P2-02-06: Inhibition of CDK4 Phosphorylation of Smad3 Decreases Cyclin D Overexpressing Breast Cancer Cell Proliferation and Migration.
Authors: Tarasewicz E , Hardy A , Straehla J , Foucar C , Zelivianski S , Jeruss J 
Published: June 2012

Introduction: Several aspects of breast cancer onset and disease progression have been linked to members of the TGFβ superfamily and their associated downstream signaling components, the Smads. Alterations in Smad signaling have been directly implicated in the dichotomous role of TGFβ in malignancy, enacting both tumor suppressant and tumor promoting behaviors in breast carcinogenesis. Our previous work characterized Smad3 as a tumor suppressor and found that Smad3 action is inhibited upon phosphorylation by cyclin D/CDK4 in breast cancer cells. Others have shown that CDK phosphorylation of Smad3 can affect cell motility. Based on these findings, we hypothesized that activation of CDK4 leads to phosphorylation and inhibition of Smad3, thus releasing cell cycle arrest and promoting cell proliferation and metastasis.

Methods: MCF7 parental and cyclin D overexpressing cells (MCF7 CD1) were treated with a CDK4 inhibitor alone or in combination with doxorubicin or paclitaxel and cell proliferation was determined using the MTS assay. MCF7 study cells were also transduced with lentiviral empty vector, wild-type (WT) Smad3, or Smad3 containing mutant CDK sites (T179V and 5M) resistant to inhibitory cyclin D/CDK phosphorylation. Stably transduced cells were plated in Matrigel 3-dimensional (3D) culture and treated with CDK4 inhibitor alone or with doxorubicin over a 12 day time course. Colony area was measured using MetaMorph software. Transwell migration and Matrigel invasion assays were used to determine the effect that transfection with Smad3 CDK phosphorylation site mutants or treatment with a CDK4 inhibitor had on highly metastatic cyclin D expressing MDA-MB-231 cells. Study wells were fixed/stained and the number of migrated or invaded cells was counted. Results: Treatment with the CDK4 inhibitor alone resulted in a growth inhibitory effect on proliferation of MCF7 parental and CD1 cells. Treatment with the CDK4 inhibitor in combination with chemotherapy resulted in the greatest inhibition of cell proliferation. In 3D culture, colonies formed by MCF7 study cells transduced with T179V or 5M Smad3 mutant constructs had smaller acinar size when compared with vector-transduced control cells. When compared to untreated cells, the MCF7 CD1 cells treated with CDK4 inhibitor or chemotherapy alone had smaller acinar size, with greatest decrease in size observed when the treatments were combined. Lastly, MDA-MB-231 cells transfected with the 5M Smad3 mutant construct, containing mutations in all 5 CDK phosphorylation sites, resulted in the greatest decrease in cell migration and invasion, when compared with cells transfected with both vector control or WT Smad3. Treatment of the MDA-MB-231 cells with the CDK4 inhibitor also resulted in a significant decrease in cell migration and invasion.

Conclusions: Mutation of CDK phosphorylation sites in the Smad3 construct or direct inhibition of CDK4 resulted in a decrease in breast cancer cell proliferation, 3D colony formation and cell migration and invasion. Treatment with the CDK4 inhibitor in combination with chemotherapy further augmented these results. Inhibition of CDK4 and restoration of Smad3 activity may have a role in the treatment of breast cancers overexpressing cyclin D.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-06.