Inhibition of the HER2 pathway by n-3 polyunsaturated fatty acids prevents breast cancer in fat-1 transgenic mice

Zuquan Zou,Sandrine Bellenger,Karen A Massey,Anna Nicolaou,Audrey Geissler,Célia Bidu,Bernard Bonnotte,Anne-Sophie Pierre,Mélaine Minville-Walz,Michaël Rialland,John Seubert,Jing X Kang,Laurent Lagrost,Michel Narce,Jérôme Bellenger

doi:10.1194/jlr.m042754

Abstract

Overexpression of the tyrosine kinase receptor, ErbB2/HER2/Neu, occurs in 25-30% of invasive breast cancer (BC) with poor patient prognosis. Due to confounding factors, inconsistencies still remain regarding the protective effects of n-3 polyunsaturated fatty acids (PUFAs) on BC. We therefore evaluated whether fat-1 transgenic mice, endogenously synthesizing n-3 PUFAs from n-6 PUFAs, were protected against BC development, and we then aimed to study in vivo a mechanism potentially involved in such protection. E0771 BC cells were implanted into fat-1 and wild-type (WT) mice. After tumorigenesis examination, we analyzed the expression of proteins involved in the HER2 signaling pathway and lipidomic analyses were performed in tumor tissues and plasma. Our results showed that tumors totally disappeared by day 15 in fat-1 mice but continued to grow in WT mice. This prevention can be related in part to significant repression of the HER2/β-catenin signaling pathway and formation of significant levels of n-3 PUFA-derived bioactive mediators (particularly 15-hydroxyeicosapentaenoic acid, 17-hydroxydocosahexaenoic acid, and prostaglandin E3) in the tumors of fat-1 mice compared with WT mice. All together these data demonstrate an anti-BC effect of n-3 PUFAs through, at least in part, HER2 signaling pathway downregulation, and highlight the importance of gene-diet interactions in BC.

Highlights

Overexpression of the tyrosine kinase receptor, ErbB2/HER2/Neu, occurs in 25–30% of invasive breast cancer (BC) with poor patient prognosis
In order to validate the potential link between the observed differences in polyunsaturated fatty acids (PUFAs)-derived mediators and the differential tumorigenicities of E0771 cells in the mice, we studied the effects of PGE2, PGE3, and 17-hydroxydocosahexaenoic acid (HDHA) on the protein expression of HER2, HER3, and c-Myc in these cells in culture
Treatment of BT-474 and SK-BR-3 BC cells with n-3 PUFAs suppresses the expression of HER2 oncoprotein via regulation of HER2/neu gene transcription [30]

Summary

MATERIALS AND METHODS

Materials RPMI 1640, fetal bovine serum (FBS), glutamine, and antibiotics were purchased from PAA Laboratories. After blocking nonspecific binding sites with 5% BSA in Trisbuffered saline (TBS) (0.1% Tween-20 in TBS), blots were probed overnight at 4°C with primary antibody against p-HER2 (Tyr1248), HER2, p-HER3 (Tyr1289), HER3, p-Akt (Ser473), p-GSK-3␤ (Ser9), E-cadherin, ␤-catenin (Cell Signaling, Ozyme), c-Myc (Santa Cruz Biotechnology), and ␤-actin (Sigma-Aldrich) at a concentration of 1/2,000, washed in Tween-TBS (T-TBS), and incubated for 1 h at room temperature with horseradish peroxidaseconjugated goat anti-rabbit IgG for all the antibodies, except ␤-actin was incubated with goat anti mouse IgG (Jackson ImmunoResearch Laboratories). P-HER3 (two slides per block at two different levels) immunohistofluorescence was performed using an automated Leica BondMax. Briefly, after dewax, antigen retrieval with EDTA (pH 9) buffer, and inhibition of endogenous peroxidases with H2O2 (3%), slides were saturated in BSA (3%) in PBS for 20 min, incubated with an avidin and biotin blockage kit The statistical study of the tumor p-HER3 immunohistofluorescence quantification was performed using the Mann-Whitney U test by Tanagra software (**P < 0.01)

RESULTS

Findings

DISCUSSION