The Human Adenocarcinoma-associated Gene, AGR2, Induces Expression of Amphiregulin through Hippo Pathway Co-activator YAP1 Activation

Abstract

Anterior Gradient Homolog 2 (AGR2) is expressed by the normal intestine and by most human adenocarcinomas, including those derived from the esophagus, pancreas, lung, breast, ovary, and prostate. Xenografts of human adenocarcinoma cell lines in nude mice previously demonstrated that AGR2 supports tumor growth. In addition, AGR2 is able to induce in vitro a transformed phenotype in fibroblast and epithelial cell lines. The mechanism underlying the growth promoting effects of AGR2 is unknown. The present study shows that AGR2 induces expression of amphiregulin (AREG), a growth promoting EGFR ligand. Induced AREG expression in adenocarcinoma cells is able to rescue the transformed phenotype that is lost when AGR2 expression is reduced. Additional experiments demonstrate that AGR2 induction of AREG is mediated by activation of the Hippo signaling pathway co-activator, YAP1. Thus AGR2 promotes growth by regulating the Hippo and EGF receptor signaling pathways. Anterior Gradient Homolog 2 (AGR2)2 encodes a 17 kDa protein that is highly conserved in vertebrates. AGR2 was first described in Xenopus laevis, where its expression is responsible for the development of a glandular organ called the cement gland (1). A significant role in tissue regeneration was established for AGR2 in salamanders where it functions in nerve-dependent limb regeneration (2). AGR2 is also expressed by secretory cells in the normal murine intestine (3). In humans, enhanced AGR2 expression was first described in breast cancer, which was followed by similar observations in most human adenocarcinomas, including those derived from the esophagus, pancreas, lung, ovary, and prostate (4 -11). Both in vitro and in vivo studies demonstrated that AGR2 promotes tumor growth and metastasis (3,6,12). In adenocarcinoma cell lines and nontransformed fibroblasts, AGR2 induces cell proliferation and anchorage-independent growth in soft agar. Human adenocarcinoma cell lines grown in vivo as mouse xenografts result in smaller tumors when AGR2 expression is reduced (3, 6). In vitro studies examining cell migration suggested that AGR2 may function in a non-cell autonomous fashion (3).The mechanisms responsible for AGR2 effects on growth and transformation are unknown. AGR2 expression in humans is restricted to epithelial cells, for which the EGF signaling pathway serves a regulatory role in controlling cell growth. The present study tested the hypothesis that AGR2 affects cell signaling, and potentially that of the EGFR pathway, which has established significance in epithelial cancers. EXPERIMENTAL PROCEDURESCell Lines-H460 lung adenocarcinoma cells obtained from Dr. David Beer (University of Michigan, Ann Arbor, MI) were previously known as SEG-1 and was the focus of a previous publication (3). A recent report revealed that SEG-1 cells are actually H460 lung adenocarcinoma cells (13). The H460 cells used in this study were reassessed by Winand Dinjens, Erasmus Medical Center, Rotterdam, the Netherlands using the Powerplex 16 TM system (Promega Corp., Madison, WI) to...

Loss of Anterior Gradient 2 (Agr2) Expression Results in Hyperplasia and Defective Lineage Maturation in the Murine Stomach

“…Because a definitive marker for stem cells in the gastric corpus has yet to be identified, the hypothesis is inferred from previous studies that have impli- The results concerning cell proliferation may be viewed as contrary to previously published data in which Agr2 enhanced the growth and cell division of adenocarcinoma cell lines. Previous work demonstrated that Agr2 expression activates the Hippo and EGF signaling pathways in lung and esophageal adenocarcinoma cell lines (28). These results may be reconciled if there are different stimuli for cell division among stem cells and progenitors.…”

“…Several studies have also demonstrated that Agr2 supports many of the transformed properties of adenocarcinoma cell lines (26,27). Potential mechanisms include activation of the Hippo signaling pathway through the co-activator Yap1 and the induced expression of the EGF receptor ligand amphiregulin (28). The present study explored the in vivo function of Agr2 by generating a null mouse.…”

The aneurogenic limb identifies developmental cell interactions underlying vertebrate limb regeneration

“…HQ676606). The activity of human AGR2 protein on lung adenocarcinoma cells in culture depends on stimulation of amphiregulin expression by activation of the Hippo signaling pathway coactivator YAP1 (33). When the axons regenerate after amputation of the newt limb, the nAG protein is up-regulated first in the Schwann cells of the distal nerve sheath and then in gland cells underlying the wound epidermis (26).…”

AGR2 Gene Function Requires a Unique Endoplasmic Reticulum Localization Motif

“…AGR2 expression in esophageal and lung adenocarcinoma cells induces expression of the EGF receptor ligand amphiregulin (AREG), which is responsible for the transformed phenotype observed in cultured cells in vitro (21). In addition, AGR2 stimulation of AREG expression required activation of the Hippo signaling pathway co-activator, YAP1.…”