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 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 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.

Keywords: BCSC, breast cancer stem cells; CDK; CDK, cyclin dependent kinase; CDKi, cyclin dependent kinase inhibitor; CK, cytokeratin; EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PR, progesterone receptor; Pin1, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1; Smad3; TNBC, triple negative breast cancer; cyclin; paclitaxel; triple negative breast cancer.

Figure 1.

TNBC cells show increased Smad3 activity and decreased cell migration in response to CDKi treatment. (A) Immunoblotting confirmed expression of study proteins in TNBC cells. *P < 0.05 for indicated cell lines (B) Smad3 reporter activity was measured in study cells after increasing doses of CDK2i and CDK4i. Study cells were (C) treated with control DMSO (NT), CDK2i or CDK4i or (D) transfected with Vec, WT or 5M Smad3 and cell migration was assessed. (E) Representative images of Vec and 5M transfected cells are shown at 10× magnification.

Figure 2.

MDA-MB-231 cells expressing 5M Smad3 show decreased cell invasion and CDK inhibition results in decreased MMP2 expression, decreased CDK-mediated phosphorylation of Smad3 at the T179 site, and restoration of proteins related to cell cycle arrest. (A) MDA-MB-231 cells were transfected with Vec, WT, or 5M Smad3 and invasion was assessed (B and C) MDA-MB-231 cells were treated with control DMSO (NT), CDK2i, or CDK4i and immunoblotting was performed to assess expression of study proteins.

Figure 3.

Paclitaxel/CDK2i induced apoptosis in vitro and decreased tumor volume and Ki67 staining in vivo in a MDA-MB-231 xenograft model. (A) MDA-MB-231 cells were treated and analyzed for apoptosis using TUNEL staining. (B) Female athymic nu/nu mice were inoculated with MDA-MB-231 cells to establish tumors, and treated for 21 d Tumor volume was measured at indicated time points. The tumor volumes from treated groups were compared with the volume from the respective control treated group at Day 21, and significance was then determined; *P < 0.05, **P < 0.005. (C) Representative images from Ki67 stained xenografts from each treatment group at 20X magnification.

Figure 4.

CDK2i treatment of MDA-MB-231 alters activity of transcription factors associated with oncogenic processes. MDA-MB-231 cells were transduced with TF reporters and treated with CDK2i. Bioluminescence was normalized to TA control at (A) 1, 2, and 4 hours (early timepoints) and (B) at 1, 3, and 6 d (late timepoints).

Figure 5.

Proposed model for impact of CDK2/4 on dichotomous TGFβ/Smad3 signaling in breast cancer. Canonical Smad3 signaling, where phosphorylation occurs at the C-terminus, results in tumor suppressive events, including cell cycle arrest and apoptosis. Cyclin overexpression in breast cancer cells leads to hyperactive CDK activity that results in CDK-mediated noncanonical phosphorylation of Smad3 in the linker region and downstream oncogenic events such as cell proliferation and migration/invasion. Inhibiting CDK activity through use of pharmacological CDK inhibitors may prevent noncanonical phosphorylation of Smad3 and favor tumor suppressive Smad signaling.