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 4

Smad activation and induction of the HDM2 promoter.

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Smad activation and induction of the HDM2 promoter. (A) Western blot...
(A) Western blot of p-Smad3 in 293T, SKOV3, Vaco400:RII, HCT116, and HCT116:3-6 cells. Tubulin and Ku70 were used as internal controls for loading. (B) A schematic of 2 putative SBEs designated SBE1 and SBE2 and the p53-binding elements in the P2 promoter of HDM2. Reporter assays were conducted using dominant negative Smad4 (dnSmad4) or control vector only (VO) in transient transfection assays. HCT116:3-6 cells were transfected with an SBE2X2 reporter as a positive control or HDM2 P2 reporter. All samples were transfected with renilla to use as an internal control. Cells were treated with vehicle or TGF-β1 (10 ng/ml). Reporter activity was determined relative to renilla to generate relative activity. Fold induction was determined relative to vehicle control and SD was calculated relative to the mean. (C) Western blot of FLAG-Smad3 and Ku70 that bound to the HDM2 promoter. 293T cells were transfected with FLAG-Smad3 and treated with vehicle or TGF-β1 (10 ng/ml). Nuclear extracts were prepared from the latter mentioned cell treatments. Biotinylated PCR fragments of the HDM2 promoter, SBE1, SBE2, ΔHDM2, or a positive control, SBE2X2, were mixed with the nuclear extracts. Biotinylated DNA fragments bound to streptavidin beads were used to purify FLAG-Smad3 and Ku70 (internal control).