Fig. 3.

Effects of cetuximab on downstream phosphorylation events. Immunoblotting was used to determine the effects of EGF on ERBB1 (tyrosine-1173) and ERK1/2 (threonine 202/tyrosine 204) in nine cell lines with varying sensitivity to cetuximab. HDC82 and SW48 were sensitive (S); HCA7, CAR1, COLO678, GP2D, and SW403 were partial responders (PR); LOVO and HCT116 were resistant (R). HDC82, SW48, HCA7, and CAR1 are WT for KRAS/BRAF/PIK3CA, whereas the remainder have codons 12/13 KRAS mutations. E−, E+, C−, C+ refer to the absence or presence of EGF and cetuximab respectively. (A) EGF increased ERBB1 phosphorylation (P-ERBB1) in all sensitive and resistant cell lines apart from HCA7. Preincubation with cetuximab reduced tyrosine phosphorylation in all cell lines. ERK1/2 phosphorylation (P-ERK1/2) was stimulated in all of the responsive but not resistant cell lines after EGF stimulation. Preincubation with cetuximab reduced P-ERK1/2 only slightly in HDC82, SW48 and COLO678 with no real reduction in CAR1, GP2D, and SW403. No effect was seen in HCT116 and LOVO. (B) The effects of EGF on ERBB2 (tyrosine 1221/1222) and ERBB3 (tyrosine 1289) phosphorylation (P-ERBB2 and P-ERBB3) were also investigated (all cell lines expressed ERBB2 and ERBB3). Addition of EGF resulted in increased P-ERBB2 in HDC82, SW48, GP2D, and LOVO with significant residual activity in the presence of cetuximab in all these cell lines apart from LOVO. A similar effect was seen with P-ERBB3 in GP2D. No P-ERBB3 was seen in SW48, a cell line with low ERBB3 expression.