity. [ABSTRACT FROM AUTHOR]

There is growing epidemiological evidence indicating an association between diabetes mellitus and the increased incidence of colorectal cancer (CRC). The preferred initial and most widely used pharmacological agent for the treatment of type 2 diabetes is metformin, which in parallel reduces the risk of CRC and improves patient prognosis. AMP-activated protein kinase (AMPK) appears to be tightly associated with the beneficial metabolic effects of metformin, serving as a cellular energy sensor activated in response to a variety of conditions that deplete cellular energy levels. Such conditions include nutrient starvation (particularly glucose), hypoxia and exposure to toxins that inhibit the mitochondrial respiratory chain complex. The aim of the present study was to determine the effect of metformin on CRC cell lines, with different levels of anterior gradient 2 (AGR2) expression, exposed to 5-fluorouracil (5-FU) and oxaliplatin, alone or in combination with metformin. AGR2 has recently emerged as a factor involved in colon carcinogenesis. In AGR2-knockout cells, markedly higher levels of phosphorylated-AMPK were observed in comparison with control cells transfected with GFP-scrambled guide RNA, which indicated that the presence of AGR2 may interfere with the metformin-dependent activation of AMPK. In addition, metformin in combination with 5-FU and oxaliplatin induced ROS production and attenuated autophagy. This effect was enhanced in AGR2-knockout cells. [ABSTRACT FROM AUTHOR]

In recent years, the discovery of 'tumour niche', a microenvironment that favours tumour development has changed our perspective of cancer. This microenvironment generated by the tumour cells itself and surrounding cells is capable of providing essential elements for its growth. Consequently, the homoeostasis of the secretory pathway (SP) has become an essential player in cancer development. The SP not only promotes cellular adaptation to protein misfolding due to oncogenic transformation or challenging tumour niche but also allows tumour cells to produce specific secretomes. This impacts tumour cells in cis‐ or trans‐ as well as stromal cells in the tumour niche. In this context, the Anterior GRadient 2 (AGR2) protein has been identified as a key player. AGR2 is a protein disulphide isomerase that resides in the endoplasmic reticulum (ER) and mediates the formation of disulphide bonds, catalyses the cysteine‐based redox reactions and assists the quality control of proteins. AGR2 not only plays an essential role in the homoeostasis of the SP but also exerts pro‐oncogenic gain‐of‐function due to its reported mislocalisation in the tumour niche microenvironment. In this review, we summarise the dual role of AGR2, inside and outside the ER, on the tumour niche and its microenvironment. Review: Anterior Gradient 2 (AGR2), an ER‐resident protein (iAGR2), is also a secreted molecule (eAGR2) that plays a pivotal role in tumor niche establishment and, as a consequence, participates in inflammation, immunogenicity, Extracellular matrix (ECM) remodeling, Epithelial‐Mesenchymal transition (EMT) and angiogenesis. Here, we provide an in‐depth description of e/iAGR2 in the context of the tumor niche and how e/iAGR2, through the Tumor Niche Secretome (TNS), might play a role in these functions. [ABSTRACT FROM AUTHOR]

AGR2 is a well-studied secreted protein that is involved in multiple biological processes including cell proliferation and migration. The mechanism by which AGR2 increases the growth and migration of non-small cell lung cancer cells (NSCLC) is still unknown. In this study, we report that AGR2 is directly targeted by miR-342-3p. Functional studies suggest that overexpression of miR-342-3p inhibits the proliferation and migration of non-small cell lung cancer cells. Overexpression of AGR2 counteracts the phenotypes induced by miR-342-3p. Moreover, AGR2 expression is up-regulated and negatively correlated with miR-342-3p levels in NSCLC cells and tissues. A meta-analysis of survival data indicates that NSCLC patients with high levels of AGR2 in their tumors have a worse prognosis. Collectively, the identification of miR-342-3p and AGR2 might facilitate the development of biomarkers and therapeutic targets for this devastating disease. [ABSTRACT FROM AUTHOR]

Background: Anterior gradient 2 (AGR2) has been implicated in tumor-associated phenotypes such as cell viability, invasion and metastasis in various human cancers. However, the tumor promoting activity of AGR2 has not yet been determined in biliary tract cancers. Thus, we examined the expression of AGR2 and its tumor-promoting activity in biliary tract cancer cells in this study. Methods: Expression of AGR2 mRNA and protein was analyzed by real time RT-PCR and western blotting, respectively. MTT assay was employed to measure cell viability and pulsed BrdU incorporation by proliferating cells was monitored by flow cytometry. Soft agar colony formation assay and transwell invasion assay were employed to determine anchorage-independent growth and in vitro invasion of the tumor cells, respectively. In vivo tumor formation was examined by injection of tumor cells into immunocompromised mice subcutaneously. Statistical analysis was performed with 2-tailed unpaired Student's t-test for continuous data and with one-way ANOVA for multiple group comparisons. Bonferroni tests were used for post hoc 2-sample comparisons. Results: AGR2 mRNA was detected in SNU-245, SNU-478, and SNU-1196 cell lines, and its protein expression was confirmed in SNU-478 and SNU-245 cell lines by western blot analysis. Knockdown of AGR2 expression with an AGR2-specific short hairpin RNA (shRNA) in SNU-478, an ampulla of Vater cancer cell line resulted in decreased cell viability and in decreased anchorage-independent growth by 98%. The AGR2 knockdown also increased the sensitivity of the cells to chemotherapeutic drugs, including gemcitabine, 5-fluorouracil and cisplatin. In addition, SNU-478 cells expressing AGR2-shRNA failed to form detectable tumor xenografts in nude mice, whereas control cells formed tumors with an average size of 179 ± 84 mm³ in 3 weeks. Overexpression of AGR2 in SNU-869 cells significantly increased cell viability through enhanced cell proliferation and the number of Matrigel™-invading cells compared with AGR2-negative SNU-869 cells. Conclusions: Our findings implicate that AGR2 expression augments tumor-associated phenotypes by increasing proliferative and invasive capacities of the ampulla of Vater cancer cells. [ABSTRACT FROM AUTHOR]

Background Through a transcriptome microarray analysis, we have isolated Anterior gradient protein 2 (AGR2) as a gene up-regulated in papillary thyroid carcinoma (PTC). AGR2 is a disulfide isomerase over-expressed in several human carcinomas and recently linked to endoplasmic reticulum (ER) stress. Here, we analyzed the expression of AGR2 in PTC and its functional role. Methods Expression of AGR2 was studied by immunohistochemistry and real time PCR in normal thyroids and in PTC samples. The function of AGR2 was studied by knockdown in PTC cells and by ectopic expression in non-transformed thyroid cells. The role of AGR2 in the ER stress was analyzed upon treatment of cells, expressing or not AGR2, with Bortezomib and analyzing by Western blot the expression levels of GADD153. Results PTC over-expressed AGR2 at mRNA and protein levels. Knockdown of AGR2 in PTC cells induced apoptosis and decreased migration and invasion. Ectopic expression of AGR2 in non-transformed human thyroid cells increased migration and invasion and protected cells from ER stress induced by Bortezomib. Conclusions AGR2 is a novel marker of PTC and plays a role in thyroid cancer cell survival, migration, invasion and protection from ER stress. [ABSTRACT FROM AUTHOR]

Abstract: The pro-metastatic protein anterior gradient-2 (AGR2) was previously demonstrated as a predictive factor of poor response to tamoxifen treatment. In this study we aimed to delineate the key signalling pathway that may contribute to regulation of AGR2 protein induction in order to identify novel targets to overcome tamoxifen resistance in tumour cells. Together, our data identify PDPK1-AKT as a pro-oncogenic signalling pathway that triggers AGR2 protein induction in response to tamoxifen and suggest that AKT inhibitors could be used as part of a therapeutic strategy to treat tamoxifen resistant, AGR2 over-expressing cancers. [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: The Anterior Gradient (AGR) genes AGR2 and AGR3 are part of the Protein Disulfide Isomerase (PDI) family and harbour core thioredoxin folds (CxxS motifs) that have the potential to regulate protein folding and maturation. A number of proteomics and transcriptomics screens in the fields of limb regeneration, cancer cell metastasis, pro-oncogenic oestrogen-signalling, and p53 regulation have identified AGR2 as a novel component of these signalling pathways. Curiously, despite the fact that the AGR2 and AGR3 genes are contiguous on chromosome 7p21.1-3, the AGR3 protein has rarely been identified in such OMICs screens along with AGR2 protein. Therefore there is little information on how AGR3 protein is expressed in normal and diseased states. A panel of three monoclonal antibodies was generated towards AGR3 protein for identifying novel clinical models that can be used to define whether AGR3 protein could play a positive or negative role in human cancer development. One monoclonal antibody was AGR3-specific and bound a linear epitope that could be defined using both pep-scan and phage-peptide library screening. Using this monoclonal antibody, endogenous AGR3 protein expression was shown to be cytosolic in four human ovarian cancer subtypes; serous, endometrioid, clear cell, and mucinous. Mucinous ovarian cancers produced the highest number of AGR3 positive cells. AGR3 expression is coupled to AGR2 expression only in mucinous ovarian cancers, whereas AGR3 and AGR2 expressions are uncoupled in the other three types of ovarian cancer. AGR3 expression in ovarian cancer is independent of oestrogen-receptor expression, which is distinct from the oestrogen-receptor dependent expression of AGR3 in breast cancers. Isogenic cancer cell models were created that over-express AGR3 and these demonstrated that AGR3 mediates cisplatin-resistance in mouse xenografts. These data indicate that AGR3 is over-expressed by a hormone (oestrogen-receptor α)-independent mechanism and identify a novel protein-folding associated pathway that could mediate resistance to DNA-damaging agents in human cancers. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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 ERα. 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 ERα-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. [ABSTRACT FROM AUTHOR]

Pancreatic cancer is a serious malignancy accompanied by a well-documented poor prognosis. Accumulating studies have indicated the crucial roles played by long non-coding RNAs (lncRNAs) in proliferation, apoptosis and invasion of cancer cells. The aim of the current study was to investigate the role of lncRNA LINC01207 in autophagy and apoptosis of pancreatic cancer cells and its regulatory mechanism interacting with miR-143-5p. Initially, expression profiles of lncRNAs and genes associated with pancreatic cancer were identified. The expression patterns of LINC01207, miR-143-5p and AGR2 in both pancreatic cancer and adjacent tissues were then determined. The binding relationship of LINC01207 to miR-143-5p and targeting relationship of miR-143-5p to AGR2 were subsequently verified. Silencing of LINC01207, or up-regulation or down-regulation of miR-143-5p was introduced into the pancreatic cancer cells, so as to analyze their effects on the cell growth, apoptosis and autophagy. Besides, these regulatory effects were further explored with the determination of the autophagy- and apoptosis-related gene or proteins. LINC01207 and AGR2 were highly expressed while miR-143-5p was poorly expressed in pancreatic cancer. Functionally, LINC01207 can bind to miR-143-5p, and AGR2 was a target gene of miR-143-5p. Importantly, silencing of LINC01207 down-regulated the expression of AGR2 by up-regulating miR-143-5p. Moreover, silencing of LINC01207 and up-regulation of miR-143-5p promoted cell apoptosis and autophagy, corresponding to increased expression of autophagy- and apoptosis-related proteins, in addition to inhibited cell growth. Taken together, silencing of LINC01207 prevents the progression of pancreatic cancer by impairing miR-143-5p-targeted AGR2 expression, providing a potential target for pancreatic cancer treatment. • Effects of lncRNA on pancreatic cancer cell autophagy and apoptosis are explored. • LINC01207 and AGR2 are elevated while miR-143-5p is declined in pancreatic cancer. • LINC01207 increases AGR2 expression through inhibiting miR-143-5p expression. • LINC01207 silencing promotes autophagy and apoptosis in pancreatic cancer cells. • This study provides an attractive target for the treatment of pancreatic cancer. [ABSTRACT FROM AUTHOR]