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Id2 is a target of the beta-catenin/T cell factor pathway in colon carcinoma
S P Rockman1, S A Currie, M Ciavarella
1Surgical Oncology Research Laboratory, Peter MacCallum Cancer Institute, East Melbourne, Victoria, Australia.
Abstract:
Activation of beta-catenin/T cell factor (TCF) transcription as a result of mutations in the adenomatous polyposis coli (APC) and/or beta-catenin genes occurs in the majority of colon tumors. An increasing number of genes, including c-myc and cyclin D1, have been implicated as targets of this pathway. We now report that the dominant negative helix-loop-helix regulator Id2 is also a target of the beta-catenin/TCF transcription pathway in colon adenocarcinoma. Investigation of the mechanism for the overexpression of Id2 in colon carcinoma cells demonstrated that the Id2 promoter is activated, and the Id2 protein is up-regulated by beta-catenin. Conversely, reducing free beta-catenin blocked this induction of promoter activity. We have also used an electrophoretic mobility shift assay and supershift to identify a motif in the Id2 promoter that binds to TCF4 protein. Site-directed mutagenesis of this motif abolished promoter reporter activity. Both transfection of Id2 into SW480 cells and induction of Id2 in HT29 colon cells was found to increase anchorage-independent survival of these cells. Growing evidence associates disruption to Id2 expression with tumorigenesis, and our findings suggest that this dysregulation of Id2 expression is due to the activation of the beta-catenin/TCF pathway.
Insights
Mutations in APC and beta-catenin genes activate the beta-catenin/TCF pathway, leading to Id2 overexpression in colon cancer. This Id2 dysregulation promotes tumor cell survival and anchorage-independent growth.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The beta-catenin/T cell factor (TCF) transcription pathway is frequently activated in colon tumors due to mutations in adenomatous polyposis coli (APC) and/or beta-catenin genes.
- Genes such as c-myc and cyclin D1 are known targets of this oncogenic pathway.
Purpose of the Study:
- To investigate whether the Id2 gene is a target of the beta-catenin/TCF transcription pathway in colon adenocarcinoma.
- To elucidate the mechanism of Id2 overexpression in colon cancer cells.
Main Methods:
- Electrophoretic mobility shift assay (EMSA) and supershift assays were used to identify TCF4 binding motifs in the Id2 promoter.
- Site-directed mutagenesis was employed to assess the functional significance of the identified TCF4 binding site.
- Reporter assays were conducted to measure Id2 promoter activity.
- Transfection and induction experiments were performed in colon cancer cell lines (SW480 and HT29) to evaluate the functional consequences of Id2 modulation.
Main Results:
- The Id2 gene was identified as a direct transcriptional target of the beta-catenin/TCF pathway in colon adenocarcinoma.
- Beta-catenin activation leads to the up-regulation of Id2 protein and activation of the Id2 promoter.
- A specific motif in the Id2 promoter was found to bind TCF4 protein, and its mutation abolished promoter activity.
- Increased Id2 expression enhanced anchorage-independent survival in colon cancer cells.
Conclusions:
- Dysregulation of Id2 expression in colon cancer is driven by the aberrant activation of the beta-catenin/TCF pathway.
- Id2 plays a role in promoting tumor cell survival and anchorage-independent growth, contributing to colon tumorigenesis.