Inhibition of CDK-mediated Smad3 phosphorylation reduces the Pin1-Smad3 interaction and aggressiveness of triple negative breast cancer cells

ABSTRACT

Triple negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. Although TNBC is not defined by specific therapeutic targets, a subset of patients have tumors that overexpress cyclins. High cyclin D/E expression catalyzes CDK4/2 activity. In turn, CDK4/2 can non-canonically phosphorylate Smad3, a key TGFβ signaling intermediate, and this phosphorylation has been associated with the shift from tumor-suppressive to oncogenic TGFβ pathway action in breast oncogenesis. Additionally, CDK-mediated Smad3 phosphorylation facilitates an interaction between Smad3 and Pin1, a cis-trans isomerase that is also overexpressed in aggressive breast cancers. Treatment with CYC065, a CDK2/9 inhibitor, decreased non-canonical Smad3 phosphorylation and inhibited the Pin1-Smad3 interaction. We hypothesized that the interaction of Pin1 and Smad3, facilitated by CDK-mediated Smad3 phosphorylation, promotes TNBC cell aggressiveness. Inhibition of the Pin1-Smad3 interaction in TNBC cell lines, through depletion of Pin1 or CYC065 treatment, resulted in decreased cell migration/invasion and impeded the EMT program. Inhibition of CDK-mediated phosphorylation of Smad3 by mutagenesis also decreased cell migration, underscoring the importance of non-canonical CDK2 phosphorylation of Smad3 to enable cell motility. Pin1 depletion restored Smad3 protein levels and tumor-suppressive activity, suggesting that the Pin1-Smad3 interaction has a negative impact on canonical Smad3 action. Collectively, the data show that the Pin1-Smad3 interaction, facilitated by CDK-mediated Smad3 phosphorylation, is associated with oncogenic TGFβ signaling and breast cancer progression. Inhibition of this interaction with CYC065 treatment may provide an important therapeutic option for TNBC patients.

KEYWORDS:

• CDK2
• CDK9
• CDK inhibitor
• cyclin
• cell cycle
• EMT
• invasion
• migration
• Pin1
• Smad3
• TGFβ
• triple negative breast cancer

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Dr. David Blake and Dr. Daniella Zheleva (Cyclacel Ltd.) for generously providing CYC065, and for helpful discussions regarding the dosage requirements for CYC065.

Funding

The authors acknowledge financial support from the Society of Surgical Oncology - Clinical Investigator Award (to J.S. Jeruss) and NIH R01GM097220 (to J.S. Jeruss), A Sister's Hope Foundation, and the Saslow Family (J.S.Jeruss). Alexandra Thomas is supported by NIH 5F31CA206259–02.