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

Koichi Matsuzaki,Kazuichi Okazaki,Miki Murata,Go Sekimoto,Katsunori Yoshida,Toshihito Seki,Shigeru Taketani,Jun-Ichi Fujisawa,Chiaki Kitano,Yoshiko Uemura

doi:10.1158/0008-5472.can-08-4203

Abstract

Transforming growth factor (TGF)-beta initially inhibits growth of mature epithelial cells. Later, however, autocrine TGF-beta signaling acts in concert with the Ras pathway to induce a proliferative and invasive phenotype. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) 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-beta inhibited cell growth by down-regulating c-Myc oncoprotein through the pSmad2C and pSmad3C pathway; TGF-beta 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, TbetaRI 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-beta-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-beta 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.

Highlights

Transforming growth factor (TGF)-h, a potent inhibitor of epithelial cell growth, acts as a tumor suppressor in early cancer
We initially studied the kinetics of Smad2 and Smad3 phosphorylation in mouse embryo–derived fibroblasts (MEF) after stimulation by platelet-derived growth factor (PDGF)
TGF-h signal in the presence of PDGF induced an increase in phosphorylation of Smad2L/C: pSmad2L/C appeared at 15 minutes, reached a maximum at 30 minutes, and returned to the basal level by 2 hours (Fig. 1D)

Summary

Introduction

Transforming growth factor (TGF)-h, a potent inhibitor of epithelial cell growth, acts as a tumor suppressor in early cancer. Tumor-promoting effects of TGF-h have been shown by the ability of agents that block TGF-h signaling to inhibit invasiveness of cancer cell lines in vitro and their metastatic potential in vivo [4,5,6] and by data showing that TGF-h can directly stimulate growth and motility of cancer cells [7]. Active TGF-h type I receptor (ThRI) phosphorylates the COOH-terminal serine residues of receptor-activated Smads [10], which include Smad and the highly similar protein Smad. The linker domain undergoes regulatory phosphorylation by Ras-associated kinases, including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and cyclin-dependent kinase (CDK) 4 [11,12,13]. Phosphorylated Smad and Smad rapidly oligomerize with Smad and translocate to the nucleus, where they regulate transcription of target genes [16]

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