Abstract B37: Analysis of the role of JNK and therapeutic effect of JNK inhibition on pancreatic cancer.

Introduction: c-Jun NH2-terminal kinase (JNK) is a member of mitogen-activated protein kinase (MAPK) family, and its signaling pathway is known to regulate a variety of cellular activities including apoptosis, survival, differentiation, and proliferation. Recently, it has been suggested that JNK is involved in the development of several cancers, but the role of JNK in pancreatic cancer is not fully elucidated. In this study, we examined the role of JNK in tumorigenesis and proliferation of pancreatic cancer. We also evaluated the therapeutic effect of JNK inhibition on pancreatic cancer.

Methods: The status of JNK was examined by immunohistochemical staining of human pancreatic cancer specimens. The activity of JNK in pancreatic cancer cell lines was determined by western blotting. The effect of JNK inhibition by JNK inhibitor (SP600125) or by siRNA on the growth of pancreatic cancer cell lines was determined by Cell Counting Kit-8. Cell cycle status was analyzed by flow cytometry. To examine the effect of oncogenic Ras on JNK activity, oncogenic or wild-type Ras expressing plasmid was transfected to pancreatic cancer cell lines. Pancreas-specific Kras^G12D expression and type II TGFβ receptor knockout mice (Kras^G12D+Tgfbr2^KO mice) was used as a mouse model of pancreatic cancer. The effect of JNK inhibitor was evaluated in vivo by i.p. injection of SP600125 to Kras^G12D+Tgfbr2^KO mice. We evaluated the involvement of apoptosis by transferase-mediated dUTP nick end labeling (TUNEL) assay for pancreatic cancer specimens of Kras^G12D+Tgfbr2^KO mice, and tumor angiogenesis using human umbilical vein endothelial cells (HUVEC).

Results: In the immunohistochemical staining, JNK activation was observed in over 90% of human pancreatic cancer specimens. Growth inhibition was observed in pancreatic cancer cell lines by treatment with JNK inhibitor in a dose dependent manner. Knockdown of JNK1 and JNK2 by siRNA showed suppressed proliferation and decreased expression of cyclin D1 in pancreatic cancer cell lines. Cell cycle analysis showed accumulation of cells in G0/G1 phase by JNK1 and JNK2 inhibition. It was suggested that oncogenic Ras promotes JNK activation, and cyclinD1 expression is regulated through JNK activation. Pancreatic tissue from Kras^G12D+Tgfbr2^KO mice showed higher activation of JNK than pancreatic tissue from Kras^G12D mice or wild-type mice. Treating Kras^G12D+Tgfbr2^KO mice with JNK inhibitor for 4 weeks led to less progression of pancreatic cancer, and immunohistochemical staining showed reduced phosphorylated JNK, c-jun, cyclin D1 and PCNA in the pancreatic cancer tissues compared to Kras^G12D+Tgfbr2^KO mice treated with vehicle. The survival time of Kras^G12D+Tgfbr2^KO mice was significantly prolonged by SP600125 treatment. Apoptosis observed in pancreatic cancer tissue was not decreased by treating with JNK inhibitor. Angiogenesis by HUVEC was decreased when incubated in the supernatant of pancreatic cancer cell lines treated by SP600125.

Conclusion: These data indicate that JNK is involved in the development of pancreatic cancer, and inhibiting JNK may be a potential therapy for pancreatic cancer.

Citation Format: Takahashi Ryota, Nakata Wachiko, Kinoshita Hiroto, Hayakawa Yoku, Nakagawa Hayato, Ijichi Hideaki, Hirata Yoshihiro, Maeda Shin, Koike Kazuhiko. Analysis of the role of JNK and therapeutic effect of JNK inhibition on pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B37.