Abstract P5-11-01: Impact of CDK mediated Smad3 phosphorylation and Pin1 in triple negative breast cancer cell migration

Abstract

Abstract Introduction: Estrogen receptor (ER), progesterone receptor (PR), and Human Epidermal Growth Factor Receptor 2 (HER2) negative breast cancers, also known as triple negative breast cancers can overexpress cyclins, such as cyclin D and E, which have been linked to cancer progression and metastasis. Cyclin/CDK complexes can phosphorylate and regulate Smads, downstream signaling proteins in the transforming growth factor-beta (TGF-b) superfamily. CDKs phosphorylate Smad3, predominantly in the linker region, resulting in the decreased tumor suppressive role of this protein. The peptidyl-prolyl cis/trans isomerase Pin1 is also overexpressed in breast cancer. Pin1 can bind phosphorylated Smads contributing to TGF-β induced cell migration and invasion. Inhibition of CDK-mediated Smad3 phosphorylation has been shown to restore Smad3 activity and decrease tumor cell migration in vitro. Based on these findings, we hypothesized that inhibition of Pin1 or CDK-mediated Smad3 phosphorylation would result in decreased triple negative breast cancer cell migration. Methods: Hs578T, MDA-MB-436, and MDA-MB-231 triple negative breast cancer cells (TN) were transfected with either an si control (siCN) or an siPin1 plasmid to knockdown Pin1 protein expression. Transwell migration assays were used to determine the effect of Pin1 knockdown on migration. TN cells were treated with CDK4 or CDK2 inhibitors alone or in combination. The study cells were also stably transduced with lentiviral empty vector, wild-type (WT) Smad3, or a Smad3 mutant (5M) resistant to CDK-mediated phosphorylation. The effect of pharmacologically or genetically inhibiting CDK-mediated Smad3 phosphorylation on Pin1 binding to phosphorylated Smad3 (p-Smad3) or total Smad3 was determined using a co-immunoprecipitation assay (Co-IP). Results: Transfecting the study cells with siPin1 resulted in decreased cell migration when compared to the siCN transfected cells. Treatment with the CDK4 or CDK2 inhibitors alone or in combination did not affect Pin1 protein levels, but resulted in decreased Pin1 binding to p-Smad3 with the greatest decrease found after treatment with combination therapy. Similarly, transduction of the study cells with the WT or the 5M Smad3 constructs had no effect on Pin1 levels but resulted in decreased Pin1 binding to Smad3. The greatest decrease was observed in the 5M transduced cells. Concusions: Inhibition of CDK-mediated Smad3 phosphorylation and knock down of Pin1 resulted in decreased migration of TN breast cancer cells. Inhibition of CDK-mediated Smad3 phosphorylation by mutation of the CDK phosphorylation sites or by treatment with CDK4 and CDK2 inhibitors negatively impacted Pin1 binding to Smad3. Together, these findings indicate that CDK-mediated Smad3 phosphorylation enhances Pin1 binding to Smad3 and promotes tumor cell migration in triple negative breast cancer cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-11-01.