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.

CD155 contributes to the mesenchymal phenotype of triple‐negative breast cancer

“…In TNBC, TGF‐β was shown to induce EMT and metastasis . Smad family members such as Smad2, Smad3 and Smad4 mediated these processes . Our in silico analysis showed that CD155 was positively correlated with TGF‐β and Smad3 but not Smad2 and Smad4.…”

Prolyl Isomerase Pin1 in Human Cancer: Function, Mechanism, and Significance

“…Phosphorylated Thr179-Pro motif of Smad2/3 interacts with Pin1 in a TGF-β-dependent manner, inducing migration and invasion via N-cadherin in prostate cancer cells (Matsuura et al, 2010). In turn, Pin1-Smad3 interaction is reduced by the inhibition of CDK-mediated Smad3 phosphorylation, leading to the suppression of triple negative breast cancer cells (Thomas et al, 2017).…”

Pin1 promotes pancreatic cancer progression and metastasis by activation of NF‐κB‐IL‐18 feedback loop

“…Pin1 promotes cell proliferation by upregulating cyclins in hepatocellular carcinoma cells 27 . Pin1 also interacts with Smad3 to drive oncogenic TGFβ signalling and promote breast cancer progression 28 . Our RNA sequencing results from the different Pin1‐expressing cell lines provide support for the idea that Pin1 might implement its oncogenic effect by regulating NF‐κB signalling in PDAC.…”