TGF-β1–induced expression of human Mdm2 correlates with late-stage metastatic breast cancer
Shinako Araki, … , David A. Boothman, Lindsey D. Mayo
Shinako Araki, … , David A. Boothman, Lindsey D. Mayo
Published December 1, 2009
Citation Information: J Clin Invest. 2010;120(1):290-302. https://doi.org/10.1172/JCI39194.
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Research Article Oncology

TGF-β1–induced expression of human Mdm2 correlates with late-stage metastatic breast cancer

Abstract

The E3 ubiquitin ligase human murine double minute (HDM2) is overexpressed in 40%–80% of late-stage metastatic cancers in the absence of gene amplification. Hdm2 regulates p53 stability via ubiquitination and has also been implicated in altering the sensitivity of cells to TGF-β1. Whether TGF-β1 signaling induces Hdm2 expression leading to HDM2-mediated destabilization of p53 has not been investigated. In this study, we report that TGF-β1–activated SMA- and MAD3 (Smad3/4) transcription factors specifically bound to the second promoter region of HDM2, leading to increased HDM2 protein expression and destabilization of p53 in human cancer cell lines. Additionally, TGF-β1 expression led to Smad3 activation and murine double minute 2 (Mdm2) expression in murine mammary epithelial cells during epithelial-to-mesenchymal transition (EMT). Furthermore, histological analyses of human breast cancer samples demonstrated that approximately 65% of late-stage carcinomas were positive for activated Smad3 and HDM2, indicating a strong correlation between TGF-β1–mediated induction of HDM2 and late-stage tumor progression. Identification of Hdm2 as a downstream target of TGF-β1 represents a critical prosurvival mechanism in cancer progression and provides another point for therapeutic intervention in late-stage cancer.

Authors

Shinako Araki, Jacob A. Eitel, Christopher N. Batuello, Khadijeh Bijangi-Vishehsaraei, Xian-Jin Xie, David Danielpour, Karen E. Pollok, David A. Boothman, Lindsey D. Mayo

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Figure 2

TGF-β1 increases Hdm2 mRNA and protein levels.

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TGF-β1 increases Hdm2 mRNA and protein levels. (A) Western blot analysis...
(A) Western blot analysis of Hdm2 and p53 in HCT116 and HCT116:3-6 treated with TGF-β1 (10 ng/ml) for 0, 24, 48, and 72 hours. (B) Vaco400 cells with control plasmid (Vaco400:neo) or expressing the functional TGF-β1 receptor II (Vaco400:RII) cells were treated with vehicle (veh) or TGF-β1 (10 ng/ml) for 24 or 48 hours. Western blot was prepared from the extracts, and Hdm2, p53, and tubulin were detected. (C) HCT116 and HCT116:3-6 were transfected with the HDM2 promoter-luciferase reporter construct and renilla expression plasmid; then cells were treated with TGF-β1 (10 ng/ml) or vehicle. After 48 hours, extracts were prepared for analysis. Fold induction represents vehicle to TGF-β1 treatments and error bars represent SD generated from the mean. (D) Real-time PCR was performed on HCT116:3-6 cells treated with vehicle or TGF-β1 (10 ng/ml) at 6 or 24 hours.