The estrogen-regulated anterior gradient 2 (AGR2) protein in breast cancer: a potential drug target and biomarker

Abstract

Initially discovered as an estrogen-responsive gene in breast cancer cell lines, anterior gradient 2 (AGR2) is a developmentally regulated gene belonging to the protein disulfide isomerase (PDI) gene family. Developmentally, AGR2 is expressed in the mammary gland in an estrogen-dependent manner, and AGR2 knockout and overexpression mouse models indicate that the gene promotes lobuloalveolar development by stimulating cell proliferation. Although AGR2 overexpression alone seems insufficient for breast tumorigenesis in mice, several lines of investigations suggest that AGR2 promotes breast tumorigenesis. Overexpression of AGR2 in several breast cancer cell lines increases cell survival in clonogenic assays and cell proliferation, whereas AGR2 loss of function leads to decreased cell cycle progression and cell death. In addition, AGR2 was shown to promote metastasis of breast epithelial cells in an in vivo metastasis assay. As a PDI, AGR2 is thought to be involved in the unfolded protein response that alleviates endoplasmic reticulum stress. Since cancer has to overcome proteotoxic stress due to excess protein production, AGR2 may be one of many pro-survival factors recruited to assist in protein folding or degradation or both. When AGR2 is secreted, it plays a role in cellular adhesion and dissemination of metastatic tumor cells. In breast cancer, AGR2 expression is associated with estrogen receptor (ER)-positive tumors; its overexpression is a predictor of poor prognosis. The AGR2 gene is directly targeted by ER-alpha, which is preferentially bound in tumors with poor outcome. Whereas aromatase inhibitor therapy decreases AGR2 expression, tamoxifen acts as an agonist of AGR2 expression in ER-positive tumors, perhaps contributing to tamoxifen resistance. AGR2 is also overexpressed in a subset of ER-negative tumors. Furthermore, AGR2 expression is associated with the dissemination of metastatic breast cancer cells and can be used as a marker to identify circulating tumor cells and metastatic cells in sentinel lymph nodes. In conclusion, AGR2 is a promising drug target in breast cancer and may serve as a useful prognostic indicator as well as a marker of breast cancer metastasis.

Figure 1

Overexpression of AGR2 and AGR3 in the ER-positive luminal A and B breast cancer subtypes. (a) Primary breast tumor gene expression data from three independent studies [29-31] with a total of 1,169 samples were log2-normalized and mean centered and then combined into one large data set. AGR2 is expressed significantly higher in the ER-positive luminal A (LumA) and B (LumB) subtypes and is lowly expressed in the ER-negative basal-like (Basal), Her2, and normal breast-like (Normal-Like) tumors. (b) AGR3 gene expression was analyzed in the same way as AGR2 and similarly was expressed significantly higher in LumA and LumB tumors. *P <0.05; **P <0.01; ****P <0.0001. AGR2, anterior gradient 2; ER, estrogen receptor; ns, not significant.

Figure 2

ER-binding and chromatin looping surrounding the AGR2 and AGR3 genes. (a) ER-binding sites surrounding the AGR2 and AGR3 are displayed for ER-positive cell lines and primary tumors. ChIA-Pet data in MCF-7 cells provided by Fullwood and colleagues [23] define an estradiolinduced loop mediated by ER-binding near AGR2 and AGR3 (ER-loop region indicated with red bar). Peaks defined by Welboren and colleagues [34] identify ER-binding upon treatment with estradiol in the proximal promoter of AGR2 and the first intron of AGR3 (MCF-7 minus ligand versus MCF-7 +E2). Treatment with tamoxifen alone (MCF-7 +Tamoxifen) induces ER binding to AGR2 but not AGR3, whereas treatment with fulvestrant alone (MCF-7 +Fulvestrant) induces ER binding to AGR3 but not AGR2. Ross-Innes and colleagues [33] profiled ER binding in tamoxifen-responsive (MCF-7, T47D, ZR75) and tamoxifen-resistant (BT474, TamR) cell lines and ER-positive primary breast tumors (M1-3, metastases; P1-7, poor-outcome tumors; G1-8, good-outcome tumors; ERneg, ER-negative tumor). Using the published peak data, we identified ER binding in only one good-prognosis tumor, whereas poor-prognosis and metastatic primary tumors display increased ER binding. Tamoxifen-resistant and -responsive cell lines display pervasive ER-binding throughout the region. (b) ChIA-PET data from Fullwood and colleagues [23] identify an ER-mediated chromatin loop in MCF-7 cells spanning a 130-kb region that includes the AGR2 and AGR3 genes. The loop region, displayed as a red bar in (a), contains multiple putative ER-binding sites indicated by the location of the bound receptors in the loop. AGR2, anterior gradient 2; E2, estradiol; ER, estrogen receptor; ERE, estrogen response element.

Figure 3

Mechanisms of AGR2 activation and subsequent cellular effects of AGR2 expression. Green molecules represent activators of AGR2 expression, and red molecules represent inhibitors. Blue proteins represent targets that AGR2 works through to mediate tumor progression, metastasis, drug resistance, invasion, cell survival, and cell proliferation. AREG, amphiregulin; AGR2, anterior gradient 2; C4.4A, LY6/PLAUR domain containing 3; CTSB/D, cathepsin B and cathepsin D; DAG1, dystroglycan 1; EBP1, ErbB3-binding protein 1; EGFR, epidermal growth factor receptor; ER, estrogen receptor, ERet, endoplasmic reticulum; FOXA, forkhead box family members A1 and A2; HSP90, heat-shock protein 90; Tam, tamoxifen; TGF-β, transforming growth factor-beta; YAP1, yes-associated protein 1.