Comparative analysis of human, rodent and snake deltavirus replication.

The recent discovery of Hepatitis D (HDV)-like viruses across a wide range of taxa led to the establishment of the Kolmioviridae family. Recent studies suggest that kolmiovirids can be satellites of viruses other than Hepatitis B virus (HBV), challenging the strict HBV/HDV-association dogma. Studying whether kolmiovirids are able to replicate in any animal cell they enter is essential to assess their zoonotic potential. Here, we compared replication of three kolmiovirids: HDV, rodent (RDeV) and snake (SDeV) deltavirus in vitro and in vivo. We show that SDeV has the narrowest and RDeV the broadest host cell range. High resolution imaging of cells persistently replicating these viruses revealed nuclear viral hubs with a peculiar RNA-protein organization. Finally, in vivo hydrodynamic delivery of viral replicons showed that both HDV and RDeV, but not SDeV, efficiently replicate in mouse liver, forming massive nuclear viral hubs. Our comparative analysis lays the foundation for the discovery of specific host factors controlling Kolmioviridae host-shifting.

CDK4 Phosphorylates AMPKα2 to Inhibit Its Activity and Repress Fatty Acid Oxidation.

The roles of CDK4 in the cell cycle have been extensively studied, but less is known about the mechanisms underlying the metabolic regulation by CDK4. Here, we report that CDK4 promotes anaerobic glycolysis and represses fatty acid oxidation in mouse embryonic fibroblasts (MEFs) by targeting the AMP-activated protein kinase (AMPK). We also show that fatty acid oxidation (FAO) is specifically induced by AMPK complexes containing the α2 subunit. Moreover, we report that CDK4 represses FAO through direct phosphorylation and inhibition of AMPKα2. The expression of non-phosphorylatable AMPKα2 mutants, or the use of a CDK4 inhibitor, increased FAO rates in MEFs and myotubes. In addition, Cdk4-/- mice have increased oxidative metabolism and exercise capacity. Inhibition of CDK4 mimicked these alterations in normal mice, but not when skeletal muscle was AMPK deficient. This novel mechanism explains how CDK4 promotes anabolism by blocking catabolic processes (FAO) that are activated by AMPK.

Antagonistic functions of LMNA isoforms in energy expenditure and lifespan.

Alternative RNA processing of LMNA pre-mRNA produces three main protein isoforms, that is, lamin A, progerin, and lamin C. De novo mutations that favor the expression of progerin over lamin A lead to Hutchinson-Gilford progeria syndrome (HGPS), providing support for the involvement of LMNA processing in pathological aging. Lamin C expression is mutually exclusive with the splicing of lamin A and progerin isoforms and occurs by alternative polyadenylation. Here, we investigate the function of lamin C in aging and metabolism using mice that express only this isoform. Intriguingly, these mice live longer, have decreased energy metabolism, increased weight gain, and reduced respiration. In contrast, progerin-expressing mice show increased energy metabolism and are lipodystrophic. Increased mitochondrial biogenesis is found in adipose tissue from HGPS-like mice, whereas lamin C-only mice have fewer mitochondria. Consistently, transcriptome analyses of adipose tissues from HGPS and lamin C-only mice reveal inversely correlated expression of key regulators of energy expenditure, including Pgc1a and Sfrp5. Our results demonstrate that LMNA encodes functionally distinct isoforms that have opposing effects on energy metabolism and lifespan in mammals.

FGF19 and its analog Aldafermin cooperate with MYC to induce aggressive hepatocarcinogenesis.

FGF19 hormone has pleiotropic metabolic functions, including the modulation of insulin sensitivity, glucose/lipid metabolism and energy homeostasis. On top of its physiological metabolic role, FGF19 has been identified as a potentially targetable oncogenic driver, notably in hepatocellular carcinoma (HCC). Nevertheless, FGF19 remained an attractive candidate for treatment of metabolic disease, prompting the development of analogs uncoupling its metabolic and tumor-promoting activities. Using pre-clinical mice models of somatic mutation driven HCC, we assessed the oncogenicity of FGF19 in combination with frequent HCC tumorigenic alterations: p53 inactivation, CTNNB1 mutation, CCND1 or MYC overexpression. Our data revealed a strong oncogenic cooperation between FGF19 and MYC. Most importantly, we show that this oncogenic synergy is conserved with a FGF19-analog Aldafermin (NGM282), designed to solely mimic the hormone's metabolic functions. In particular, even a short systemic treatment with recombinant proteins triggered rapid appearance of proliferative foci of MYC-expressing hepatocytes. The fact that FGF19 analog Aldafermin is not fully devoid of the hormone's oncogenic properties raises concerns in the context of its potential use for patients with damaged, mutation-prone liver.

Ras/MAPK signalling intensity defines subclonal fitness in a mouse model of hepatocellular carcinoma.

Quantitative differences in signal transduction are to date an understudied feature of tumour heterogeneity. The MAPK Erk pathway, which is activated in a large proportion of human tumours, is a prototypic example of distinct cell fates being driven by signal intensity. We have used primary hepatocyte precursors transformed with different dosages of an oncogenic form of Ras to model subclonal variations in MAPK signalling. Orthotopic allografts of Ras-transformed cells in immunocompromised mice gave rise to fast-growing aggressive tumours, both at the primary location and in the peritoneal cavity. Fluorescent labelling of cells expressing different oncogene levels, and consequently varying levels of MAPK Erk activation, highlighted the selection processes operating at the two sites of tumour growth. Indeed, significantly higher Ras expression was observed in primary as compared to secondary, metastatic sites, despite the apparent evolutionary trade-off of increased apoptotic death in the liver that correlated with high Ras dosage. Analysis of the immune tumour microenvironment at the two locations suggests that fast peritoneal tumour growth in the immunocompromised setting is abrogated in immunocompetent animals due to efficient antigen presentation by peritoneal dendritic cells. Furthermore, our data indicate that, in contrast to the metastatic-like outgrowth, strong MAPK signalling is required in the primary liver tumours to resist elimination by NK (natural killer) cells. Overall, this study describes a quantitative aspect of tumour heterogeneity and points to a potential vulnerability of a subtype of hepatocellular carcinoma as a function of MAPK Erk signalling intensity.

Fibroblast growth factor 19 stimulates water intake.

Fibroblast growth factor 19 (FGF19) is a hormone with pleiotropic metabolic functions, leading to ongoing development of analogues for treatment of metabolic disorders. On the other hand, FGF19 is overexpressed in a sub-group of hepatocellular carcinoma (HCC) patients and has oncogenic properties. It is therefore crucial to precisely define FGF19 effects, notably in the context of chronic exposure to elevated concentrations of the hormone. Here, we used hydrodynamic gene transfer to generate a transgenic mouse model with long-term FGF19 hepatic overexpression. We describe a novel effect of FGF19, namely the stimulation of water intake. This phenotype, lasting at least over a 6-month period, depends on signaling in the central nervous system and is independent of FGF21, although it mimics some of its features. We further show that HCC patients with high levels of circulating FGF19 have a reduced natremia, indicating dipsogenic features. The present study provides evidence of a new activity of FGF19, which could be clinically relevant in the context of FGF19 overexpressing cancers and in the course of treatment of metabolic disorders by FGF19 analogues.

Lamin C Counteracts Glucose Intolerance in Aging, Obesity, and Diabetes Through ß-Cell Adaptation.

Aging-dependent changes in tissue function are associated with the development of metabolic diseases. However, the molecular connections linking aging, obesity, and diabetes remain unclear. Lamin A, lamin C, and progerin, products of the Lmna gene, have antagonistic functions on energy metabolism and life span. Lamin C, albeit promoting obesity, increases life span, suggesting that this isoform is crucial for maintaining healthy conditions under metabolic stresses. Because ß-cell loss during obesity or aging leads to diabetes, we investigated the contribution of lamin C to ß-cell function in physiopathological conditions. We demonstrate that aged lamin C only-expressing mice (Lmna LCS/LCS ) become obese but remain glucose tolerant due to adaptive mechanisms including increased ß-cell mass and insulin secretion. Triggering diabetes in young mice revealed that Lmna LCS/LCS animals normalize their fasting glycemia by both increasing insulin secretion and regenerating ß-cells. Genome-wide analyses combined to functional analyses revealed an increase of mitochondrial biogenesis and global translational rate in Lmna LCS/LCS islets, two major processes involved in insulin secretion. Altogether, our results demonstrate for the first time that the sole expression of lamin C protects from glucose intolerance through a ß-cell-adaptive transcriptional program during metabolic stresses, highlighting Lmna gene processing as a new therapeutic target for diabetes treatment.

Interleukin-8 expression is regulated by histone deacetylases through the nuclear factor-kappaB pathway in breast cancer.

We have reported recently that the chemokine interleukin 8 (IL-8)/CXCL8 was overexpressed in invasive estrogen receptor (ERalpha)-negative breast cancer cells compared with ERalpha-positive breast cancer cells. We now demonstrate that histone deacetylases (HDACs) play an essential role in the regulation of IL-8 gene expression in ERalpha-positive MCF-7 breast cancer cells. Treatment of MCF-7 cells with the HDAC inhibitor trichostatin A (TSA) led to a strong up-regulation of IL-8 protein and RNA levels in MCF-7 cells. The up-regulation of IL-8 in MCF-7 cells was time- and concentration-dependent. Moreover, run-on and transfection experiments demonstrated that IL-8 induction by HDAC inhibitors was transcriptional and involved mainly the nuclear factor-kappaB (NF-kappaB) site of the IL-8 promoter. These observations are corroborated by an up-regulation of NF-kappaB activity in MCF-7 cells in the presence of TSA. In addition, blocking NF-kappaB pathway by adenoviral delivery of a dominant-negative IkappaBorIkappaB kinase complex 2 (IKK2) mutant abolished IL-8 gene induction by histone deacetylase inhibitors. HDAC inhibitors triggered IKK phosphorylation and up-regulated p65 nuclear translocation, although they decreased the protein levels of IkappaBalpha, which accounts for NF-kappaB activation. TSA increased binding of acetylated histone 3 to the IL-8 gene promoter. In summary, our results demonstrate that NF-kappaB pathway repression by HDAC is responsible for the low expression of IL-8 in ERalpha-positive breast cancer cells.

Regulator of G protein signaling-4 controls fatty acid and glucose homeostasis.

Circulating free fatty acids are a reflection of the balance between lipogenesis and lipolysis that takes place mainly in adipose tissue. We found that mice deficient for regulator of G protein signaling (RGS)-4 have increased circulating catecholamines, and increased free fatty acids. Consequently, RGS4-/- mice have increased concentration of circulating free fatty acids; abnormally accumulate fatty acids in liver, resulting in liver steatosis; and show a higher degree of glucose intolerance and decreased insulin secretion in pancreas. We show in this study that RGS4 controls adipose tissue lipolysis through regulation of the secretion of catecholamines by adrenal glands. RGS4 controls the balance between adipose tissue lipolysis and lipogenesis, secondary to its role in the regulation of catecholamine secretion by adrenal glands. RGS4 therefore could be a good target for the treatment of metabolic diseases.

SPARC represses E-cadherin and induces mesenchymal transition during melanoma development.

During progression of melanoma, loss of the cell-cell adhesion molecule E-cadherin contributes to uncontrolled growth and invasive behavior of transformed melanocytes. Secreted protein acidic and rich in cysteine (SPARC) is a nonstructural matricellular protein that regulates cell-matrix interactions leading to alterations in cell adhesion and proliferation. Overexpression of SPARC has been associated with progression of various cancers, including melanoma; however, its role in primary tumor development is not well defined. We show that normal human melanocytes overexpressing SPARC adopt a fibroblast-like morphology, concomitant with loss of E-cadherin and P-cadherin expression, and increased expression of mesenchymal markers. Concurrent with these changes, SPARC expression stimulates melanocyte motility and melanoma cell invasion. Expression of SPARC results in transcriptional down-regulation of E-cadherin that correlates with induction of Snail, a repressor of E-cadherin. Conversely, SPARC depletion leads to up-regulation of E-cadherin and reduces Snail levels, and SPARC-null cells exhibit a marked change in their mesenchymal phenotype. Finally, analysis of SPARC, Snail, and E-cadherin levels in melanocytes and malignant melanoma cell lines further supports the functional relationship among these proteins during melanoma progression. Our findings provide evidence for the role of SPARC in early transformation of melanocytes and identify a novel mechanism, whereby tumor-derived SPARC promotes tumorigenesis by mediating Snail induction and E-cadherin suppression.

CXC chemokines located in the 4q21 region are up-regulated in breast cancer.

Recent data suggest that chemokines could be essential players in breast carcinogenesis. We previously showed that the CXC chemokine CXCL8 (interleukin-8) was overexpressed in estrogen receptor alpha (ERalpha)-negative breast cell lines. Analysis of CXCL8 chromosomal location showed that several CXC chemokines (CXCL1, CXCL2, CXCL3, CXCL4, CXCL4V1, CXCL5, CXCL6, CXCL7, and CXCL8) were localized in the same narrow region (360 kb in size) of chromosome 4. We thus hypothesized that they could belong to the same cluster. Quantification of these chemokines in breast tumors showed that samples expressing high CXCL8 also produced elevated levels of CXCL1, CXCL3, and CXCL5, and displayed low content of ERalpha. CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8 were co-regulated both in tumors and in breast cancer cell lines. CXCL5 and CXCL8 were mainly produced by epithelial cells, whereas CXCL1, CXCL2, and CXCL3 had a high expression in blood cells. The overexpression of these chemokines in tumor cells was not the result of gene amplification, but rather of an enhanced gene transcription. Our data suggest that high CXCL8 expression in tumors is mainly correlated to activating protein-1 (AP-1) pathway and to a minor extent to NF-kappaB pathway. Interestingly, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8 chemokines were present at higher levels in metastases when compared with grade I and III biopsies. High levels of CXCL8, CXCL1, and CXCL3 accounted for a shorter relapse-free survival of ERalpha-positive patients treated with tamoxifen. In summary, we present evidences that multiple CXC chemokines are co-expressed in CXCL8-positive breast tumors. In addition, these chemokines could account for the higher aggressiveness of these types of tumors.

Oestrogen receptor negative breast cancers exhibit high cytokine content.

INTRODUCTION: An emerging hypothesis suggests that cytokines could play an important role in cancer as potential modulators of angiogenesis and leucocyte infiltration. METHODS: A novel multiplexed flow cytometry technology was used to measure the expression of 17 cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 [p70], IL-13, IL-17, granulocyte colony-stimulating factor [CSF], granulocyte-macrophage CSF, IFN-gamma, monocyte chemoattractant protein [MCP]-1, macrophage inflammatory protein [MIP]-1beta, tumour necrosis factor [TNF]-alpha) at the protein level in 105 breast carcinoma. B lymphocyte, T lymphocyte and macrophage levels were determined by immunohistochemistry. RESULTS: Fourteen of the 17 cytokines were expressed in breast carcinoma, whereas only nine cytokines could be detected in normal breast. Most cytokines were more abundant in breast carcinoma than in normal breast, with IL-6, IL-8, granulocyte CSF, IFN-gamma, MCP-1 and MIP-1beta being very abundant. IL-2, IL-6, IL-8, IL-10, IFN-gamma, MCP-1, MIP-1beta and TNF-alpha, and to a lesser extent IL-1beta and IL-13 exhibited levels of expression that were inversely correlated to oestrogen receptor and progesterone receptor status. Most cytokines were not correlated with age at cancer diagnosis, tumour size, histological type, or lymph node status. However, IL-1beta, IL-6, IL-8, IL-10, IL-12, MCP-1 and MIP-1beta were more abundant in high-grade tumours than in low-grade tumours. In addition, IL-8 and MIP-1beta were expressed to a greater degree in HER2-positive than in HER2-negative patients. The expression of most of the studied cytokines was correlated to levels of activator protein-1, which is known to regulate numerous cytokines. Overexpression of MCP-1 and MIP-1beta were linked to B lymphocyte, T lymphocyte and macrophage infiltration, whereas high levels of IL-8 were correlated with high macrophage content in tumour. Moreover, IL-8 positive tumours exhibited increased vascularization. CONCLUSION: We found that multiple cytokines were overexpressed in oestrogen receptor negative breast carcinoma, and that the three major cytokines--MCP-1, MIP-1beta and IL-8--were correlated with inflammatory cell component, which could account for the aggressiveness of these tumours.

Peroxisome proliferator-activated receptor gamma recruits the positive transcription elongation factor b complex to activate transcription and promote adipogenesis.

Positive transcription elongation factor b (P-TEFb) phosphorylates the C-terminal domain of RNA polymerase II, facilitating transcriptional elongation. In addition to its participation in general transcription, P-TEFb is recruited to specific promoters by some transcription factors such as c-Myc or MyoD. The P-TEFb complex is composed of a cyclin-dependent kinase (cdk9) subunit and a regulatory partner (cyclin T1, cyclin T2, or cyclin K). Because cdk9 has been shown to participate in differentiation processes, such as muscle cell differentiation, we studied a possible role of cdk9 in adipogenesis. In this study we show that the expression of the cdk9 p55 isoform is highly regulated during 3T3-L1 adipocyte differentiation at RNA and protein levels. Furthermore, cdk9, as well as cyclin T1 and cyclin T2, shows differences in nuclear localization at distinct stages of adipogenesis. Overexpression of cdk9 increases the adipogenic potential of 3T3-L1 cells, whereas inhibition of cdk9 by specific cdk inhibitors, and dominant-negative cdk9 mutant impairs adipogenesis. We show that the positive effects of cdk9 on the differentiation of 3T3-L1 cells are mediated by a direct interaction with and phosphorylation of peroxisome proliferator-activated receptor gamma (PPARgamma), which is the master regulator of this process, on the promoter of PPARgamma target genes. PPARgamma-cdk9 interaction results in increased transcriptional activity of PPARgamma and therefore increased adipogenesis.

The nuclear receptor liver receptor homolog-1 is an estrogen receptor target gene.

Liver receptor homolog-1 (LRH-1) is a nuclear receptor previously known to have distinct functions during mouse development and essential roles in cholesterol homeostasis. Recently, a new role for LRH-1 has been discovered in tumor progression, giving LRH-1 potential transforming functions. In order to identify critical factors stimulating LRH-1 expression leading to deregulated cellular proliferation, we studied its expression and its regulation in several breast cancer cell lines. We observed that LRH-1 expression was increased in estrogen receptor (ER) alpha expressing cell lines, whereas weak-to-no expression was found in nonexpressing ERalpha cell lines. In MCF7, LRH-1 expression was highly induced after treatment with 17beta-estradiol (E2). This transcriptional regulation was the result of a direct binding of the ER to the LRH-1 promoter, as demonstrated by gelshift and chromatin immunoprecipitation assays. Interestingly, siRNA-mediated inactivation of LRH-1 decreased the E2-dependent proliferation of MCF7 cells. Finally, LRH-1 protein expression was detected by immunohistochemistry in tumor cells of human mammary ductal carcinomas. Altogether, these data demonstrate that LRH-1 is transcriptionally regulated by the ER alpha and reinforce the hypothesis that LRH-1 could exert potential oncogenic effects during breast cancer formation.

KAT2B Is Required for Pancreatic Beta Cell Adaptation to Metabolic Stress by Controlling the Unfolded Protein Response.

The endoplasmic reticulum (ER) unfolded protein response (UPR(er)) pathway plays an important role in helping pancreatic ß cells to adapt their cellular responses to environmental cues and metabolic stress. Although altered UPR(er) gene expression appears in rodent and human type 2 diabetic (T2D) islets, the underlying molecular mechanisms remain unknown. We show here that germline and ß cell-specific disruption of the lysine acetyltransferase 2B (Kat2b) gene in mice leads to impaired insulin secretion and glucose intolerance. Genome-wide analysis of Kat2b-regulated genes and functional assays reveal a critical role for Kat2b in maintaining UPR(er) gene expression and subsequent ß cell function. Importantly, Kat2b expression is decreased in mouse and human diabetic ß cells and correlates with UPR(er) gene expression in normal human islets. In conclusion, Kat2b is a crucial transcriptional regulator for adaptive ß cell function during metabolic stress by controlling UPR(er) and represents a promising target for T2D prevention and treatment.

CDK4 is an essential insulin effector in adipocytes.

Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4(R24C)). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT.

Mechanisms underlying differential expression of interleukin-8 in breast cancer cells.

We have recently reported that interleukin-8 (IL-8) expression was inversely correlated to estrogen receptor (ER) status and was overexpressed in invasive breast cancer cells. In the present study, we show that IL-8 overexpression in breast cancer cells involves a higher transcriptional activity of IL-8 gene promoter. Cloning of IL-8 promoter from MDA-MB-231 and MCF-7 cells expressing high and low levels of IL-8, respectively, shows the integrity of the promoter in both cell lines. Deletion and site-directed mutagenesis of the promoter demonstrate that NF-kappaB and AP-1 and to a lesser extent C/EBP binding sites play a crucial role in the control of IL-8 promoter activity in MDA-MB-231 cells. Knockdown of NF-kappaB and AP-1 activities by adenovirus-mediated expression of an NF-kappaB super-repressor and RNA interference, respectively, decreased IL-8 expression in MDA-MB-231 cells. On the contrary, restoration of Fra-1, Fra-2, c-Jun, p50, p65, C/EBPalpha and C/EBPbeta expression levels in MCF-7 cells led to a promoter activity comparable to that observed in MDA-MB-231 cells. Our data constitute the first extensive study of IL-8 gene overexpression in breast cancer cells and suggest that the high expression of IL-8 in invasive cancer cells requires a complex cooperation between NF-kappaB, AP-1 and C/EBP transcription factors.