We previously demonstrated that pancreatic
adenocarcinoma BxPC-3 xenografts display resistance to treatment with
Erbitux,
gemcitabine, and radiation, whereas MIA PaCa-2 xenografts are highly sensitive to the same
therapy. Here, we elucidate in vitro mechanisms that may explain the observed differential response of
epidermal growth factor receptor (EGFR) expressing pancreatic
adenocarcinoma xenografts to
Erbitux-based combination
therapy in vivo. MIA PaCa-2 and BxPC-3
protein lysates were probed with
antibodies to EGFR, ErbB2, ErbB3, and ErbB4. Constitutive ErbB3 activity was visualized by immunoblot analysis using anti-
phosphotyrosine antibodies and receptor-specific immunoprecipitates. erbB2 and erbB3 gene expression in both cell lines was quantified with real-time polymerase chain reaction.
Erbitux-induced internalization of EGFR was determined by flow cytometry following
Erbitux treatment for different incubation times at 0 degrees C and 37 degrees C. MIA PaCa-2 and BxPC-3
protein extracts were also probed with anti-phospho-
mitogen-activated protein kinase antibody after stimulation with
EGF and in the presence of
Erbitux. Although both cell lines expressed EGFR and ErbB2
protein, ErbB3
protein was selectively expressed by BxPC-3 cells, where it also showed evidence of constitutive phosphorylation. There was a 10-fold increase of erbB3 transcript levels in BxPC-3 cells compared with MIA PaCa-2. ErbB4
protein was not detectable in either cell line.
Erbitux mediated EGFR internalization in MIA PaCa-2 cells after 2 hours of incubation, whereas it did not promote EGFR internalization in BxPC-3 cells. Likewise,
EGF-dependent phosphorylation of MAPK p44/42 was blocked by
Erbitux treatment in MIA PaCa-2 but not BxPC-3 cells.
Erbitux selectively interfered with
EGF-induced MAPK activation in MIA PaCa-2 but not BxPC-3 cells. Persistent MAPK activation and impaired in vitro internalization of EGFR by BxPC-3
pancreatic cancer cells may be due to constitutive ErbB3 signaling, facilitated by heterodimerization with EGFR, which may explain resistance to
Erbitux-based combination
therapy in vivo.