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

Abstract

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]

Molecular features of triple negative breast cancer cells by genome-wide gene expression profiling analysis

“…Purified RNA from each clinical sample and cell line, as well as poly-A RNA from normal human heart, lung, liver, and kidney (Takara, Otsu, Japan) was reverse transcribed for single-stranded cDNA using oligo(dT) [12][13][14][15][16][17][18] primers with Superscript II reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA). qRT-PCR analysis was performed using an ABI PRISM 7500 Real-Time PCR system (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) and SYBR Premix Ex Taq (Takara) according to the manufacturer's instructions.…”

“…Vector-based shRNAs and the psiU6BX3 expression system were constructed as previously described (13). The shRNA target sequences were the same as those of the siRNA oligonucleotides.…”

Involvement of C12orf32 overexpression in breast carcinogenesis

“…Through the genome-wide expression analysis of a large number of microdissected clinical cancer materials, we have identified dozens of genes that function as oncogenes in the process of development and/or progression of breast cancer (10)(11)(12)(13)(14)(15)(16)(17), bladder cancer (18,19), synovial sarcoma (20,21), testicular seminoma (22), and renal cell carcinoma (23,24). Such molecules are considered to be candidate molecular targets for development of new therapeutic modalities.…”

Mutations in the Gene Encoding the E2 Conjugating Enzyme UBE2T Cause Fanconi Anemia

“…21 It is common for an E2 to function with a set of E3 ligases, since far fewer E2s (~38) are encoded in the genome than E3 ligases (600-1,000). 22 UBE2T might have a partner other than FANCL, such as BRCA1 23 or other E3s, as has been suggested by yeast two-hybrid assays, 24,25 raising the possibility that UBE2T might have a function outside the FA pathway. Although a siUBE2T knockdown in AP65P-hTERT modestly sensitized cells to UV ( Figure 3C), we detected only a marginal impact of UBE2T lentiviral transduction on UV survival ( Figure 3D).…”