Introduction: The transcription factor Smad3, a component of the TGF\#946; signaling cascade, contributes to G1 arrest in breast cancer cells. Cyclin D1, together with its catalytic partner CDK4, promotes G1/S-phase transition. Overexpression of cyclin D1 is associated with poor prognosis in breast cancer. Cyclin D regulates phosphorylation of Smad3 through CDK4, and inhibits Smad3 activity in normal mouse fibroblasts and epithelial cells. We hypothesize that overexpression of cyclin D1 exerts tumorigenic effects in breast cancer cells through a CDK4 mediated inhibition of Smad3. Methods: Western blotting was used to evaluate expression of Smad3, phosphorylated Smad3, and CDK4 in parental, vector control and cyclin D1 overexpressing MCF7 and T47D cells. To examine the impact of cyclin D1 overexpression on Smad3 function, constructs containing empty vector (V), WT Smad3 (WT), and Smad3 with single or multiple CDK phosphorylation site mutations were cotransfected with a Smad3 reporter into the study cells. Smad3 activity was then evaluated by luciferase reporter assays. Study cells were also transfected with V, WT or mutant Smad3 and treated with a CDK4 inhibitor. Smad3 function was examined by luciferase reporter assays and cell cycle analysis. Specificity of CDK4 inhibition was assessed by cotransfection of Smad3 constructs and CDK4 siRNA. Results: Study cells expressed Smad3, P-Smad 3, and CDK4. Transfection of WT Smad3 elicited an increase in Smad3 reporter activity above control in study panel of cells. In parental and vector control MCF7 and T47D cells, transfection of all single site Smad3 mutant constructs resulted in increased reporter activity above WT Smad3. In MCF7 and T47D cyclin D1 overexpressing cells, transfection of Smad3 Thr8 and Thr178 site mutation constructs induced a Smad3 reporter response above WT, whereas independent transfection of the S203, 207 and 212 mutations resulted in a response comparable to WT construct. Transfection with 4 and 5-mutation Smad3 constructs resulted in hightest overall Smad3 reporter activity, compared to WT Smad3, in cyclin D overexpressing cells. MCF7 and T47D cells treated with CDK4 inhibitor demonstrated an increase in G1 cell cycle population. MCF7 parental and cyclin D1 overexpressing cells transfected with Thr8 or Thr178 mutants and CDK4 siRNA showed highest Smad3 reporter activity when compared to study cells transfected with WT Smad3 alone. Conclusions: Mutation of multiple CDK phosphorylation sites in the Smad3 protein appear to work synergistically to facilitate release of cyclin D1 mediated inhibition of Smad3 in breast cancer cells. The direct role of CDK4 in deregulation of Smad3 activity was demonstrated by rescue of Smad 3 function found in cyclin D1 overexpressing cells transfected with CDK4 siRNA or treated with the CDK4 inhibitor. Inhibition of CDK4 activity may have a role in treatment of primary and metastatic breast cancers overexpressing cyclin D.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3318.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO