Functional characterization of miR-708 microRNA in telomerase positive and negative human cancer cells.

Activation of a telomere length maintenance mechanism (TMM), including telomerase and alternative lengthening of telomeres (ALT), is essential for replicative immortality of tumor cells, although its regulatory mechanisms are incompletely understood. We conducted a microRNA (miRNA) microarray analysis on isogenic telomerase positive (TEP) and ALT cancer cell lines. Amongst nine miRNAs that showed difference in their expression in TEP and ALT cancer cells in array analysis, miR-708 was selected for further analysis since it was consistently highly expressed in a large panel of ALT cells. miR-708 in TEP and ALT cancer cells was not correlated with C-circle levels, an established feature of ALT cells. Its overexpression induced suppression of cell migration, invasion, and angiogenesis in both TEP and ALT cells, although cell proliferation was inhibited only in TEP cells suggesting that ALT cells may have acquired the ability to escape inhibition of cell proliferation by sustained miR-708 overexpression. Further, cell proliferation regulation in TEP cells by miR708 appears to be through the CARF-p53 pathway. We demonstrate here that miR-708 (i) is the first miRNA shown to be differentially regulated in TEP and ALT cancer cells, (ii) possesses tumor suppressor function, and (iii) deregulates CARF and p21WAF1-mediated signaling to limit proliferation in TEP cells.

Stress-induced changes in CARF expression determine cell fate to death, survival, or malignant transformation.

CARF (Collaborator of ARF) was discovered as an ARF-interacting protein that activated ARF-p53-p21WAF1 signaling involved in cellular response to a variety of stresses, including oxidative, genotoxic, oncogenic, or telomere deprotection stresses, leading to senescence, growth arrest, or apoptosis. Of note, whereas suppression of CARF was lethal, its enrichment was associated with increased proliferation and malignant transformation of cells. These reports have predicted that CARF could serve as a multi-stress marker with a predictive value for cell fates. Here, we recruited various in vitro stress models and examined their effect on CARF expression using human normal fibroblasts. We demonstrate that CARF levels in stress and post-stress conditions could predict the fate of cells towards either death or enhanced proliferation and malignant transformation. We provide extensive molecular evidence that (i) CARF expression changes in response to stress, (ii) it modulates cell death or survival signaling and determines the fate of cells, and (iii) it may serve as a predictive measure of cellular response to stress and an important marker for biosafety.

What makes a bad egg? Egg transcriptome reveals dysregulation of translational machinery and novel fertility genes important for fertilization.

BACKGROUND: Egg quality can be defined as the egg ability to be fertilized and subsequently develop into a normal embryo. Previous research has shed light on factors that can influence egg quality. Large gaps however remain including a comprehensive view of what makes a bad egg. Initial development of the embryo relies on maternally-inherited molecules, such as transcripts, deposited in the egg during its formation. Bad egg quality is therefore susceptible to be associated with alteration or dysregulation of maternally-inherited transcripts. We performed transcriptome analysis on a large number (N = 136) of zebrafish egg clutches, each clutch being split to monitor developmental success and perform transcriptome analysis in parallel. We aimed at drawing a molecular portrait of the egg in order to characterize the relation between egg transcriptome and developmental success and to subsequently identify new candidate genes involved in fertility. RESULTS: We identified 66 transcript that were differentially abundant in eggs of contrasted phenotype (low or high developmental success). Statistical modeling using partial least squares regression and genetics algorithm demonstrated that gene signatures from transcriptomic data can be used to predict developmental success. The identity and function of differentially expressed genes indicate a major dysregulation of genes of the translational machinery in poor quality eggs. Two genes, otulina and slc29a1a, predominantly expressed in the ovary and dysregulated in poor quality eggs were further investigated using CRISPR/Cas9 mediated genome editing. Mutants of each gene revealed remarkable subfertility whereby the majority of their eggs were unfertilizable. The Wnt pathway appeared to be dysregulated in the otulina mutant-derived eggs. CONCLUSIONS: Here we show that egg transcriptome contains molecular signatures, which can be used to predict developmental success. Our results also indicate that poor egg quality in zebrafish is associated with a dysregulation of (i) the translational machinery genes and (ii) novel fertility genes, otulina and slc29a1a, playing an important role for fertilization. Together, our observations highlight the diversity of the possible causes of egg quality defects and reveal mechanisms of maternal origin behind the lack of fertilization and early embryonic failures that can occur under normal reproduction conditions.

Double maternal-effect: duplicated nucleoplasmin 2 genes, npm2a and npm2b, with essential but distinct functions are shared by fish and tetrapods.

BACKGROUND: Nucleoplasmin 2 (npm2) is an essential maternal-effect gene that mediates early embryonic events through its function as a histone chaperone that remodels chromatin. Recently, two npm2 (npm2a and npm2b) genes have been annotated in zebrafish. Thus, we examined the evolution of npm2a and npm2b in a variety of vertebrates, their potential phylogenetic relationships, and their biological functions using knockout models via the CRISPR/cas9 system. RESULTS: We demonstrated that the two npm2 duplicates exist in a wide range of vertebrates, including sharks, ray-finned fish, amphibians, and sauropsids, while npm2a was lost in coelacanth and mammals, as well as some specific teleost lineages. Using phylogeny and synteny analyses, we traced their origins to the early stages of vertebrate evolution. Our findings suggested that npm2a and npm2b resulted from an ancient local gene duplication, and their functions diverged although key protein domains were conserved. We then investigated their functions by examining their tissue distribution in a wide variety of species and found that they shared ovarian-specific expression, a key feature of maternal-effect genes. We also demonstrated that both npm2a and npm2b are maternally-inherited transcripts in vertebrates, and that they play essential, but distinct, roles in early embryogenesis using zebrafish knockout models. Both npm2a and npm2b function early during oogenesis and may play a role in cortical granule function that impact egg activation and fertilization, while npm2b is also involved in early embryogenesis. CONCLUSION: These novel findings will broaden our knowledge on the evolutionary history of maternal-effect genes and underlying mechanisms that contribute to vertebrate reproductive success. In addition, our results demonstrate the existence of a newly described maternal-effect gene, npm2a, that contributes to egg competence, an area that still requires further comprehension.

foxr1 is a novel maternal-effect gene in fish that is required for early embryonic success.

The family of forkhead box (Fox) transcription factors regulates gonadogenesis and embryogenesis, but the role of foxr1 in reproduction is unknown. Evolutionary history of foxr1 in vertebrates was examined and the gene was found to exist in most vertebrates, including mammals, ray-finned fish, amphibians, and sauropsids. By quantitative PCR and RNA-seq, we found that foxr1 had an ovarian-specific expression in zebrafish, a common feature of maternal-effect genes. In addition, it was demonstrated using in situ hybridization that foxr1 was a maternally-inherited transcript that was highly expressed even in early-stage oocytes and accumulated in the developing eggs during oogenesis. We also analyzed the function of foxr1 in female reproduction using a zebrafish CRISPR/cas9 knockout model. It was observed that embryos from the foxr1-deficient females had a significantly lower survival rate whereby they either failed to undergo cell division or underwent abnormal division that culminated in growth arrest at around the mid-blastula transition and early death. These mutant-derived eggs contained dramatically increased levels of p21, a cell cycle inhibitor, and reduced rictor, a component of mTOR and regulator of cell survival, which were in line with the observed growth arrest phenotype. Our study shows for the first time that foxr1 is an essential maternal-effect gene and may be required for proper cell division and survival via the p21 and mTOR pathways. These novel findings will broaden our knowledge on the functions of specific maternal factors stored in the developing egg and the underlying mechanisms that contribute to reproductive success.

Cyclin A2: At the crossroads of cell cycle and cell invasion.

Cyclin A2 is an essential regulator of the cell division cycle through the activation of kinases that participate to the regulation of S phase as well as the mitotic entry. However, whereas its degradation by the proteasome in mid mitosis was thought to be essential for mitosis to proceed, recent observations show that a small fraction of cyclin A2 persists beyond metaphase and is degraded by autophagy. Its implication in the control of cytoskeletal dynamics and cell movement has unveiled its role in the modulation of RhoA activity. Since this GTPase is involved in both cell rounding early in mitosis and later, in the formation of the cleavage furrow, this suggests that cyclin A2 is a novel actor in cytokinesis. Taken together, these data point to this cyclin as a potential mediator of cell-niche interactions whose dysregulation could be taken as a hallmark of metastasis.

CARF (Collaborator of ARF) overexpression in p53-deficient cells promotes carcinogenesis.

Collaborator of ARF (CARF), initially identified as a binding partner of ARF (Alternate Reading Frame), has been shown to activate ARF-p53 pathway by multiple ways including stabilization of ARF and p53 tumor suppressor proteins, and transcriptional repression of a p53 antagonist, HDM2. Level of CARF expression was shown to determine fate of cells. Whereas its knockdown caused apoptosis, its over- and super-expressions caused senescence and increase in malignant properties of cancer cells, respectively, and were closely linked to increase and decrease in p53 activity. Using p53-compromised cancer cells, we demonstrate that CARF induces growth arrest when wild type p53 is present and in p53-absence, it promotes carcinogenesis. Biochemical analyses on CARF-induced molecular signaling revealed that in p53-null cells, it caused transcriptional repression of p21(WAF1) leading to increase in CDK4, CDK6, pRb and E2F1 resulting in continued cell cycle progression. Furthermore, it instigated increase in migration and invasion of cancer cells that was marked by upregulation of MMP2, MMP3, MMP9, uPA, several interleukins and VEGF expression. Consistent with these findings, we found that human clinical samples of epithelial and glial cancers (frequently marked by loss of p53 function) possessed high level of CARF expression showing a relationship with cancer aggressiveness. The data demonstrated that CARF could be considered as a diagnostic marker and a therapeutic target in p53-compromised malignancies.

Cyclin A2 modulates EMT via ß-catenin and phospholipase C pathways.

We have previously demonstrated that Cyclin A2 is involved in cytoskeletal dynamics, epithelial-mesenchymal transition (EMT) and metastasis. This phenotype was potentiated by activated oncogenic H-Ras. However, the mechanisms governing EMT in these cells have not yet been elucidated. Here, we dissected the pathways that are responsible for EMT in cells deficient for Cyclin A2. In Cyclin A2-depleted normal murine mammary gland (NMuMG) cells expressing RasV12, we found that ß-catenin was liberated from the cell membrane and cell-cell junctions and underwent nuclear translocation and activation. Components of the canonical wingless (WNT) pathway, including WNT8b, WNT10a, WNT10b, frizzled 1 and 2 and TCF4 were upregulated at the messenger RNA and protein levels following Cyclin A2 depletion. However, suppression of the WNT pathway using the acetyltransferase porcupine inhibitor C59 did not reverse EMT whereas a dominant negative form of TCF4 as well as inhibition of phospholipase C using U73122 were able to do so. This suggests that a WNT-independent mechanism of ß-catenin activation via phospholipase C is involved in the EMT induced by Cyclin A2 depletion. Our findings will broaden our knowledge on how Cyclin A2 contributes to EMT and metastasis.

Cyclin A2, a novel regulator of EMT.

Our previous work showed that Cyclin A2 deficiency promotes cell invasion in fibroblasts. Given that the majority of cancers emerge from epithelia, we explored novel functions for Cyclin A2 by depleting it in normal mammary epithelial cells. This caused an epithelial to mesenchymal transition (EMT) associated with loss of cell-to-cell contacts, decreased E-Cadherin expression and increased invasive properties characterized by a reciprocal regulation of RhoA and RhoC activities, where RhoA-decreased activity drove cell invasiveness and E-Cadherin delocalization, and RhoC-increased activity only supported cell motility. Phenotypes induced by Cyclin A2 deficiency were exacerbated upon oncogenic activated-Ras expression, which led to an increased expression of EMT-related transcriptional factors. Moreover, Cyclin A2-depleted cells exhibited stem cell-like properties and increased invasion in an in vivo avian embryo model. Our work supports a model where Cyclin A2 downregulation facilitates cancer cell EMT and metastatic dissemination.

Collaborator of ARF (CARF) regulates proliferative fate of human cells by dose-dependent regulation of DNA damage signaling.

Collaborator of ARF (CARF) has been shown to directly bind to and regulate p53, a central protein that controls tumor suppression via cellular senescence and apoptosis. However, the cellular functions of CARF and the mechanisms governing its effect on senescence, apoptosis, or proliferation are still unknown. Our previous studies have shown that (i) CARF is up-regulated during replicative and stress-induced senescence, and its exogenous overexpression caused senescence-like growth arrest of cells, and (ii) suppression of CARF induces aneuploidy, DNA damage, and mitotic catastrophe, resulting in apoptosis via the ATR/CHK1 pathway. In the present study, we dissected the cellular role of CARF by investigating the molecular pathways triggered by its overexpression in vitro and in vivo. We found that the dosage of CARF is a critical factor in determining the proliferation potential of cancer cells. Most surprisingly, although a moderate level of CARF overexpression induced senescence, a very high level of CARF resulted in increased cell proliferation. We demonstrate that the level of CARF is crucial for DNA damage and checkpoint response of cells through ATM/CHK1/CHK2, p53, and ERK pathways that in turn determine the proliferative fate of cancer cells toward growth arrest or proproliferative and malignant phenotypes. To the best of our knowledge, this is the first report that demonstrates the capability of a fundamental protein, CARF, in controlling cell proliferation in two opposite directions and hence may play a key role in tumor biology and cancer therapeutics.

A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity.

Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12(-/-) but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrate's gene exons and by MMP-12-mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3-infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.

Molecular characterization of collaborator of ARF (CARF) as a DNA damage response and cell cycle checkpoint regulatory protein.

CARF is an ARF-binding protein that has been shown to regulate the p53-p21-HDM2 pathway. CARF overexpression was shown to cause growth arrest of human cancer cells and premature senescence of normal cells through activation of the p53 pathway. Because replicative senescence involves permanent withdrawal from the cell cycle in response to DNA damage response-mediated signaling, in the present study we investigated the relationship between CARF and the cell cycle and whether it is involved in the DNA damage response. We demonstrate that the half-life of CARF protein is less than 60 min, and that in cycling cells CARF levels are highest in G2 and early prophase. Serially passaged normal human skin and stromal fibroblasts showed upregulation of CARF during replicative senescence. Induction of G1 growth arrest and senescence by a variety of drugs was associated with increase in CARF expression at the transcriptional and translational level and was seen to correlate with increase in DNA damage response and checkpoint proteins, ATM, ATR, CHK1, CHK2, γH2AX, p53 and p21. Induction of growth arrest by oncogenic RAS and shRNA-mediated knockdown of TRF2 in cancer cells also caused upregulation of CARF. We conclude that CARF is associated with DNA damage response and checkpoint signaling pathways.

Molecular bridging of aging and cancer: A CARF link.

Collaborator of ARF (CARF) was first cloned as an ARF partner in yeast two-hybrid screens. It enhances ARF-dependent and -independent p53 functions, which are central to the control of cell growth and tumor suppression in human cells. CARF interacts with ARF, p53, and MDM2 proteins, and in turn gets regulated by MDM2-mediated degradation, suggesting a self-regulatory loop. CARF is upregulated during replicative, oncogenic, and stress-induced senescence. Overexpression of CARF induced premature senescence in normal human fibroblasts that was mediated by upregulation of p53-p21(CIP1/WAF1) and p16(INK4a)- pRB pathways. Knockdown of CARF resulted in mitotic arrest leading to excessive chromosomal condensation, aneuploidy, and apoptosis, suggesting that CARF is essential for cell survival. Most recently, we have found that CARF causes bidirectional regulation of p53 and pRB pathways, either arresting or promoting growth, and thus, it could be a potential threshold link between aging and cancer.

CARF: an emerging regulator of p53 tumor suppressor and senescence pathway.

Replicative senescence, a major outcome of normal cells with finite lifespan, is a widely accepted in vitro model for ageing studies. Limited repair and defense mechanisms of normal cells, in addition to DNA alterations and oncogene inductions under stress, are believed to result in senescence as a protective mechanism to prevent undesirable proliferation of cells. The ARF/p53/p21(cip1/waf1) tumor suppression pathway acts as a molecular sensor and regulator of cellular stress, senescence, and immortalization. Understanding the molecular regulation of this pathway by intrinsic and extrinsic signals is extremely important to address unsolved questions in senescence and cancer. CARF was first discovered as a binding partner of ARF and has since been shown to have both ARF-dependent and -independent functions that converge to regulate p53 pathway. CARF directly binds to p53 and HDM2, and functions in a negative feedback pathway. Whereas CARF transcriptionally represses HDM2 to increase p53 activity, HDM2 in return degrades CARF. Thus, CARF may act as a novel key regulator of the p53 pathway at multiple checkpoints. The aim of this article is to discuss the current knowledge about functions of CARF and its impact on p53 pathway in regulation of senescence and carcinogenesis.

Neutralizing anti-4-1BBL treatment improves cardiac function in viral myocarditis.

Coxsackievirus B3 (CVB3) is the most common causative agent of infectious myocarditis. Chronic inflammation, loss of contractile tissue, and maladaptive remodeling all contribute to dilated cardiomyopathy and heart failure. The 4-1BB receptor is a costimulatory molecule expressed by T cells and cardiomyocytes. We infected mice with CVB3 to examine if virus infection triggers 4-1BB activation and whether inhibition of this pathway will reduce inflammation and improve heart function. Echocardiography was performed on days 3, 9, 30 and at 10 weeks post-infection (pi) and ejection fraction (EF), left ventricular (LV) wall thickness, contractility, and internal cardiac dimensions were measured. At day 9, reduced rate of wall thickening (30+/-17 vs 70+/-19%), increased LV wall thickness (0.15+/-0.04 vs 0.09+/-0.01 cm in diastole and 0.19+/-0.04 vs 0.15+/-0.02 cm in systole), and reduced cardiac volume (0.013+/-0.004 vs 0.023+/-0.003 ml in diastole and 0.004+/-0.002 ml vs 0.007+/-0.001 ml in systole) were observed in infected hearts as compared with shams. At 14 days pi, CVB3-infected mice were randomly assigned to receive either anti-4-1BBL neutralizing (M522) or control antibodies (Ab) for 8 weeks. Cardiac damage, fibrosis, and inflammation were assessed by histological stains and immunohistochemistry. Polymerase chain reaction (PCR) was utilized to detect matrix metalloproteinase (MMP)-2, MMP-9, and MMP-12 expressions. At 10 weeks pi, M522 treatment improved LV wall thickening rate (-10+/-13 vs -49+/-16%, expressed as percentage change from baseline) and reduced diastolic LV posterior wall thickness (17+/-10 vs 57+/-47%, expressed as percentage change from baseline), cardiac damage as assessed by histological scores (0 vs 1.3+/-1.5), fibrosis by collagen volume fraction (3.2+/-0.6 vs 4.9+/-2.2%), overall inflammation (5.9+/-1.3 vs 8.5+/-4.1%), and T-cell infiltration (1.3+/-0.9 vs 4.3+/-3.8%) as compared to control. MMP-12 was highly increased during acute and chronic myocarditis, but was significantly decreased by M522 treatment. Thus, long-term inhibition of the 4-1BB pathway reduces cardiac damage, remodeling, and inflammation during viral myocarditis.