Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial–mesenchymal transition

Abstract

Anterior gradient 2 (AGR2), a member of the protein disulfide isomerase family, has been implicated in various cancers including pancreatic ductal adenocarcinoma (PDAC) and is known to promote cancer progression. However, the prognostic value of AGR2 expression and the interaction with epithelial-mesenchymal transition (EMT) remain unclear. We investigated the clinical significance of AGR2 and EMT markers in PDAC patients by immunohistochemical analyses. Although AGR2 expression was not observed in normal pancreas, all pancreatic precursor neoplastic lesions were positive for AGR2, even at the earliest stages, including pancreatic intraepithelial neoplasia-1A, AGR2 expression was reduced in 27.7% (54/195 cases) of PDAC patients. AGR2 downregulation correlated with EMT markers (vimentin overexpression and reduced membranous E-cadherin expression), high Union for International Cancer Control stage (Po0.0001), high histological cellular grade (Po0.0001), and adverse outcome (Po0.0001). In vitro, targeted silencing of AGR2 in cancer cells using siRNA reduced cell proliferation, colony formation, cell invasiveness, and migration, but did not alter EMT markers. To confer a more aggressive phenotype and induce EMT in PDAC cells, we co-cultured PDAC cell lines with primary-cultured pancreatic stellate cells (PSCs) and found that AGR2 was downregulated in co-cultured PDAC cells compared with PDAC monocultures. Treatment with transforming growth factor beta-1 (TGF-b), secreted from PSCs, decreased AGR2 expression, whereas inhibition of TGF-b signaling using recombinant soluble human TGF-b receptor type II and TGF-b-neutralizing antibodies restored AGR2 expression. We conclude that AGR2 downregulation is a useful prognostic marker, induced by EMT, and that secreted TGF-b from PSCs may partially contribute to AGR2 downregulation in PDAC patients. AGR2 downregulation does not induce EMT or a more aggressive phenotype, but is a secondary effect of these processes in advanced PDAC.

Regulation of Gene Expression by Sodium Valproate in Epithelial-to-Mesenchymal Transition

“…Moreover, although ID2 has been previously associated with EMT and shown to suppress the SMAD pathway [39][40][41], decreasing of ID2 expression by TGF-b1 stimulation was confirmed by RNA-Seq and real-time RT-PCR in the present study. Previously, a role for AGR2 in EMT was investigated [42][43][44]. We also confirmed that AGR2 expression was down-regulated by TGF-b1.…”

Downregulation of AGR2, p21, and cyclin D and alterations in p53 function were associated with tumor progression and chemotherapy resistance in epithelial ovarian carcinoma

“…In tumorigenesis, AGR2 plays an important role by activating survival and metastasis pathways by interacting with cyclin D1, cathepsin B, D, Myc, p‐Src, and EGFR and by blocking cell death inhibiting p53 function. The overexpression of AGR2 was observed in several tumor types, which supports the hypothesis of AGR2 be an oncogene, but the downregulation of this gene also was observed in prostate tumors, ovarian, colorectal, and pancreas …”

“…Nevertheless, original studies of AGR2 show that this protein has an important function in the preservation of cellular homeostasis, by promoting the differentiation of secretory epithelial cells . Downregulation of AGR2 seems to be related to tumor progression of tumors originating in secretory organs . Thus, hypothetically, in the early stage of the tumor, increased AGR2 expression could activate proliferative mechanisms, such as increased cyclin D1, but with tumor progression, the reduction in AGR2 could activate mechanisms of invasion and metastasis by reducing adhesion molecules .…”

Mechanisms of anterior gradient-2 regulation and function in cancer

“…In addition, K-ras(G12D)/Smad4(lox/lox) mice that are heterozygous for agr2 are attenuated in initiation and progression to pancreatic ductal cell carcinoma [74]. Consistent with this study, secretion of TGF-␤ from pancreatic stellate cells might reduce agr2 expression in pancreatic ductal adenocarcinoma [75]. Another cancer type where the TGF-␤/AGR2 axis might play a role in carcinogenesis is oesophageal adenocarcinoma [68].…”