IMPAIRED INTERNALIZATION OF EGFR AS A MECHANISM FOR RESISTANCE TO ERBITUX (ANTI-EGFR AB) COMBINATION THERAPY IN BXPC-3 PANCREATIC ADENOCARCINOMA XENOGRAFTS
Publishing Number: 281
Juan P. Arnoletti, Donald Buchsbaum, Zhi-Qiang Huang, Ashley Hawkins, Selwyn Vickers, University of Alabama at Birmingham, Birmingham, AL
We have previously demonstrated that pancreatic adenocarcinoma BxPC-3 xenografts are resistant to treatment with Erbitux, gemcitabine and radiation, while MiaPaCa-2 xenografts respond favorably to the same therapy. The objective of this study is to elucidate the intra-cellular mechanisms that could explain pancreatic adenocarcinoma xenograft differential response to Erbitux combination therapy.
Methods: MiaPaCa-2 and BxPC-3 cells were cultured in serum-free medium containing Erbitux or human IgG, 5
mg/ml, for 36 hrs. Cells cultured without Erbitux were used as control. Cells were stimulated with EGF, 60 ng/ml for 10 min. Cell lysate was harvested after cells were washed and lysed. Equal amounts of cell lysate were separated with SDS-PAGE and transferred to PVDF membrane. The membranes were probed with rabbit anti-phospho MAPK p44/42. Similarly, cells in serum-rich media were lysed, analyzed and probed with antibodies anti-EGFR, ErbB2, ErbB3 and ErbB4. ErbB2 and ErbB3 gene expression levels in both cell lines were quantified with real-time PCR following RNA extraction. Erbitux-induced internalization of EGFR was determined from the difference between EGFR protein expression levels following Erbitux treatment at 0oC and 37oC for different incubation times (1-4 hrs). Cell-associated EGFR was measured by flow cytometry after Erbitux treatment (0.1 mg/ml), followed by FITC conjugated with goat anti-human IgG antibody.Results: EGFR and ErbB2 proteins and the ErbB2 gene were expressed by both cell lines. ErbB3 protein was selectively expressed by BxPC-3 cells. There was a 10-fold increase in the ErbB3 gene expression levels of BxPC-3 cells when compared to MiaPaCa-2. ErbB4 protein was not present in either cell line. Erbitux induced internalization of EGFR in MiaPaCa-2 cells after 2 hrs of incubation. Erbitux did not promote EGFR internalization in BxPC-3 cells. EGF induced phosphorylation of MAPK p44/42 and this was blocked by Erbitux treatment in MiaPaCa-2, but not in BxPC-3 cells.
Conclusions: 1) Erbitux blocked EGF-induced MAPK activation in MiaPaCa-2 cells but not in BxPC-3 cells; 2) Impaired internalization of EGFR on BxPC-3 pancreatic cancer cells may be due to ErbB3 protein expression, which results in heterodimerization of EGFR, persistent MAPK activation and resistance to Erbitux-based combination therapy.
