Delineation of a FOXA1/ERα/AGR2 regulatory loop that is dysregulated in endocrine therapy-resistant breast cancer

Abstract

Tamoxifen, a selective estrogen receptor (ER) modulator (SERM), remains a frontline clinical therapy for patients with ERα-positive breast cancer. However, the relatively rapid development of resistance to this drug in the metastatic setting remains an impediment to a durable response. Although drug resistance likely arises by many different mechanisms, the consensus is that most of the implicated pathways facilitate the outgrowth of a subpopulation of cancer cells that can either recognize tamoxifen as an agonist or bypass the regulatory control of ERα. Notable in this regard is the observation here and in other studies that expression of anterior gradient homology 2 (AGR2), a known proto-oncogene and disulfide isomerase, was induced by both estrogen (17β-estradiol, E2) and 4-hydroxytamoxifen (4OHT) in breast cancer cells. The importance of AGR2 expression is highlighted here by the observation that (i) its knockdown inhibited the growth of both tamoxifen-sensitive and -resistant breast cancer cells and (ii) its increased expression enhanced the growth of ERα-positive tumors in vivo and increased the migratory capacity of breast cancer cells in vitro. Interestingly, as with most ERα target genes, the expression of AGR2 in all breast cancer cells examined requires the transcription factor FOXA1. However, in tamoxifen-resistant cells, the expression of AGR2 occurs in a constitutive manner, requiring FOXA1, but loses its dependence on ER. Taken together, these data define the importance of AGR2 in breast cancer cell growth and highlight a mechanism where changes in FOXA1 activity obviate the need for ER in the regulation of this gene.

Implications: These findings reveal the transcriptional interplay between FOXA1 and ERα in controlling AGR2 during the transition from therapy-sensitive to -resistant breast cancer and implicate AGR2 as a relevant therapeutic target.

Figure 1. AGR2 is constitutively expressed in tamoxifen resistant ERα-positive breast cancer

A. AGR2 mRNA expression is associated with a worse prognosis in breast cancer. Forest plot analysis indicating genes associated with prognosis in Luminal A breast cancer. B. AGR2 expression is constitutively expressed in a cellular model of tamoxifen resistance. MCF7 and TamR cells were seeded in phenol red free media for 48 hours with CFS and then treated with either Vehicle, 1nM E2, 100nM 4OHT alone or in combination with 100nM ICI. AGR2 mRNA expression was analyzed by qRT-PCR, normalized to the expression of the 36B4 housekeeping gene and presented as fold change relative to Vehicle (Veh) treated cells. C. AGR2 is upregulated by antiestrogens in MCF7 cells, but is constitutively expressed in TamR cells. MCF7 and TamR cells were plated for 48 hours in phenol red free media supplemented with CFS and then treated with 10nM E2, 100nM 4OHT or 100nM ICI for 24 hrs. Whole cell extracts were immunoblotted for ERα, AGR2 and cytokeratin 18 (KRT18) expression and the Relative Density (Rel Dens) of the extracts determined.

Figure 2. Functional activities of AGR2 in tamoxifen sensitive and tamoxifen resistant ERα-positive breast cancer cells

A. AGR2 is required for 17β-estradiol induced proliferation. MCF7 and TamR cells were treated with siAGR2 and plated for 48 hours in phenol red free media supplemented with CFS. 0.2 × 10⁴ Control and siAGR2 cells were then plated on 96-well plates and treated with either Vehicle or 17β-estradiol (E2) as indicated for 5 days. B. AGR2 affects TamR cell migration. TamR cells were treated with siAGR2 for 48hrs in phenol red free media and then serum starved for 24hrs. 7.5×10⁴ cells were plated and then allowed to migrate towards 10% FBS for 16hrs. C. Overexpressed AGR2 is secreted and enhances breast cancer cell migration. MCF7 cells overexpressing AGR2 (MCF7 AGR2) or MCF7 Gal4 control cells were plated in phenol red free media with CFS for 24 hours and then changed to phenol red free media containing insulin, transferrin and selenium for 48 hours. Proteins from the supernatant were then precipitated and whole cell extracts and precipitated supernatant immunoblotted for AGR2 expression. For the migration assay, MCF7 and TamR cells were plated for 48 hours in phenol red free media containing CFS before being serum starved for 24 hours. 7.5×10⁴ cells were plated and then allowed to migrate for 16 hours towards spent media harvested from either MCF7 Gal4 or MCF7 AGR2 overexpressing cells.

Figure 3. AGR2 overexpression induces E2 induced growth in vivo

A. Overexpressed AGR2 increases tumor growth in vivo. MCF7 Gal4 and MCF7 AGR2 cells were injected into the axial mammary fat pads of ovariectomiz edestrogenized NU/NU mice. After tumor volume growth to 0.2 cm³, MCF7 Gal4 and MCF7 AGR2 tumors were randomized (10 mice per group) to continue receiving E2 treatment with and without tamoxifen co-treatment (* indicates statistically significant differences with the MCF7 Gal4 E2 group, P<0.05). B. Survival curves of MCF7 Gal4 and MCF7 AGR2 tumors, which were randomized (10 mice per group) to continue receiving E2 treatment with and without tamoxifen co-treatment (Gal4 E2 vs AGR2 E2, p=0.0009; Gal4 E2 vs Gal4 E2+Tam, p=0.0156; AGR2 E2 vs AGR2 E2+Tam, p=0.0009; AGR2 E2+Tam vs Gal4 E2+Tam, p=0.367). C. Survival curves of MCF7 Gal4 and MCF7 AGR2 tumors, which were randomized (10 mice per group) and had E2 treatment withdrawn, or had E2 treatment withdrawn but with continued tamoxifen treatment (p=0.2044).

Figure 4. AGR2 expression in tamoxifen resistant breast cancer cells loses its dependence on ERα

A. AGR2 expression is regulated by ERα in MCF7 cells. MCF7 cells were treated with siERα and plated for 48 hours in phenol red free media supplemented with CFS and then treated with 1nM E2 for 24hrs. AGR2 expression was analyzed by qRT-PCR, normalized to the expression of the 36B4 housekeeping gene and presented as fold change relative to siLuc-Vehicle treated cells. B. ERα regulation of AGR2 is diminished in TamR cells. TamR cells were treated with siERα and plated for 48 hours in phenol red free media supplemented with CFS and then treated with 1nM E2 for 24hrs. AGR2 expression was analyzed by qRT-PCR. C. ERα regulation of AGR2 is diminished in TamR cells. TamR cells were treated with siERα for 48hrs and then treated with E2 for 24 hrs. The resulting whole cell extracts were immunoblotted for ERα, AGR2 and cytokeratin 18 (KRT18) expression.

Figure 5. FOXA1 drives AGR2 expression in both tamoxifen sensitive and tamoxifen resistant estrogen receptor positive breast cancer

A. Definition of the AGR2 expression network identifies FOXA1 as a regulator of AGR2 expression. A comparative analysis of CCLE and TCGA was performed to predict genes whose expression is highly associated with AGR2. From the genes identified, FOXA1 was one of the top represented genes of the list. CCLE correlation is represented by size with larger circles predicting higher co-expression and TCGA correlation is represented based on color, Red>Purple>Blue>White. B. FOXA1 regulates AGR2 mRNA expression. MCF7 and TamR cells were treated with siFOXA1 and plated for 4 days in phenol red free media supplemented with CFS. FOXA1 and AGR2 expression was analyzed by qRT-PCR, normalized to the expression of the 36B4 housekeeping gene and presented as fold change relative to siCtrl treated cells. C. FOXA1 regulates AGR2 expression in MCF7 and TamR cells. MCF7 and TamR cells were treated with siFOXA1 for 4 days in phenol red free media with CFS. The resulting whole cell extracts were immunoblotted for FOXA1, AGR2, Erα and β-Actin (ACTB) expression. D. FOXA1 regulates AGR2 expression when ERα expression is induced. MCF7 and MCF7 GPS ER cells were treated with siFOXA1 for 4 days in phenol red free media with CFS. The resulting whole cell extracts were immunoblotted for FOXA1, AGR2, ERα and β-Actin (ACTB) expression. E. FOXA1 regulates AGR2 mRNA expression with induced ER expression. MCF7 and MCF7 GPS ER cells were treated with siFOXA1 and plated for 4 days in phenol red free media supplemented with CFS. FOXA1, ERα 3′ UTR, ERα and AGR2 expression was analyzed by qRT-PCR, normalized to the expression of the 36B4 housekeeping gene and presented as fold change relative to siCtrl treated cells.