HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR

Abstract

The members of the epidermal growth factor receptor (EGFR) kinase family are important players in breast morphogenesis and cancer. EGFR2/HER2 and EGFR expression have a prognostic value in certain subtypes of breast cancer such as HER2-amplified, basal-like and luminal type B. Many clinically approved small molecular inhibitors and monoclonal antibodies have been designed to target HER2, EGFR or both. There is, however, still limited knowledge on how the two receptors are expressed in normal breast epithelium, what effects they have on cellular differentiation and how they participate in neoplastic transformation. D492 is a breast epithelial cell line with stem cell properties that can undergo epithelial to mesenchyme transition (EMT), generate luminal- and myoepithelial cells and form complex branching structures in three-dimensional (3D) culture. Here, we show that overexpression of HER2 in D492 (D492(HER2)) resulted in EMT, loss of contact growth inhibition and increased oncogenic potential in vivo. HER2 overexpression, furthermore, inhibited endogenous EGFR expression. Re-introducing EGFR in D492(HER2) (D492(HER2/EGFR)) partially reversed the mesenchymal state of the cells, as an epithelial phenotype reappeared both in 3D cultures and in vivo. The D492(HER2/EGFR) xenografts grow slower than the D492(HER2) tumors, while overexpression of EGFR alone (D492(EGFR)) was not oncogenic in vivo. Consistent with the EGFR-mediated epithelial phenotype, overexpression of EGFR drove the cells toward a myoepithelial phenotype in 3D culture. The effect of two clinically approved anti-HER2 and EGFR therapies, trastuzumab and cetuximab, was tested alone and in combination on D492(HER2) xenografts. While trastuzumab had a growth inhibitory effect compared with untreated control, the effect of cetuximab was limited. When administered in combination, the growth inhibitory effect of trastuzumab was less pronounced. Collectively, our data indicate that in HER2-overexpressing D492 cells, EGFR can behave as a tumor suppressor, by pushing the cells towards epithelial differentiation.

Figure 1

Expression of EGFR and HER2 in normal human breast gland. (a) HER2 and EGFR are expressed in luminal and myoepithelial cells, respectively. Confocal microscopy images of human mammary gland tissue sections. The sections were stained with antibodies against CK19, CK14, EGFR and HER2 in various combinations, and the images show part of an intralobular duct. Cytokeratin 19 and 14 (top left) identify luminal and myoepithelial cells, respectively. Bar=50 μm. (b) Expression of EGFR and HER2 in cultured primary breast epithelial cells. Western blot of lysates made from purified primary myoepithelial (MEP) and luminal (LEP) epithelial cells from normal human mammary gland, stained with antibodies against EGFR, HER2, CK14 and CK19. GAPDH=loading control.

Figure 2

HER2 overexpression reduces EGFR expression. (a) Expression of endogenous EGFR in D492^HER2 is reduced compared with D492^ctrl. Confocal microscopy images of D492 cells expressing ctrl, HER2, EGFR and HER2/EGFR grown on culture flasks and analyzed by immunofluorescence staining for EGFR and HER2 expression. Bar=50 μm. (b) HER2 overexpression leads to reduced EGFR transcription levels. Quantitative reverse transcriptase–PCR was performed using monolayer RNA isolates from all four cell lines. Transcription of HER2 and EGFR was analyzed, and normalized to GAPDH. (c) HER2 overexpression leads to ligand-independent EGFR and HER2 phosphorylation. Cells on monolayer were starved for 24 h in media without EGF. Cells were then given control media or EGF-containing culture media. Protein lysates were collected at +180 min and blotted for total and phosphorylated EGFR and HER2. Actin=loading control.

Figure 3

HER2 overexpression induces mesenchymal transition. (a) D492^HER2 cells lose epithelial phenotype in monolayer culture. Confocal microscopy images of D492 cells expressing ctrl, HER2, EGFR and HER2/EGFR grown on culture flasks and analyzed by immunofluorescence staining for CK14, CK19, E-, N- and P-cadherin expression. Bar=50 μm. (b) D492^HER2 and D492^HER2/EGFR gain mesenchymal phenotype in monolayer culture. Western blotting performed using lysates from monolayer cultures. Membranes were blotted for CK14, CK19, p63, E-, P-, N-cadherin and Axl expression. Actin=Loading control. (c) In D492^HER2, miR-200c and miR141 are strongly downregulated. Quantitative reverse transcriptase–PCR was performed using monolayer RNA isolates from all four cell lines. Transcription of miR-200c, miR-141 and ZEB1was analyzed and normalized to GAPDH.

Figure 4

HER2 overexpression leads to loss of growth arrest in monolayer and increased survival in low attachment. (a) D492^HER2 cells have lost contact inhibition in monolayer culture. D492 cells expressing ctrl, HER2, EGFR and HER2/EGFR were seeded on 24-well plates and cell proliferation measured bi-daily by trypsinizing cells, counting and measuring viability. (b) HER2 leads to higher colony formation in low attachment. D492 cells expressing ctrl, HER2, EGFR and HER2/EGFR were seeded on low-attachment plates and colonies were counted. Bar=200 μm.

Figure 5

EGFR partially reverses HER2-induced EMT in 3D culture. (a) D492^HER2 cells show disrupted branching morphogenesis and generate spindle- and grape-like colonies in 3D culture. D492 cells expressing ctrl, HER2, EGFR and HER2/EGFR were seeded into rBM and cultured for 2 weeks. Top picture panel shows representative bright-field images of branching structures found in 3D organotypic cultures of D492 cells. Bar=100 μm. Representative isolated structures were fixed and stained for E- and N-cadherin, CK14 and CK19. Bar=50 μm. After the culture period, colonies were counted into four groups, branching/budding, solid round, grape like and spindle. The left table demonstrates the frequencies of each of these phenotypic groups within each D492 subline. The right table demonstrates the same ratios when cultures were treated with the small-molecule HER2 inhibitor CP724,714. (b) EGFR restores the epithelial phenotype in 3D culture in D492^HER2 (D492^HER2/EGFR). Western immunoblot for E- and N-cadherin, CK14 and Ck19 of protein lysates isolated from 3D cultures after 14 days of cultivation.

Figure 6

Overexpression of HER2 in D492 leads to increased tumor growth in NSG mice which is partially reversed by EGFR signaling. (a) D492^HER2 and D492^HER2/EGFR generate tumors efficiently in NSG mice. D492 cells expressing ctrl, HER2, EGFR and HER2/EGFR were injected into NSG mouse mammary fat pads. Numbers represent appearance palpable tumors over total number over injections performed during the course of the experiment. (b) EGFR negatively affects HER2-induced tumor growth. Changes to tumor volumes of D492 cell lines. (c) Cetuximab reduces trastuzumab sensitivity in D492^HER2 tumors. D492^HER2 cells were allowed to form tumors in NSG mice. After 2 weeks, mice were randomized into four groups and treated with trastuzumab, cetuximab and a combination of both drugs. Changes to tumor volumes were measured two times weekly.

Figure 7

EGFR expression reverses HER2-induced loss of cytokeratin expression in xenografts tumors. (a) Mosaic picture of sections from H&E staining of D492^HER2 and D492^HER2/EGFR tumors. Tumors were paraffin embedded, sliced and stained with H&E. Squares denote areas represented in immunofluorescence in (b). Bar=1 mm. (b) EGFR reverses HER2-induced loss of cytokeratin expression. Confocal images showing xenograft slices from D492^HER2 and D492^HER2/EGFR tumors stained with HER2, EGFR, CK14 and E-cadherin. Bar=100 μm. (c) Apoptosis levels are higher in close proximity to epithelial, CK14-expressing tumor areas. Confocal images showing xenograft slices from D492^HER2 and D492^HER2/EGFR tumors stained with CK14, Ki67 and cleaved caspase-3 antibodies. Bar=100 μm.