LMW-E MEDIATES MAMMARY TUMORIGENESIS BY DEREGULATING ACINAR MORPHOGENESIS & GENERATING CANCER STEM CELLS

Cyclin E is the regulatory subunit of the cyclin E/CDK2 complex that mediates the G1-S phase transition. N-terminal cleavage of cyclin E by elastase in breast cancer generates two low molecular weight (LMW) isoforms that exhibit both enhanced kinase activity and resistance to p21 and p27 inhibition compared to fulllength cyclin E. Clinically, approximately 27% of breast cancer patients overexpress LMW-E and associate with poor survival. Therefore, we hypothesize that LMW-E disrupts normal mammary acinar morphogenesis and serves as the initial route into breast tumor development. We first demonstrate that LMW-E overexpression in non-tumorigenic hMECs is sufficient to induce tumor formation in athymic mice significantly more than overexpression of full-length cyclin E and requires CDK2- associated kinase activity. Further in vivo passaging of these tumors augments LMW-E expression and tumorigenic potential. When subjected to acinar morphogenesis in vitro, LMW-E mediates significant morphological disruption by generating hyperproliferative and multi-acinar complexes. Proteomic analysis of patient tissues and tumor cells with high LMW-E expression reveals that the activation of the b-Raf-ERK1/2-mTOR pathway in concert with high LMW-E expression predicts poor patient survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (b-raf inhibitor) effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing the G1/S cell cycle arrest. In addition, the LMW-E-expressing tumor cells exhibit phenotypes characteristic of the EMT and enhanced cellular invasiveness. These tumor cells also enrich for cells with CSC phenotypes such as increased CD44hi/CD24lo population, enhanced mammosphere formation, and upregulation of ALDH expression and enzymatic activity. Furthermore, the CD44hi/CD24lo population also shows positive correlation with LMW-E expression in both the tumor cell line model and breast cancer patient samples (p\u3c0.0001 & p=0.0435, respectively). Combination treatment using doxorubicin and salinomycin demonstrates synergistic cytotoxic effects in cells with LMW-E expression but not in those with full-length cyclin E expression. Finally, ProtoArray microarray identifies Hbo1 as a novel substrate of the cyclin E/CDK2 complex and its overexpression results in enrichment for CSCs. Collectively, these data emphasize the strong oncogenic potential of LMW-E in mammary tumorigenesis and suggest possible therapeutic strategies to treat breast cancer patients with high LMW-E expression

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

RAP1: Protector of Telomeres, Defender against Obesity

Telomere dysfunction has previously been linked to metabolic disorders. In this issue of Cell Reports, Martínez et al. (2013) and Yeung et al. (2013) now extend this link, demonstrating that deletion of the telomere binding protein RAP1 leads to obesity and insulin resistance

Low-Molecular-Weight Cyclin E in Human Cancer: Cellular Consequences and Opportunities for Targeted Therapies

Cyclin E, a regulatory subunit of cyclin-dependent kinase 2 (CDK2), is central to the initiation of DNA replication at the G1/S checkpoint. Tight temporal control of cyclin E is essential to the coordination of cell-cycle processes and the maintenance of genome integrity. Overexpression of cyclin E in human tumors was first observed in the 1990s and led to the identification of oncogenic roles for deregulated cyclin E in experimental models. A decade later, low-molecular-weight cyclin E (LMW-E) isoforms were observed in aggressive tumor subtypes. Compared with full-length cyclin E, LMW-E hyperactivates CDK2 through increased complex stability and resistance to the endogenous inhibitors p21CIP1 and p27KIP1 LMW-E is predominantly generated by neutrophil elastase-mediated proteolytic cleavage, which eliminates the N-terminal cyclin E nuclear localization signal and promotes cyclin E's accumulation in the cytoplasm. Compared with full-length cyclin E, the aberrant localization and unique stereochemistry of LMW-E dramatically alters the substrate specificity and selectivity of CDK2, increasing tumorigenicity in experimental models. Cytoplasmic LMW-E, which can be assessed by IHC, is prognostic of poor survival and predicts resistance to standard therapies in patients with cancer. These patients may benefit from therapeutic modalities targeting the altered biochemistry of LMW-E or its associated vulnerabilities. Cancer Res; 78(19); 5481-91. ©2018 AACR

LMW-E renders hMECs tumorigenic, and LMW-E expression is selected with increased <i>in vivo</i> passaging.

<p>(A) Schematic of the generation of <i>in vivo</i> passaged clones with 3 successive injections (T1G2, T1G3, and T1G4). (B) Tumors from <i>in vivo</i> passaging were removed from mice, minced, and cultured on monolayer plates. Lysates were extracted and subjected to Western blot analysis with antibodies against cyclin E, elafin, and β-actin. EL (C), LMW-E (D), and elafin (E) protein levels were quantified by densitometry and compared between different generations of <i>in vivo</i> passaged cells (Student <i>t</i> test, *p<0.05). (F) Paraffin-embedded slides of 4 representative tumors were stained with hematoxylin and eosin (top panel) and cyclin E antibody (bottom panel).</p

Combination drug treatment prevents induction of aberrant acinar development by LMW-E.

<p>(A) Cells were seeded on Matrigel for 24 hours and then treated with rapamycin, sorafenib, and roscovitine as indicated. Medium containing drugs was replaced every 4 days, and lysates were collected on day 15 for Western blot analysis with the indicated antibodies. (B) On day 15 of Matrigel culture, cells grown as in (A) were fixed and stained with E-cadherin (red) and Ki67 (green), and nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. (C) The diameters of the acini were measured and averaged from three independent experiments. Error bars = SEM (Student <i>t</i> test, *p<0.05). (D) The number of Ki67-positive cells per acinus was counted and averaged from three independent experiments. Error bars = SEM (Student <i>t</i> test, *p<0.05). Kaplan-Meier survival plots demonstrating association between full length and high LMW-E on disease-specific survival. Association of:</p

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

LMW-E is tumorigenic.

<p>Athymic mice were injected subcutaneously with 1×10⁷ 76NE6 cells stably transfected with empty vector, EL, LMW-E and MDA-MB-468 cells. After 10 weeks, the tumors were removed for expansion in culture for further <i>in vivo</i> passaging and also for IHC analysis. Tumor incidence rate was estimated with exact 95% confidence intervals and Fisher's exact tests were used to compare tumor incidence rate between/among groups (*p<0.0001: 76NE6-vector vs. 76NE6-LMW-E; 76NE6-EL vs. 76NE6-LMW-E; 76NE6-vector vs. TDCs; 76NE6-EL vs. TDCs).</p