Growth inhibition of human lung adenocarcinoma cells by antibodies against epidermal growth factor receptor and by ganglioside GM3: involvement of receptor-directed protein tyrosine phosphatase(s)

Abstract

Summary Growth of the EGF receptor-expressing non-small-cell lung carcinoma cell line H125 seems to be at least partially driven by autocrine activation of the resident EGF receptors. Thus, the possibility of an EGF receptor-directed antiproliferative treatment was investigated in vitro using a monoclonal antibody (cxEGFR ior egf/r3) against the human EGF receptor and gangliosides which are known to possess antiproliferative and anti-tyrosine kinase activity. The moderate growth-inhibitory effect of aEGFR ior egf/r3 was strongly potentiated by the addition of monosialoganglioside G M3 Likewise, the combination of aEGFR ior egf/r3 and GM3 inhibited EGF receptor autophosphorylation activity in Hi 25 cells more strongly than either agent alone. A synergistic inhibition of EGF receptor autophosphorylation by aEGFR ior egf/r3 and GM3 was also observed in the human epidermoid carcinoma cell line A431. In both cell lines, the inhibition of EGF receptor autophosphorylation by GM3 was prevented by pretreatment of the cells with pervanadate, a potent inhibitor of protein tyrosine phosphatases (PTPases). Also, GM3 accelerated EGF receptor dephosphorylation in isolated A431 cell membranes. These findings indicate that GM3 has the capacity to activate EGF receptor-directed PTPase activity and suggest a novel possible mechanism for the regulation of cellular PTPases.

Modulation of Growth Factor Signaling by Gangliosides: Positive Or Negative?

“…This diversity has, not surprisingly, led to equally wide-ranging results, including reports delineating opposing effects of gangliosides on the same receptor-ligand pair. Inhibitory effects include inhibition of EGFR phosphorylation by GM3 (Alves et al, 2002;Bremer et al, 1986;Mirkin et al, 2002;Rebbaa et al, 1996;Suarez Pestana et al, 1997) and inhibition of PDGFR phosphorylation by GM1, GM2, GD1a, and GT1b (Farooqui et al, 1999;Hynds et al, 1995). In contrast, enhancement of growth factor receptor phosphorylation has been demonstrated for GD1a and GM1 in a number of systems including EGFR Liu et al, 2004), and FGF (Rusnati et al, 2002).…”

Interaction of the Extracellular Domain of the Epidermal Growth Factor Receptor with Gangliosides

“…Second, ganglioside activation of phosphatase-has been shown to dephosphorylate the EGFR. A role for GM3 in the activation of phosphatase-was clearly shown using phosphatase inhibitors, antisense treatment, and transfection experiments (20,21). It is likely that gangliosides affect a number of protein functions in the membrane, including phosphatases and other growth factor receptors (2-11).…”

“…The ability of A431 membrane preparations rich in EGFR to bind to GM3-coated plates (18) or to GM3-coated beads (19) suggests a direct interaction with GM3. But, because the EGFR was not the only component of the membrane preparations, we cannot rule out the possibility that GM3 also binds to other membrane components, such as a tyrosine-specific phosphatase, as suggested by Suarez Pestana et al (20,21). Purified recombinant domains of the EGFR provide a means to determine whether GM3 interacts directly with the EGFR and localizes this binding site to the extracellular domain or ectodomain of the EGFR.…”

The transmembrane protein tyrosine phosphatase RPTPσ modulates signaling of the epidermal growth factor receptor in A431 cells

“…The cells were stimulated with 100 ng/ ml EGF for 5 min at 378C. Cell extracts were prepared as described (Tomic et al, 1995) and normalized protein amounts were analysed directly by immunoblotting with anti-phosphotyrosine antibodies or subsequent to immunoprecipitation using anti-EGF receptor antibody 425 as described (Sua rez Pestana et al, 1997). To evaluate inducible PTP expression in stably transfected cells, the cells were grown for 5 days in the absence or presence of ATc (100 ng/ ml).…”

“…The functional relevance of these interactions is, however, in most cases not well characterized. In previous investigations we found that the EGF receptor dephosphorylation can be activated by lipids as phosphatidic acid (Tomic et al, 1995) and the ganglioside G M3 (Sua rez Pestana et al, 1997). While the phosphatidic acid eect seems to be mediated by SHP-1, the possible target PTP for G M3 has been unknown.…”