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.