RESUMO
BACKGROUND & AIMS: The APC tumor suppressor is well known for its ability to regulate Wnt signaling through mediation of beta-catenin levels in the cell. Transient over expression of the tumor suppressor gene APC in colon cancer cells prevents entry into S phase of the cell cycle, a phenotype only partially restored by cotransfection of a transcriptionally active form of beta-catenin. In an attempt to define its transcription-independent tumor suppressor functions, we tested whether APC directly affects DNA replication. METHODS: A transcriptionally quiescent in vitro DNA replication system, the polymerase chain reaction, DNA binding assays, and transient transfections in colon cancer cell lines were used to determine the effects of APC on DNA replication and the mechanism by which it works. RESULTS: We report that exogenous full-length APC inhibits replication of template DNA through a function that maps to amino acids 2140-2421, a region of the protein commonly lost by somatic or germline mutation. This segment of APC directly interacts with DNA, while mutation of the DNA-binding S(T)PXX motifs within it abolishes DNA binding and reduces inhibition of DNA replication. Phosphorylation of this segment by cyclin-dependent kinases also reduces inhibition of DNA replication. Furthermore, transient transfection of an APC segment encoding amino acids 2140-2421 into a colon cancer cell line with mutant APC prevents cell cycle progression into or through S phase. CONCLUSIONS: Our results suggest that APC can negatively regulate cell cycle progression through inhibition of DNA replication by direct interaction with DNA.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Neoplasias Colorretais/genética , Replicação do DNA/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteína da Polipose Adenomatosa do Colo/química , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/fisiopatologia , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fase G1/fisiologia , Humanos , Oócitos/fisiologia , Fosforilação , Estrutura Terciária de Proteína , Fase S/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Xenopus laevisRESUMO
Activation of Wnt signaling through beta-catenin dysregulation occurs in numerous human tumors, including gastric cancer. The specific consequences of Wnt signaling in gastric cancer, however, are not well characterized. This study shows that the introduction of mutant beta-catenin into gastric cancer cell lines by adenoviral infection enhances invasiveness and proliferation and up-regulates the expression of the gene encoding the matrix metalloproteinase (MMP) family member membrane type 3 MMP (MT3-MMP). Up-regulation of MT3-MMP is critical to the invasive phenotype as shown by small interfering RNA (siRNA) studies. Immunohistochemical staining also showed that MT3-MMP was highly expressed in gastric cancers with activating beta-catenin mutations. These observations suggest that Wnt activation may contribute to gastric cancer progression by increasing the invasiveness of neoplastic cells in the stomach via up-regulation of MT3-MMP expression.