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Smad2 and Smad3 Phosphorylated at Both Linker and COOH-Terminal Regions Transmit Malignant TGF-β Signal in Later Stages of Human Colorectal Cancer

Departments of ¹ Gastroenterology and Hepatology, ² Surgical Pathology, and ³ Microbiology, Kansai Medical University, Osaka, Japan; and ⁴ Department of Biotechnology, Kyoto Institute of Technology, Kyoto, Japan

Requests for reprints: Koichi Matsuzaki, Department of Gastroenterology and Hepatology, Kansai Medical University, 10-15 Fumizonocho, Moriguchi, Osaka 570-8506, Japan. Phone: 81-6-6992-1000, ext. 41201; Fax: 81-6-6996-4874; E-mail: matsuzak{at}takii.kmu.ac.jp.

Key Words: metastasis • transforming growth factor-β • Smad • cyclin-dependent kinase • c-Jun NH₂-terminal kinase

Transforming growth factor (TGF)-β initially inhibits growth of mature epithelial cells. Later, however, autocrine TGF-β signaling acts in concert with the Ras pathway to induce a proliferative and invasive phenotype. TGF-β activates not only TGF-β type I receptor (TβRI) but also Ras-associated kinases, which differentially phosphorylate the mediators Smad2 and Smad3 to create distinct phosphorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C) and both linker and COOH-terminally phosphorylated Smad2/3 (pSmad2L/C and pSmad3L/C). In this study, we investigated actions of pSmad2L/C and pSmad3L/C in cancer progression. TGF-β inhibited cell growth by down-regulating c-Myc oncoprotein through the pSmad2C and pSmad3C pathway; TGF-β signaling, in turn, enhanced cell growth by up-regulating c-Myc through the cyclin-dependent kinase (CDK) 4–dependent pSmad2L/C and pSmad3L/C pathways in cell nuclei. Alternatively, TβRI and c-Jun NH₂-terminal kinase (JNK) together created cytoplasmic pSmad2L/C, which entered the nucleus and stimulated cell invasion, partly by up-regulating matrix metalloproteinase-9. In 20 clinical samples, pSmad2L/C and pSmad3L/C showed nuclear localization at invasion fronts of all TGF-β–producing human metastatic colorectal cancers. In vitro kinase assay confirmed that nuclear CDK4 and cytoplasmic JNK obtained from the tumor tissue could phosphorylate Smad2 or Smad3 at their linker regions. We suggest that CDK4, together with JNK, alters tumor-suppressive TGF-β signaling to malignant characteristics in later stages of human colorectal cancer. The linker phosphorylation of Smad2 and Smad3 may represent a target for intervention in human metastatic cancer. [Cancer Res 2009;69(13):5321–30]