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MicroRNAs (miRs) are a large class of small regulatory RNAs that function as nodes of signaling networks. This implicates that miRs expression has to be finely tuned, as observed during cell cycle progression. Here, using an expression profiling approach, we provide evidence that the CDK inhibitor p27Kip1 regulates miRs expression following cell cycle exit. By using wild type and p27KO cells harvested in different phases of the cell cycle we identified several miRs regulated by p27Kip1 during the G1 to S phase transition. Among these miRs, we identified miR-223 as a miR specifically upregulated by p27Kip1 in G1 arrested cells. Our data demonstrate that p27Kip1 regulated the expression of miR-223, via two distinct mechanisms. p27Kip1 directly stabilized mature miR-223 expression, acting as a RNA binding protein and it controlled E2F1 expression that, in turn, regulated miR-223 promoter activity. The resulting elevated miR-223 levels ultimately participated to arresting cell cycle progression following contact inhibition. Importantly, this mechanism of growth control was conserved in human cells and deranged in breast cancers. Here, we identify a novel and conserved function of p27Kip1 that, by modulating miR-223 expression, contributes to proper regulation of cell cycle exit following contact inhibition. Thus we propose a new role for miR-223 in the regulation of breast cancer progression.

Coordinated cell proliferation and ability to form intercellular seals are essential features of epithelial tissue function. Tight junctions (TJs) classically act as paracellular diffusion barriers. More recently, their role in regulating epithelial cell proliferation in conjunction with scaffolding zonula occludens (ZO) proteins has come to light. The kidney collecting duct (CD) is a model of tight epithelium that displays intense proliferation during embryogenesis followed by very low cell turnover in the adult kidney. Here, we examined the influence of each ZO protein (ZO-1, -2 and -3) on CD cell proliferation. We show that all 3 ZO proteins are strongly expressed in native CD and are present at both intercellular junctions and nuclei of cultured CD principal cells (mCCDcl1). Suppression of either ZO-1 or ZO-2 resulted in increased G0/G1 retention in mCCDcl1 cells. ZO-2 suppression decreased cyclin D1 abundance while ZO-1 suppression was accompanied by increased nuclear p21 localization, the depletion of which restored cell cycle progression. Contrary to ZO-1 and ZO-2, ZO-3 expression at intercellular junctions dramatically increased with cell density and relied on the presence of ZO-1. ZO-3 depletion did not affect cell cycle progression but increased cell detachment. This latter event partly relied on increased nuclear cyclin D1 abundance and was associated with altered β1-integrin subcellular distribution and decreased occludin expression at intercellular junctions. These data reveal diverging, but interconnected, roles for each ZO protein in mCCDcl1 proliferation. While ZO-1 and ZO-2 participate in cell cycle progression, ZO-3 is an important component of cell adhesion.

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.

Asymmetric division plays a crucial role during oogenesis, characterized by extrusion of a small polar body in the first meiosis.1 Oocytes preserve most of their maternal stores by expelling a minimal cytoplasmic content with half chromosomes, which is essential for early embryonic development by maintaining nutrition and energy resources due to extremely asymmetric division.2 Spindle positioning is important for the asymmetric or symmetric division in cell cycle.3 Cytoplasmic actin is involved in chromosome gathering and spindle positioning in mitosis, because randomization of spindle positioning occurs after disassembling actin with disrupting drugs.4 Also, astral microtubules emanating from centrosomes interact with cortical anchor proteins decide the spindle position along the longest cell axis, which helps the symmetric division.5 However, this rule is not suitable for oocytes because they are round and lack of centrosome and astral microtubules. In contrast to symmetric mitotic divisions, oocytes undergo asymmetric divisions with half homologous chromosomes segregated to the small polar body during anaphase of the first meiotic division. To compensate for the lack of centrosome and astral microtubules, actin assembly is very important for the spindle positioning by transmitting forces over long distances during oocytes meiosis. A recent paper in Cell Cycle describes a key factor, Formin-like 1 (FMNL1), associated with RhoA signal pathway, regulates asymmetric division by controlling actin assembly and spindle positioning in mouse oocytes (Fig. 1).6 FMNL1 is a member of Formin family proteins which are the actin nucleators. It has been shown that FMNL1 is essential for cell adhesion, cytokinesis, cell polarization and migration in mitosis. The authors discovered the functions of FMNL1 in meiotic mouse oocytes by using specific morpholino microinjection and live cell imaging. FMNL1 knockdown caused disrupted asymmetric division, characterized by the large polar bodies and low rate of polar body extrusion. Immunofluorescent staining showed that in addition to its cytoplasmic distribution, FMNL1 was primarily localized at the spindle poles after germinal vesicle breakdown (GVBD), which implies that it may be involved in spindle organization and positioning. Next, the authors found various morphologically defective spindles in FMNL1 knockdown oocytes by co-staining FMNL1, α-tubulin and DNA. The wrong localization and lower expression level of phospho-p44/42 mitogen-activated protein kinase (p-MAPK) further confirmed the FMNL1s roles in spindle organization, because MAPK has been previously shown to be required for proper spindle formation. In addition, same results were observed in mammalian diaphanous1 (mDia1) knockdown oocytes. FMNL1 depletion resulted in reduced mDia1 expression, which implied that FMNL1 might be upstream to mDia1.7 Subsequently, time-lapse microscopic and immunofluorescence intensity analysis demonstrated that the wrong spindle positioning was due to the aberrant actin assembly and expression level in FMNL1 knockdown oocytes. Similar results have been observed in other members of the Formin family members. Meantime, cortical polarity was disrupted as shown by a loss of actin cap and cortical granule-free domain (CGFD) formation. Because spindle migration is actin-dependent, FMNL1 may regulate spindle positioning by its effect on actin nucleation, which can further affect oocyte cortical polarity formation. Also, FMNL1 depletion resulted in aberrant localization and expression patterns of a cis-Golgi marker protein, GM130, which caused disrupted Golgi apparatus.6 Because Golgi mediates the long-range transport of vesicles, FMNL1 may modulate vesicle-based mechanism of actin network for asymmetric division by regulating Golgi complex. In addition, mDia1 and Profilin1 had similar localization patterns in mouse oocytes and mDia1 knockdown resulted in reduced Profilin1 expression, which also partially explained the wrong actin assembly.7 Inhibition of the small GTPase RhoA activity resulted in decreased FMNL1 expression, which indicates that RhoA may be the upstream molecule of FMNL1.6 However, RhoA inhibition did not alter mDia1 expression, which indicates that there is a FMNL1-mDia1-Profilin1 signaling pathway. Taken together, there are RhoA-FMNL1/GM130 and RhoA-FMNL1-dMia1- Profilin1 pathways for the spindle positioning and actin assembly during mouse oocyte meiosis.7 In summary, the identification of FMNL1 as a key regulator for spindle positioning and actin assembly in the current study strongly implies that the actin-based spindle positioning is vital for asymmetric division during oocyte maturation. These recent studies, on one hand clarify the mechanism for asymmetric division in mouse oocytes, and on the other hand show a RhoA-FMNL1 pathway. It will contribute to understanding how physical parameters including cell size and shape are controlled in the cell cycle. Figure 1. FMNL1 regulates asymmetric division by controlling actin assembly and spindle positioning in mouse oocytes. Actin network modulation is essential for the asymmetric positioning of meiotic spindle. FMNL1 is important for spindle organization with the help ...

Expression of oncogenes or short telomeres can trigger an anticancer response known as cellular senescence activating the p53 and RB tumor suppressor pathways. This mechanism is switched off in most tumor cells by mutations in p53 and RB signaling pathways. Surprisingly, p53 disabled tumor cells could be forced into senescence by expression of a mutant allele of the nuclear envelope protein lamin A. The pro-senescence lamin A mutant contains a deletion in the sequence required for processing by the protease ZMPSTE24 leading to accumulation of farnesylated lamin A in the nuclear envelope. In addition, the serine at position 22, a target for CDK1-dependent phosphorylation, was mutated to alanine, preventing CDK1-catalyzed nuclear envelope disassembly. The accumulation of this mutant lamin A compromised prophase to prometaphase transition leading to invaginations of the nuclear lamina, nuclear fragmentation and impaired chromosome condensation. Cells exited this impaired mitosis without cytokinesis and re-replicated their DNA ultimately arresting in interphase as polyploid cells with features of cellular senescence including increased expression of inflammatory gene products and a significant reduction of tumorigenicity in vivo.

OCT4A is best known as a “master” transcription factor involved in stem cell pluripotency and self renewal in concert with SOX2 and NANOG. Although there has been some indication of a dialog between OCT4 and elements of DNA repair,1 a recent article published in Cell Cycle by Huna and colleagues2 establishes OCT4A as a multifunctional protein with a novel role as a major p53-dependent regulator of the DNA damage response (DDR) pathway. The latter activities occur in the absence of SOX2/NANOG response, i.e., independently of OCT4A's role in the stemness network. The report2 sheds considerable light on the innermost workings of the DDR and its integration, a subject that has long been of great curiosity and ambiguity.3 The findings (Fig. 1) provide insight into the nature of the pre-senescent transition state from which cells undergoing DDR ultimately execute cell-fate decisions – in this case, mitotic recovery versus “death” by terminal senescence or apoptosis. By silencing OCT4A and p21Cip1 in PA-1 ovarian teratocarcinoma cells (which have a functional p53 axis) followed by exposure to etoposide, OCT4A was shown to suppress the levels and activity of p21Cip1, whereas the reciprocal effect was not observed.2 Like OCT4A, p21Cip1 is a multi-functional protein; it is involved in activating cell cycle checkpoints, DNA repair, activating the “accelerated” senescence response to DNA damage, blocking apoptosis, and regulating the WIP1 phosphatase.6 These observations indicate that OCT4A is an important regulator in the p53/DDR pathway that especially controls the p21Cip1 protein and its various effects on the cell; however, it clearly exerts additional p21Cip1-independent activities, such as in establishing the G2/M-arrested pre-senescent transition state itself. Figure 1. Cell-autonomous events in PA-1 cells treated with etoposide, based on Huna et al.2 The G2/M-arrested transition state (OCT4A[+]/p21Cip1[+]) has characteristics of both self renewal and senescence. OCT4A suppresses p21Cip1 and senescence while ... The results further suggest that OCT4A functions in tandem with AMPK in directing cell-fate decisions in this cancer model, notably the evolution of the senescence phenotype; this complex process in part involves the AMPK/autophagy-driven cytoplasmic sequestration of p16Ink4a, which is a mediator of the transition from pre- to terminal senescence, redirecting some cells toward mitotic recovery. These events represent a compelling target for therapeutic intervention by overcoming the resistance of cancer stem cells to DNA-damaging anticancer agents. Indeed, Huna et al.2 note that in some scenarios the application of drugs that modulate AMPK/autophagy might serve to redirect chemotherapy-refractory tumor cells into an apoptotic death pathway. This strategy is certainly worth testing. The findings also inform treatment strategies that target the therapy-induced senescence phenotype in cancer cells which has a complex, often negative, relationship to therapeutic outcome.7 Molecular insight such as that provided by Huna et al2 might ultimately allow such decisions to be made on a rational mechanistic basis. As Huna and colleagues2 emphasize, these OCT4A-mediated contributions to the DDR have to date only been characterized for a single cancer cell line, PA-1, and a single anticancer drug, etoposide. It will thus be vital to establish the generality or exclusivity of these roles in cell lines derived from other tumor sites (including those with differing p53/p16Ink4a status) as well as in response to other classes of DNA-damaging agents, and indeed to other stress stimuli. It should also be noted that the current study focuses on a high dose of etoposide resulting in a population of cells with extensively damaged genomes of which few (∼1%) are destined to emerge from the hypothetical metastable “bipotential” pre-senescent state, undergo mitotic recovery and “survive;” what will happen over a range of lower doses will be of great interest in the clinical context. An intriguing observation from this study2 is the considerable molecular heterogeneity of the early damage-signaling response among individual cells that is suggested to underlie the bifurcation of cell fate decisions. Such heterogeneity reaffirms the criticality of undertaking such molecular time-course studies at the single-cell level, as was indeed done by Huna et al.2 The outstanding contribution of this research team that demonstrates a pivotal role for OCT4A and autophagy in cell-autonomous DDR-related cell fate decisions will hopefully encourage meaningful discussion and further unraveling of the complex signaling dynamics and relationships between self renewal, senescence and cell cycle checkpoints/DDR in cancer cells. Such insight into the responses triggered by therapeutic agents in different cancer cell types will be crucial for designing effective approaches for preventing and treating this devastating disease.

Exonuclease 1 (EXO1) is one of several nucleic acid-trimming enzymes required for the repair of double strand breaks that constitute the most toxic form of DNA damage. By resecting DNA ends, this processive 5′ to 3′ exonuclease helps to produce recombinogenic 3′ overhangs, which are than captured by the homologous recombination machinery to repair double strand breaks, i.e., to reestablish an intact DNA double helix.1 This mechanism raises the question of how EXO1 is prevented from carrying out excessive DNA digestions at strand breaks, possibly generating even more deleterious intermediates responsible for chromosomal aberrations. Clearly, further research is needed to understand the regulation of EXO1 in the maintenance of genome stability but, in the current issue of Cell Cycle, Bologna et al.2 provide important new insights into this challenging problem. They report that, in conjunction, 2 related peptide modifiers control the level and enzymatic activity of EXO1 in human cells. A first hint for this newly discovered regulatory circuit came from the finding that EXO1 undergoes ubiquitin-dependent proteasomal degradation in response to agents like hydroxyurea, aphidicolin or camptothecin that cause replication stress by stalling DNA replication forks. Interestingly, the authors discovered that, in such genome-threatening situations like replication stress, both the ubiquitination of EXO1 and its degradation are triggered by a preceding conjugation with SUMO (for Small Ubiquitin-like MOdifier). The SUMO cascade is an intensively studied protein modification system with key roles in diverse cellular processes. Like for posttranscriptional ubiquitination, the toolbox for protein sumoylation comprises a hierarchy of E1, E2 and E3 enzymes. The SUMO moiety is activated in an ATP-dependent manner by the E1 activating enzyme and then transferred to an E2 conjugating enzyme. This E2 conjugates SUMO to target lysine residues either by direct substrate recognition or with the assistance of E3 ligases acting as substrate adapters. Recent proteomic screens revealed a high prevalence of SUMO substrates in DNA-metabolic pathways.3,4 By demonstrating sumoylation of EXO1, Bologna and colleagues extend this list of sumoylated DNA-resecting enzymes (already including other nucleases like Rad1, FEN1, Sae2/CtIP and the Mre11 complex) by one additional entry and confirm the paradigm of SUMO-dependent processing of DNA double strand breaks. In fact, the E2 and E3 enzymes (UBC9 and PIAS1, respectively) involved in EXO1 sumoylation had been shown previously to co-localize with sites of DNA double strand break repair.5,6 The finding that EXO1 is de-sumoylated by the SENP6 protease completes this regulatory circuit. To address the biological relevance of EXO1 sumoylation, Bologna and colleagues identified the lysine acceptor sites and generated an EXO1 triple mutant that is catalytically active, but refractory to sumoylation. Since a central arena for SUMO actions relates to pathways promoting genome stability, the authors next tested the impact of EXO1 overexpression on chromosomal aberrations in camptothecin-treated cells. Overexpression of wild-type EXO1 increases the prevalence of chromosomal breaks, presumably due to a disproportionate DNA-resecting activity. However, this negative effect of EXO1 on genome stability is attenuated when, instead of the wild-type protein, the SUMO-refractory triple mutant was overexpressed at the same protein level and under the same conditions. Evidently, interference with sumoylation protects cells experiencing replication stress from the deleterious consequences of an excess level of EXO1 Based on these findings, Bologna and colleagues.2 proposed a model of how the conjugation of EXO1 with peptide modifiers is linked to its function in handling replication stress (Fig. 1). Their scheme involves recruitment of constitutively sumoylated EXO1 to DNA ends at stalled replication forks. SUMO stimulates the catalytic activity of EXO1 during DNA resection in the context of stalled replication forks and, upon full substrate engagement, induces its ubiquitination. Thus, this combination of constitutive sumoylation and on-site ubiquitination ensures fit-to-purpose resection but prevents excessive digestion of free DNA ends by EXO1. A challenge for the future will be to understand how SUMO is able to fine-tune the enzymatic activity of EXO1 independently of ubiquitin-dependent degradation. In general terms, SUMO can have effects either as a molecular “glue” or as an “anti-glue” in modulating protein-protein or protein-DNA interactions.7 Thus, one testable possibility is that SUMO moieties mediate the association of EXO1 to binding partners displaying SUMO-interacting motifs in structures assembled at stalled replication forks. Another possibility is that SUMO residues might facilitate the displacement of DNA-binding proteins that sterically hinder accessibility and DNA resection by EXO1. Figure 1. Dual control of EXO1 activity by conjugation with SUMO (S) and ubiquitin (U) reported by Bologna et al.2 Constitutively sumoylated EXO1 is recruited to stalled replication forks induced by treatment with hydroxyurea (HU; a ribonucleotide reductase ...

In naive T lymphocytes, the outcome of TCR stimulation is regulated by the simultaneous binding of CD28 to its natural ligand, expressed on APCs. Zynda et al. found that a mild, sustained increase in temperature not only enhances IL-2 production by CD4+ T cells receiving the canonical two 2 activation signals, but it also enhances IL-2 production under conditions of insufficient co-stimulation of CD28. Since co-stimulation via CD28 is considered necessary for full activation and IL-2 production at 37°C, these data suggest that mild hyperthermia may be able to mimic at least some of the responses elicited by CD28-mediated co-stimulation. Numerous studies show that optimal T cell activation involves reorganization of the plasma membrane, both in the order of membrane lipids and membrane cortex associated proteins, such as actin, spectrin, and vimentin.1,2 The authors provide evidence that reversible changes in membrane fluidity occur immediately after exposing cells to mild hyperthermia and were able to duplicate these effects using a known membrane fluidizer, 0.05% ethanol. They also demonstrated that co-stimulation through CD28 affects membrane fluidization in an identical manner. Collectively, these data suggest the intriguing possibility that heat can replace or mimic the co-stimulation via CD28 through its ability to alter membrane fluidity (see Fig. 1). The observed effects of fever-range hyperthermia on T-cell stimulation persist over hours indicating that the changes in lipid fluidity likely modulate the molecular order in the membrane. Figure 1. Comparing co-stimulation, temperature elevation and isothermal membrane fluidization effects on T cell activation threshold. CD4+ T cells need co-stimulation via CD28 receptors for sufficient survival, activation and IL-2 production. Fever-range heat ... Under resting conditions, distinct sphingomyelin and cholesterol containing microdomains with selective confinement of signaling molecules occur in the plasma membrane of T cells, but generally, these individual membrane domains are only a few nanometers in diameter and difficult to distinguish by microscopy.3 However, when T cells are activated using TCR/CD28-receptors at 37°C, the nanoscale membrane domains appear to cluster into larger aggregates that can be visualized. The authors observed that CD4+ T lymphocytes exposed to mild heating alone exhibit significant, yet reversible, clustering of plasma membrane GM1 enriched membrane domains. Similarly, it has previously been shown that CD28 mediated co-stimulation enhances the clustering of macromolecular signaling platforms in the plasma membrane, and that CD28s function is actually dependent on this membrane reorganization. In this way, perhaps mild hyperthermia and CD28 can each lower the T cell activation threshold. Whether mild heating acts as a co-stimulator itself, or through the enhancement of a signaling pathway downstream of CD28 signaling is an important question. The data of Zynda et al. show that the greatest amount of IL-2 is produced when both heat and CD28 co-stimulation are used, suggesting that there may be some non-overlapping or additive effects of heat and CD28 ligation in addition to observed similarities. The authors, using CD4+ T cells, demonstrated that mild heating and ethanol (both of which increased membrane fluidity) caused an identical rearrangement of ankyrin and spectrin. In addition to the spectrin and ankyrin cytoskeletal systems, actin polymerization has also been shown to play a prominent role in cholesterol dependent signal domain aggregation and synapse formation during T cell activation.4,5 The authors also found that actin polymerization was necessary for enhancement of IL-2 production by mild heating and ethanol. These data provide additional support for the hypothesis that thermal signals influence cytoskeletal reorganization in order to lower the threshold for T-cell activation. These findings fit perfectly with the “Membrane Sensor Hypothesis” which postulates that subtle changes in the fluidity and/or organization of cell membranes can be the most upstream event of temperature-sensing and signaling during mild heat shock.6 Hyperfluidization by mild heat is coupled with specific reorganization of membrane domains and results in heat shock protein expression, similar to isothermal hyperfluidization gained by the administration of alcohols6 or with lipid interacting drugs.7 The central role that membrane fluidity and reorganization of the plasma membrane microdomains play in T cell activation strongly supports a fundamental role for heat sensitive changes at the plasma membrane to mediate many different functional effects of temperature. Finally, the possibility that a sustained increase in temperature could reduce the activation threshold of T lymphocytes may help to explain multiple studies that demonstrate a significant survival benefit associated with fever following infection in multiple vertebrates.

In their active hypophosphorylated state, members of the retinoblastoma family of pocket proteins negatively regulate cell cycle progression at least in part by repressing expression of E2F-dependent genes. Mitogen-dependent activation of G1 and G1/S Cyclin Dependent Kinases (CDKs) results in coordinated hyperphosphorylation and inactivation of these proteins, which no longer bind and repress E2Fs. S and G2/M CDKs maintain pocket protein hyperphosphorylated through the end of mitosis. The inactivating action of inducible CDKs is opposed by the Ser/Thr protein phosphatases PP2A and PP1. Various trimeric PP2A holoenzymes have been implicated in dephosphorylation of pocket proteins in response to specific cellular signals and stresses or as part of an equilibrium with CDKs throughout the cell cycle. PP1 has specifically been implicated in dephosphorylation of pRB in late mitosis and early G1. This review is particularly focused on the emerging role of PP2A as a major hub for integration of growth suppressor signals that require rapid inactivation of pocket proteins. Of note, activation of particular PP2A holoenzymes triggers differential activation of pocket proteins in the presence of active CDKs.

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.

Stem cells have a high and constitutive endogenous activity of the DNA damage response (DDR) pathway1 which may differ in some aspects from the function of the DDR pathway in somatic cells. It is likely that such stemness-related functions of the DDR pathway may play a role in the resistance to genotoxic insult seen in stem cells and cancer cells.2 Lately, it has become clear that the context of the DDR pathway is more complex than first anticipated, and as many other signaling pathways it plays roles beyond its first recognized function as guardian of the genome. One example is the finding that the DDR pathway axis ATR-Chk1 plays a critical function in undamaged cells during mitosis.3 An additional function for H2AX in the DDR pathway, was provided by Andang et al,1 who showed that it mediates proliferation control in stem cells under non-genome damaging conditions. In recent publications by the Hallbook research group, another unexpected function of components of the DDR pathway was reported, again unrelated to direct genome protection. The Hallbook group previously showed that Chk1 in the DDR signaling axis controls the last mitosis in cells undergoing interkinetic nuclear migration (INM) in the developing eye neuroepithelium.4 However, not all cells undergo INM in the neuroepithelium, and the group has now in a carefully executed study, continued to show that Lim1+ horizontal progenitor cells (HPCs), that undergo their last mitosis on the basal side of the retina without INM, have a significantly attenuated dependence on the DDR pathway (Fig. 1).5 Figure 1. Schematic of signaling pathways involving the DNA damage response (DDR) pathway in apical and basal cells in the developing neuroepithelium. Only apical cells undergo interkinetic nuclear migration (INM) and are cell cycle arrested by an activated DDR ... Interestingly, during activation of the ATR-Chk1 (DDR pathway) axis in the developing retinal progenitor cells by Cisplatin, an inducer of ATR/p53 signaling,6 the apical cells were cell cycle arrested as expected, while the basal HPCs cells were not.5 The HPCs nevertheless had a functional p53/p21 pathway regulating cell cycle and apoptosis indicating an inability of the activated DDR pathway to engage the p53 pathway and ultimately apoptosis. However, despite the lack of cell cycle arrest and apoptosis in response to Cisplatin, the DDR pathway was induced and HPCs had elevated γH2AX levels indicating a robust induction of a DDR pathway response. This situation, which has previously been reported in other stem cells1 and in cancer pre-lesions,7 suggests an ability of the HPCs to withstand genotoxic insult and maintain proliferation. Such ability may be a factor that under for example environmental challenging conditions, allows for accumulation of mutations that later may drive development of cancer initiating cells. On the other hand, critical progenitor cell types may maintain capacity to divide and generate new cells during developmental or repair processes – a last resort for survival when most other more sensitive cells are lost to apoptosis. The attenuated DDR pathway response is then a trade off between immediate survival and risking cancer at a later time.

Epigenetic alterations during cellular differentiation are a key molecular mechanism which both instructs and reinforces the process of lineage commitment. Within the haematopoietic system, progressive changes in the DNA methylome of haematopoietic stem cells (HSCs) are essential for the effective production of mature blood cells. Inhibition or loss of function of the cellular DNA methylation machinery has been shown to lead to a severe perturbation in blood production and is also an important driver of malignant transformation. HSCs constitute a very rare cell population in the bone marrow, capable of life-long self-renewal and multi-lineage differentiation. The low abundance of HSCs has been a major technological barrier to the global analysis of the CpG methylation status within both HSCs and their immediate progeny, the multipotent progenitors (MPPs). Within this Extra View article, we review the current understanding of how the DNA methylome regulates normal and malignant hematopoiesis. We also discuss the current methodologies that are available for interrogating the DNA methylation status of HSCs and MPPs and describe a new data set that was generated using tagmentation-based whole genome bisulfite sequencing (TWGBS) in order to comprehensively map methylated cytosines using the limited amount of genomic DNA that can be harvested from rare cell populations. Extended analysis of this data set clearly demonstrates the added value of genome-wide sequencing of methylated cytosines and identifies novel important cis-acting regulatory regions that are dynamically remodeled during the first steps of haematopoietic differentiation.

The primary cilium is a specialized dynamic signaling compartment that regulates cellular differentiation by the vertebrate hedgehog pathway. The microtubular axoneme of the primary cilium is built from the mother centriole (basal body) by a conserved process called intraflagellar transport (IFT).1 IFT consist of trains of intraflagellar particles, biochemically characterized as IFT-A and IFT-B that are transported in the anterograde direction by kinesin II, and in the retrograde direction by dynein 2.1 Mutations in IFT-A and dynein 2 result in accumulation of IFT-B at ciliary tips, and axonemal bulges, characteristic of disrupted retrograde transport inside cilia.1 Defects in primary cilia and in trafficking of proteins to the cilia are associated with a heterogeneous group of newly described human diseases affecting multiple tissues known as “ciliopathies." One particular class of skeletal ciliopathies affects hedgehog-regulated bone and cartilage morphogenesis, and results in chondrodysplastic phenotypes. These include the short-rib polydactyly syndrome (SRPS), asphyxiating thoracic dystrophy or Jeune syndrome (JATD), and Sensenbrenner syndrome, and result from mutations mainly in IFT-A2 or dynein 2.3 Recently, two genes, WDR34 and WDR60, have been implicated in SRPS and JATD,4,5 and could function as dynein intermediate chains in retrograde IFT.6,7 A recent paper in Cell Cycle describes a candidate dynein light chain protein, TCTEX1D2, with a conserved domain similar to the C-terminus of the dynein light chain TCTEX1 subunit (Fig. 1), to be associated with WDR34/60, and the authors propose that TCTEX1D2 could be functioning as a light chain subunit in dynein complexes, particularly in relation to ciliogenesis. Figure 1. Cartoon showing dynein motor subunits. LT, LL and RB represent dynein light chain subunits. TCTEX1D2 has a conserved domain similar to the C-terminus of the dynein light chain TCTEX1 subunit (LT), and could be functioning as a light chain subunit in dynein ... While identifying TCTEX1D2-binding proteins using tandem affinity purification and mass spectrometry, the authors discovered TCTEX1D2 to be associated with dynein complexes. Interestingly, in nonciliated cells, TCTEX1D2 was associated with the cytoplasmic dynein 1 heavy chain (DYNC1H1), whereas, in ciliated cells it was exclusively associated with the dynein 2 heavy chain (DYNC2H1). In both cases, TCTEX1D2 was associated with WDR34 and WDR60. Similar to WDR34 and WDR60, TCTEX1D2 was localized to the basal body, and the primary cilium. In addition, TCTEX1D2 was also localized to pericentriolar satellites around the basal body. The localization of TCTEX1D2 to the basal body was dependent on WDR60. While its unclear if TCTEX1D2 is involved in retrograde IFT in cilia, lack of this protein resulted in decreased ciliogenesis. Defects in IFT-A and dynein 2 characteristically results in accumulation of IFT-B proteins in ciliary tips, and is indicative of disrupted retrograde IFT. The Chlamydomonas ortholog of WDR60 (FAP163) has been shown to be present in the flagellar matrix and is actively trafficked by IFT, and knockdown of the Planaria ortholog results in severe defects in ciliary assembly with accumulated electron-dense material, characteristic of defective retrograde IFT.7 In addition, the Chlamydomonas ortholog of WDR34 (FAP133) has been shown to associate with the LC8 dynein light chain and the IFT dynein heavy chain and light intermediate chain motor complex.6 Surprisingly, fibroblasts from WDR60 patients do not show accumulation of IFT-B in ciliary tips,5 and fibroblasts from WDR34 patients do not show defects in ciliogenesis,4 possibly because of the hypomorphic nature of the alleles. Alternatively, defects in WDR34/WDR60/TCTEX1D2 may cause less severe accumulation of IFT trains in cilia. Exome capture and parallel sequencing efforts in the past few years have resulted in the identification of multiple novel cilia/centrosome-associated genes in the complex ciliopathy disease spectrum. Proteomic analyses of proteins associated with cilia localized factors also provide a rich trove of information and missing links in identification of novel disease-associated genes. The identification of TCTEX1D2 as a dynein-associated protein in the current study strongly implies that defects in this protein could be associated with skeletal ciliopathies. The identification of TCTEX1D2 as a dynein subunit could also provide links to novel dynein-associated cargos important in ciliogenesis.

Hepatocyte odd protein shuttling (HOPS) moves between nucleus and cytoplasm. HOPS overexpression leads to cell cycle arrest in G 0/G 1, and HOPS knockdown causes centrosome alterations, with subsequent abnormal cell division. Recently, we demonstrated that HOPS acts as a functional bridge in NPM-p19(Arf) interactions. Here we show that HOPS is present in 3 different isoforms that play distinct intracellular functions. Although HOPS is a transmembrane ubiquitin, an isoform with intermediate molecular weight is cleaved from the membrane and released into the cytosol, to act as the shuttling protein. We identified a signal peptide peptidase structure in N-terminal membrane-bound HOPS that allows the regulated intramembrane proteolysis (RIP) system to control the relative amounts of the released, shuttling isoform capable of binding NPM. These results argue for distinct, isoform-specific functions of HOPS in the nucleolus, nucleus, and cytoplasm and provide insight into the dynamics of HOPS association with NPM, whose mutation and subsequent delocalization is found in 30% of acute myeloid leukemia patients.

Despite recent advances in medical procedures, cardiovascular disease remains a clinical challenge and the leading cause of mortality in the western world. The condition causes progressive smooth muscle cell (SMC) dedifferentiation, proliferation, and migration that contribute to vascular restenosis. The incidence of disease of the internal mammary artery (IMA), however, is much lower than in nearly all other arteries. The etiology of this IMA disease resistance is not well understood. Here, using paired primary IMA and coronary artery SMCs, serum stimulation, siRNA knockdowns, and verifications in porcine vessels in vivo, we investigate the molecular mechanisms that could account for this increased disease resistance of internal mammary SMCs. We show that the residue-specific phosphorylation profile of the retinoblastoma tumor suppressor protein (Rb) appears to differ significantly between IMA and coronary artery SMCs in cultured human cells. We also report that the differential profile of Rb phosphorylation may follow as a consequence of differences in the content of cyclin-dependent kinase 2 (CDK2) and the CDK4 phosphorylation inhibitor p15. Finally, we present evidence that siRNA-mediated CDK2 knockdown alters the profile of Rb phosphorylation in coronary artery SMCs, as well as the proliferative response of these cells to mitogenic stimulation. The intrinsic functional and protein composition specificity of the SMCs population in the coronary artery may contribute to the increased prevalence of restenosis and atherosclerosis in the coronary arteries as compared with the internal mammary arteries.

Endocytic vesicle fusion is inhibited during mitosis, but the molecular pathways that mediate the inhibition remain unclear. Here we uncovered an essential role of Polo-like kinase 1 (Plk1) in this mechanism. Phosphoproteomic analysis revealed that Plk1 phosphorylates the intermediate filament protein vimentin on Ser459, which is dispensable for its filament formation but is necessary for the inhibition of endocytic vesicle fusion in mitosis. Furthermore, this mechanism is required for integrin trafficking toward the cleavage furrow during cytokinesis. Our results thus identify a novel mechanism for fusion inhibition in mitosis and implicate its role in vesicle trafficking after anaphase onset.

Cell size is under genetic control, and size at division may be mutationally changed so that cells divide at a larger or smaller size without any alteration in the generation time.1 Larger and smaller cells thus double their size during the same time period, illustrating that larger cells grow faster than smaller cells. However, the increase of cellular growth rate with cell size in certain genetic backgrounds could amplify size heterogeneity of the population in the absence of additional regulation. Therefore, growing cells possess a homeostatic size control mechanism, which shortens the cycle time of larger cells, while it extends the cycle time for smaller ones (Fig. 1). This homeostasis works through the size control mechanisms that regulate the length of G1 or G2 phase, thereby controlling the timing of entry into DNA replication or mitosis, respectively (G1/S and G2/M size controls). The molecular mechanisms underlying these size controls are still obscure. Figure 1. Mechanism of size homeostasis. The average mass doubling time (red curve) is independent of birth size, if larger cells can double their size during the same time as small ones. The size control mechanism modulates the time between 2 ... A few years ago, 2 papers published in Nature demonstrated an intracellular concentration gradient of Pom1 kinase in rod-shaped fission yeast cells.2,3 The level of the mitotic inhibitory Pom1 kinase was found to be highest at cell tips and minimal around the nucleus in the middle of the cell. As cells grow, the Pom1 concentration at the cell center decreases, thus relieving its mitotic inhibitory effect. These observations lead to the proposal that the Pom1 gradient generated by a reaction–diffusion mechanism could serve as the basis of mitotic size control in fission yeast.2-4 A straightforward prediction of this hypothesis, that balanced growth and division should be compromised in Pom1-deleted cells, has been tested now by Wood and Nurse5 and published in the October 1, 2013 issue of Cell Cycle. By using live-cell imaging, the cycle time vs. birth size relationship (for example the green curve on Fig. 1) of Pom1-deleted cells was determined. This analysis showed no significant difference compared with wild-type cells, suggesting that Pom1 and its intracellular gradient cannot play a central role in cell size control. The existence of size control in Pom1-deleted cells has been also confirmed by analysis of cells returning to the normal cell size after a reversible cell cycle block. In this situation, cells are born at a larger size than normal, and their subsequent cycle time is shorter than the time required to double their size (see Fig. 1). As a consequence, cells become progressively smaller in each cycle, until they recover their normal size. The rate of reversion to normal cell size was indistinguishable in pom1Δ and wild-type cells, confirming the existence of size control in the mutant cells. Back-up mechanisms are common in biology, and size control is no exception. Fission yeast cells also have a G1/S size control, which provides size homeostasis in cells with short G2 phase, e.g., wee1 mutants.6 However, the kinetics of cell division after nutritional-shift experiments with Pom1-deleted cells is consistent with these cells still having a G2/M size control rather than depending on the G1/S size control.5 Pom1 regulates Cdk1 inhibitory phosphorylation,2,3 which is normally essential for cell viability of fission yeast. However, cells with non-phosphorylable Cdk1 are viable if the oscillation of Cdk1 activity is driven only by a single Cdk1- mitotic B-type cyclin (Cdc13) fusion protein.7 Despite having a more variable size at cell division, these cells still have a homeostatic size control mechanism.5,7 These observations suggest that inhibitory Cdk1 phosphorylation regulated by Pom1 is dispensable for fission yeast size control. It is very likely that cells with non-phosphorylable Cdk1 regulate their cell size by G1/S control, because they have an extended G1 phase.7 The length of G1 phase in fission yeast is controlled by the stoichiometric Cdk1 inhibitor, Rum1.8 Rum1-deleted cells, after inactivation of the major Cdk1-inhibiting kinase (Wee1), become progressively smaller at each cell division.8 The lack of size homeostasis in the absence of any G1- and G2-specific Cdk1 inhibitors suggest that balanced growth and division is a systems-level property of the Cdk1 activity control system.

Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells.

Polycomb group protein BMI1 is an important regulator of senescence, aging, and cancer. On one hand, it is overexpressed in cancer cells and is required for self-renewal of stem cells. On the other hand, it is downregulated during senescence and aging. MicroRNAs have emerged as major regulators of almost every gene associated with cancer, aging, and related pathologies. At present, very little is known about the miRNAs that regulate the expression of BMI1. Here, we report that miR-141 posttranscriptionally downregulates BMI1 expression in human diploid fibroblasts (HDFs) via a miR-141 targeting sequence in the 3′ untranslated region of BMI1 mRNA. We also show that overexpression of miR-141 induces premature senescence in HDFs via targeting of BMI1 in normal but not in exogenous BMI1-overexpressing HDFs. Induction of premature senescence in HDFs was accompanied by upregulation of p16INK4a, an important downstream target of BMI1 and a major regulator of senescence. Our results suggest that miR-141-based therapies could be developed to treat pathologies where BMI1 is deregulated.

One outcome of persistent activation of the DNA damage response (DDR) is cellular senescence; a programmed, permanent, cell cycle exit. DDR activation serves as a block to malignant transformation with senescence as a core feature of this effect. Diverse conditions that induce senescence, for example ionizing radiation, oncogene activation and telomere attrition, themselves induce DDR activation. In virtually all of these instances, senescence can be rescued by ablation of p53 function, thereby allowing the cells to re-enter the cell cycle in the presence of DNA damage. In this issue, Monasor and colleagues provide new evidence suggesting that the well-known tumor suppressor INK4a/ARF enables replicative stress (RS)-induced senescence.1 This is notable, since, in contrast to p53, INK4a/ARF has generally been thought not to play a role in DNA damage-induced senescence. Loss of the INK4a/ARF locus on chromosome 9p21 is one of the most frequent abnormalities observed in human tumors, second only to loss and/or mutation of TP53 (p53). The INK4a/ARF locus encodes two proteins, p16(INK4a) and ARF (p16Ink4A and p19ARF in mice), which regulate the Retinoblastoma and p53 pathways respectively.2 In normal cells, INK4a/ARF levels are usually barely detectable. Monasor and colleagues report that persistent RS is also associated with elevated INK4a/ARF expression, and that INK4a/ARF deficient cells can sustain growth in the presence of substantial levels of RS. Furthermore, upon interrogation of the Cancer Cell Line Encyclopaedia Project (http://www.broadinstitute.org/ccle/home), they found a correlation between copy number variation (CNV) and p16INK4A levels; cell lines with high levels of CNVs (RS has been shown to cause CNV3) exhibited low levels of p16INK4A. The authors consequently propose INK4a/ARF as a bona fide RS-checkpoint activator.1 ATR is an activating protein kinase of the DDR. Persistent ATR activation can drive cells to senescence, although independently of INK4a/ARF.4 Previous work from this team in modeling ATR-defective Seckel syndrome in mice (AtrS/S) showed that impaired ATR function also results in RS-induced DNA damage, senescence and cell death.5 AtrS/S mice exhibit severe embryo-wide cellular attrition with the surviving postnatal animals exhibiting a profound growth restriction, progeria and early death. AtrS/S MEFs fail to cycle, instead undergoing premature senescence. They do not undergo spontaneous immortalization and are even impervious to oncogene-induced immortalization. Genetic ablation of TP53 did not rescue senescence in either the AtrS/S animal or their derived MEFs.5 In fact, synthetic lethality was observed, a serendipitous observation now being actively pursued in the context of ATR kinase inhibitors (ATRi) as an anticancer therapy.6 The characteristic premature senescence seen in AtrS/S MEFs is overcome by reduced INK4a/ARF, a rescue not concomitant with a restoration of ATR expression or a reduction of RS.1 Indeed, ATRS/S + INK4a/ARF−/− cells effectively cycle with persistent RS. In contrast to these MEFs, deletion of INK4a/ARF does not rescue the phenotype of the AtrS/S mouse. The authors argue that INK4a/ARF-dependent senescence is the likely outcome of modest RS, while in the context of high levels of RS (e.g., during AtrS/S embryonic development and/or following high doses of ATRi), significant cell death occurs. This is significant, as it implies that ATR inhibitors would still be toxic toward INK4a/ARF-deficient tumors, as, indeed, demonstrated by the authors here using a mouse pancreatic cancer cell model. While INK4a/ARF deletion cannot rescue the ATRS/S phenotype, the authors suggest that this does not exclude the fact that the RS-checkpoint defined here could be operative in other contexts associated with more modest levels of RS. One situation where low but persistent RS likely occurs, as discussed by Monasor and colleagues, is oncogene-induced senescence. Oncogene-induced RS, which has long been associated with p16INK4a expression,7 represents a barrier, characterized by DDR activation,8 that must be overcome on the route to frank malignant transformation. The model proposed by Monasor and colleagues is that a key role of the p16 INK4a -Rb pathway would be to promote senescence in cells experiencing chronic RS, thus stopping them on the march toward transformation (Fig. 1). Still, how persistent RS activates INK4a/ARF remains a provocative, open question. Figure 1. A schematic summarizing the contrasting outcomes of elevated replicative stress in the presence or absence of INK4a/ARF, as proposed by Monasor and colleagues. Prolonged replicative stress, which can occur under various non-mutually ...

Abrogation of DNA damage-induced cell cycle checkpoints represents a promising concept for chemosensitization during anticancer treatment. A prominent example is the Chk1 inhibitor UCN-01 that is currently used in clinical trials in combination with various DNA damaging or DNA replication inhibiting drugs. It is well-established that inhibition of Chk1 in the presence of DNA damage leads to an override of the G2 cell cycle checkpoint, resulting in a premature mitotic entry.1 Damaged cells that are forced to enter mitosis show a transient mitotic arrest mediated by the spindle assembly checkpoint (SAC). At the same time, massive chromosome fragmentation is commonly observed, and this seems to be a prerequisite for the subsequent mitotic catastrophe, leading to the induction of apoptosis during mitosis, which involves a Mad2-dependent proapoptotic pathway2 (Fig. 1). However, the exact nature of mitotic defects triggering mitotic catastrophe remains unclear, but it is investigated in the work presented by Beeharry et al.3 Figure 1. Model illustrating mitotic centromere fragmentation after UCN-01-mediated S and G2 checkpoint override. Replication-stress (gemcitabine treatment) or inhibition of topoisomerase II (etoposide or Adriamycin treatment) leads to cell cycle ... In the June 1, 2013 issue of Cell Cycle, Beeharry and colleagues provide interesting new insights into the mitotic defects that result from abrogation of a DNA damage-induced S- or G2-phase checkpoint arrest. Immunofluorescence and electron microscopy analysis of a panel of different pancreatic cancer cell lines revealed that sequential treatment with the DNA replication inhibitor gemcitabine or with topoisomerase II inhibitors such as etoposide and adriamycin in combination with UCN-01 leads to the generation of MUGs (mitosis with unreplicated genomes). Here, MUGs are characterized by severe fragmented mitotic chromosomes with unreplicated centromeres and kinetochores detached from the bulk chromosomes. MUGs were first described by Brinkley et al. some 30 years ago,4 but Beeharry and colleagues now provide the first evidence that MUGs might be the primary outcome of G2 checkpoint override after treatment with chemotherapeutic DNA replication inhibitors. In contrast, G2 checkpoint override after treatment of cells with alkylating agents such as methyl methanesulfonate (MMS), albeit inducing DNA damage and arresting cells in G2 phase of the cell cycle, do not induce MUGs, and accordingly, cells progress though mitosis without further delay. Interestingly, inhibition of topoisomerase II by etoposide and adriamycin, although not grossly inhibiting DNA replication, still causes MUGing. By using FISH analysis with a centromere-specific probe hybridizing to the centromere region of chromosome 7, the authors found that inhibition of topoisomerase II, like inhibition of DNA replication by gemcitabine, results in unreplicated centromeres.3 It was previously shown that replication of centromeric DNA proceeds until metaphase,5 supporting the notion that the replication of centromeres might be sensitive toward topoisomerase II inhibition. However, it remains an open question how topoisomerase II inhibition blocks centromere replication. The work presented by Beeharry et al. suggests that the underlying cause for mitotic catastrophe observed after G2 checkpoint override might be a lack of replication and subsequent fragmentation of the centromere, resulting in acentric genomes. These interesting results might also explain why cells cannot satisfy the SAC after forced entry into mitosis and arrest in a metaphase-like state.2 Furthermore, it would be interesting to address the question of whether this mitotic arrest or the centromere fragmentation is directly related to the massive chromosome fragmentation that is commonly seen in mitotic cells after checkpoint override. In fact, it might be possible that chromosome condensation that occurs on chromosomes after forced entry into mitosis can generate forces that tear chromosomes apart. From the clinical point of view, this study provides insights on the choice of chemotherapeutic drugs that are to be combined with G2 checkpoint inhibitors. At least in pancreatic cancer cells, as shown here, the combination of inhibitors of centromere replication and G2 checkpoint inhibitors might be the most powerful combination, in particular for cancer cells lacking functional p53. Appropriate clinical trials will show whether this holds true in a clinical setting. Also, additional studies investigating other cancer entities are needed to provide a more general view on the efficacy of chemotherapeutic drugs that affect centromere replication and S or G2 checkpoint inhibitors.

Increasing epidemiological evidence suggests a role of steroid hormones in the development and progression of certain molecular subtypes of ovarian cancer. In general, the data suggest that estrogens are associated with increased risk of ovarian cancer, while progesterone and progestins may have a protective role (for recent review on this topic, see ref. 1). Despite these epidemiological associations, there remains a surprisingly limited understanding of the underlying biology and mechanisms of steroid receptor action in early and late stages of ovarian tumorigenesis. However, given that hormonal therapies represent the best of “targeted therapy in personalized medicine,” with limited toxicities, there is renewed interest in the static and maintenance roles for hormonal therapy in ovarian cancer recurrence. In addition, there might be a role for endocrine therapy in treatment of early-stage ovarian cancer, when expression of nuclear receptors is high. In the May 1, 2013 issue of Cell Cycle, Carol Lange and colleagues published an exciting study, which provides mechanistic insight into how progesterone receptor (PR) mediates the protective effects of progestins in ovarian cancer.2 The authors used a number of established ovarian cancer cell lines of different histological subtypes to show that progestins activate nuclear PR, leading to elevated FOXO1 expression and ultimately cellular senescence. Senescence is at least in part due to the induction of the cell cycle inhibitor p21. It is important to point out that in addition to the elegant mechanistic studies, the authors provide very valuable and extensive data on PR protein expression in the different molecular subtypes of human ovarian cancer, showing low PR expression in clear cell and mucinous tumors (3 and 18%, respectively) and moderate to high expression in serous (35%), low-grade serous (64%) and endometriod tumors (67%) (Fig. 1). Figure 1. Schematic representation of PR action as target in different subtypes of ovarian cancer. SMS, senescence messaging secretome. As with other scientific studies, this manuscript provides clues to mechanism of action of progestins but at the same time raises many new questions. For example, what is the cause for the biphasic dose response to progestins? Does this imply a critical role for the other progesterone-binding proteins? And can this explain prior discrepant results showing both progesterone-induced apoptosis and proliferation? Importantly, given the increasing realization of diverse roles of the two PR isoforms, PR-A and PR-B, what can we expect to see from loss (or overexpression) of the PR-A isoform? And, finally, what is the role of PR action in the tumor microenvironment? This study has important translational implications, in that PR agonists (and antagonists) are clinically available. However, to date, small clinical trial cohorts have demonstrated modest activity in ovarian cancer.3 Many of these trials have limitations, and it might be time to revisit the potential use of progestins and anti-progestins in ovarian cancer therapy. Several steps can be taken. First and foremost is the need to use biomarkers to predict active PR signaling in the tumor (and possibly the stroma). In addition, clinical trials mostly occurred in chemotherapy-resistant, late-stage disease; however, data at hand suggest that there is progressive loss of PR during tumor progression. This might imply a place for progestin in early-stage tumorigenesis, perhaps from precursors like endometriosis, which predispose to endometrioid, clear cell and low-grade serous ovarian tumors (Fig. 1). In support of this rationale is the fact that PR expression is highest in low-grade serous and endometrioid (although not clear cell) tumors,2,4 further suggesting the early roles of PR-mediated pathogenesis and the potential for PR-targeted therapy or prevention, at least in some histological subtypes. And finally, the idea of using synthetic lethal approaches, i.e., combining progestin with drugs showing increased efficacy against senescent cells would be warranted. One approach that comes to mind is the use of immune therapies. Senescence of epithelial tumor cells leads to the release of inflammatory proteins and mediators of extracellular matrix (also called the senescence- messaging secretome or SMS),5,6 which can act as activators of the innate immune response, leading to tumor cell clearance. In addition, active immunization with senescent cell-derived, tumor antigen-based vaccines may act in synergy to the potentially immunogenic in vivo inducers of senescence (like progestins, for example), providing additional benefits in both therapeutic as well as preventive approaches, although the efficacy of such approaches remains to be determined. In summary, a renewed effort to understand steroid hormone action in ovarian cancer (in both the epithelium and stroma) may lead to novel approaches for targeted therapies with limited toxicities and therapeutic benefit.

Due to its role in aging and antitumor defense, cellular senescence has recently attracted increasing interest. However, there is currently no single specific marker that can unequivocally detect senescent cells. Here, we identified α-L-fucosidase (α-Fuc) as a novel sensitive biomarker for cellular senescence. Regardless of the stress stimulus and cell type, α-Fuc activity was induced in all canonical types of cellular senescence, including replicative, DNA damage- and oncogene-induced senescence. Strikingly, in most models the degree of α-Fuc upregulation was higher than the induction of senescence-associated β-galactosidase (SA-β-Gal), the current gold standard for senescence detection. As α-Fuc is convenient and easy to measure, we suggest its utility as a valuable marker, in particular in cells with low SA-β-Gal activity.

Glioblastoma multiforme (GBM) arises from cells in the brain called astrocytes and can form in many different parts of the brain, including the cerebellum and spinal cord. GBM is both the most frequent and also the most deadly adult brain tumor, with an incidence rate of between two to three per 1,000 people. Post-surgical standard of care usually consists of radiation combined with temozolomide and dexamethasone.1 However, even with aggressive intervention, GBM continues to be an aggressive, progressive disease with extremely high mortality rates. Because of the severity of the disease and the poor median and overall survival statistics for GBM patients, the need for identifying new and more effective targets and pathways to treat GBM is obvious and critical. Caveolae are submicroscopic invaginations found in the cell membranes of a variety of tissue types and play an ever-expanding role in multiple cellular processes.2 The predominant structural components of caveolae is the trans-membrane-bound protein caveolin-1 (Cav-1). Cav-1 has been extensively studied and its activities characterized in a number of cancers, for which it has been shown to function as either a tumor suppressor or tumor promoter depending on tissue type and the underlying cellular proteome. Cav-1 has only recently received increased attention in the brain cancer field, with approximately 25 published papers appearing in PubMed on Cav-1 and human brain cancers. The in vitro characterizations of the role of Cav-1 in GBM have largely been undertaken by Martin and colleagues, where Cav-1 was identified it as a tumor suppressor, affecting proliferation in part through modulating TGFB/SMAD signaling.3 In a new study, Quann et al.4 expanded upon this previous work by creating a stable Cav-1-overexpressing cell line based on the common GBM-derived cell line U-87MG. Microarray analyses comparing Cav-1-overexpressing cells to control cells established that critical cell cycle genes and cell survival proteins and pathways, such as cyclin D1 and AKT/mTOR, respectively, were downregulated. Perhaps more importantly, using a mouse xenograft model, they found that Cav-1-overexpressing tumors were significantly less proliferative and less invasive when compared with control cells, with explanted tumors displaying marked silencing of cell cycle and protein biosynthetic pathways. Finally, Cav-1-overexpressing cells were found to be sensitized to the antitumor effects of the most commonly used chemotherapy agent, temozolomide, and were significantly more likely to undergo apoptosis after treatment as compared with controls cells. These results extend the role of Cav-1 into the prognosis and possibly the treatment of GBM. Interestingly, one of the most frequent point mutants in GBM occurs in the tumor suppressor protein, p53.5 In certain p53-mutant tumors, glucose restriction, which induces oxidative stress, resulted in activation of autophagy and an autophagy-dependent degradation of mutant p53, leading to a feedforward acceleration of autophagy and tumor inhibition.6 Furthermore, in the tumor stroma, Cav-1 expression has been found to be similarly downregulated by oxidative stress when autophagy was activated, which, in turn, resulted in a feedforward upregulation of stromal autophagy.7 Termed the “reverse Warburg effect,” this tumor microenvironment is defined by enhanced stromal aerobic glycolysis, oxidative stress and localized inflammation, which, in turn, promotes tumor cell survival through cancer cell parasitism of nutrients released from the autophagic stromal cells. Collectively, these results suggest that the expression levels of Cav-1, and certain mutant forms of p53, may be regulated in a similar fashion by autophagy, leading, however, to different phenotypic outcomes depending upon whether their expression occurs in the tumor or in the stromal component. Thus, it will be very important to determine whether and how Cav-1 and mutant forms of p53 cross-talk with the stroma and define their relationship with autophagy and the metabolism of tumor cells. While the current study was limited to one cell line and an ectopic xenograft mouse model, the observations are extremely interesting, and further investigations are clearly warranted and encouraged. Given the great advances in mouse modeling of brain malignancies and the recent focus on perfecting non-invasive imaging of drug sensitivity/responses,8 it is likely that more dynamic and comprehensive investigations into Cav-1 as an etiological mediator of GMB progression and treatment will be forthcoming. (Fig. 1) Figure 1. Caveolin-1 plays a central role in Glioblastoma multiforme onset and progression and may be a biomarker for sensitivity to chemotherapy. Red lines denote genes or pathways inhibited by Cav-1, while green lines indicate those that are ...

A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects.

Neuronal survival is dependent upon the retinoblastoma family members, Rb1 (Rb) and Rb2 (p130). Rb is thought to regulate gene repression, in part, through direct recruitment of chromatin modifying enzymes to its conserved LXCXE binding domain. We sought to examine the mechanisms that Rb employs to mediate cell cycle gene repression in terminally differentiated cortical neurons. Here, we report that Rb loss converts chromatin at the promoters of E2f-target genes to an activated state. We established a mouse model system in which Rb-LXCXE interactions could be induciblely disabled. Surprisingly, this had no effect on survival or gene silencing in neuronal quiescence. Absence of the Rb LXCXE-binding domain in neurons is compatible with gene repression and long-term survival, unlike Rb deficiency. Finally, we are able to show that chromatin activation following Rb deletion occurs at the level of E2fs. Blocking E2f-mediated transcription downstream of Rb loss is sufficient to maintain chromatin in an inactive state. Taken together our results suggest a model whereby Rb-E2f interactions are sufficient to maintain gene repression irrespective of LXCXE-dependent chromatin remodeling. © 2013 Landes Bioscience.

Epidemiological data suggest an important role of vitamin D signaling in cancer development and progression, and experimental studies demonstrate that the active vitamin D metabolite 1α, 25-dihydroxyvitamin D₃ (1,25D₃) has broad spectrum antitumor activity. Hypercalcemia has often been suggested to limit the clinical application of these data. The 14-epi-analog of 1,25D₃, inecalcitol [19-nor-14-epi-23-yne-1,25-(OH)₂D₃; TX522], was developed to have superagonistic antitumor activities but low hypercalcemia potential. We examined the antitumor activity of inecalcitol and the underlying mechanisms in a murine squamous cell carcinoma (SCC) model system. In vitro, compared with 1,25D₃, inecalcitol showed enhanced vitamin D receptor (VDR)-mediated transcriptional activity. Inecalcitol suppressed SCC cell proliferation in a dose-dependent manner with an IC50 value 30 times lower than that of 1,25D₃. Both inecalcitol and 1,25D₃ induced a comparable level of G0/G₁ cell cycle arrest in SCC cells. The level of apoptosis induced by inecalcitol was markedly higher than that of 1,25D₃. Apoptosis was mediated through the activation of the caspase 8/10- caspase 3 pathway. Further, inecalcitol markedly inhibited the mRNA and protein expression of c-IAP1 and XIAP compared with 1,25D₃. In vivo, inecalcitol inhibits SCC tumor growth in a dose-dependent fashion. Notably, inecalcitol induced a significantly higher level of apoptosis in the SCC xenograft model. While in vitro inecalcitol demonstrates apparent enhanced VDR binding and antiproliferative effects compared to 1,25D₃, in vivo these advantages disappear; at doses of inecalcitol that have equivalent antitumor effects, similar hypercalcemia is seen. This may be explained by the pharmacokinetics of 1,25D₃ vs. inecalcitol and attributed to the much shorter serum half-life of inecalcitol.We show that inecalcitol has potent antitumor activity in the SCC model system, and this is associated with a strong induction of apoptosis. These findings support the further development of inecalcitol in cancer treatment.

Although intensive regimens of antiretroviral therapy (HAART) reduce viral loads to undetectable levels in the circulation, HIV quickly resumes active replication when treatment is interrupted due to the emergence of the virus from latent reservoirs.1,2 Although it is difficult to exclude the possibility that slowly replicating viruses persist in sanctuary sites that are poorly accessed by the antiviral drugs, the consensus in the field is that the virus emerges from a small population of resting memory CD4 T cells (~1 in 106 cells) harboring silenced HIV proviruses. Eliminating this latent reservoir is particularly challenging since it is established early during infection, is extremely stable (with an estimated half-life of 44 mo), and can be replenished during episodes of viremia or by homeostatic replacement of latently infected cells. Since latently infected cells express minimal levels of viral proteins they are invisible to the immune system and unaffected by antiretroviral drugs. Recent curative strategies have therefore focused on developing pharmaceutical agents that can induce HIV expression in latently infected cells and then purging these cells by antiviral immune responses, viral cytopathic effects or even cell-targeted killing strategies (the rhetorically named “shock and kill” strategy).1,2 In the last three months there has been a flurry of provocative papers, including the report by Boehm et al.3 published in a previous issue of Cell Cycle, demonstrating that JQ1 and other clinically useful bromodomain (BET family) inhibitors can efficiently reverse HIV latency in established cell lines and in certain primary cell models for HIV latency.4-7 Thus the BET proteins have been validated as potential new targets for HIV induction strategies. But, how do these compounds work? All BET proteins are highly conserved transcriptional regulators capable of binding to acetyl-lysine residues found on histones and many transcription factors through tandem bromodomains. Perhaps the best-studied BET family member is BRD4, which binds the positive transcription elongation factor b (P-TEFb) the essential cofactor for the HIV Tat gene (Fig. 1). It was therefore postulated that BET family inhibitors induce HIV expression by inhibiting BRD4 interactions with P-TEFb and thereby favoring enhanced Tat binding.4,6,8 Consistent with this model, and the inhibitor studies, knockdown of BRD4 by shRNA also results in potent HIV induction. Figure 1. Models for HIV induction by BET inhibitors. (A) Inhibition of BRD4 blocks its association with P-TEFb and permits enhanced association with the HIV transactivator protein Tat. The Tat:P-TEFb complex is recruited to the HIV promoter and ... Boehm et al.3 have now discovered that in addition to BRD4, a second BET protein, BRD2, also regulates HIV latency. Knockdown of BRD2 by shRNA activates HIV transcription to an even higher extent than knockdown of BRD4 and to levels comparable to JQ1 treatment of cells. In contrast to BRD4, BRD2 associates directly with transcription complexes and proteins required for chromatin remodeling. Thus it seems likely that BRD2 can enhance HIV transcription in response to JQ1 and other BET inhibitors. But how can it act as a repressor in the absence of BET inhibitors? One clue comes from the observation that BRD2 interacts directly with the E2F1 transcription factor. Earlier studies showed that E2F1 can bind together with NFκB p50 to the HIV enhancer and block HIV transcription mediated by the NFκB p50/p65 heterodimer.9 It seems reasonable to postulate that BRD2 is recruited to the HIV LTR by E2F1/p50 heterodimers, and then recruits repressor complexes carrying acetylated lysine residues (Fig. 1). Thus when BRD2 is inhibited, these interactions are blocked, and repressor complexes are exchanged for activators. Although further work will be needed to confirm the molecular details of the two mechanisms outlined in Figure 1, it is important to note at this stage that the high potency of JQ1 and related BET inhibitors in mediating HIV induction could be due to the targeting of multiple bromodomain proteins that regulate HIV transcription. Indeed, preliminary data emerging from several laboratories suggests that several other bromodomain proteins in addition to BRD4 and BRD2 can also contribute to the maintenance of HIV latency. It’s a safe bet that studies of these important regulatory mechanisms will reshape our understanding of HIV latency in the years to come.

Our genomes are attacked constantly by reactive oxygen species generated as by-products of metabolic processes or induced by exogenous sources, such as UV light and cigarette smoke. Tens of thousands of DNA lesions are estimated to occur daily in each human cell. To cope with these genomic insults and preserve their genetic information, cells have evolved a set of conserved mechanisms collectively called the DNA damage response (DDR). These highly orchestrated signaling and DNA repair networks are tightly controlled by various post-translational modifications (PTMs), including, predominantly, phosphorylation and ubiquitination.1 Double-strand breaks (DSBs) are the most deleterious type of DNA lesion. Under physiological conditions, the majority of DNA breakages that enter or arise in mitosis originate during S phase, when DNA replication machinery approaches unstable or challenging DNA structures, such as fragile sites.2 Once a DSB occurs, the protein kinases ataxia-telangiectasia mutated protein (ATM) and DNA-dependent protein kinase (DNA-PK) activate the DDR by phosphorylating H2AX to generate γ-H2AX. This factor recruits mediator of DNA damage checkpoint 1 (MDC1), which mediates the DDR in the vicinity of a DSB. MDC1 promotes the phosphorylation-dependent recruitment of DDR-sensor proteins, such as the Mre11-Rad50-Nbs1 complex (MRN).1 This formation is deemed the “proximal” DDR, and its components are early markers of DSBs. MDC1 also functions as a platform for the integration of ubiquitination events. As a second PTM, ubiquitination orchestrates the “distal” DDR. Phosphorylated MDC1 recruits the E3 ubiquitin ligase, RNF8, which mobilizes two parallel ubiquitin chain cascades: Lys48-linked ubiquitin and Lys63-linked ubiquitin.3 These chains are essential for the recruitment of “caretaker” gene products and DNA repair proteins, such as 53BP1, BRCA1 and Rad51, which constitute the fully activated DDR.4,5 Precise spatiotemporal synchronization of proximal and distal DDR events is essential for efficient DNA repair. The fully activated DDR, coordinated by phosphorylation and ubiquitination, can occur during G1, S or G2 phases of the cell cycle. In contrast, only the proximal DDR exists in mitosis.6 This truncated DDR can sense and mark DNA damage but cannot execute DNA repair, owing to its inability to activate ubiquitin signaling cascades. Cells in mitosis preserve their damaged DNA until mitotic exit, when the DDR resumes full activation, and the lesion can be repaired.7 Inactivation of DNA repair in mitosis likely occurs because of compacted and condensed mitotic chromosomes that obstruct RNF8 and the other distal DDR components to the proximal DDR assembly. In addition, the complexity of mitotic processes and the limited duration of mitosis could explain why chromosomal segregation and cytokinesis preclude DNA repair. In a recent issue of Cell Cycle, Macurek et al.8 described the maintenance of the proximal DDR during mitosis and the mechanisms by which damaged mitotic DNA is marked and preserved but not repaired. These authors identified the strict, cell cycle-dependent regulation of Wip1/PPM1D, a phosphatase that dephosphorylates γ-H2AX and other elements of the DDR.9 Wip1 protein expression and enzymatic activity are low during G1, increase gradually as cells enter S phase and peak during G2. When cells enter mitosis, Wip1 phosphatase activity is downregulated rapidly by ubiquitin-dependent Wip1 proteasomal degradation coordinated by APC/CCdc20 (Fig. 1). Macurek et al.8 demonstrated that the Wip1 catalytic domain is phosphorylated at multiple sites during mitosis by cyclin B/Cdk1 and other unidentified kinases. Phosphorylation of Wip1 inactivates its phosphatase activity. Endogenous Wip1 phosphorylation may regulate its phosphatase activity or may only be a prerequisite for ubiquitination and consequent proteasomal degradation. Nevertheless, the work of Macurek et al.8 provides evidence that phosphorylation and proteasomal degradation occur cooperatively to inactivate and remove Wip1 from the cellular milieu during mitosis. Wip1 is essential for the timely inactivation of the DDR. After damaged DNA is repaired, the removal of Wip1 as the cell proceeds to mitosis allows only the proximal DDR to assemble on existing DNA lesions. Macurek et al.8 elucidate the function of Wip1 phosphatase during the cell cycle and underscore the importance of Wip1 downregulation in the preservation of a truncated DDR during mitosis. Additional research is warranted to address the molecular details of distal DDR inactivation during mitosis. Figure 1. Wip1 inactivation and degradation lead to truncated DDR in mitosis. Wip1 phosphatase terminates the DDR. Here, the concept of yin-yang is used to represent the harmony of phosphorylation (p = Yin-dark) and ubiquitination (Ub = Yang-white) ...

Primary cilia are microtubule-based solitary sensing structures on the cell surface that play crucial roles in cell signaling and development. Abnormal ciliary function leads to various human genetic disorders, collectively known as ciliopathies. Outer dense fiber protein 2 (Odf2) was initially isolated as a major component of sperm-tail fibers. Subsequent studies have demonstrated the existence of many splicing variants of Odf2, including Cenexin1 (Odf2 isoform 9), which bears an unusual C-terminal extension. Strikingly, Odf2 localizes along the axoneme of primary cilia, whereas Cenexin1 localizes to basal bodies in cultured mammalian cells. Whether Odf2 and Cenexin1 contribute to primary cilia assembly by carrying out either concerted or distinct functions is unknown. By taking advantage of odf2-/- cells lacking endogenous Odf2 and Cenexin1, but exogenously expressing one or both of these proteins, we showed that Cenexin1, but not Odf2, was necessary and sufficient to induce ciliogenesis. Furthermore, the Cenexin1-dependent primary cilia assembly pathway appeared to function independently of Odf2. Consistently, Cenexin1, but not Odf2, interacted with GTP-loaded Rab8a, localized to the distal/subdistal appendages of basal bodies, and facilitated the recruitment of Chibby, a centriolar component that is important for proper ciliogenesis. Taken together, our results suggest that Cenexin1 plays a critical role in ciliogenesis through its C-terminal extension that confers a unique ability to mediate primary cilia assembly. The presence of multiple splicing variants hints that the function of Odf2 is diversified in such a way that each variant has a distinct role in the complex cellular and developmental processes.

Lung carcinoma is the primary cause of death by cancer. In the last decades, together with improvements in anesthetic and surgical techniques, several new drugs for chemotherapy or biotherapy have been made available. Thus, while metastatic patients are treated by chemotherapy and/or biologically targeted therapies, in initial stages of disease (I−II), surgery remains the cornerstone of treatment. Locally advanced disease is generally treated in the setting of multimodal combinations, including chemotherapy, radiotherapy and surgery, whose respective indications have been refined in the last years. However, these developments provided only limited increase in survival, and a large room for improvement persists.1 If clinical stage of non-small cell lung cancer (NSCLC) is the major determinant of treatment strategy, pathologic stage is currently considered the most important determinant of prognosis in resected patients and is the most important parameter determining the choice of adjuvant treatments. However, within the same stage of disease, large prognostic variability obviously exists and sensitivity to adjuvant treatments is heterogeneous. Furthermore, at present there is no consensus regarding the usefulness of post-operative follow-up. Repeated chest X-ray, CT scan, fiberoptic bronchoscopy or PET scans have been proposed, with enormous variations in medical resource utilization and costs. The identification of patients with poor prognosis within a determined stage would be useful for individual tailoring follow-up procedures.2 The identification of prognostic factors is of major importance to develop adequate management strategies. For instance, subgroups of patients who benefit more from peri-operative chemotherapy still need to be identified more precisely, to define those for whom the benefit/risk ratio of neo-adjuvant or adjuvant treatments is the most favorable. This issue is of particular importance in early stages, especially stage IB, in which no strict guidelines are available and which represent the majority of stage I-II resected lung cancer. Several prognostic markers have been proposed in the last years, including characteristics of tumoral immune microenvironment, growth factors and their receptors, markers of systemic inflammation, peptides/proteins/enzymes produced by tumor cells with impact on cell cycle, metabolism and sensitivity to chemotherapy, suggesting the paramount importance of this topic.3-5 The study by Galluzzi et al.6 reports evidence that intratumoral levels of hepatic lipase LIPC, as assessed by immunohistochemistry, positively correlate with disease outcome in two independent series of non-metastatic NSCLC patients treated by surgery, possibly in a multimodality setting. Furthermore, the authors found that in one of two series, patients with tumors expressing low levels of LIPC had better survival when receiving adjuvant cis-platinum-based chemotherapy, whereas patients with tumors expressing high levels of LIPC had survival unaffected by post-operative chemotherapy. Although validation by specifically designed prospective clinical trials is mandatory to draw definitive conclusion on possible clinical applications, LIPC seems an extremely interesting marker in resected NSCLC from both a prognostic and a predictive point of view. The same team previously identified LIPC as a cisplatinum response modifier by a genome-wide siRNA-based screen in human NSCLC A549 cells.7 Together with LIPC, 84 other functional cisplatinum response modifiers were identified, including several proteins known to regulate platinum-induced cell death (e.g., the pro-apoptotic cytoplasmic adaptor APAF-1 and the anti-apoptotic Bcl-2 family member BCL-XL) as well as factors with no obvious links with platinum-elicited signaling pathways, including another enzyme (pyrixodal kynase) and the hepatic lipase LIPC. Thus the mechanisms responsible for the impact of LIPC on the effect of platinum-based chemotherapy remain to be elucidated. Although discovered as a platinum-response modifier with possible impact on survival in patients receiving platinum-based chemotherapy, LIPC was shown to have a strong prognostic impact also in patients who did not undergo chemotherapy. Thus, the mechanisms explaining the impact on prognosis of LIPC are even more obscure and probably intriguing. Response to therapy and survival in cancer patients probably depends not only on the ability of treatments in removing or killing proliferating tumor cells, but on a complex interaction between disease, treatments and respective impacts on host reaction, whose determinants are not completely elucidated.8 Identification of mechanisms responsible for LIPC impact on response to chemotherapy as well as on survival in chemotherapy naive patients could provide interesting insights in the general knowledge of mechanisms of survival in cancer patients.

miR-206, a member of the so-called myomiR family, is largely acknowledged as a specific, positive regulator of skeletal muscle differentiation. A growing body of evidence also suggests a tumor suppressor function for miR-206, as it is frequently downregulated in various types of cancers. In this study, we show that miR-206 directly targets cyclin D1 and contributes to the regulation of CCND1 gene expression in both myogenic and non-muscle, transformed cells. We demonstrate that miR-206, either exogenous or endogenous, reduces cyclin D1 levels and proliferation rate in C2C12 cells without promoting differentiation, and that miR-206 knockdown in terminally differentiated C2C12 cells leads to cyclin D1 accumulation in myotubes, indicating that miR-206 might be involved in the maintenance of the post-mitotic state. Targeting of cyclin D1 might also account, at least in part, for the tumor-suppressor activity suggested for miR-206 in previous studies. Accordingly, the analysis of neoplastic and matched normal lung tissues reveals that miR-206 downregulation in lung tumors correlates, in most cases, with higher cyclin D1 levels. Moreover, gain-of-function experiments with cancer-derived cell lines and with in vitro transformed cells indicate that miR-206-mediated cyclin D1 repression is directly coupled to growth inhibition. Altogether, our data highlight a novel activity for miR-206 in skeletal muscle differentiation and identify cyclin D1 as a major target that further strengthens the tumor suppressor function proposed for miR-206.

Metformin is a well-established diabetes drug that prevents the onset of most types of human cancers in diabetic patients, especially by targeting cancer stem cells. Metformin exerts its protective effects by functioning as a weak "mitochondrial poison," as it acts as a complex I inhibitor and prevents oxidative mitochondrial metabolism (OXPHOS). Thus, mitochondrial metabolism must play an essential role in promoting tumor growth. To determine the functional role of "mitochondrial health" in breast cancer pathogenesis, here we used mitochondrial uncoupling proteins (UCPs) to genetically induce mitochondrial dysfunction in either human breast cancer cells (MDA-MB-231) or cancer-associated fibroblasts (hTERT-BJ1 cells). Our results directly show that all three UCP family members (UCP-1/2/3) induce autophagy and mitochondrial dysfunction in human breast cancer cells, which results in significant reductions in tumor growth. Conversely, induction of mitochondrial dysfunction in cancer-associated fibroblasts has just the opposite effect. More specifically, overexpression of UCP-1 in stromal fibroblasts increases β-oxidation, ketone body production and the release of ATP-rich vesicles, which "fuels" tumor growth by providing high-energy nutrients in a paracrine fashion to epithelial cancer cells. Hence, the effects of mitochondrial dysfunction are truly compartment-specific. Thus, we conclude that the beneficial anticancer effects of mitochondrial inhibitors (such as metformin) may be attributed to the induction of mitochondrial dysfunction in the epithelial cancer cell compartment. Our studies identify cancer cell mitochondria as a clear target for drug discovery and for novel therapeutic interventions.

Squamous cell carcinomas of the head and neck originate in the mucosal linings of the oral cavity, the oropharynx, the larynx and hypopharynx. Most patients present with advanced stages of disease and are treated by surgery with postoperative (chemo) radiotherapy or chemoradiation, the combination of systemic cisplatin with concomitant locoregional radiotherapy, sometimes followed by salvage surgery. Patients who are unfit to receive chemoradiation are treated with a combination of cetuximab, an antibody inhibiting the EGF receptor, combined with locoregional radiotherapy, also called bioradiation.1 Since last decades it has been shown that human papillomaviruses (HPV), known as the cause of cervical cancer, also cause a subgroup of head and neck cancers, most particularly those arising in the oropharynx. The proportion of HPV-attributable disease varies over the world, but appears to increase rapidly. In the USA and Canada the proportion of HPV+ve tumors is between 50 and 70%,2,3 and in Europe usually somewhat less (30%). However, both the proportion and incidence rates of HPV+ve tumors are increasing.4 In the Netherlands the proportion increased from 5% in 1990 to 30% in 2010.5 Tumors caused by HPV infection form a different disease entity. They are different at the molecular and clinical level. HPV+ve tumors have a very favorable prognosis when compared with HPV−ve tumors. In fact the predominant factor that predicts prognosis of oropharyngeal cancer is the presence of HPV.2,3 The prognosis is so favorable that two studies have been initiated to de-intensify therapy: the RTOG1016 trial in the USA and the De-ESCALaTE HPV study in Europe. In both studies patients with HPV+ve tumors are randomized between a chemoradiation arm and a bioradiation arm, with the aim to reduce toxicity in the bioradiation arm while maintaining favorable prognosis. However, it could be questioned whether cetuximab is the optimal substitute of cisplatin for HPV+ve tumors. In this issue Li and Johnson6 provide data that there might be other options at the horizon, also less toxic than cisplatin and more targeted to HPV. The HPV genome encodes two viral oncoproteins named E6 and E7. The virus uses these proteins to create an S-phase environment in the host cell to allow viral replication using the host cell DNA replication machinery. The E6 protein binds and inactivates p53 and the E7 protein the pocket proteins pRb, p107 and p130, key proteins in the regulation of the G1 and G2 cell cycle checkpoints. These host proteins are subsequently ubiquinated and targeted for proteosomal degradation. Previously it has been shown that the knockdown of these E6/E7 genes inhibits proliferation of HPV-positive head and neck cancer cell lines.7 In this issue Li and Johnson,6 focusing on E6 and the p53 pathway, convincingly show that the cell cycle arrest and apoptosis caused by E6(/E7) knockdown is p53 and p21 mediated, as expected. Intriguingly, they also showed that bortezomib, a drug that inhibits proteosomal protein degradation and approved for treatment of multiple myeloma, was able to liberate and restore p53 and p21 expression specifically in HPV+ve cell lines, also causing cell death. This would suggest that bortezomib might be an interesting alternative for cisplatin in the combination with radiotherapy for HPV+ve tumors. Bortezomib is considerably less toxic than cisplatin. Obviously a lot of additional work needs to be done before considering a clinical study. First, all data reported by Li and Johnson were collected by in vitro experiments. Therapy experiments in mouse models of xenografted cell lines or even better, human-in-mouse tumor models,8 are required. In addition, other questions remain. Although the authors convincingly showed that p53 and p21 are upregulated by bortezomib in HPV+ve cell lines and cause apoptosis, the HPV+ve cell lines are not more sensitive to bortezomib than the HPV-ve cell lines. That is somewhat unexpected. Moreover, one of the HPV+ve cell lines showed a G1 arrest and the other two a G2 arrest after bortezomib treatment. Although p53 and p21 play a role in both checkpoints, the nature of this difference should be solved, and also whether it is relevant in the context of irradiation. Bortezomib-based treatment protocols would include radiotherapy. Notwithstanding, irrespective of these considerations that clearly deserve attention, the observation is very interesting and should be further evaluated.

Emerging evidence suggests that the tumor microenvironment plays a critical role in regulating cancer stem cells (CSCs) and tumor progression through both autocrine and paracrine signaling. Elevated production of bone morphogenetic proteins (BMPs) from human ovarian cancer cells and stroma has been shown to increase CSC proliferation and tumor growth. Here, we report that Lin28, a stem cell factor, binds to BMP4 mRNA in epithelial ovarian carcinoma cells, thereby promoting BMP4 expression at the post-transcriptional level. As co-expression of Lin28 and Oct4 (another stem cell factor) has been implicated in ovarian cancer CSCs, we also determined that high levels of Lin28 are associated with an unfavorable prognosis when co-expressed with high levels of Oct4. Together, these findings uncover a new level of regulation of BMP4 expression and imply a novel Lin28/Oct4/BMP4-mediated mechanism of regulating ovarian tumor cell growth, thus holding potential for the development of new strategies for the diagnosis and treatment of ovarian cancer.

Urothelial cell carcinoma (UCC) represents the most common malignancy of the urinary tract. It is estimated that there were 73,500 new cases of UCC and 15,000 deaths for both sexes in the United States in 2012.1 As the final recipient and reservoir of urine, the urothelium is inevitably exposed to carcinogens present in tobacco, which can create stepwise molecular alterations that eventually lead to transformation of urothelial cells. This concept is supported by epidemiologic studies that state that tobacco consumption is the most important factor for the development of this disease, contributing to approximately 50% of all cases.2 UCC is a heterogeneous disease; 70% of newly diagnosed bladder tumors are non-muscle invasive (NMIBC) and show a much better prognosis compared with those that invade the detrusor muscle (MIBC).3 From the molecular point of view, evidence in the literature supports the existence of two distinct pathogenetic pathways involved in UCC development, corresponding to these two distinct (NMIBC and MIBC) biological and clinical phenotypes. In fact, while disruption to the PI3K-AKT-mTOR pathway and alterations in the tyrosine kinase receptor gene FGFR3 and the oncogene HRAS are associated with NMIBC, the main genetic alterations underlying MIBC involve tumor suppressor genes encoding proteins that regulate cell cycle and apoptosis pathways, including TP53, CDKN2A, CCND1, CDKN1B and RB1.4 Recent works have also suggested epigenetic mechanisms like promoter methylation in the pathogenesis of this disease.5,6 Understanding the multistep accumulation of genetic and epigenetic alterations related to environmental factors in the development and progression of this disease is crucial for the discovery of biomarkers that might be useful in predicting the behavior and prognosis of UCC in individual patients. In an intent to understand the genetic/epigenetic alterations that accumulates in the process of UCC development, the group led by Hoque has developed a very interesting cellular model for smoking-induced UCC.7 In this study, SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. The authors observed morphological alterations and increased cell proliferation after 4 mo of exposure to CSE. After 6 mo the treated cells showed anchorage-independent growth and an increase in the migratory and invasive potential. The observed properties after 6 mo of CSE treatment were not noticeable at 4 mo of treatment, suggesting that some driver gene/genes might alter due to prolonged exposure to tobacco. In order to assess key molecular alterations occurring in CSE-treated cells, the authors evaluated the expression level of specific genes involved in the PI3K-AKT pathway and found upregulation of AKT1, AKT2, HRAS, RAC1 and downregulation of PTEN, FOXO1, MAPK1 and PDK1 among altered genes. Interestingly, immunohistochemistry for FOXO1 performed on UCC samples showed higher level and frequency of expression in the smokers group compared with non-smokers. In their view, this might reflect the fact that FOXO1 in smokers is subjected to an enhanced phosphorylation by AKT with consequent cytoplasmic translocation. Using genome-wide methylation analysis, the authors also found differentially methylated genes in CSE-treated and untreated HUC1 cell lines. They further confirmed methylation status of MCAM, DCC and HIC1 in CSE-treated and untreated HUC1 cell lines by a complementary approach (QMSP). These findings support that epigenetic alterations are simultaneously related to smoke-associated UCC. As stated above, P53 represents the most frequently dysregulated gene in UCC, especially in the pathway related to muscle-invasive tumors. However, in this work, translational and transcriptional levels of P53 were unchanged after 6 mo of CSE treatment. In this regard, the authors speculate that it could be possible that a prolonged period of exposure might be necessary to alter the P53 pathway that is involved in the progression of NMIBC to MIBC. It would be therefore be useful to specifically investigate the mechanisms and the alterations necessary for this to happen, especially on a structural basis like LOH and copy number alterations. Detailed molecular studies using this cellular model will eventually help to identify related genes and pathways that are altered due to smoking in a stepwise fashion. Ultimately, accumulated knowledge will help to develop personalized management of UCC patients.

Analysis of microarrays performed in p53-, TAp63α- and ΔNp63α-inducible SaOs-2 cell lines allowed the identification of NCF2 mRNA upregulation in response to p53 induction. NCF2 gene encodes for p67phox, the cytosolic subunit of the NADPH oxidase enzyme complex. The recruitment of p67phox to the cell membrane causes the activation of the NADPH oxidase complex followed by the generation of NADP+ and superoxide from molecular oxygen. The presence of three putative p53 binding sites on the NCF2 promoter was predicted, and the subsequent luciferase and chromatin immunoprecipitation assays showed the activation of NCF2 promoter by p53 and its direct binding in vivo to at least one of the sites, thus confirming the hypothesis. NCF2 upregulation was also confirmed by real-time PCR in several cell lines after p53 activation. NCF2 knockdown by siRNA results in a significant reduction of ROS production and stimulates cell death, suggesting a protective function of Nox2-generated ROS in cells against apoptosis. These results provide insight into the redox-sensitive signaling mechanism that mediates cell survival involving p53 and its novel target NCF2/p67phox.

Growth-promoting and nutrient/mitogen-sensing pathways such as mTOR convert p21- and p16-induced arrest into senescence (geroconversion). We have recently demonstrated that hypoxia, especially near-anoxia, suppresses geroconversion. This gerosuppressive effect of hypoxia correlated with inhibition of the mTOR/S6K pathway but not with modulation of the LKB1/AMPK/eEF2 pathway. Here we further show that mTOR inhibition is required for gerosuppression by hypoxia, at least in some cellular models, because depletion of TSC2 abolished mTOR inhibition and gerosupression by hypoxia. Also, in two cancer cell lines resistant to inhibition of mTOR by both p53 and hypoxia, hypoxia did not suppress geroconversion. Therefore, the effects of hypoxia on the oxygen-sensing mTOR pathway and geroconversion are cell type-specific. We also briefly discuss replicative senescence, organismal aging and free radical theory.

FBXW7, a component of E3 ubiquitin ligase, plays an important role in mitotic checkpoint, but its role remains unclear. Aurora B is a mitotic checkpoint kinase that plays a pivotal role in mitosis by ensuring correct chromosome segregation and normal progression through mitosis. Whether Aurora B and FBXW7 are coordinately regulated during mitosis is not known. Here, we show that FBXW7 is a negative regulator for Aurora B. Ectopic expression of FBXW7 can suppress the expression of Aurora B. Accordingly, FBXW7 deficiency leads to Aurora B elevation. Mechanistic studies show that all FBXW7 isoforms are negative regulators of Aurora B expression through ubiquitination-mediated protein degradation. Aurora B interacts with R465 and R505 residues of WD 40 domain of FBXW7. Significantly, inverse correlation between FBXW7 and Aurora B elevation is translated into the deregulation of mitosis. FBWX7 expression mitigates Aurora B-mediated cell growth and mitotic deregulation. In addition, FBXW7 reduces the percentage of multinucleated cells caused by Aurora B overexpression. These data suggest that FBXW7 is an important negative regulator of Aurora B, and that the loss or mutation of FBXW7 as seen in many types of cancer could lead to an abnormal elevation of Aurora B and result in deregulated mitosis, which accelerates cancer cell growth.

The circadian clock is a global regulatory mechanism that confers daily rhythmicity on many biochemical and physiological functions, including DNA excision repair in mammalian organisms. Here, we investigated the effect of the circadian clock on the major DNA damage response pathways by using mouse cell lines mutated in genes encoding proteins in the positive (Bmal1, CLOCK) or negative (Cry 1/2, Per 1/2) arms of the transcription-translation feedback loop that generates the circadian clock. We find that cells mutated in these genes are indistinguishable from wild-type in their response to UV, ionizing radiation and mitomycin C. We conclude that either the majority of DNA damage response reactions are not controlled by the circadian clock or that, even if such a control exists at the organism level, it is supplanted by homeostatic control mechanisms at the cellular level in tissue culture. We suggest that caution must be exercised in extrapolating from experiments in tissue culture to whole animals with respect to the effect of the circadian clock on cellular response to DNA damaging agents.

Origin recognition complex (ORC) is highly dynamic, with several ORC subunits getting posttranslationally modified by phosphorylation or ubiquitination in a cell cycle-dependent manner. We have previously demonstrated that a WD repeat containing protein ORC-associated (ORCA/LRWD1) stabilizes the ORC on chromatin and facilitates pre-RC assembly. Further, ORCA levels are cell cycle-regulated, with highest levels during G(1), and progressively decreasing during S phase, but the mechanism remains to be elucidated. We now demonstrate that ORCA is polyubiquitinated in vivo, with elevated ubiquitination observed at the G(1)/S boundary. ORCA utilizes lysine-48 (K48) ubiquitin linkage, suggesting that ORCA ubiquitination mediates its regulated degradation. Ubiquitinated ORCA is re-localized in the form of nuclear aggregates and is predominantly associated with chromatin. We demonstrate that ORCA associates with the E3 ubiquitin ligase Cul4A-Ddb1. ORCA is ubiquitinated at the WD40 repeat domain, a region that is also recognized by Orc2. Furthermore, Orc2 associates only with the non-ubiquitinated form of ORCA, and Orc2 depletion results in the proteasome-mediated destabilization of ORCA. Based on the results, we suggest that Orc2 protects ORCA from ubiquitin-mediated degradation in vivo.

Heat shock protein 90 (Hsp90) is an essential, evolutionarily conserved molecular chaperone. Cancer cells rely on Hsp90 to chaperone mutated and/or activated oncoproteins, and its involvement in numerous signaling pathways makes it an attractive target for drug development. Surprisingly, however, the impact of Hsp90 inhibitors on cancer cells is frequently cytostatic in nature, and efforts to enhance the antitumor activity of Hsp90 inhibitors in the clinic remain a significant challenge. In agreement with previous data obtained using Wee1 siRNA, we show that dual pharmacologic inhibition of Wee1 tyrosine kinase and Hsp90 causes cancer cells to undergo apoptosis in vitro and in vivo. Gene expression profiling revealed that induction of the intrinsic apoptotic pathway by this drug combination coincided with transcriptional downregulation of Survivin and Wee1, an outcome not seen in cells treated separately with either agent. At the translational level, expression of these two proteins, as well as activated Akt, was completely abrogated. These data support the hypothesis that Wee1 inhibition sensitizes cancer cells to Hsp90 inhibitors; they establish combined Wee1/Hsp90 inhibition as a novel therapeutic strategy; and they provide a mechanistic rationale for enhancing the pro-apoptotic activity of Hsp90 inhibitors.

Neither the molecular mechanisms whereby cancer cells intrinsically are or become resistant to the DNA-damaging agent cisplatin nor the signaling pathways that account for cisplatin cytotoxicity have thus far been characterized in detail. In an attempt to gain further insights into the molecular cascades elicited by cisplatin (leading to resistance or underpinning its antineoplastic properties), we comparatively investigated the ability of cisplatin, C2-ceramide and cadmium dichloride, alone or in the presence of an array of mitochondrion-protective agents, to trigger the permeabilization of purified mitochondria. In addition, we compared the transcriptional response triggered by cisplatin, C2-ceramide and cadmium dichloride in non-small cell lung carcinoma A549 cells. Finally, we assessed the capacity of cisplatin, C2-ceramide and cadmium dichloride to reduce the clonogenic potential of a battery of yeast strains lacking proteins involved in the regulation of cell death, DNA damage signaling and stress management. This multipronged experimental approach revealed that cisplatin elicits signaling pathways that are for the most part "private," i.e., that manifest limited overlap with the molecular cascades ignited by other inducers of mitochondrial apoptosis, and triggers apoptosis mainly in a transcription-independent fashion. Indeed, bona fide cisplatin-response modifiers that we have recently identified by a functional genome-wide siRNA screen are either not transcriptionally regulated during cisplatin-induced cell death or their transcriptional modulation reflects the activation of an adaptive response promoting cisplatin resistance.

Macroautophagy is an ancient, evolutionarily conserved catabolic process involving the degradation of organelles and long-lived proteins, an important process for maintaining cellular homeostasis. Many cellular stresses, including hypoxia/anoxia and the lack of growth factors and nutrients, trigger autophagy to either mediate survival or cell death. Numerous intracellular factors have been implicated in promoting autophagy, including the tumor suppressor p53. Interestingly, p53 can promote cell fate decisions such as autophagy through transcriptional and post-transcriptional pathways. The complexity of how p53 can regulate cellular fate may be driven by specific pathways that are activated in response to cellular cues, while the understanding of intra- and extracellular signaling that promotes post-translational modifications to p53 still remains incomplete. For example, various enzymes lead to phosphorylation, acetylation, glycosylation, ubiqutination, neddylation, sumoylation and methylation of p53, which are implicated in regulating the activity of p53. These specific posttranslational modifications would most likely change the recruitment of specific proteins, DNA binding or changes in compartmentalization of p53. The combination of specific modifications that are necessary to fine-tune p53 activity are still not well-defined. The signaling pathways that would activate specific enzymes to direct p53 to mediate cellular processes such as autophagy is important to understand, since manipulating these enzymes pharmacologically would be of therapeutic value. A recent paper by Naidu, et al. examines how post-translational modifications to p53 direct its activity to promote autophagy.1 Since post-translational modifications to p53 in the C terminus (ubiquitin and sumoylation) have been implicated in redistribution of p53 to the cytoplasm,2 the authors rationalized that post-transcriptional activity of p53 in the cytoplasm would be a key event in regulating autophagy. They show that in order for p53 to mediate autophagy sumoylation at K386 and acetylation of lysine 120 are necessary for facilitating the conversion of LC3, a key protein involved in autophagy. TIP60 can acetylate p53 at lysine 120, which is necessary for induction of the p21 gene and the pro-apoptotic gene, puma.3 Recent work by Lin, et al. showed that TIP60 was necessary for the induction of autophagy,4 which is also reported by Naidu, et al. herein.1 Collectively, Naidu, et al.’s studies show that PIASγ can modify and activate TIP60, resulting in TIP60 and PIASγ post-translational modifications to p53 that redirects its activity to induce autophagy In light of these new findings, some additional biochemical questions remain, including how the regulator of p53, Mdm2, is involved in this pathway. Interestingly, Mdm2 forms a complex with, and is acetylated and sumoylated, by TIP60 and PIASγ, respectively.5,6 Both modifications independently inactivate the ubiquitin ligase activity of Mdm2. Since TIP60/PIASγ regulate p53 and TIP60/PIASγ can regulate Mdm2, this suggests that Mdm2 may be serving as a scaffold to mediate these modifications to p53. It would be interesting to determine if Mdm2 can facilitate these modifications, as Mdm2 has been reported to have cellular-suppressor activity.7 Additionally, considering that the Mdm2 family member Mdmx is found with p53 in the cytoplasm,8 Mdmx may also be playing a role in regulating p53-mediated autophagy. Further work is also needed to establish a biochemical understanding of the events necessary to direct p53 for induction of autophagy and if preventing the p53-Mdm2 or p53-Mdmx complex using small-molecule inhibitors would promote or halt the induction of autophagy.

p53 plays an important role in mitotic checkpoint, but what its role is remains enigmatic. Aurora A is a Ser/Thr kinase involved in correcting progression of mitosis. Here, we show that p53 is a negative regulator for Aurora A. We found that p53 deficiency leads to Aurora A elevation. Ectopic expression of p53 or DNA damage-induced expression of p53 can suppress the expression of Aurora A. Mechanistic studies show that p53 is a negative regulator for Aurora A expression through both transcriptional and posttranslational regulation. p53 knockdown in cancer cells reduces the level of p21, which, in turn, increases the activity of CDK2 followed by induction of Rb1 hyperphosphorylation and its dissociation with transcriptional factor E2F3. E2F3 can bind to Aurora A gene promoter, potentiating Aurora A gene expression and p53 deficiency, enhancing the binding of E2F3 on Aurora A promoter. Also, p53 deficiency leads to decelerating Aurora A's turnover rate, due to the fact that p53 deficiency causes the downregulation of Fbw7α, a component of E3 ligase of Aurora A. Consistently, p53 knockdown-mediated Aurora A elevation is mitigated when Fbw7α is ectopically expressed. Thus, p53-mediated Aurora A degradation requires Fbw7α expression. Significantly, inverse correlation between p53 and Aurora A elevation is translated into the deregulation of centrosome amplification. p53 knockdown leads to high percentages of cells with abnormal amplification of centrosome. These data suggest that p53 is an important negative regulator of Aurora A, and that loss of p53 in many types of cancer could lead to abnormal elevation of Aurora A and dysregulated mitosis, which provides a growth advantage for cancer cells.

Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.

Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver.

Ubiquitination of histones plays a critical role in the regulation of several processes within the nucleus, including maintenance of genome stability and transcriptional regulation. The only known ubiquitination site on histones is represented by a conserved Lys residue located at the C terminus of the protein. Here, we describe a novel ubiquitin mark at the N-terminal tail of histone H2As consisting of two Lys residues at positions 13 and 15 (K13/K15). This "bidentate" site is a target of the DNA damage response (DDR) ubiquitin ligases RNF8 and RNF168. Histone mutants lacking the K13/K15 site impair RNF168- and DNA damage-dependent ubiquitination. Conversely, inactivation of the canonical C-terminal site prevents the constitutive monoubiquitination of histone H2As but does not abolish the ubiquitination induced by RNF168. A ubiquitination-defective mutant is obtained by inactivating both the N- and the C-terminal sites, suggesting that these are unique, non-redundant acceptors of ubiquitination on histone H2As. This unprecedented result implies that RNF168 generates a qualitatively different Ub mark on chromatin.

Numerous stimuli, including oncogenic signaling, DNA damage or eroded telomeres trigger proliferative arrest, termed cellular senescence. Accumulating evidence suggests that cellular senescence is a potent barrier to tumorigenesis in vivo, however oncogene induced senescence can also promote cellular transformation.1,2 Several oncogenes, whose overexpression results in cellular senescence, converge on the TOR (target of rapamycin) pathway. We therefore examined whether attenuation of TOR results in delay or reversal of cellular senescence. By using primary human fibroblasts undergoing either replicative or oncogenic RAS-induced senescence, we demonstrated that senescence can be delayed, and some aspects of senescence can be reversed by inhibition of TOR, using either the TOR inhibitor rapamycin or by depletion of TORC1 (TOR Complex 1). Depletion of TORC2 fails to affect the course of replicative or RAS-induced senescence. Overexpression of REDD1 (Regulated in DNA Damage Response and Development), a negative regulator of TORC1, delays the onset of replicative senescence. These results indicate that TORC1 is an integral component of the signaling pathway that mediates cellular senescence.