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 1

p53 destabilization by TGF-β1 stimulation.

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p53 destabilization by TGF-β1 stimulation. (A) HCT116 and HCT116:3-6 cel...
(A) HCT116 and HCT116:3-6 cells were treated with vehicle or 10 ng/ml of TGF-β1 for 48 and 72 hours. Cellular extracts were prepared for Western blot, and p53 and Ku70 (internal control) levels were detected. (B) Western blot of p53 laddering, an indication of ubiquitination (Ub) (arrows) in cellular extracts of HCT116:3-6 cells treated with 10 ng/ml of TGF-β1 for 0, 24, and 48 hours. (C) HCT116, Hct116 p53–/–, and HCT116:3-6 cells were treated with 10 ng/ml of TGF-β1 with or without the proteasome inhibitor MG132 (30 μM). Cellular extracts were immunoprecipitated with control IgG or p53 antibodies. Precipitates were separated on an SDS-PAGE gel, and a Western blot was prepared. The left side was probed for ubiquitin. The right side was probed for p53. (D) HCT116:3-6 cells were transfected with HA-Ub. Cells were then treated with MG132 (30 μM) with or without TGF-β1 (10 ng/ml); cellular extracts were immunoprecipitated with p53 or IgG antibodies and prepared for Western blot analysis of HA. (E) Western blot of p53 in extracts isolated from HCT116:3-6 cells treated with TGF-β1 (10 ng/ml) with or without Nutlin3 (10 μM).