Abstract P6-04-12: CDK inhibition mediates tumor suppressive Smad3 action and decreased metastatic phenotypes in triple negative breast cancer cells

Introduction: Breast cancer onset and disease progression has been linked to members of the TGFb superfamily and their downstream signaling components, the Smads. Changes in Smad signaling have been associated with the dichotomous role of TGFβ in malignancy, mediating both tumor suppressant and tumor promoting behaviors in breast cancer. Our previous work showed that cyclin/CDK mediated phosphorylation of Smad3 resulted in the inhibition of canonical tumor suppressant Smad3 action. We thus hypothesized that activation of CDKs leads to phosphorylation and inhibition of Smad3, releasing cell cycle arrest and promoting cell proliferation and metastasis. Using triple negative breast cancer cell lines, we examined the impact of specific CDK inhibitors (CDKis) on signaling patterns, metastatic phenotypes, and protein expression profiles. Methods: Triple negative cell lines Hs578T, MDA-MB-231 and MDA-MB-436 were treated with CDK2i or CDK4i and Smad3 activity was measured using a luciferase assay. Transwell migration and Matrigel invasion assays were used to show the effect of transfection with Smad3 CDK phosphorylation site mutants, resistant to inhibitory CDK phosphorylation, or treatment with CDK2i or CDK4i on the study cells. Immunoblotting was performed to measure protein expression levels of Smad3, pSmad3 T179, and MMP2. A novel live cell array was implemented to quantify changes in activity of 14 cancer-related transcription factors over a period of 3 days. TUNEL and Ki67 staining analysis and a xenograft mouse model were used to determine impact of CDKis, in combination with paclitaxel chemotherapy, on apoptosis and tumor growth respectively. Results: Treatment of study cells with CDK2i or CDK4i resulted in increased Smad3 activity and decreased migration and invasion in the study cells. Transfection with a 5M Smad3 mutant construct, containing mutations in all 5 CDK phosphorylation sites, resulted in the greatest decrease in cell migration, when compared with cells transfected with both vector control or WT Smad3. CDK2i or CDK4i therapy resulted in decreased Smad3 protein phosphorylation at the T179 site, while total Smad3 levels were unaffected. CDK2i/4i treatment also resulted in decreased MMP2 expression. The array studies revealed decreased activity of EMT transcription factors B-catenin, Snail, Twist, in MDA-MB-231 cells following CDK2i treatment. CDK2i and paclitaxel combination treatment resulted in increased apoptosis and decreased tumor volume and Ki67 staining compared to control treatment. Conclusions: We have shown for the first time that cyclin/CDK activity, in part, mediates the metastasis-associated role of Smad3 in triple negative breast cancer cells. Importantly, both CDK2i and CDK4i treatment resulted in decreased cell migration/invasion and also corresponded to decreased MMP2 expression. Lastly, the array studies revealed CDK2i mediated effects on key transcription factors associated with the promotion of EMT. Future studies will explore the significance of Smad3 interaction with these EMT factors and will pursue the clinical application of CDK2i for patients with triple negative/cyclin overexpressing breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-12.