CDK4 Phosphorylation Regulates Smad3 Activity in Cyclin D1 Overexpressing Breast Cancer Cells.

Introduction: Overexpression of cyclin D1 is associated with poor prognosis in breast cancer. Cyclin D1 regulates the noncanonical phosphorylation of Smad3, a member of the TGFΒ signaling cascade, 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 cell cycle control.Methods: 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 cyclin D1 overexpressing MCF-7 and T47D cells. Smad3 containing mutant CDK sites is resistant to inhibitory CyclinD1/CDK4 phosphorylation. 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. Levels of mRNA for cell cycle regulators cyclin dependent kinase inhibitor (cdki) p15 and c-myc were measured by qRT-PCR.Results: As expected, higher amounts of c-myc mRNA and lower amounts of cdki p15 mRNA were observed in parental, vector control and cyclin D1 overexpressing cells. While subsequent transfection with WT Smad3 resulted in a decrease in c-myc and an increase in p15 mRNA levels, the greatest decrease in c-myc and increase in p15 mRNA was found after transfection with CDK mutated Smad3, most prominently in the cyclin D1 overexpressing cells. Transfection of all individual Smad3 CDK phosphorylation site mutations resulted in an increase in Smad3 reporter activity as compared to transfection with WT Smad3. Transfection of Smad3 constructs with 4 and 5 CDK sites mutated resulted in highest overall Smad3 reporter activity, compared to WT Smad3, in the study cells. A dose dependent increase in Smad3 reporter activity was shown when MCF-7 and T47D cells were transfected with WT Smad3 and treated with increasing concentrations of a specific CDK4 inhibitor. Comparatively higher Smad3 reporter activity was found when the study cells were transfected with individual Smad3 mutant constructs and treated with the CDK4 inhibitor. Interestingly, transfection with the Smad3 construct with all CDK sites mutated and treatment with the CDK4 inhibitor resulted in inhibition of Smad3 reporter activity in the study cells. Lastly, cell cycle analysis revealed that MCF-7 and T47D cells treated with the CDK4 inhibitor showed an increase in the G1 cell cycle population.Conclusions: Mutation of CDK phosphorylation sites in the Smad3 construct or direct inhibition of CDK4 appear to facilitate release of cyclin D1/CDK4 mediated inhibition of Smad3 in breast cancer cells. To this end, restoration of Smad3 activity resulted in increasing p15 and decreasing c-myc mRNA levels in the study cells. However, mutation of all Smad3 CDK phosphorylation sites and treatment with the CDK4 inhibitor resulted in a decrease in Smad3 reporter activity, indicating that some CDK activity is necessary for Smad3 function. By helping to restore cell cycle control, inhibition of CDK4 activity may have a role in the treatment of primary and metastatic breast cancers overexpressing cyclin D. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2152.