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Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells.

ABSTRACT: Breast cancer onset and disease progression have been linked to members of the TGF? superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGF? in malignancy, mediating both tumor suppressant and pro-metastatic behaviors. Overexpression of cell cycle regulators, cyclins D and E, renders cyclin-dependent kinases (CDKs) 4/2 hyperactive. Noncanonical phosphorylation of Smad3 by CDK4/2 inhibits tumor suppressant actions of Smad3. We hypothesized that CDK inhibition (CDKi) would restore Smad3 action and help promote cancer cell regression. Treatment of triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-436, Hs578T) with CDK2i or CDK4i resulted in increased Smad3 activity and decreased cell migration. Transfection with a 5M Smad3 construct containing inhibitory mutations in 5 CDK phosphorylation sites also resulted in decreased TNBC cell migration and invasion. MDA-MB-231 cells treated with CDK2i or CDK4i resulted in decreased Smad3 protein phosphorylation at the CDK phosphorylation T179 site, decreased MMP2 and c-myc expression, and increased p15 and p21 expression. Using a novel transfected cell array, we found that CDK2i treatment decreased activity of the epithelial-to-mesenchymal transition related transcription factors Snail and Twist. In vivo studies in an MDA-MB-231 tumor model showed that individual and combination treatment with paclitaxel and CDK2i resulted in decreased tumor volume and Ki67 staining. Collectively, these data support further investigation of targeted CDK inhibitors as a promising therapeutic strategy for TNBC, a breast cancer subtype with limited treatment options.

SUBMITTER: Tarasewicz E

PROVIDER: S-EPMC4614520 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells.

Tarasewicz Elizabeth E Rivas Lisbi L Hamdan Randala R Dokic Danijela D Parimi Vamsi V Bernabe Beatriz Penalver BP Thomas Alexandra A Shea Lonnie D LD Jeruss Jacqueline S JS

Cell cycle (Georgetown, Tex.) 20140101 20

Breast cancer onset and disease progression have been linked to members of the TGFβ superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFβ in malignancy, mediating both tumor suppressant and pro-metastatic behaviors. Overexpression of cell cycle regulators, cyclins D and E, renders cyclin-dependent kinases (CDKs) 4/2 hyperactive. Noncanonical phosphorylation of Smad3 by CDK4/2 inhibits tumor suppressant ac ...[more]

PMID: 25485498

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Project description:Simple Summary The cyclin E/CDK2 complex may present a promising target axis for the treatment of triple-negative breast cancers (TNBC); however, therapeutically relevant doses of CDK2 inhibitors have been associated with toxicities. Here, we report that the suboptimal dosing of the CDK 2, 7 and 9 inhibitor SNS-032 reduced the viability of TNBC cells and upregulated the checkpoint ligand PD-L1 expression in surviving cancer cells in vitro and in human orthotopic MDA-MB-231 TNBC xenografts grown in immunodeficient mice. Moreover, in immunodeficient, TNBC xenograft-bearing mice engrafted with human immune cells, SNS-032 treatment was associated with the infiltration of CD45+ human immune cells in tumors. In these orthotopic MDA-MB-231 TNBC-bearing mice, suboptimal SNS-032 doses given sequentially ahead of dosing with the anti-PD-L1 antibody avelumab significantly restricted tumor growth compared with monotherapy. These findings suggest that surviving cancer cells following suboptimal CDK inhibitor treatment may be responsive to checkpoint immunotherapy. Abstract Triple-negative breast cancers (TNBC) expressing PD-L1 qualify for checkpoint inhibitor immunotherapy. Cyclin E/CDK2 is a potential target axis in TNBC; however, small-molecule drugs at efficacious doses may be associated with toxicity, and treatment alongside immunotherapy requires investigation. We evaluated CDK inhibition at suboptimal levels and its anti-tumor and immunomodulatory effects. Transcriptomic analyses of primary breast cancers confirmed higher cyclin E/CDK2 expression in TNBC compared with non-TNBC. Out of the three CDK2-targeting inhibitors tested, the CDK 2, 7 and 9 inhibitor SNS-032 was the most potent in reducing TNBC cell viability and exerted cytotoxicity against all eight TNBC cell lines evaluated in vitro. Suboptimal SNS-032 dosing elevated cell surface PD-L1 expression in surviving TNBC cells. In mice engrafted with human immune cells and challenged with human MDA-MB-231 TNBC xenografts in mammary fat pads, suboptimal SNS-032 dosing partially restricted tumor growth, enhanced the tumor infiltration of human CD45+ immune cells and elevated cell surface PD-L1 expression in surviving cancer cells. In tumor-bearing mice engrafted with human immune cells, the anti-PD-L1 antibody avelumab, given sequentially following suboptimal SNS-032 dosing, reduced tumor growth compared with SNS-032 alone or with avelumab without prior SNS-032 priming. CDK inhibition at suboptimal doses promotes immune cell recruitment to tumors, PD-L1 expression by surviving TNBC cells and may complement immunotherapy.

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Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells.

Publications

Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells.

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