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DNA Damage and Cellular Stress Responses

Essential Role of T-Cell Factor/ß-Catenin in Regulation of Rad6B: A Potential Mechanism for Rad6B Overexpression in Breast Cancer Cells

¹ Breast Cancer Program, Karmanos Cancer Institute and ² Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan

Requests for reprints: Malathy P.V. Shekhar, Breast Cancer Program, Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, MI 48201. Phone: 313-833-0715, ext. 2326; Fax: 313-831-7518. E-mail: shekharm{at}karmanos.org

We have previously shown that the postreplication DNA repair gene Rad6B plays a critical role in the maintenance of genomic integrity of human breast cells. Whereas normal breast cells express low levels of Rad6B, increases in Rad6B expression occur in hyperplasia with overexpression in breast carcinomas. Here, we show that the human Rad6B gene is a transcriptional target of T-cell factor (TCF)-4/ß-catenin/p300. Rad6B promoter activity is subject to negative regulation in normal human MCF10A breast cells whereas it is constitutively active in metastatic MDA-MB-231 breast cancer cells. Derepression and activation of Rad6B promoter in MCF10A cells require coexpression of ß-catenin and p300. Using electrophoresis mobility shift assay, Western blot analysis of electrophoresis mobility shift assay, UV cross-linking, and chromatin immunoprecipitation assay, we show that Rad6B transcriptional repression in MCF10A cells is due to paucity of transcriptionally active ß-catenin assembled on the TCF binding sequence in the Rad6B promoter rather than to a deficit/decreased affinity of TCF-4 for the TCF binding element in Rad6B promoter. Three-dimensional epithelial acini generated in vitro from MCF10A cells cotransfected with ß-catenin and p300 showed ß-catenin expression on the membrane, cytoplasm, and/or nuclei with concomitant Rad6 overexpression, whereas control acini showed ß-catenin on the membranes and negligible Rad6 expression. Immunohistochemical analysis of 12 breast carcinomas showed an 80% correlation between Rad6 and ß-catenin expression, and combined nuclear and cytoplasmic staining of ß-catenin and Rad6 was detected in 25% of the breast carcinomas. In vivo implantation of MCF10A-Rad6B cells produced hyperplastic lesions. These data reveal a potentially important role for transcriptionally active ß-catenin in the regulation of Rad6B gene expression, and link aberrant ß-catenin signaling with transcriptional deregulation of Rad6B and breast cancer development. (Mol Cancer Res 2006;4(10):729–45)

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				M. P.V. Shekhar, B. Gerard, R. J. Pauley, B. O. Williams, and L. Tait Rad6B Is a Positive Regulator of {beta}-Catenin Stabilization Cancer Res., March 15, 2008; 68(6): 1741 - 1750. [Abstract] [Full Text] [PDF]