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

Abstract

Keywords: CDK, cyclin, paclitaxel, Smad3, triple negative breast cancer Abbreviations: BCSC, breast cancer stem cells; 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; Pin1, peptidyL-prolyl cis-trans isomerase NIMA-interacting 1; PR, progesterone receptor; TNBC, triple negative breast cancerBreast cancer onset and disease progression have been linked to members of the TGFb superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFb 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.

Influence of gap junction intercellular communication composed of connexin 43 on the antineoplastic effect of adriamycin in breast cancer cells

“…Multiples studies have revealed that, in breast cancer patients, the expression of connexin proteins is correlated with clinicopathological biomarkers, including estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), which are normally used to predict the response of mammary cancer patients to therapy (33,53). The Hs578T cell line used in the current study is a type of triple-negative breast cancer (TNBC), while the MCF-7 cell line is non-TNBC (ER+ and PR+) (54,55). ER and PR status have traditionally been used to select patients suitable for tamoxifen treatment (56).…”

The Expression and Clinical Outcome of pCHK2-Thr68 and pCDC25C-Ser216 in Breast Cancer

“…In addition, we observed a higher expression of pCHK2-Thr68 in TNBC tissues than non-TNBC tissues. Greater activation of the cell cycle checkpoint protein (e.g., CDK) may be associated with the increased activity of TNBC stem cells [25,26] as accumulated p53 tumor suppressor gene and BRCA1 gene mutations may trigger ATM/ATR–CHK1/2–CDC25C pathways [27,28]. Such increases in pCHK2-Thr68 also occur in other tumors such as mucinous adenocarcinomas and colorectal cancer [29,30,31].…”

Phase II neoadjuvant clinical trial of carboplatin and eribulin in women with triple negative early-stage breast cancer (NCT01372579)

“…Overexpression of cyclins, and the consequent impact on CDK activity, induces inhibitory noncanonical phosphorylation of Smad3. Collectively, these events result in the blockade of canonical, Smad3-mediated tumor suppression, thereby contributing to breast cancer progression and metastasis [19,21,22]. In pre-clinical studies, pharmacologic CDK inhibition has restored the canonical tumor suppressant actions of Smad3 signaling.…”

“…Additional predictive markers and novel therapeutics directed toward cyclins/CDKs and TGFb signaling are also in active development [19]. Smad3 is a transcriptional regulator that canonically functions through phosphorylation at the C-terminus to facilitate tumor suppression [20].…”