Abstract

The human c-fes locus encodes a non-receptor protein-tyrosine kinase implicated in myeloid, vascular endothelial, and neuronal cell differentiation. A recent analysis of the tyrosine kinome in colorectal cancer identified c-fes as one of only seven genes with consistent kinase domain mutations. Although four mutations were identified (M704V, R706Q, V743M, S759F), the consequences of these mutations on Fes kinase activity were not explored. To address this issue, Fes mutants with these substitutions were co-expressed with STAT3 in human 293T cells. Surprisingly, the M704V, R706Q, and V743M mutations substantially reduced Fes autophosphorylation and STAT3 Tyr-705 phosphorylation compared with wild-type Fes, whereas S759F had little effect. These mutations had a similar impact on Fes kinase activity in a yeast expression system, suggesting that they inhibit Fes by affecting kinase domain structure. We have also demonstrated for the first time that endogenous Fes is strongly expressed at the base of colonic crypts where it co-localizes with epithelial cells positive for the progenitor cell marker Musashi-1. In contrast to normal colonic epithelium, Fes expression was reduced or absent in colon tumor sections from most individuals. Fes protein levels were also low or absent in a panel of human colorectal cancer cell lines, including HT-29 and HCT 116 cells. Introduction of Fes into these lines with a recombinant retrovirus suppressed their growth in soft agar. Together, our findings strongly implicate the c-Fes protein-tyrosine kinase as a tumor suppressor rather than a dominant oncogene in colorectal cancer.

Highlights

  • Oncogenic homologues of c-fes are found in avian and feline retroviruses, and active Fes mutants cause hyperplasia in some tissues and are highly transforming in rodent fibroblasts, suggesting that c-fes is a proto-oncogene [1, 2]

  • A human chronic myelogenous leukemia-derived cell line (K-562) that lacks Fes expression can be induced to undergo growth arrest and terminal differentiation following the re-introduction of Fes, implicating this kinase as a potential suppressor of chronic myelogenous leukemia progression [7]

  • The colorectal cancer-associated mutations found in the Fes kinase domain (M704V, R706Q, V743M, S759F) were assessed for their effects on Fes kinase activity

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Summary

MATERIALS AND METHODS

Plasmids and Retroviruses—Coding sequences for wild-type human Fes, a kinase-active coiled-coil domain mutant (L145P), and a kinaseinactive mutant (KE) were fused to the enhanced yellow-shifted variant of green fluorescent protein (YFP) in the plasmid vector pEYFP-C1 (BD Bioscience-Clontech) as described elsewhere [4]. Codons for the colon cancer-associated mutations M704V, R706Q, V743M, and MARCH 31, 2006 VOLUME 281 NUMBER 13

Fes Tyrosine Kinase Suppresses Colon Cancer Cell Growth
RESULTS
Fes expression in normal colon versus tumor
Fes staining
Full Text
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