Conversion of normal epithelial cells to
tumors is associated with a shift in
transforming growth factor-beta (
TGF-beta) function: reduction of
tumor suppressor activity and increase of oncogenic activity. However, specific mechanisms of this functional alteration during human colorectal
carcinogenesis remain to be elucidated.
TGF-beta signaling involves Smad2/3 phosphorylated at linker regions (pSmad2/3L) and COOH-terminal regions (pSmad2/3C). Using
antibodies specific to each phosphorylation site, we herein showed that Smad2 and Smad3 were phosphorylated at COOH-terminal regions but not at linker regions in normal colorectal epithelial cells and that pSmad2/3C were located predominantly in their nuclei. However, the linker regions of Smad2 and Smad3 were phosphorylated in 31 sporadic colorectal
adenocarcinomas. In particular, late-stage invasive and metastatic
cancers typically showed a high degree of phosphorylation of Smad2/3L. Their extent of phosphorylation in 11
adenomas was intermediate between those in normal epithelial cells and
adenocarcinomas. Whereas pSmad2L remained in the cytoplasm, pSmad3L was located exclusively in the nuclei of Ki-67-immunoreactive
adenocarcinomas. In contrast, pSmad3C gradually decreased as the
tumor stage progressed. Activated c-Jun NH(2)-terminal
kinase in
cancers could directly phosphorylate Smad2/3L. Although Mad homology 2 region sequencing in the Smad4 gene revealed a G/A substitution at
codon 361 in one
adenocarcinoma, the mutation did not correlate with phosphorylation. No mutations in the
type II TGF-beta receptor and Smad2 genes were observed in the
tumors. In conclusion, pSmad3C, which favors
tumor suppressor activity of
TGF-beta, was found to decrease, whereas c-Jun NH(2)-terminal
kinase tended to induce the phosphorylation of Smad2/3L in human colorectal
adenoma-
carcinoma sequence.