FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2
Tao Zuo, … , Pan Zheng, Yang Liu
Tao Zuo, … , Pan Zheng, Yang Liu
Published November 15, 2007
Citation Information: J Clin Invest. 2007;117(12):3765-3773. https://doi.org/10.1172/JCI32538.
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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 2

Foxp3 represses Skp2 transcription.

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Foxp3 represses Skp2 transcription. (A) Transfection of...
(A) Transfection of Foxp3-V5 into TSA cells repressed expression of the Skp2 gene. The mRNA levels of Foxp3, Skp2, and p27 were measured by real-time PCR for the vector control cells, polyclonal Foxp3-V5 transfectants (CL30), and 2 stable Foxp3-V5 transfectant clones, CL302 and CL305. Data shown are relative amounts of transcripts after normalizing against the amounts of total RNA based on the levels of Hprt mRNA. The means of the vector group are artificially defined as 1.0. Data shown are means ± SD of 3 independent experiments. (B) Foxp3 reduces Skp2 with a corresponding increase in p27. Lysates of Foxp3-V5 or vector-transfected TSA cell lines were analyzed by Western blot using SKP2, p27, β-actin, and anti-V5 (which recognize V5-tagged Foxp3) antibodies. (C) Foxp3 increased stability of p27. Vector or Foxp3-V5 transfectants were treated with cycloheximide (CHX, 100 μM) for the indicated intervals. Cells were collected and p27 protein levels were detected by Western blot. The upper panel shows representative experiments while the lower panel shows the decay of p27, using time 0 as 1.0. Protein loading equivalence was assessed by the expression of β-actin. The relative intensity of bands was measured relative to their respective β-actin bands using BandScan software (version 4.3; Glyco). (D) Polyubiquitination of the p27 in vector or Foxp3 transfectants. Vector or Foxp3-V5–transfected TSA cell lines were either left untreated or treated with proteasome inhibitor MG132 (40 μM) for 3 hours. Equal aliquot of the cellular lysates were immunoprecipitated with anti-p27 antibodies. The precipitates were separated by SDS-PAGE and transferred into nitrocellulose membrane, which were then autoclaved in water for 30 minutes and blotted with HRP-conjugated anti-polyubiquitin antibody Ub P4D1 (33).