Abstract P3-07-11: Inhibition of Pin1 or CDK-mediated Smad3 phosphorylation reduces triple negative breast cancer cell EMT, migration and invasion
Authors: Thomas AL , Hamdan R , Hong A , Lind H , Oppat K , Rosenthal E , Thomas AJ , Jeruss JS 
Published: March 2017

Abstract
Introduction: Triple negative breast cancer (TNBC) is an aggressive subtype associated with poor outcomes. Accordingly, there is an urgent need to develop novel and targeted therapeutics for patients with this disease subtype. Cyclins D and E and the corresponding activation of CDK4/2 represent promising therapeutic targets for the treatment of TNBC. CDK4/2 can non-canonically phosphorylate Smad3, a key TGFβ signaling intermediate, and this phosphorylation is associated with the promotion of cell migration and EMT in cyclin-overexpressing breast cancers. Additionally, CDK-mediated Smad3 phosphorylation facilitates an interaction between Smad3 and Pin1. Pin1 is a cis-trans isomerase that is also overexpressed in aggressive breast cancers and can enable TNBC cell migration. Based on these findings, we hypothesized that blockade of the CDK-mediated Smad3-Pin1 interaction, either through inhibition of Pin1 or CDK-mediated Smad3 phosphorylation, would abrogate TNBC cell migration and invasion.
Methods: Pin1 expression was knocked-down (KD) in MDA-MB-231, MDA-MB-436, and Hs578T TNBC cells by transfection with Pin1-targeting siRNA (siPin1) or control non-specific siRNA (siNS). KD efficiency was confirmed with immunoblotting. Pin KD/TNBC cell migration and invasion assays were performed on uncoated or Matrigel-coated trans-wells, respectively. Media containing 10% FBS was used as a chemoattractant. Following Pin1 KD, immunoblotting was used to evaluate EMT-associated protein expression. To inhibit CDK-mediated Smad3 phosphorylation, TNBC cells were treated with 600 nM of CDK2 inhibitor (CDK2i) for 72 hours. Immunoblotting was then performed to determine Smad3 phosphorylation and EMT-associated protein expression. Co-immunoprecipitation assays were used to examine the impact of CDK2i treatment on the Smad3-Pin1 interaction. Finally, following CDK2i treatment, assays were performed to determine the ability of TNBC cells to migrate and invade.
Results: KD of Pin1 expression in TNBC cells resulted in a decrease in cell migration and invasion when compared to control cells in all the study cell lines. This corresponded with changes in EMT-associated protein expression, including increased levels of ZO-1 and claudin and decreased β-catenin. CDK2i treatment produced a decrease in Smad3 T179 site non-canonical phosphorylation and inhibited Smad3-Pin1 binding. CDK2i treatment also abrogated TNBC cell migration and invasion, paralleling expression changes in EMT-associated proteins with an increase in claudin and decrease in β-catenin.
Conclusions: Inhibition of the Smad3-Pin1 interaction, through KD of Pin1 expression or CDK2i-mediated blockade of non-canonical Smad3 phosphorylation, reduced TNBC cell EMT-type changes, demonstrated by increased expression of the tight junction proteins ZO-1 and claudin and decreased β-catenin, a key player in the WNT pathway. These findings also correlated to a reduction in TNBC cell migration and invasion. Collectively, these data show that the Smad3-Pin1 interaction, facilitated by CDK-mediated Smad3 phosphorylation, is associated with pro-migratory TGFβ signaling. Inhibition of this interaction, with CDK2 inhibitor treatment, may provide an important therapeutic option for TNBC patients.
Citation Format: Thomas AL, Hamdan R, Hong A, Lind H, Oppat K, Rosenthal E, Thomas AJ, Jeruss JS. Inhibition of Pin1 or CDK-mediated Smad3 phosphorylation reduces triple negative breast cancer cell EMT, migration and invasion [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-11.