Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-α and survivin

Abstract

IntroductionAnterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein. Its upregulation has been well documented in a number of cancers, particularly breast cancer, for which mixed data exist on the prognostic implications of AGR2 expression. Although emerging evidence indicates that AGR2 is associated with poor prognosis, its function and impact on cancer-relevant pathways have not been elucidated in breast cancer.MethodsTo investigate the biologic role of AGR2 in breast cancer, AGR2 was transiently knocked down, by using siRNA, in T47 D and ZR-75-1 (estrogen receptor-α (ER)-positive) and MDA-MB-231 and SK-BR-3 (ER-negative) human breast cancer cell lines. The impact of silencing AGR2 was evaluated in both anchorage-dependent and anchorage-independent growth (soft agar, spheroid) assays. Cell-cycle profiles in ER-positive cell lines were determined with BrdU incorporation, and cell death was measured with Annexin V, JC-1, and F7-26 staining. After transiently silencing AGR2 or stimulating with recombinant AGR2, modulation of key regulators of growth and survival pathways was assessed with Western blot. Combination studies of AGR2 knockdown with the antiestrogens tamoxifen and fulvestrant were carried out and assessed at the level of anchorage-dependent growth inhibition and target modulation (cyclin D1, ER).ResultsAGR2 knockdown inhibited growth in anchorage-dependent and anchorage-independent assays, with a more-pronounced effect in ER-positive cell lines. Cyclin D1 levels and BrdU incorporation were reduced with AGR2 knockdown. Conversely, cyclin D1 was induced with recombinant AGR2. AGR2 knockdown induced cell death in ZR-75-1 and T47 D cells, and also downregulated survivin and c-Myc. Evidence of AGR2-ER crosstalk was demonstrated by a reduction of ER at the protein level after transiently silencing AGR2. AGR2 knockdown in combination with fulvestrant or tamoxifen did not preclude the efficacy of the antiestrogens, but enhanced it. In addition, p-Src, implicated in tamoxifen resistance, was downregulated with AGR2 knockdown.ConclusionsTransiently silencing AGR2 in ER-positive breast cancer cell lines inhibited cell growth and cell-cycle progression and induced cell death. Breast cancer drivers (ER and cyclin D1) as well as cancer-signaling nodes (pSrc, c-Myc, and survivin) were demonstrated to be downstream of AGR2. Collectively, the data presented support the utility of anti-AGR2 therapy in ER-positive breast cancers because of its impact on cancer-relevant pathways.

Genome-scale analysis of DNA methylation in lung adenocarcinoma and integration with mRNA expression

“…Also of interest are AGR2, KRT8, and SFN. AGR2 is a known proto-oncogene recently confirmed to be overexpressed in lung adenocarcinoma (Pizzi et al 2012), and has been shown to promote cell proliferation and migration in several different cancers (Ramachandran et al 2008;Vanderlaag et al 2010;Park et al 2011). KRT8, encoding keratin 8, was previously reported to be up-regulated in lung adenocarcinoma (Wikman et al 2002); and SFN, encoding stratifin, has been reported to be overexpressed in early invasive lung adenocarcinoma (Shiba-Ishii et al 2011).…”

Agr2 Mediates Paracrine Effects on Stromal Fibroblasts That Promote Invasion by Gastric Signet-Ring Carcinoma Cells

“…Indeed, the effect of Agr2 on cell proliferation is controversial. For example, overexpression of Agr2 in NIH3T3 cells promotes their proliferation (18), and Agr2 exerts a proproliferative effect in various cancers (17,18,39). However, proliferation of stem or progenitor cells in the gastric mucosa is elevated in Agr2-deficient mice (40).…”

Anterior gradient 2 profiling in Barrett columnar epithelia and adenocarcinoma

“…AGR2 belongs to the protein disulfide isomerase family, which modulates a number of cell functions, from cell adhesion to DNA binding [8,9]. Being part of this enzyme family, AGR2 is considered an important oncogene with a role in the promotion, growth, and dissemination of many adenocarcinomas [10][11][12][13][14].…”

“…Preliminary studies point to AGR2 overexpression in several adenocarcinomas [11][12][13][14][15] as well as in BAc [10,16,17]. AGR2 has also recently been described as one of the most overexpressed genes involved in esophageal metaplastic transformation in the p63-deficient mouse model [18].…”