Therapeutic advances in BIG3-PHB2 inhibition targeting the crosstalk between estrogen and growth factors in breast cancer

Abstract

Our previous studies demonstrated that specific inhibition of the BIG3-PHB2 complex, which is a critical modulator in estrogen (E2) signaling, using ERAP, a dominant negative peptide inhibitor, leads to suppression of E2-dependent estrogen receptor (ER) alpha activation through the reactivation of the tumor suppressive activity of PHB2. Here, we report that ERAP has significant suppressive effects against synergistic activation caused by the crosstalk between E2 and growth factors associated with intrinsic or acquired resistance to anti-estrogen tamoxifen in breast cancer cells. Intrinsic PHB2 released from BIG3 by ERAP effectively disrupted each interaction of membrane-associated ERα and insulin-like growth factor 1 receptor beta (IGF-1Rβ), EGFR, PI3K or human epidermal growth factor 2 (HER2) in the presence of E2 and the growth factors IGF or EGF, followed by inhibited the activation of IGF-1Rβ, EGFR or HER2, and reduced Akt, MAPK and ERα phosphorylation levels, resulting in significant suppression of proliferation of ERα-positive breast cancer cells in vitro and in vivo. More importantly, combined treatment with ERAP and tamoxifen led to a synergistic suppression of signaling that was activated by crosstalk between E2 and growth factors or HER2 amplification. Taken together, our findings suggest that the specific inhibition of BIG3-PHB2 is a novel potential therapeutic approach for the treatment of tamoxifen-resistant breast cancers activated by the crosstalk between E2 and growth factor signaling, especially in premenopausal women.

Keywords: Crosstalk; PHB2; estrogen; growth factor; tamoxifen-resistance.

Fig 1

ERAP suppresses non-genomic signaling by the crosstalk between E2 and insulin-like growth factor 1 (IGF-1). (a) Luciferase assays showing the inhibitory effect of ERAP on the estrogen receptor alpha (ERα) transcriptional activity of MCF-7 cells by 10 nM E2 and 50 ng/mL IGF-1. These data represent the mean ± SD of three independent experiments (***P < 0.001). (b) The inhibitory effects of ERAP on the interactions of ERα with IGF-1Rβ and PI3K in the presence of E2 and IGF-1. (c–f) The inhibitory effects of ERAP on Akt (c, d), MAPK (c, d), and multiple ERα phosphorylations (e, f) induced by E2 and IGF-1 stimulation in MCF-7 (c, e) and BT-474 (d, f) cells. The data are expressed as the fold increase over untreated cells at 0 h. The above all blot were cropped, and the full-length blots are included in Supplementary Figure S4.

Fig 2

ERAP suppresses the cell growth induced by the crosstalk between E2 and insulin-like growth factor 1 (IGF-1). (a, b) MTT assays evaluating the inhibitory effect of ERAP on the growth of MCF-7 (a) and BT-474 (b) cells by 10 nM E2 and/or 50 ng/mL IGF-1 stimulation. These data represent the mean ± SD of three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001). (c) Flow cytometric analyses showing the effect of ERAP and tamoxifen on the cell cycle in the presence of E2 and IGF-1 in MCF-7 cells.

Fig 3

ERAP suppresses in vivo tumor growth induced by the crosstalk between E2 and insulin-like growth factor 1 (IGF-1). (a) ERAP inhibits tumor growth in a human breast cancer KPL-3C xenograft mouse model. The tumor volume represents the mean ± SE of each group (n = 5) (*P < 0.05; ** P < 0.01; NS, no significance). (b) The body weights of the KPL-3C xenograft mice. The body weight represents the mean ± SD of each group (n = 5). (c) The effects of ERAP on the phosphorylation levels of Akt, MAPK and estrogen receptor alpha (ERα) proteins in tumors. The blots were cropped, and the full-length blots are included in Supplementary Figure S4. (d) Representative immunohistochemical staining of nuclear Ki67 (upper) and TUNEL staining (lower) in tumors at day 21.

Fig 4

ERAP regulates signaling pathways by the crosstalk of E2 and a variety of growth factors. (a) An MTT assay showing the inhibitory effect of ERAP on the growth of BT-474 cells by E2 and/or EGF stimulation. These data represent the mean ± SD of three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001). (b) The inhibitory effects of ERAP on Akt, MAPK, and estrogen receptor alpha (ERα) phosphorylation induced by E2 and/or EGF stimulation in BT-474 cells. The data are expressed as the fold increase over untreated cells at 0 h. (c) Inhibitory effects of ERAP on the interactions of estrogen receptor alpha (ERα) with human epidermal growth factor 2 (HER2), epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor beta (IGF-1Rβ) or PI3K. (d, e) The inhibitory effects of ERAP on the interactions of EGFR (d) or HER2 (e) with ERα and Shc in BT-474 cells. The above all blot were cropped, and the full-length blots are included in Supplementary Figure S4. (f) Schematic illustrations of inhibitory effects of ERAP on cell functions caused by the crosstalk of ERα and IGF-1R, EGFR or HER2.