HSP90 inhibitor AUY922 can reverse Fulvestrant induced feedback reaction in human breast cancer cells

Abstract

Hormone therapy has become one of the main strategies for breast cancer, however, many estrogen receptor (ER) positive patients end in tumor collapse due to initial or acquired resistance to hormone treatment, which includes Fulvestrant. Here we report that ErbB receptors and downstream PI3K/AKT and ERK pathway have been reactivated after treatment of Fulvestrant in ER positive MCF-7 and T47D cells, which are related to Fulvestrant resistance. HSP90 is a universally expressed chaperone protein and plays a vital role in both normal and cancer cells, HSP90 inhibitor AUY922 can reverse this feedback reactivation effect of Fulvestrant by targeting multiple proteins related in ErbB receptors, PI3K/AKT and ERK pathway, which is much better than single targeting inhibitors. We also consolidate these effects in human fresh breast tumors. Combination of AUY922 and Fulvestrant may become a promising therapy strategy in breast cancer treatment.

Keywords: AUY922; ErbB receptors; PI3K/AKT; fulvestrant; hormone resistance.

Figure 1

Fulvestrant decreases estrogen receptor (ER)‐α in breast cancer cells. Western blotting was used to test the effect of Fulvestrant on ER‐α after treatment of indicated concentration (vehicle control, 100, 200 nM) for 48 h in MCF‐7 cells (a) and T47D cells (b).

Figure 2

Feedback reactivation of Fulvestrant on ErbB receptors and downstream PI3K/AKT and ERK pathway. Western blotting was used to test changes of EGFR, HER2, HER3, HER4 and their phosphorylation types after treatment with Fulvestrant of indicated concentration (vehicle control, 100, 200 nM) for 48 h in (a, b) MCF‐7 and (c, d) T47D cells. Proliferation related total protein and phosphorylated ones were tested were tested in downstream PI3K/AKT and ERK pathway (e, f).

Figure 3

HSP90 inhibitor AUY922 inhibits PI3K/AKT and ERK pathway. (a) AUY922 could inhibit HSP90 activity after 24 h treatment at indicated concentration in MCF‐7 and T47D cells. (b) Fulvestrant (control, 100, 200 nM) didn't affect HSP90 activity after 48 h treatment. (c) PI3K/AKT and ERK pathway were inhibited after 24 h treatment of AUY922.

Figure 4

AUY922 can reverse the feedback reactivation effect of Fulvestrant in breast cancer cells. Cells were treated with Fulvestrant (100 nM) for 24 h and then added AUY922 (30 nM) for another 24 h together with Fulvestrant, total protein level of EGFR, HER2, HER3, HER4 and their phosphorylation status were tested, and down stream PI3K/AKT, ERK pathway were checked with western blotting in MCF‐7 cells (a, b, e) and T47D cells (c, d, f).

Figure 5

Combination use of AUY922 and Fulvestrant inhibits proliferation and causes cell cycle arrest in breast cancer cells. (a) Cell growth assay. Cells were treated with Fulvestrant (100 nM) alone or AUY922 alone at indicated concentration (0–50 nM) or combined with Fulvestrant (100 nM) for 72 h, MTT was added and incubated for 4 h before OD value measurement at 570 nm. (b) Colony forming assay. Cells were seeded in 6‐well plates on the first day, AUY922 (5 nM) and/or Fulvestrant (10 nM) was added in the medium and kept for 7–10 days before stained with crystal violet. Dye was solubilized and the optical density at 592 nm was measured. The results were representative of three separate experiments. (c) Cell cycle analysis. Cells were treated with Fulvestrant (100 nM) and/or AUY922 (30 nM) together for 48 h. Propidium iodide staining was detected by flow cytometry.

Figure 6

AUY922 can reverse the feedback effect of Fulvestrant in human breast tumor. Fresh tumor sample from two breast cancer patients (ER+, PR+, HER−) were treated with 100 nM Fulvestrant for 24 h, and then added 30 nM AUY922 together for another 24 h treatment in Dulbecco's modified eagle medium (DMEM) medium with 10% FBS then frozen, tissue fragments in RIPA were crushed using Ultrasonic Cell Breaker. Lysates were then analyzed by western blotting with indicated antibodies.