Cdk4 and Cdk6 Couple the Cell-Cycle Machinery to Cell Growth via mTORC1.

Cell growth is coupled to cell-cycle progression in mitotically proliferating mammalian cells, but the underlying molecular mechanisms are not well understood. CyclinD-Cdk4/6 is known to phosphorylate RB to promote S-phase entry, but recent work suggests they have additional functions. We show here that CyclinD-Cdk4/6 activates mTORC1 by binding and phosphorylating TSC2 on Ser1217 and Ser1452. Pharmacological inhibition of Cdk4/6 leads to a rapid, TSC2-dependent reduction of mTORC1 activity in multiple human and mouse cell lines, including breast cancer cells. By simultaneously driving mTORC1 and E2F, CyclinD-Cdk4/6 couples cell growth to cell-cycle progression. Consistent with this, we see that mTORC1 activity is cell cycle dependent in proliferating neural stem cells of the adult rodent brain. We find that Cdk4/6 inhibition reduces cell proliferation partly via TSC2 and mTORC1. This is of clinical relevance, because Cdk4/6 inhibitors are used for breast cancer therapy.

Cyclin D-CDK4 relieves cooperative repression of proliferation and cell cycle gene expression by DREAM and RB.

The retinoblastoma protein (RB) restricts cell cycle gene expression and entry into the cell cycle. The RB-related protein p130 forms the DREAM (DP, RB-like, E2F, and MuvB) complex and contributes to repression of cell cycle-dependent genes during quiescence. Although both RB and DREAM bind and repress an overlapping set of E2F-dependent gene promoters, it remains unclear whether they cooperate to restrict cell cycle entry. To test the specific contributions of RB and DREAM, we generated RB and p130 knockout cells in primary human fibroblasts. Knockout of both p130 and RB yielded higher levels of cell cycle gene expression in G0 and G1 cells compared to cells with knockout of RB alone, indicating a role for DREAM and RB in repression of cell cycle genes. We observed that RB had a dominant role in E2F-dependent gene repression during mid to late G1 while DREAM activity was more prominent during G0 and early G1. Cyclin D-Cyclin-Dependent Kinase 4 (CDK4)-dependent phosphorylation of p130 occurred during early G1, and led to the release of p130 and MuvB from E2F4 and decreased p130 and MuvB binding to cell cycle promoters. Specific inhibition of CDK4 activity by palbociclib blocked DREAM complex disassembly during cell cycle entry. In addition, sensitivity to CDK4 inhibition was dependent on RB and an intact DREAM complex in both normal cells as well as in palbociclib-sensitive cancer cell lines. Although RB knockout cells were partially resistant to CDK4 inhibition, RB and p130 double knockout cells were significantly more resistant to palbociclib treatment. These results indicate that DREAM cooperates with RB in repressing E2F-dependent gene expression and cell cycle entry and supports a role for DREAM as a therapeutic target in cancer.

Porcine epidemic diarrhea virus M protein blocks cell cycle progression at S-phase and its subcellular localization in the porcine intestinal epithelial cells.

Porcine epidemic diarrhea (PED) caused by virulent strains of porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric disease of swine characterized by severe enteritis, vomiting, and watery diarrhea. This study investigates the subcellular localization and function of PEDV M protein through examination of its effects on cell growth, cell cycle progression, and interleukin 8 (IL-8) expression. Our results revealed that the PEDV M protein is localized throughout the cytoplasm. The M protein altered swine intestinal epithelial cell line (IEC) growth and induced cell cycle arrest at the S-phase via the cyclin A pathway. The S-phase arrest is associated with a decrease in level of cyclin A. Furthermore, our results revealed that the M protein of PEDV does not induce endoplasmic reticulum (ER) stress and does not activate NF-κB which is responsible for IL-8 and Bcl-2 expression. This is the first report to demonstrate that the PEDV M protein is localized in the whole cell and induces cell cycle arrest at the S-phase. This study provides novel findings in the function of M proteins of PEDV.

A mathematical model of HiF-1α-mediated response to hypoxia on the G1/S transition.

Hypoxia is known to influence the cell cycle by increasing the G1 phase duration or by inducing a quiescent state (arrest of cell proliferation). This entry into quiescence is a mean for the cell to escape from hypoxia-induced apoptosis. It is suggested that some cancer cells have gain the advantage over normal cells to easily enter into quiescence when environmental conditions, such as oxygen pressure, are unfavorable [43,1]. This ability contributes in the appearance of highly resistant and aggressive tumor phenotypes [2]. The HiF-1α factor is the key actor of the intracellular hypoxia pathway. As tumor cells undergo chronic hypoxic conditions, HiF-1α is present in higher level in cancer than in normal cells. Besides, it was shown that genetic mutations promoting overstabilization of HiF-1α are a feature of various types of cancers [7]. Finally, it is suggested that the intracellular level of HiF-1α can be related to the aggressiveness of the tumors [53,24,4,10]. However, up to now, mathematical models describing the G1/S transition under hypoxia, did not take into account the HiF-1α factor in the hypoxia pathway. Therefore, we propose a mathematical model of the G1/S transition under hypoxia, which explicitly integrates the HiF-1α pathway. The model reproduces the slowing down of G1 phase under moderate hypoxia, and the entry into quiescence of proliferating cells under severe hypoxia. We show how the inhibition of cyclin D by HiF-1α can induce quiescence; this result provides a theoretical explanation to the experimental observations of Wen et al. (2010) [50]. Thus, our model confirms that hypoxia-induced chemoresistance can be linked, for a part, to the negative regulation of cyclin D by HiF-1α.

[Cyclins D in regulation and dysregulation of the cell cycle in multiple myeloma]. / Cykliny D v regulaci a dysregulaci bunecného cyklu u mnohocetného myelomu.

Multiple myeloma is the second most common hematooncological disease characterized by clonal proliferation of plasma cells and monoclonal immunoglobulin production. It is a heterogenous disease; however, dysregulation of cyclins D seems to be an early unifying pathogenic event in multiple myeloma. In almost all patients, there is increased expression level of at least one of the cyclins D. Nevertheless, the mechanism of this increase is unknown in many cases. Next to wellknown roles of cyclins D in the cell cycle, they have many other functions contributing to tumor cell progression. Cyclins D are prognostic markers and are also used for subclassification of multiple myeloma. In this review, we focus on significance of cyclins D in multiple myeloma.

The requirement for cyclin D function in tumor maintenance.

D-cyclins represent components of cell cycle machinery. To test the efficacy of targeting D-cyclins in cancer treatment, we engineered mouse strains that allow acute and global ablation of individual D-cyclins in a living animal. Ubiquitous shutdown of cyclin D1 or inhibition of cyclin D-associated kinase activity in mice bearing ErbB2-driven mammary carcinomas triggered tumor cell senescence, without compromising the animals' health. Ablation of cyclin D3 in mice bearing Notch1-driven T cell acute lymphoblastic leukemias (T-ALL) triggered tumor cell apoptosis. Such selective killing of leukemic cells can also be achieved by inhibiting cyclin D associated kinase activity in mouse and human T-ALL models. Inhibition of cyclin D-kinase activity represents a highly-selective anticancer strategy that specifically targets cancer cells without significantly affecting normal tissues.

A two-dimensional ERK-AKT signaling code for an NGF-triggered cell-fate decision.

Growth factors activate Ras, PI3K, and other signaling pathways. It is not well understood how these signals are translated by individual cells into a decision to proliferate or differentiate. Here, using single-cell image analysis of nerve growth factor (NGF)-stimulated PC12 cells, we identified a two-dimensional phospho-ERK (pERK)-phospho-AKT (pAKT) response map with a curved boundary that separates differentiating from proliferating cells. The boundary position remained invariant when different stimuli were used or upstream signaling components perturbed. We further identified Rasa2 as a negative feedback regulator that links PI3K to Ras, placing the stochastically distributed pERK-pAKT signals close to the decision boundary. This allows for uniform NGF stimuli to create a subpopulation of cells that differentiates with each cycle of proliferation. Thus, by linking a complex signaling system to a simpler intermediate response map, cells gain unique integration and control capabilities to balance cell number expansion with differentiation.

Cyclin D as a therapeutic target in cancer.

Cyclin D1, and to a lesser extent the other D-type cyclins, is frequently deregulated in cancer and is a biomarker of cancer phenotype and disease progression. The ability of these cyclins to activate the cyclin-dependent kinases (CDKs) CDK4 and CDK6 is the most extensively documented mechanism for their oncogenic actions and provides an attractive therapeutic target. Is this an effective means of targeting the cyclin D oncogenes, and how might the patient subgroups that are most likely to benefit be identified?

Dynamic modelling of oestrogen signalling and cell fate in breast cancer cells.

Cancers of the breast and other tissues arise from aberrant decision-making by cells regarding their survival or death, proliferation or quiescence, damage repair or bypass. These decisions are made by molecular signalling networks that process information from outside and from within the breast cancer cell and initiate responses that determine the cell's survival and reproduction. Because the molecular logic of these circuits is difficult to comprehend by intuitive reasoning alone, we present some preliminary mathematical models of the basic decision circuits in breast cancer cells that may aid our understanding of their susceptibility or resistance to endocrine therapy.

Induction of human beta-cell proliferation and engraftment using a single G1/S regulatory molecule, cdk6.

OBJECTIVE: Most knowledge on human beta-cell cycle control derives from immunoblots of whole human islets, mixtures of beta-cells and non-beta-cells. We explored the presence, subcellular localization, and function of five early G1/S phase molecules-cyclins D1-3 and cdk 4 and 6-in the adult human beta-cell. RESEARCH DESIGN AND METHODS: Immunocytochemistry for the five molecules and their relative abilities to drive human beta-cell replication were examined. Human beta-cell replication, cell death, and islet function in vivo were studied in the diabetic NOD-SCID mouse. RESULTS: Human beta-cells contain easily detectable cdks 4 and 6 and cyclin D3 but variable cyclin D1. Cyclin D2 was only marginally detectable. All five were principally cytoplasmic, not nuclear. Overexpression of the five, alone or in combination, led to variable increases in human beta-cell replication, with the cdk6/cyclin D3 combination being the most robust (15% versus 0.3% in control beta-cells). A single molecule, cdk6, proved to be capable of driving human beta-cell replication in vitro and enhancing human islet engraftment/proliferation in vivo, superior to normal islets and as effectively as the combination of cdk6 plus a D-cyclin. CONCLUSIONS: Human beta-cells contain abundant cdk4, cdk6, and cyclin D3, but variable amounts of cyclin D1. In contrast to rodent beta-cells, they contain little or no detectable cyclin D2. They are primarily cytoplasmic and likely ineffective in basal beta-cell replication. Unexpectedly, cyclin D3 and cdk6 overexpression drives human beta-cell replication most effectively. Most importantly, a single molecule, cdk6, supports robust human beta-cell proliferation and function in vivo.