Induction of cancer cell migration by epidermal growth factor is initiated by specific phosphorylation of tyrosine 1248 of c‐erbB‐2 receptor via epidermal growth factor receptor

Abstract

Induction of tumor cell migration is a key step in invasion and metastasis. Here we report that the epidermal growth factor (EGF)-induced cell migration of breast cancer cells is attributed to a transient, rather than a sustained, activation of phospholipase C (PLC)-gamma1 due to c-erbB-2 signaling. EGF stimulation of EGF receptor (EGFR) overexpressing cells resulted in long-term PLC-gamma1 tyrosine phosphorylation and sustained levels of inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG) producing sinusoidal calcium oscillations. In contrast, c-erbB-2/EGFR expressing cells displayed baseline transient calcium oscillations after EGF treatment due to short-term PLC-gamma1 tyrosine phosphorylation and short-term IP3 and DAG turnover. A third cell line expressing a point-mutated c-erbB-2 receptor that lacks the autophosphorylation Y1248 was generated to investigate whether the different PLC-gamma1 activation was attributed to this structure. Neither PLC-gamma1 tyrosine phosphorylation nor IP3 and DAG turnover and calcium oscillations were observed in this cell line, indicating the modulation of the PLC-g1 activation time course by c-erbB-2 signaling. Induction of cell migration was solely observable in the c-erbB-2-positive cell line as proved by the mode of actin reorganization and a cell migration assay, using a 3D-collagen lattice. In summary, c-erbB-2 up-regulation switches on the cell migration program by modulating the time course of PLC-gamma1 activation.

Antitumour effects of PLC-γ1-(SH2)2-TAT fusion proteins on EGFR/c-erbB-2-positive breast cancer cells

“…The breast adenoma carcinoma cell line MDA-MB-468-HER2 (MDA-HER2; EGFR and c-erbB-2 positive) (Dittmar et al, 2002) was maintained in Leibowitz L-15 media (PAA, Linz, Austria) supplemented with 10% heat-inactivated foetal calf serum (PAA), 10 U ml À1 penicillin, 10 mg ml À1 streptomycin and 400 mg ml À1 G418 (Merck BioSciences GmbH, Schwalbach, Germany) in 371C humidified atmosphere without CO 2 supplementation.…”

“…The analysis of PLC-g1 tyrosine phosphorylation by Western Blot was performed as previously described (Dittmar et al, 2002) with minor modifications. MDA-HER2 cells (4 Â 10 6 ) were pretreated with PS1/2-TAT (133 mM, 30 min) prior to EGF treatment (100 ng ml À1 , 2 min).…”

“…In addition to this, it was recently shown that the EGFinduced cell migration of EGFR/c-erbB-2-positive breast cancer cells, but not of solely EGFR-positive breast cancer cells, could be blocked with moderate concentrations of the PLC-g1 inhibitor U73122 (Dittmar et al, 2002). Thus, inhibition of PLC-g1 activity is an appropriate way to block EGF-driven tumour cell migration and invasiveness.…”

“…A key role in this process is the ability of single tumour cells or tumour cell clusters to become motile, thereby detaching from the primary tumour and start to migrate through the surrounding extracellular matrix. A decisive step in the initiation of migration of epidermal growth factor receptor (EGFR)/c-erbB-2-positive breast cancer cells has been attributed to the specific phosphorylation of tyrosine residue 1248 of c-erbB-2 via EGFR, thereby modulating the time course of the adaptor protein phospholipase C-g1 (PLC-g1) (Dittmar et al, 2002). Epidermal growth factor receptor and c-erbB-2 belong to the erbB receptor family and they are activated via ligand-induced homodimerisation as well as heterodimerisation (Alroy and Yarden, 1997), whereby c-erbB-2 is the preferred heterodimerisation partner of all erbB receptors (Graus-Porta et al, 1997).…”

“…The relationship between EGFR/c-erbB-2 heterodimer signalling and gelsolin activation in breast cancer cells has been called the 'master switch' hypothesis (Brandt et al, 1999;Thor et al, 2001). Epidermal growth factor receptor/c-erbB-2 heterodimers are constitutively formed on breast cancer cells, thereby determining a motogenic phenotype (Brandt et al, 1999) through the abovementioned mechanism (Dittmar et al, 2002). In addition to its participation in overall cell migration, there is evidence that the directionality of cell migration is also PLC-g1 regulated.…”