Differential responses by pancreatic carcinoma cell lines to prolonged exposure to Erbitux (IMC-C225) anti-EGFR antibody.
BACKGROUND
Pancreatic cancer remains a devastating disease, with 95% of all patients diagnosed with the disease dying within 2 years. The combined therapy using Erbitux, gemcitabine, and radiation caused complete tumor regression using a nude mouse model inoculated with pancreatic MiaPaCa-2 cells but only a delay in tumor growth with BxPC-3. We investigated the effect of prolonged Erbitux treatment to the sensitivity to gemcitabine and/or radiation and the epidermal growth factor receptor (EGFR) signal transduction pathway.


METHODS
MiaPaCa-2 and BxPC-3 cells were cultured with or without Erbitux for 6 weeks. Cells were then treated with gemcitabine and/or radiation, harvested 48 h after treatment, and counted. Differences in EGFR expression after exposure to Erbitux were analyzed by FACS. Internalization rates of EGFR induced by Erbitux on these cell lines were determined using 125I-EGF binding assay after removal of Erbitux by acidic wash. Cell lysates were harvested after cells were stimulated with EGF, FGF, or IGF-1 respectively, and EGFR was immunoprecipitated using Erbitux. Samples were separated using SDS-PAGE and transferred to PVDF membrane. The membranes were probed with antibody against human growth factor receptor binding protein (Grb2) to detect the association of this Ras-MAPK upstream adaptor protein to EGFR. Cell lysates were also separated with SDS-PAGE and probed with rabbit anti-human PARP after samples were transferred to PVDF membrane. Expression of BAX and Bcl-(XL) were probed in the cells treated with or without Erbitux.


RESULTS
Proliferation assays indicated that prolonged exposure to Erbitux increased the sensitivities of MiaPaCa-2 to gemcitabine and radiation therapy (41 +/- 16% vs 52 +/- 9% for gemcitation, 28 +/- 9 vs 39 +/- 9% for combination; P = 0.015) but not for BxPC-3. FACS analysis showed that the expressed EGFR level decreased by about 42% on MiaPaCa 2 whereas no loss was seen on BxPC-3. Expression of BAX was upregulated on MiaPaCa-2. Poly (ADP-ribose) polymerase cleavage indicated the killing was mediated by apoptosis. Immunoblots showed that Grb2 was co-immunoprecipitated with EGFR after EGF stimulation. Incubation with Erbitux blocked Grb2 binding in MiaPaCa-2 but not BxPC 3. FGF transactivated EGFR down stream Ras-MAPK in the presence or absence of Erbitux. Internalization of EGFR induced by Erbitux did not differ between MiaPaCa-2 and BxPC-3.


CONCLUSIONS
1) Association of Grb2 to EGFR in BxPC-3 induced by EGF in the presence of Erbitux indicates an alternate pathway of Ras-MAPK activation, which may be related with the tumor resistance to treatment; 2) transactivation of EGFR downstream Ras-MAPK pathway by FGF contributes the resistance to treatment; and 3) downregulation of EGFR may increase the response to therapy.
Huang
Zhi-qiang
ZQ
Division of General Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294-0016, USA.
Buchsbaum
Donald J
DJ
Raisch
Kevin P
KP
Bonner
James A
JA
Bland
Kirby I
KI
Vickers
Selwyn M
SM
D048868:Adaptor Proteins, Signal Transducing; D000230:Adenocarcinoma / Q000737:chemistry / Q000378:metabolism / Q000473:pathology*; D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D061067:Antibodies, Monoclonal, Humanized; D017209:Apoptosis; D002455:Cell Division / Q000187:drug effects / Q000528:radiation effects; D000068818:Cetuximab; D003037:Cobalt Radioisotopes; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000032:analysis / Q000235:genetics / Q000502:physiology; D005434:Flow Cytometry; D051380:GRB2 Adaptor Protein; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007163:Immunosorbent Techniques; D007457:Iodine Radioisotopes; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000737:chemistry / Q000378:metabolism / Q000473:pathology*; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D000093542:Gemcitabine
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D003037:Cobalt Radioisotopes; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D007457:Iodine Radioisotopes; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D003841:Deoxycytidine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases; D000068818:Cetuximab; D000093542:Gemcitabine