To determine whether integrin-induced clustering of EGFR affects tumor cell response to EGF, MDA-MB-231 cells were exposed to mouse monoclonal anti-β4 on ice, followed by control rabbit IgG or rabbit anti-mouse IgG to induce integrin and EGFR clustering, in the presence or absence of EGF (10 ng/ml). Western blots were prepared from cell lysates and probed for phospho-Akt and phospho-Erk1,2, then stripped, and probed again for total Akt and total Erk1,2 (Figure 3A). In suspended cells, there was only a very minimal, if any, effect of EGFR clustering

on EGF-stimulated Akt and Erk1,2 phosphorylation. AZD9291 in vitro Crosslinking α6β4 by itself resulted in only a very small to equivocal increase in phospho-Akt (lane 2). EGF in the absence of α6β4 crosslinking did stimulate Akt phosphorylation (lane 3), but the effect appeared to be abrogated in the presence of α6β4 crosslinking (lane 4). Crosslinking α6β4 produced MLN2238 in vivo a small increase in phospho-Erk1,2 (lane 2), as did the addition of EGF (lane 3), but the two together did not clearly have more than an additive effect (lane 4). Figure 3 The effect of α6β4 crosslinking on EGFR signaling following treatment with EGF (A) or HB-EGF (B). A) MDA-MB-231 cells in suspension were exposed to anti-β4 on ice, followed by control rabbit IgG (lanes 1 and 3) or rabbit anti-mouse IgG (lanes 2 and 4) at 37°C for 30 min to crosslink α6β4,

with (lanes 3 and 4) or without (lanes 1 and 2) subsequent addition of EGF (10 ng/ml) for 5 min. B) MDA-MB-231 cells were exposed to

anti-β4 on ice, then added to plates coated with control rabbit IgG (lanes 1, 3, Selleck GANT61 5, 7, 9 and 11) or rabbit anti-mouse IgG (lanes 2, 4, 6, 8, 10, or 12) at 37°C to crosslink α6β4, in the presence (lanes 3, 4, 7, 8, 11, and 12) or absence(lanes 1, 2, 5, 6, 9, and 10) of simultaneous coating with HB-EGF. Western blots prepared from cell lysates were probed for phospho-Akt and phospho-Erk1,2, then stripped and probed for total Akt and total Erk1,2. Alternatively, to evaluate effects on adherent cells, the cells were exposed to mouse monoclonal anti-β4 in suspension on ice, then added to plates coated with control rabbit IgG or rabbit anti-mouse IgG to crosslink α6β4, with or without a substrate of HB-EGF (Figure 3B). Crosslinking α6β4 in adherent cells in the absence of HB-EGF produced a slight increase in phosphorylation of Erk1,2 at 1 hr (lane 10). However, P-type ATPase crosslinking the integrin in adherent cells did not appear to enhance phosphorylation of either Akt or Erk1,2 in response to HB-EGF. In contrast, crosslinking α6β4 integrin on cells in suspension to induce cell surface clustering of EGFR had a marked effect on Rho activation in response to EGF (Figure 4). EGF in the absence of α6β4 crosslinking did not induce Rho activation in suspended MDA-MB-231 cells at 15 and 30 min (lanes 5 and 9), and crosslinking α6β4 in the absence of EGF even produced a slight decrease in activated Rho after 15 min and 30 min of integrin crosslinking (lanes 4 and 8).