HER receptor-mediated dynamic signalling in breast cancer cells

Abstract

The dynamics of cell signalling are critical to cell fate decisions. Human Epidermal growth factor Receptors (HERs)-mediated Ras/Raf/MEK/ERK and PI3K/Akt signalling cascades relay extracellular signals from the plasma membrane to targets in the nucleus and cytoplasm and play pivotal roles in cell fate determination including proliferation, differentiation and cell survival. Both pathways, once activated, are further regulated by complex feedback loops which may exert either positive or negative effects on cascade components and can result in signalling oscillation. In this study, heregulin (HRG) - and epidermal growth factor (EGF)- stimulated oscillation of both p-ERK1/2 and p-Akt expression in breast cancer cell lines was demonstrated. The oscillation was cell line dependent and was observed in MCF-7 and MCF-7/HER2-18 cells but not in BT474 cells. The oscillation was augmented by cycloheximide implicating transcriptional involvement. Gene expression analysis identified 29 genes as possible candidates involved in the transcriptional feedback regulation. Apart from the feedback regulation, feedforward regulation was also observed. To expedite the analyses In-cell Western and Reverse Phase Protein Array (RPPA) assays were developed. A scheme of transcriptional feedback loops regulating the oscillation in the ERK1/2 pathway is proposed, including negative feedback loops to ERK1/2 from DUSPs, early positive and late negative feedback loops to MEK1/2 and positive feedback loops to HER-3 from AREG, HB-EGF, CYR61 and CTGF. Two HER-2-targeted inhibitory monoclonal antibodies were investigated – trastuzumab and pertuzumab. Trastuzumab not only inhibited the growth of HER-2 over-expressing MCF-7/HER2- 18 cells and BT474 cells but also that of EGF-driven MCF-7 cells which expressed low/moderate HER-2 levels. Pertuzumab blocked the growth of both MCF-7 and MCF-7/HER2-18 driven by either EGF or HRG. When used in combination with trastuzumab, pertuzumab had much more potent activity in inhibiting cell growth and signalling than either single drug. Trastuzumab and pertuzumab had opposing effects on immediate p-ERK1/2 signalling and trastuzumab’s effects on signalling could be mimicked by the PI3K inhibitor LY294002. PTPN13, a non-receptor type tyrosine protein phosphatase, is a proposed tumour suppressor in breast cancer. This was investigated as a candidate regulator of the signalling oscillation and although not observed as a transcriptional modulator of the oscillation, its high expression level was observed to be associated with cell growth inhibition in MCF-7/HER2-18 cells by trastuzumab. Moreover, immunohistochemical analysis of 121 clinical tumours which had received trastuzumab treatment revealed the correlation between the expression level of PTPN13 and the mutation status of PIK3CA. In conclusion, the observed oscillation may contribute to the elucidation of the complex regulation of signalling pathways, which is vital to the different cell fate decision made through the same core pathway. The synergy between trastuzumab and pertuzumab supports the clinical use of the combination treatment and suggested PI3K/Akt pathway as the major pathway in controlling tumour growth.