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Ubiquitination and Downregulation of BRCA1 by Ubiquitin-Conjugating Enzyme E2T Overexpression in Human Breast Cancer Cells

¹ Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; ² 1st Department of Surgery, Sapporo Medical University, Sapporo, Japan; and ³ Division of Genome Medicine, Institute for Genome Research, The University of Tokushima, Tokushima, Japan

Requests for reprints: Yusuke Nakamura, Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan. E-mail: yusuke{at}ims.u-tokyo.ac.jp.

Breast cancer is generated through a multistep genetic and epigenetic process including activations of oncogenes and inactivations of tumor suppressor genes. Here, we report a critical role of ubiquitin-conjugating enzyme E2T (UBE2T), an E2 ubiquitin-conjugating enzyme, in mammary carcinogenesis. Immunocytochemical staining and in vitro binding assay revealed that UBE2T interacted and colocalized with the BRCA1/BRCA1-associated RING domain protein (BARD1) complex. Knocking down of UBE2T expression with small interfering RNA drastically suppressed the growth of breast cancer cells. Interestingly, in vivo ubiquitination assay indicated BRCA1 to be polyubiquitinated by incubation with wild-type UBE2T protein, but not with C86A-UBE2T protein, an E2 activity–dead mutant, in which the 86th residue of cysteine was replaced with alanine. Furthermore, knocking down of UBE2T protein induced upregulation of BRCA1 protein in breast cancer cells, whereas its overexpression caused the decrease of the BRCA1 protein. Our data imply a critical role of UBE2T in development and/or progression of breast cancer through the interaction with and the regulation of the BRCA1/BARD1 complex. [Cancer Res 2009;69(22):8752–60]

Key Words: breast cancer • ubiquitination • BRCA1