title: 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.
authors.affiliation: Department of Surgery; Northwestern University Feinberg School of Medicine; Chicago, IL USA; Robert H. Lurie Comprehensive Cancer Center; Chicago, IL USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Tarasewicz
authors.affiliation: Department of Surgery; Northwestern University Feinberg School of Medicine; Chicago, IL USA; Robert H. Lurie Comprehensive Cancer Center; Chicago, IL USA.
authors.forename: Randala
authors.initials: R
authors.lastname: Hamdan
authors.affiliation: Department of Surgery; Northwestern University Feinberg School of Medicine; Chicago, IL USA; Robert H. Lurie Comprehensive Cancer Center; Chicago, IL USA.
authors.forename: Joelle
authors.initials: J
authors.lastname: Straehla
authors.affiliation: Department of Surgery; Northwestern University Feinberg School of Medicine; Chicago, IL USA; Robert H. Lurie Comprehensive Cancer Center; Chicago, IL USA.
authors.forename: Ashley
authors.initials: A
authors.lastname: Hardy
authors.affiliation: Department of Surgery; Northwestern University Feinberg School of Medicine; Chicago, IL USA; Robert H. Lurie Comprehensive Cancer Center; Chicago, IL USA.
authors.forename: Omar
authors.initials: O
authors.lastname: Nunez
authors.affiliation: Department of Surgery; Northwestern University Feinberg School of Medicine; Chicago, IL USA; Robert H. Lurie Comprehensive Cancer Center; Chicago, IL USA.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Zelivianski
authors.affiliation: Department of Surgery; Northwestern University Feinberg School of Medicine; Chicago, IL USA; Robert H. Lurie Comprehensive Cancer Center; Chicago, IL USA.
authors.forename: Danijela
authors.initials: D
authors.lastname: Dokic
authors.affiliation: Department of Surgery; Northwestern University Feinberg School of Medicine; Chicago, IL USA; Robert H. Lurie Comprehensive Cancer Center; Chicago, IL USA.
authors.forename: Jacqueline S
authors.initials: JS
authors.lastname: Jeruss
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D049109:Cell Proliferation / Q000187:drug effects; D019938:Cyclin D1 / Q000235:genetics; D051358:Cyclin-Dependent Kinase 4 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology*; D004357:Drug Synergism; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism*; D061986:MCF-7 Cells / Q000187:drug effects; D009154:Mutation; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D000077022:Survivin; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism
chemical_list: D000903:Antibiotics, Antineoplastic; D051017:Apoptosis Regulatory Proteins; C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D047428:Protein Kinase Inhibitors; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D000077022:Survivin; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D019938:Cyclin D1; D004317:Doxorubicin; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4
keywords: CDK4; Smad3; TGFβ; apoptosis; breast cancer; doxorubicin; survivin