Ewa Sicinska

Cyclin E Ablation in the Mouse

E type cyclins (E1 and E2) are believed to drive cell entry into the S phase. It is widely assumed that the two E type cyclins are critically required for proliferation of all cell types. Here, we demonstrate that E type cyclins are largely dispensable for mouse development. However, endoreplication of trophoblast giant cells and megakaryocytes is severely impaired in the absence of cyclin E. Cyclin E-deficient cells proliferate actively under conditions of continuous cell cycling but are unable to reenter the cell cycle from the quiescent G(0) state. Molecular analyses revealed that cells lacking cyclin E fail to normally incorporate MCM proteins into DNA replication origins during G(0)-->S progression. We also found that cyclin E-deficient cells are relatively resistant to oncogenic transformation. These findings define a molecular function for E type cyclins in cell cycle reentry and reveal a differential requirement for cyclin E in normal versus oncogenic proliferation.

Mouse Development and Cell Proliferation in the Absence of D-Cyclins

D-type cyclins (cyclins D1, D2, and D3) are regarded as essential links between cell environment and the core cell cycle machinery. We tested the requirement for D-cyclins in mouse development and in proliferation by generating mice lacking all D-cyclins. We found that these cyclin D1(-/-)D2(-/-)D3(-/-) mice develop until mid/late gestation and die due to heart abnormalities combined with a severe anemia. Our analyses revealed that the D-cyclins are critically required for the expansion of hematopoietic stem cells. In contrast, cyclin D-deficient fibroblasts proliferate nearly normally but show increased requirement for mitogenic stimulation in cell cycle re-entry. We found that the proliferation of cyclin D1(-/-)D2(-/-)D3(-/-) cells is resistant to the inhibition by p16(INK4a), but it critically depends on CDK2. Lastly, we found that cells lacking D-cyclins display reduced susceptibility to the oncogenic transformation. Our results reveal the presence of alternative mechanisms that allow cell cycle progression in a cyclin D-independent fashion.

Cyclins D2 and D1 Are Essential for Postnatal Pancreatic β-Cell Growth

ABSTRACT Regulation of adult β-cell mass in pancreatic islets is essential to preserve sufficient insulin secretion in order to appropriately regulate glucose homeostasis. In many tissues mitogens influence development by stimulating D-type cyclins (D1, D2, or D3) and activating cyclin-dependent kinases (CDK4 or CDK6), which results in progression through the G1 phase of the cell cycle. Here we show that cyclins D2 and D1 are essential for normal postnatal islet growth. In adult murine islets basal cyclin D2 mRNA expression was easily detected, while cyclin D1 was expressed at lower levels and cyclin D3 was nearly undetectable. Prenatal islet development occurred normally in cyclin D2− / − or cyclin D1 +/ − D2 − / − mice. However, β-cell proliferation, adult mass, and glucose tolerance were decreased in adult cyclin D2 − / − mice, causing glucose intolerance that progressed to diabetes by 12 months of age. Although cyclin D1 +/ − mice never developed diabetes, life-threatening diabetes developed in 3-month-old cyclin D1 − /+ D2 − / − mice as β-cell mass decreased after birth. Thus, cyclins D2 and D1 were essential for β-cell expansion in adult mice. Strategies to tightly regulate D-type cyclin activity in β cells could prevent or cure diabetes.

Requirement for cyclin D3 in lymphocyte development and T cell leukemias.

The D-type cyclins (cyclins D1, D2, and D3) are components of the core cell cycle machinery in mammalian cells. Cyclin D3 gene is rearranged and the protein is overexpressed in several human lymphoid malignancies. In order to determine the function of cyclin D3 in development and oncogenesis, we generated and analyzed cyclin D3-deficient mice. We found that cyclin D3(-/-) animals fail to undergo normal expansion of immature T lymphocytes and show greatly reduced susceptibility to T cell malignancies triggered by specific oncogenic pathways. The requirement for cyclin D3 also operates in human malignancies, as knock-down of cyclin D3 inhibited proliferation of acute lymphoblastic leukemias deriving from immature T lymphocytes. These studies point to cyclin D3 as a potential target for therapeutic intervention in specific human malignancies.

The Isopeptidase USP2a Protects Human Prostate Cancer from Apoptosis

Deubiquitinating enzymes can prevent the destruction of protein substrates prior to proteasomal degradation. The ubiquitin-specific protease 2a (USP2a) deubiquitinates the antiapoptotic proteins Fatty Acid Synthase and Mdm2. Here, we show that when USP2a is overexpressed in nontransformed cells, it exhibits oncogenic behavior both in vitro and in vivo and prevents apoptosis induced by chemotherapeutic agents. Notably, USP2a silencing in several human cancer cell lines results in apoptosis. Gene set enrichment analysis, which focuses on groups of genes sharing biological function or regulatory pathways, was done on microarray expression data from human prostate cancers. The cell death-related gene set, as well as a selected cluster of validated p53 target genes, were significantly enriched in the low USP2a expression group of tumors. Conversely, genes implicated in fatty acid metabolism were significantly associated with tumors expressing high USP2a (44%). The expression profile analysis is consistent with the effects of USP2a on its known targets, i.e., Fatty Acid Synthase and Mdm2, defining a subset of prostate tumors resistant to apoptosis. USP2a thus represents a therapeutic target in prostate cancer.

A novel direct activator of AMPK inhibits prostate cancer growth by blocking lipogenesis

5′AMP‐activated kinase (AMPK) constitutes a hub for cellular metabolic and growth control, thus representing an ideal therapeutic target for prostate cancers (PCas) characterized by increased lipogenesis and activation of mTORC1 pathway. However, whether AMPK activation itself is sufficient to block cancer cell growth remains to be determined. A small molecule screening was performed and identified MT 63–78, a specific and potent direct AMPK activator. Here, we show that direct activation of AMPK inhibits PCa cell growth in androgen sensitive and castration resistant PCa (CRPC) models, induces mitotic arrest, and apoptosis. In vivo, AMPK activation is sufficient to reduce PCa growth, whereas the allelic loss of its catalytic subunits fosters PCa development. Importantly, despite mTORC1 blockade, the suppression of de novo lipogenesis is the underpinning mechanism responsible for AMPK‐mediated PCa growth inhibition, suggesting AMPK as a therapeutic target especially for lipogenesis‐driven PCas. Finally, we demonstrate that MT 63–78 enhances the growth inhibitory effect of AR signaling inhibitors MDV3100 and abiraterone. This study thus provides a rationale for their combined use in CRPC treatment.

Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number

Genome-wide association studies (GWASs) have identified a genetic variant of moderate effect size at 6p21.1 associated with erythrocyte traits in humans. We show that this variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining human and mouse primary erythroid cells, we demonstrate that the CCND3 gene product cyclin D3 regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation, thereby controlling erythrocyte size and number. We illustrate how cell type-specific specialization can occur for general cell cycle components-a finding resulting from the biological follow-up of unbiased human genetic studies.

Androgen-Dependent Regulation of Her-2/neu in Prostate Cancer Cells

The mechanisms underlying the progression of prostate cancer to a state of resistance to hormone ablation remain poorly understood. Here, we have investigated the relationship between androgen receptor (AR) and Her-2/neu in prostate cancer cells. Overexpression of Her-2/neu (c-ErbB2) activates the AR pathway and confers a survival and growth advantage to prostate cancer cells in an androgen-deficient milieu. In vitro, the absence of androgens or AR blockade induced Her-2/neu protein expression and phosphorylation. In contrast, upon readministration of androgens, Her-2/neu mRNA, protein, and phosphorylation levels decreased linearly with increasing concentrations of dihydrotestosterone as LNCaP cells reentered the cell cycle. In vivo, induction of Her-2/neu by castration in orthotopically injected LNCaP cells resulted in a progressive increase in prostate-specific antigen secretion into the mouse serum, indicating that Her-2/neu-mediated, AR-dependent transcription occurs following castration and results in tumor cell growth. Finally, selection of LNCaP cells stably transfected with short hairpin RNA specific for AR resulted in Her-2/neu overexpression. Similarly, knockdown of Her-2/neu led to induction of AR. However, when Her-2/neu and AR were simultaneously targeted, we observed cell death, whereas surviving cells retained low level expression of Her-2/neu. Thus, induction and activation of Her-2/neu occurs in an androgen-depleted environment or as a result of AR inactivation, promoting ablation-resistant survival of prostate cancer cells. These data provide the biochemical rationale to target Her-2/neu in hormone-refractory prostate cancer.

Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer

Bacteria go the distance in cancer The bacterial species Fusobacterium nucleatum is associated with a subset of human colorectal cancers, but its role in tumorigenesis is unclear. Studying patient samples, Bullman et al. found that F. nucleatum and certain co-occurring bacteria were present not only in primary tumors but also in distant metastases. Preliminary evidence suggests that the bacterium is localized primarily within the metastatic cancer cells rather than in the stroma. Antibiotic treatment of mice carrying xenografts of F. nucleatum–positive human colorectal cancer slowed tumor growth, consistent with a causal role for the bacterium in tumorigenesis. Science, this issue p. 1443 The same bacteria present in primary tumors of patients with colorectal cancer are also present in liver metastases. Colorectal cancers comprise a complex mixture of malignant cells, nontransformed cells, and microorganisms. Fusobacterium nucleatum is among the most prevalent bacterial species in colorectal cancer tissues. Here we show that colonization of human colorectal cancers with Fusobacterium and its associated microbiome—including Bacteroides, Selenomonas, and Prevotella species—is maintained in distal metastases, demonstrating microbiome stability between paired primary and metastatic tumors. In situ hybridization analysis revealed that Fusobacterium is predominantly associated with cancer cells in the metastatic lesions. Mouse xenografts of human primary colorectal adenocarcinomas were found to retain viable Fusobacterium and its associated microbiome through successive passages. Treatment of mice bearing a colon cancer xenograft with the antibiotic metronidazole reduced Fusobacterium load, cancer cell proliferation, and overall tumor growth. These observations argue for further investigation of antimicrobial interventions as a potential treatment for patients with Fusobacterium-associated colorectal cancer.

Antiproliferative Effects of CDK4/6 Inhibition in CDK4-Amplified Human Liposarcoma In Vitro and In Vivo

Well-differentiated/dedifferentiated liposarcomas (WD/DDLPS) are among the most common subtypes of soft tissue sarcomas. Conventional systemic chemotherapy has limited efficacy and novel therapeutic strategies are needed to achieve better outcomes for patients. The cyclin-dependent kinase 4 (CDK4) gene is highly amplified in more than 95% of WD/DDLPS. In this study, we explored the role of CDK4 and the effects of NVP-LEE011 (LEE011), a novel selective inhibitor of CDK4/CDK6, on a panel of human liposarcoma cell lines and primary tumor xenografts. We found that both CDK4 knockdown by siRNA and inhibition by LEE011 diminished retinoblastoma (RB) phosphorylation and dramatically decreased liposarcoma cell growth. Cell-cycle analysis demonstrated arrest at G0–G1. siRNA-mediated knockdown of RB rescued the inhibitory effects of LEE011, demonstrating that LEE011 decreased proliferation through RB. Oral administration of LEE011 to mice bearing human liposarcoma xenografts resulted in approximately 50% reduction in tumor 18F-fluorodeoxyglucose uptake with decreased tumor biomarkers, including RB phosphorylation and bromodeoxyuridine incorporation in vivo. Continued treatment inhibited tumor growth or induced regression without detrimental effects on mouse weight. After prolonged continuous dosing, reestablishment of RB phosphorylation and cell-cycle progression was noted. These findings validate the critical role of CDK4 in maintaining liposarcoma proliferation through its ability to inactivate RB function, and suggest its potential function in the regulation of survival and metabolism of liposarcoma, supporting the rationale for clinical development of LEE011 for the treatment of WD/DDLPS. Mol Cancer Ther; 13(9); 2184–93. ©2014 AACR.