Anterior-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 there is mixed data on the prognostic implications of AGR2 expression. While there is emerging evidence that AGR2 is associated with poor prognosis, its function and impact on cancer relevant pathways has not been elucidated in breast cancer.

To investigate the biological role of AGR2 in breast cancer, AGR2 was transiently knocked down, using siRNA, in T47D and ZR-75-1 (estrogen receptor-alpha [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 by BrdU incorporation and cell death was measured by 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 by 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).

AGR2 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 T47D 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.

Transiently 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 supports the utility of anti-AGR2 therapy in ER-positive breast cancers due to its impact on cancer relevant pathways.

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