CDK4 inhibition and doxorubicin mediate breast cancer cell apoptosis through Smad3 and survivin

Abstract

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFβ superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells.

Keywords: :

• CDK4
• Smad3
• TGFβ
• apoptosis
• breast cancer
• doxorubicin
• survivin

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr Stacey Tobin and Megan Novak for their editorial assistance and Dr Vamsi Parini for assessment of TUNEL staining.

Financial Support

J.S.J. is a Lynn Sage Scholar supported by the NIH K22 CA138776 and R01GM097220 research grant, the Central Surgical Association Foundation, the Society of Surgical Oncology, the Saslow family, and A Sister’s Hope. E.T. is a Dr John N. Nicholson Fellow, a Chicago Biomedical Consortium Scholar supported by the Searle Funds at the Chicago Community Trust, and is also supported by the NIH/NCI training grant T32CA09560 and the National Cancer Institute Kirchstein-NRSA Fellowship F31 CA168106-02.