LMW-E/CDK2 Deregulates Acinar Morphogenesis, Induces Tumorigenesis, and Associates with the Activated b-Raf-ERK1/2-mTOR Pathway in Breast Cancer Patients

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

Low-molecular-weight cyclin E: the missing link between biology and clinical outcome

Cyclin E, a key mediator of transition during the G(1)/S cellular division phase, is deregulated in a wide variety of human cancers. Our group recently reported that overexpression and generation of low-molecular-weight (LMW) isoforms of cyclin E were associated with poor clinical outcome among breast cancer patients. However, the link between LMW cyclin E biology in mediating a tumorigenic phenotype and clinical outcome is unknown. To address this gap in knowledge, we assessed the role of LMW isoforms in breast cancer cells; we found that these forms of cyclin E induced genomic instability and resistance to p21, p27, and antiestrogens in breast cancer. These findings suggest that high levels of LMW isoforms of cyclin E not only can predict failure to endocrine therapy but also are true prognostic indicators because of their influence on cell proliferation and genetic instability

High LMW-E expression is associated with activated b-Raf-ERK1/2-mTOR pathway <i>in vitro</i> and in patient tissues.

<p>(A) Hierarchal cluster analysis of protein expression in 76NE6, 76NE6-LMW-E and all of the LMW-E-expressing tumor clones grown on 2D (red) and 3D (green) cultures and 276 breast cancer patient samples (blue). (B) Proteins whose expression was associated with high LMW-E levels. Red indicates that high LMW-E along with high protein expression was associated with poor prognosis; grey indicates that high LMW-E along with low protein expression was associated with poor prognosis. (C) The lysates from 3D culture were subjected to Western blot analysis to validate the RPPA data. The cell lines are 76NE6-parental (P) and with stable expression of vector (V), EL, and LMW-E and the tumor-derived cells (TDCs). (D) Linear regression analysis of the RPPA data and the densitometry values of all the proteins analyzed by Western blot analysis in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002538#pgen-1002538-g005" target="_blank">Figure 5C</a>. The same antibodies were used for the two types of analysis.</p

LMW-E induces formation of large and highly proliferative acini.

<p>(A) 76NE6, MCF-10A, HS 578T, and MDA-MB-231 cells were seeded at a density of 70 cells/mm² on 1-mm-thick Matrigel. After 15 days in 3D culture, cells were fixed and immunostained with GM-130 and α6-integrin antibodies. Nuclei were counterstained with DAPI. Scale bar = 50 µm. (B) Lysates from these cells were isolated at days 0 (d0) and 15 (d15) of acinar morphogenesis and subjected to Western blot analysis with the indicated antibodies. (C and D) 76NE6 cells stably transfected with vector, EL, and LMW-E and tumor-derived cells (TDCs) were subjected to analysis similar to that in (A) (V = vector). Scale bar = 50 µm. The diameters of the acini were measured and averaged from 3 independent experiments. Values underneath each figure represents mean (µm) ± SEM. Error bars = SEM (Student <i>t</i> test, *p<0.05). (E) Lysates from these cells were isolated at day 15 and subjected to Western blot analysis with the indicated antibodies. <i>In vitro</i> kinase assay was performed by immunoprecipitation of lysates from 3D culture using polyclonal cyclin E antibody and incubation with (γP32)ATP and GST-Rb. (F and G) Cells cultured on Matrigel for 15 days were fixed and immunostained with E-cadherin and Ki67 antibodies. Nuclei were counterstained with DAPI. Scale bar = 50 µm. The number of Ki67-positive cells per acinus was counted and averaged from 3 independent experiments. Error bars = SEM (Student <i>t</i> test, *p<0.05). (H) Linear regression of the correlation between acinar diameter and percentage of Ki67-positive cells.</p

CDK2-associated kinase activity is required for LMW-E-mediated tumorigenesis and deregulation of acinar morphogenesis.

<p>(A) 76NE6-TetR cells were cultured with or without doxycycline induction, and the lysates were extracted and subjected to Western blot analysis with antibodies against cyclin E, CDK2, and β-actin. <i>In vitro</i> kinase assay was performed by immunoprecipitation with FLAG antibody and histone H1 and GST-Rb were added as substrates. Doxycycline was administered to achieve approximately 1× and 2× cyclin E protein levels. (The doxycycline concentrations for the 76NE6-TetR-V and wild-type EL and LMW-E cells were 0, 0.2, and 0.4 ng/ml, and the doxycycline concentrations for the EL^R130A and LMW-E^R130A cells were 0, 1, and 2 ng/ml.) (B) 76NE6-TetR cells were cultured on Matrigel for 15 days with or without doxycycline induction. Bright-field images were taken at day 15. Values underneath each figure represent mean diameter (µm) ± SEM. (C) The diameters of at least 100 acini from 3 different experiments were measured. Error bars = SEM (Wilcoxon rank-sum test, *p<0.05). (D) Multi-acinar complexes were counted. Error bars = SEM (Wilcoxon rank-sum test, *p<0.05). Multi-acinar complexes were defined as complexes with more than 2 acini growing on top of each other. Logistic regression models were used to compare the rate of formation of multi-acinar complexes between/among groups (*p<0.05).</p