Defining the Role of Pin1 and CDK-mediated Smad3 Phosphorylation in Triple Negative Breast Cancer Migration and Metastasis

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 DESCRIPTION (provided by applicant): Triple negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes. Due to the heterogeneity of this subtype, there are currently no tailored therapies available for triple negative disease. Consequently, TNBC patients are treated with limited success and are prone to relapse and death because of the tendency of TNBCs to metastasize. There is an urgent need for improved therapeutic options for TNBC patients, and it is critical to investigate potential therapeutic targets to help improve patient outcomes. Cyclin D and E overexpression and the consequential impact on CDK4/2 activity correlate with aggressive breast cancer subtypes. Smad3, a key TGFβ signaling intermediate, is a substrate of CDK4/2. CDK4/2-mediated noncanonical Smad3 phosphorylation contributes to the oncogenic shift in TGFβ signaling in breast oncogenesis from promoting tumor suppression to supporting tumor growth. The impact of CDK-mediated Smad3 phosphorylation on cancer cell migration, invasion, and metastasis is an area of active study. CDK4/2 phosphorylation at the noncanonical T179 site (pT179) of Smad3 is associated with Pin1- Smad3 binding and promotion of migration and invasion. Pin1 is a peptydylprolyl cis/trans isomerase and is overexpressed in breast and other cancers. Pin1 expression levels correlate with increased breast tumor grade and poor patient outcomes. Our group has demonstrated an association with Pin1, Smad3 pT179, and CDK2 activity that impacts TNBC oncogenesis. We hypothesize that the interaction of Smad3 and Pin1, facilitated by CDK-mediated Smad3 phosphorylation, promotes cancer cell proliferation, migration and metastasis in cyclin- expressing TNBC. The specific aims of this proposal will directly test this hypothesis as follows: Aim 1: Examine the effect of Pin1 on Smad3-regulated cell cycle control in TNBC cell lines. We will knock-down (KD) Pin1 in TNBC cell lines and assay for changes in Smad3 stability and Smad3-dependent cell cycle control. We will employ a novel transcription factor reporter array to determine the impact of changes in Smad3 activity on cell proliferation and EMT-associated signaling pathways. Aim 2: Investigate the extent to which CDK2-mediated Smad3 phosphorylation impacts Pin1-Smad3 interaction and cell migration and invasion in TNBC cell lines. We will express Smad3 phosphorylation mutants or treat cells with a CDK2 inhibitor and assay for interaction with Pin1 and migration/invasion. Aim 3: Determine the extent to which inhibiting Pin1 and CDK-mediated Smad3 phosphorylation affects cancer metastasis in vivo. We will utilize mCherry-labeled MDA-MB-231 cells with Pin1 KD, transduced with Smad3 phosphorylation site mutants, or treated with CDK2 inhibitors as a xenograft model and monitor metastatic events with fluorescent imaging. Overall, this work will contribute to the growing body of evidence for the use of CDK2 inhibitors as a treatment option for TNBC patients and expand our knowledge of key signaling pathways for novel therapeutic discovery.