Abstract P5-04-13: Pin1 negatively impacts Smad3 tumor suppression in triple negative breast cancer cell lines
Authors: Thomas AL , Hamdan R , Hong A , Rosenthal E , Thomas AJ , Jeruss JS 
Published: May 2016

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, to promote the transition from tumor suppressive to oncogenic TGFβ activity in cyclin-overexpressing breast cancers. We identified a Smad3 interaction with Pin1, a cis-trans isomerase also overexpressed in aggressive breast cancers and associated with CDK-mediated Smad3 phosphorylation. Smad3 interaction with Pin1 can influence protein function and fidelity through recruitment of Smurf2 and subsequent proteasomal degradation. Based on these findings, we hypothesized that inhibition of the CDK-mediated Smad3-Pin1 interaction would stabilize Smad3 protein expression and restore tumor-suppressive Smad3 activity.

Methods: Pin1 expression was knocked-down (KD) in MDA-MB-231 TNBC cells by transfecting with Pin1-targeting siRNA (siPin1) or control non-specific siRNA (siNS). KD efficiency was confirmed by immunoblotting. To assay Smad3 transcriptional activity with Pin1 KD, luciferase reporter studies were performed. Also, following Pin1 KD, immunoblotting was used to determine expression of Smad3 and associated protein targets. MTS assays were utilized to determine cellular proliferation after Pin1 KD. Transwell migration assays were used to assay the effect of Pin1 KD or CDK2 inhibitor treatment, which blocked non-canonical Smad3 Thr179 phosphorylation, on TNBC cell migration.

Results: KD of Pin1 expression in TNBC cell lines resulted in an increase in Smad3 transcriptional activity compared to control cells, and correlated with an increase in expression of cdki p15 and a decrease in c-myc, Smad3-target genes and cell cycle regulators. Additionally, Pin1 KD resulted in a significant decrease in TNBC cell proliferation compared to siNS control TNBC cells. Smad3 protein levels increased following Pin1 KD, suggesting Pin1 action may negatively impact Smad3 stability. We also found that KD of Pin1 or treatment with a CDK2 inhibitor, which blocked Smad3 noncanonical Thr179 phosphorylation, resulted in significantly reduced TNBC cell migration.

Conclusions: Inhibiting the Smad3-Pin1 interaction by knock-down of Pin1 expression in TNBC cells restored Smad3 transcriptional activity, which correlated to an increase in expression of the Smad3 associated protein cdki p15, decrease in c-myc, and a decrease in cellular proliferation. Additionally, Pin1 KD enhanced Smad3 protein levels, suggesting a role of Pin1 in mediating Smad3 stability. Inhibiting the Smad3-Pin1 interaction with Pin1 KD or CDK2 inhibitor treatment also reduced TNBC cell migration. Collectively, these data suggest that the Smad3-Pin1 interaction, facilitated by noncanonical CDK-mediated Smad3 phosphorylation, is associated with pro-tumorigenic and pro-migratory TGFβ signaling, and inhibition of this interaction may provide an important therapeutic option for TNBC patients.

Citation Format: Thomas AL, Hamdan R, Hong A, Rosenthal E, Thomas AJ, Jeruss JS. Pin1 negatively impacts Smad3 tumor suppression in triple negative breast cancer cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-13.