FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2
Tao Zuo, Runhua Liu, Huiming Zhang, Xing Chang, Yan Liu, Lizhong Wang, Pan Zheng, Yang Liu
Tao Zuo, Runhua Liu, Huiming Zhang, Xing Chang, Yan Liu, Lizhong Wang, Pan Zheng, Yang Liu
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Research Article Oncology

FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2

Abstract

S-phase kinase-associated protein 2 (SKP2) is a component of the E3 ubiquitin ligase SKP1-Cul1-Fbox complex. Overexpression of SKP2 results in cell cycle dysregulation and carcinogenesis; however, the genetic lesions that cause this upregulation are poorly understood. We recently demonstrated that forkhead box P3 (FOXP3) is an X-linked breast cancer suppressor and an important repressor of the oncogene ERBB2/HER2. Since FOXP3 suppresses tumor growth regardless of whether the tumors overexpress ERBB2/HER2, additional FOXP3 targets may be involved in its tumor suppressor activity. Here, we show that mammary carcinomas from mice heterozygous for a Foxp3 mutation exhibited increased Skp2 expression. Ectopic expression of FOXP3 in mouse mammary cancer cells repressed SKP2 expression with a corresponding increase in p27 and polyploidy. Conversely, siRNA silencing of the FOXP3 gene in human mammary epithelial cells increased SKP2 expression. We also show that Foxp3 directly interacted with and repressed the Skp2 promoter. Moreover, the analysis of over 200 primary breast cancer samples revealed an inverse correlation between FOXP3 and SKP2 levels. Finally, we demonstrated that downregulation of SKP2 was critical for FOXP3-mediated growth inhibition in breast cancer cells that do not overexpress ERBB2/HER2. Our data provide genetic, biochemical, and functional evidence that FOXP3 is a novel transcriptional repressor for the oncogene SKP2.

Authors

Tao Zuo, Runhua Liu, Huiming Zhang, Xing Chang, Yan Liu, Lizhong Wang, Pan Zheng, Yang Liu

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Figure 3

Foxp3 binding to Skp2 is important for transcriptional repression.

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Foxp3 binding to Skp2 is important for transcriptional repression. ...
(A) Foxp3 represses mouse Skp2 promoter activity. Either Foxp3 cDNA or empty vector was transiently cotransfected with reporter vector at different ratios illustrated in the figure. Cells were transfected with either vector control or Foxp3 (1 μg/well) in conjunction with the luciferase reporter driven by 5′ promoter regions of the Skp2 gene (0.2 μg, 0.4 μg and 1.0 μg per well). Forty-eight hours later, the cell lysates were harvested and measured for luciferase activity. The luciferase activity from the cells transfected with the pGL2-basic vector was arbitrarily defined as 1.0. Data shown are means ± SD of triplicates and have been repeated at least 3 times. (B) The upper panel depicts the 5′ region of the Skp2 gene. The lower panel shows the amount of DNA precipitated by anti-V5 mAbs after subtracting a minute portion precipitated by IgG control. The data shown are a fraction of the total genomic DNA isolated from the same number of cells. (C) Deletion of 1 of the 2 Foxp3-binding sites in the Skp2 promoter region prevented FOXP3-mediated suppression. The deleted sequences are mut A: ACTAAACCAATATTCTAAT and mut B: TAAAAATAAACCATC. The promoter activity was measured in the human breast cancer line T47D.