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.
Highlights
Anterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein
These data demonstrate that AGR2 knockdown has a negative impact on anchorage-dependent and anchorage-independent growth that is more pronounced in Estrogen receptor-α (ER)-positive breast cancer cells
Removal of AGR2 has an impact on cancer-relevant pathways, including the cell cycle and 17-β estradiol (E2) signaling, resulting in cell death, demonstrating that AGR2 plays a critical role in breast cancer progression
Summary
Anterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein. Its upregulation has been well documented in a number of cancers, breast cancer, for which mixed data exist on the prognostic implications of AGR2 expression. Because of its impact on cell-growth and -survival pathways, E2 signaling has proven to be an efficacious target for ER-positive breast cancer therapy. Anterior gradient-2 (AGR2) is a secreted protein that was originally identified to be coexpressed with ER in breast cancer cell lines [10]. AGR2 has since been demonstrated to be estrogen [11,12,13] and androgen responsive [14], and its upregulation has been reported in a number of cancers, including breast, lung, ovarian, gastric, pancreatic, esophageal, and prostate cancer [11,15,16,17,18,19,20,21,22,23,24,25]. In the ER-negative breast cancer cell line, MDAMB-231, AGR2 was induced under serum starvation and hypoxia [26], suggesting a role for AGR2 in physiologically relevant stress conditions. Subsequent studies have explored the ER-positive tumor population and shown that AGR2 is inversely associated with overall and relapse-free survival [21,25], prompting us to ask whether AGR2 plays a critical role in more-invasive ER-positive tumors
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