Abstract
Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2–associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E–expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E–expressing cells by inducing G1/S cell cycle arrest. LMW-E requires CDK2–associated kinase activity to induce mammary tumor formation by disrupting acinar development. The b-Raf-ERK1/2-mTOR signaling pathway is aberrantly activated in breast cancer and can be suppressed by combination treatment with roscovitine plus either rapamycin or sorafenib.
Highlights
Cyclin E has been extensively implicated in breast cancer [1,2,3,4,5,6,7]
Using cell lines cultured on plastic and extracellular matrix (Matrigel) and comparing their proteomic profiles to breast cancer tumor samples, we demonstrated that overexpression of low molecular weight cyclin E (LMW-E) is concomitant with activation of the b-Raf-ERK1/2-mTOR pathway
We show that induction of low molecular weight (LMW)-E is sufficient to induce mammary tumor development in vivo
Summary
Cyclin E has been extensively implicated in breast cancer [1,2,3,4,5,6,7]. The function of cyclin E is modulated via association of cyclin E with CDK2, which promotes progression of cells into S phase [8,9,10]. Compared to EL, the LMW-E isoforms have higher CDK2-associated kinase activity, are more resistant to inhibition by CDK inhibitors p21 and p27, and induce higher proliferation rates when introduced into cells [14,15]. Examination of breast cancer patient samples revealed that approximately 27% of patients express high LMW-E protein levels as assessed by Western blot analysis, and high LMW-E expression significantly correlates with poor survival [16]
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