Abstract B10: Epidermal growth factor receptor inhibitor prolongs survival in pancreatic cancer by blocking gemcitabine-induced mitogen-activated protein kinase signal
Authors: Miyabayashi Koji , Ijichi Hideaki , Takahashi Ryota , Mohri Dai , Yamamoto Keisuke , Asaoka Yoshinari , Ikenoue Tsuneo , Tateishi Keisuke , Moses Harold L. , Koike Kazuhiko 
Published: May 2015

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is the most deadly cancer worldwide. Although many regimens have been tried against PDAC, an epidermal growth factor receptor (EGFR) inhibitor erlotinib in combination with gemcitabine is the only molecular target drug superior to gemcitabine alone. However, the mechanism by which PDAC with extremely frequent KRAS-mutation benefits from EGFR inhibition remains largely unknown. In this study, we evaluated the efficacy of erlotinib in combination with gemcitabine using a murine PDAC model with transforming growth factor-beta receptor II knockout plus Kras activation and investigated the mode of action.
The mice were treated using the following drug doses and treatment schedules; erlotinib was administered from 3 weeks of age and gemcitabine was administered from 4 weeks of age. We isolated PDAC cells from the murine PDAC tissues. Effects of erlotinib on the proliferation and intracellular signaling of the murine PDAC cells or human PDAC cell lines were examined in vitro. We sacrificed the mice at 7 weeks of age, and excised the pancreatic tissues and processed for western blot analysis and immunohistochemistry. We evaluated the expression of EGFR ligands by real-time PCR and the heterodimer formation of EGFR with ErbB2 by immunoprecipitaiton after incubation with gemcitabine in vitro. We assessed whether the effect of gemcitabine on EGFR/ErbB2 activation is secondary to mitogen activated protein kinase (MAPK) signal activation after incubation with or without MEK inhibitor and gemcitabine by western blot analysis and real-time PCR.
Gemcitabine + erlotinib inhibited PDAC progression and significantly prolonged the survival of the PDAC mice compared to gemcitabine alone. Gemcitabine or erlotinib also inhibited in vitro PDAC cell proliferation. Interestingly, Gemcitabine induced MAPK signaling, which was dramatically inhibited by adding erlotinib, even in the Kras-mutant PDAC cells. The suggested mechanisms were that gemcitabine induced EGFR ligand expression and also ErbB2 activation by increasing heterodimer formation with EGFR and maintaining high ErbB2 protein level in PDAC cells. We observed that the gemcitabine-induced MAPK signaling activation was in part due to induction of Egfr ligands(Egf, Tgf-a, Amphiregulin) expression by real-time PCR and ELISA. Using a phospho-RTK antibody array, we also observed that Gem induced Erbb2 activation in PDAC cells, and validated by western blot analysis, real-time PCR, and immunohistochemistry. Erlotinib inhibited the ErbB2 activation, partly by inhibiting heterodimer formation with EGFR and also decreasing ErbB2 protein expression in PDAC cells.
We observed that gemcitabine-induced EGFR ligands up-regulation and EGFR/ErbB2 activation require intact MAPK signaling and these are secondary effects of MAPK signal activation and that gemcitabine induced the activation irrespective of KRAS status and gemcitabine sensitivity.
This model helps us to evaluate an efficacy of new drugs and to investigate mechanisms of the mode of action and chemoresistance. This study provides clinical insights into potent therapeutic strategies for this difficult cancer.
Citation Format: Koji Miyabayashi, Hideaki Ijichi, Ryota Takahashi, Dai Mohri, Keisuke Yamamoto, Yoshinari Asaoka, Tsuneo Ikenoue, Keisuke Tateishi, Harold L. Moses, Kazuhiko Koike. Epidermal growth factor receptor inhibitor prolongs survival in pancreatic cancer by blocking gemcitabine-induced mitogen-activated protein kinase signal. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B10. doi: 10.1158/1557-3125.RASONC14-B10