Co-expression of HER2 and HER3 receptor tyrosine kinases enhances invasion of breast cells via stimulation of interleukin-8 autocrine secretion

Abstract

Introduction: The tyrosine kinase receptors HER2 and HER3 play an important role in breast cancer. The HER2/HER3 heterodimer is a critical oncogenic unit associated with reduced relapse-free and decreased overall survival. While signaling cascades downstream of HER2 and HER3 have been studied extensively at the level of post-translational modification, little is known about the effects of HER2/HER3 overexpression and activation on gene expression in breast cancer. We have now defined the genetic landscape induced by activation of the HER2/HER3 unit in mammary cells, and have identified interleukin (IL)8 and CXCR1 as potential therapeutic targets for the treatment of HER2/HER3-overexpressing breast cancers.

Methods: Three-dimensional (3D) cultures, invasion and migration assays were used to determine the effects of HER2 and HER3 co-expression and activation. Gene expression analysis was performed to identify the gene network induced by HER2/HER3 in 3D cultures. Bioinformatic analysis and neutralizing antibodies were used to identify key mediators of HER2/HER3-evoked invasion.

Results: Co-expression of the tyrosine kinase receptors HER2 and HER3 induced migration and invasion of MCF10A cells. Microarray analysis of these cells revealed a specific "HER2/HER3 signature" comprising 80 upregulated transcripts, with IL8 being the highest (11-fold upregulation). Notably, examination of public datasets revealed high levels of IL8 transcripts in HER2-enriched as well as basal-like primary breast tumors, two subtypes characterized by a particularly poor prognosis. Moreover, IL8 expression correlated with high tumor grade and ER-negative status. Importantly, treatment with IL8-neutralizing antibodies prevented invasion of MCF10A-HER2/HER3 and BT474 cells in 3D cultures, highlighting the importance of IL8 autocrine signaling upon HER2/HER3 activation.

Conclusions: Our findings demonstrate that HER2 and HER3 co-expression induces IL8 autocrine signaling, leading to the invasion of mammary cells. Agents targeting IL8 or its receptor CXCR1 may be useful for the treatment of HER2/HER3/IL8-positive breast cancers with invasive traits.

Figure 1

HER2 and HER3 co-expression induces mammary cell invasion, migration and proliferation. (A) Percentages of invasive structures in the presence or absence of heregulin 10 ng/ml. Results represent means ± standard error of the mean (SEM) (n = 3, *P < 0.05). (B) Normalized cell count for the migration and invasion assays. Results represent means ± SEM (n = 3, *P < 0.003, **P < 0.002). (C) Representative confocal images of equatorial cross-sections of MCF10A cells expressing empty vector, HER2, HER3 or HER2/HER3 grown in two- or three-dimensional cultures for 15 days in the presence of 10 ng/ml heregulin and stained with TO-PRO (blue) and anti-Ki67 antibodies (red). (D) Percentage of Ki67-positive cells when grown in two- or three-dimensional cultures. Results represent means ± SEM (n = 3, *P < 0.03). (E) Quantitative real-time PCR on RNA extracts of MCF10A cells expressing empty vector, HER2, HER3 or HER2/HER3 grown in two- or three-dimensional cultures for 15 days in the presence of 10 ng/ml heregulin. Quantitative real-time PCR was performed using primers against the epithelial-to-mesenchymal transition (EMT) markers E-cadherin, N-cadherin, vimentin and fibronectin1. Results represent means ± SEM (n = 3, *P < 0.05).

Figure 2

IL8 or CXCR1 blockade reduces HER2/HER3-evoked invasion of mammary cells. (A) Quantitative real-time PCR of IL8. The bar graph shows the percentages of IL8 expression in MCF10A cells expressing empty vector, HER2, HER3 or HER2/HER3, as well as in the HER2/HER3-positive breast cancer cell line BT474. Results represent means ± standard error of the mean (SEM) (n = 3, *P < 0.018; NS, not significant). (B) Percentage of invasive structures upon stimulation of MCF10A cells with IL8. Results represent means ± SEM (n = 3, *P < 0.05). (C) Representative phase-contrast pictures of MCF10A-HER2/HER3 cells grown in three-dimensional cultures for 15 days in the presence of heregulin and the indicated neutralizing antibodies. The bar graph shows the percentages of invasive structures. Results represent means ± SEM (n = 3, *P < 0.0004; NS, not significant). (D) Representative phase-contrast pictures of BT474 cells grown in three-dimensional cultures for 15 days in the presence of heregulin and neutralizing antibodies. The bar graph shows the percentages of aberrant structures. Results represent means ± SEM (n = 3; *P < 0.05; NS, not significant). CTRL, control.

Figure 3

IL8 expression correlates with HER2-enriched and basal breast cancers. Gene set analysis (GSA) of IL8 expression in a dataset comprising 1,881 primary breast tumors http://co.bmc.lu.se/gobo. (A) Box plot of IL8 expression in breast tumor samples classified into six molecular subtypes according to the classifiers of Hu et al. [16]; P < 0.0001 by analysis of variance (ANOVA). (B) Box plot of IL8 expression in breast tumor samples classified into six molecular subtypes according to the classifiers of Parker et al. [17]; P < 0.0001 by ANOVA. Numbers above the charts report the number of patients in each subtype group.

Figure 4

IL8 expression correlates with ER-negative and high-grade primary breast tumors. Gene set analysis (GSA) of IL8 in a dataset comprising 1,881 primary breast tumors http://co.bmc.lu.se/gobo. (A). IL8 expression correlates with estrogen receptor (ER)-negative tumors. Box plot of IL8 expression in ER-negative and ER-positive primary breast cancers; P < 0.0001 by analysis of variance (ANOVA). B. IL8 expression correlates with high tumor grade. Box plot of IL8 expression in grade 1, 2 and 3 primary breast cancers, respectively; P < 0.0001 by ANOVA. Numbers above the charts show the number of patients in each subtype group.

Figure 5

Model of the HER2/HER3/IL8 signaling cascade. Heregulin-induced heterodimerization and activation of HER2/HER3 causes upregulation of the IL8 transcript, which in turn mediates invasion via CXCR1.