Increased PTCHD4 expression via m6A modification of PTCHD4 mRNA promotes senescent cell survival.

RNA modifications, including N6-methyladenosine (m6A), critically modulate protein expression programs in a range of cellular processes. Although the transcriptomes of cells undergoing senescence are strongly regulated, the landscape and impact of m6A modifications during senescence are poorly understood. Here, we report a robust m6A modification of PTCHD4 mRNA, encoding Patched Domain-Containing Protein 4, in senescent cells. The METTL3/METTL14 complex was found to incorporate the m6A modification on PTCHD4 mRNA; addition of m6A rendered PTCHD4 mRNA more stable and increased PTCHD4 production. MeRIP RT-qPCR and eCLIP analyses were used to map this m6A modification to the last exon of PTCHD4 mRNA. Further investigation identified IGF2BP1, but not other m6A readers, as responsible for the stabilization and increased abundance of m6A-modified PTCHD4 mRNA. Silencing PTCHD4, a transmembrane protein, enhanced growth arrest and DNA damage in pre-senescent cells and sensitized them to senolysis and apoptosis. Our results indicate that m6A modification of PTCHD4 mRNA increases the production of PTCHD4, a protein associated with senescent cell survival, supporting the notion that regulating m6A modification on specific mRNAs could be exploited to eliminate senescent cells for therapeutic benefit.

Aberrant expression and localization of the RAP1 shelterin protein contribute to age-related phenotypes.

Short telomeres induce a DNA damage response (DDR) that evokes apoptosis and senescence in human cells. An extant question is the contribution of telomere dysfunction-induced DDR to the phenotypes observed in aging and telomere biology disorders. One candidate is RAP1, a telomere-associated protein that also controls transcription at extratelomeric regions. To distinguish these roles, we generated a knockin mouse carrying a mutated Rap1, which was incapable of binding telomeres and did not result in eroded telomeres or a DDR. Primary Rap1 knockin embryonic fibroblasts showed decreased RAP1 expression and re-localization away from telomeres, with an increased cytosolic distribution akin to that observed in human fibroblasts undergoing telomere erosion. Rap1 knockin mice were viable, but exhibited transcriptomic alterations, proinflammatory cytokine/chemokine signaling, reduced lifespan, and decreased healthspan with increased body weight/fasting blood glucose levels, spontaneous tumor incidence, and behavioral deficits. Taken together, our data present mechanisms distinct from telomere-induced DDR that underlie age-related phenotypes.

sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance.

Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in ß-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.

Eda-activated RelB recruits an SWI/SNF (BAF) chromatin-remodeling complex and initiates gene transcription in skin appendage formation.

Ectodysplasin A (Eda) signaling activates NF-κB during skin appendage formation, but how Eda controls specific gene transcription remains unclear. Here, we find that Eda triggers the formation of an NF-κB-associated SWI/SNF (BAF) complex in which p50/RelB recruits a linker protein, Tfg, that interacts with BAF45d in the BAF complex. We further reveal that Tfg is initially induced by Eda-mediated RelB activation and then bridges RelB and BAF for subsequent gene regulation. The BAF component BAF250a is particularly up-regulated in skin appendages, and epidermal knockout of BAF250a impairs skin appendage development, resulting in phenotypes similar to those of Eda-deficient mouse models. Transcription profiling identifies several target genes regulated by Eda, RelB, and BAF. Notably, RelB and the BAF complex are indispensable for transcription of Eda target genes, and both BAF complex and Eda signaling are required to open chromatin of Eda targets. Our studies thus suggest that Eda initiates a signaling cascade and recruits a BAF complex to specific gene loci to facilitate transcription during organogenesis.

Muscle cannabinoid 1 receptor regulates Il-6 and myostatin expression, governing physical performance and whole-body metabolism.

Sarcopenic obesity, the combination of skeletal muscle mass and function loss with an increase in body fat, is associated with physical limitations, cardiovascular diseases, metabolic stress, and increased risk of mortality. Cannabinoid receptor type 1 (CB1R) plays a critical role in the regulation of whole-body energy metabolism because of its involvement in controlling appetite, fuel distribution, and utilization. Inhibition of CB1R improves insulin secretion and insulin sensitivity in pancreatic ß-cells and hepatocytes. We have now developed a skeletal muscle-specific CB1R-knockout (Skm-CB1R-/-) mouse to study the specific role of CB1R in muscle. Muscle-CB1R ablation prevented diet-induced and age-induced insulin resistance by increasing IR signaling. Moreover, muscle-CB1R ablation enhanced AKT signaling, reducing myostatin expression and increasing IL-6 secretion. Subsequently, muscle-CB1R ablation increased myogenesis through its action on MAPK-mediated myogenic gene expression. Consequently, Skm-CB1R-/- mice had increased muscle mass and whole-body lean/fat ratio in obesity and aging. Muscle-CB1R ablation improved mitochondrial performance, leading to increased whole-body muscle energy expenditure and improved physical endurance, with no change in body weight. These results collectively show that CB1R in muscle is sufficient to regulate whole-body metabolism and physical performance and is a novel target for the treatment of sarcopenic obesity. -González-Mariscal, I., Montoro, R. A., O'Connell, J. F., Kim, Y., Gonzalez-Freire, M., Liu, Q.-R., Alfaras, I., Carlson, O. D., Lehrmann, E., Zhang, Y., Becker, K. G., Hardivillé, S., Ghosh, P., Egan, J. M. Muscle cannabinoid 1 receptor regulates Il-6 and myostatin expression, governing physical performance and whole-body metabolism.

MIR100 host gene-encoded lncRNAs regulate cell cycle by modulating the interaction between HuR and its target mRNAs.

Long non-coding RNAs (lncRNAs) regulate vital biological processes, including cell proliferation, differentiation and development. A subclass of lncRNAs is synthesized from microRNA (miRNA) host genes (MIRHGs) due to pre-miRNA processing, and are categorized as miRNA-host gene lncRNAs (lnc-miRHGs). Presently, the cellular function of most lnc-miRHGs is not well understood. We demonstrate a miRNA-independent role for a nuclear-enriched lnc-miRHG in cell cycle progression. MIR100HG produces spliced and stable lncRNAs that display elevated levels during the G1 phase of the cell cycle. Depletion of MIR100HG-encoded lncRNAs in human cells results in aberrant cell cycle progression without altering the levels of miRNA encoded within MIR100HG. Notably, MIR100HG interacts with HuR/ELAVL1 as well as with several HuR-target mRNAs. Further, MIR100HG-depleted cells show reduced interaction between HuR and three of its target mRNAs, indicating that MIR100HG facilitates interaction between HuR and target mRNAs. Our studies have unearthed novel roles played by a MIRHG-encoded lncRNA in regulating RNA binding protein activity, thereby underscoring the importance of determining the function of several hundreds of lnc-miRHGs that are present in human genome.

Metabolic and molecular framework for the enhancement of endurance by intermittent food deprivation.

Evolutionary considerations suggest that the body has been optimized to perform at a high level in the food-deprived state when fatty acids and their ketone metabolites are a major fuel source for muscle cells. Because controlled food deprivation in laboratory animals and intermittent energy restriction in humans is a potent physiologic stimulus for ketosis, we designed a study to determine the impact of intermittent food deprivation during endurance training on performance and to elucidate the underlying cellular and molecular mechanisms. Male mice were randomly assigned to either ad libitum feeding or alternate-day food deprivation (ADF) groups, and half of the mice in each diet group were trained daily on a treadmill for 1 mo. A run to exhaustion endurance test performed at the end of the training period revealed superior performance in the mice maintained on ADF during training compared to mice fed ad libitum during training. Maximal O2 consumption was increased similarly by treadmill training in mice on ADF or ad libitum diets, whereas respiratory exchange ratio was reduced in ADF mice on food-deprivation days and during running. Analyses of gene expression in liver and soleus tissues, and metabolomics analysis of blood suggest that the metabolic switch invoked by ADF and potentiated by exercise strongly modulates molecular pathways involved in mitochondrial biogenesis, metabolism, and cellular plasticity. Our findings demonstrate that ADF engages metabolic and cellular signaling pathways that result in increased metabolic efficiency and endurance capacity.-Marosi, K., Moehl, K., Navas-Enamorado, I., Mitchell, S. J., Zhang, Y., Lehrmann, E., Aon, M. A., Cortassa, S., Becker, K. G., Mattson, M. P. Metabolic and molecular framework for the enhancement of endurance by intermittent food deprivation.

Regulation of HuR structure and function by dihydrotanshinone-I.

The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.

Absence of cannabinoid 1 receptor in beta cells protects against high-fat/high-sugar diet-induced beta cell dysfunction and inflammation in murine islets.

AIMS/HYPOTHESIS: The cannabinoid 1 receptor (CB1R) regulates insulin sensitivity and glucose metabolism in peripheral tissues. CB1R is expressed on pancreatic beta cells and is coupled to the G protein Gαi, suggesting a negative regulation of endogenous signalling in the beta cell. Deciphering the exact function of CB1R in beta cells has been confounded by the expression of this receptor on multiple tissues involved in regulating metabolism. Thus, in models of global genetic or pharmacological CB1R blockade, it is difficult to distinguish the indirect effects of improved insulin sensitivity in peripheral tissues from the direct effects of inhibiting CB1R in beta cells per se. To assess the direct contribution of beta cell CB1R to metabolism, we designed a mouse model that allows us to determine the role of CB1R specifically in beta cells in the context of whole-body metabolism. METHODS: We generated a beta cell specific Cnr1 (CB1R) knockout mouse (ß-CB1R-/-) to study the long-term consequences of CB1R ablation on beta cell function in adult mice. We measured beta cell function, proliferation and viability in these mice in response to a high-fat/high-sugar diet and induction of acute insulin resistance with the insulin receptor antagonist S961. RESULTS: ß-CB1R-/- mice had increased fasting (153 ± 23% increase at 10 weeks of age) and stimulated insulin secretion and increased intra-islet cAMP levels (217 ± 33% increase at 10 weeks of age), resulting in primary hyperinsulinaemia, as well as increased beta cell viability, proliferation and islet area (1.9-fold increase at 10 weeks of age). Hyperinsulinaemia led to insulin resistance, which was aggravated by a high-fat/high-sugar diet and weight gain, although beta cells maintained their insulin secretory capacity in response to glucose. Strikingly, islets from ß-CB1R-/- mice were protected from diet-induced inflammation. Mechanistically, we show that this is a consequence of curtailment of oxidative stress and reduced activation of the NLRP3 inflammasome in beta cells. CONCLUSIONS/INTERPRETATION: Our data demonstrate CB1R to be a negative regulator of beta cell function and a mediator of islet inflammation under conditions of metabolic stress. Our findings point to beta cell CB1R as a therapeutic target, and broaden its potential to include anti-inflammatory effects in both major forms of diabetes. DATA AVAILABILITY: Microarray data have been deposited at GEO (GSE102027).

Altered learning, memory, and social behavior in type 1 taste receptor subunit 3 knock-out mice are associated with neuronal dysfunction.

The type 1 taste receptor member 3 (T1R3) is a G protein-coupled receptor involved in sweet-taste perception. Besides the tongue, the T1R3 receptor is highly expressed in brain areas implicated in cognition, including the hippocampus and cortex. As cognitive decline is often preceded by significant metabolic or endocrinological dysfunctions regulated by the sweet-taste perception system, we hypothesized that a disruption of the sweet-taste perception in the brain could have a key role in the development of cognitive dysfunction. To assess the importance of the sweet-taste receptors in the brain, we conducted transcriptomic and proteomic analyses of cortical and hippocampal tissues isolated from T1R3 knock-out (T1R3KO) mice. The effect of an impaired sweet-taste perception system on cognition functions were examined by analyzing synaptic integrity and performing animal behavior on T1R3KO mice. Although T1R3KO mice did not present a metabolically disrupted phenotype, bioinformatic interpretation of the high-dimensionality data indicated a strong neurodegenerative signature associated with significant alterations in pathways involved in neuritogenesis, dendritic growth, and synaptogenesis. Furthermore, a significantly reduced dendritic spine density was observed in T1R3KO mice together with alterations in learning and memory functions as well as sociability deficits. Taken together our data suggest that the sweet-taste receptor system plays an important neurotrophic role in the extralingual central nervous tissue that underpins synaptic function, memory acquisition, and social behavior.

Novel RNA-binding activity of MYF5 enhances Ccnd1/Cyclin D1 mRNA translation during myogenesis.

Skeletal muscle contains long multinucleated and contractile structures known as muscle fibers, which arise from the fusion of myoblasts into multinucleated myotubes during myogenesis. The myogenic regulatory factor (MRF) MYF5 is the earliest to be expressed during myogenesis and functions as a transcription factor in muscle progenitor cells (satellite cells) and myocytes. In mouse C2C12 myocytes, MYF5 is implicated in the initial steps of myoblast differentiation into myotubes. Here, using ribonucleoprotein immunoprecipitation (RIP) analysis, we discovered a novel function for MYF5 as an RNA-binding protein which associated with a subset of myoblast mRNAs. One prominent MYF5 target was Ccnd1 mRNA, which encodes the key cell cycle regulator CCND1 (Cyclin D1). Biotin-RNA pulldown, UV-crosslinking and gel shift experiments indicated that MYF5 was capable of binding the 3' untranslated region (UTR) and the coding region (CR) of Ccnd1 mRNA. Silencing MYF5 expression in proliferating myoblasts revealed that MYF5 promoted CCND1 translation and modestly increased transcription of Ccnd1 mRNA. Accordingly, overexpressing MYF5 in C2C12 cells upregulated CCND1 expression while silencing MYF5 reduced myoblast proliferation as well as differentiation of myoblasts into myotubes. Moreover, MYF5 silencing reduced myogenesis, while ectopically restoring CCND1 abundance partially rescued the decrease in myogenesis seen after MYF5 silencing. We propose that MYF5 enhances early myogenesis in part by coordinately elevating Ccnd1 transcription and Ccnd1 mRNA translation.

Down-regulation of eIF4GII by miR-520c-3p represses diffuse large B cell lymphoma development.

Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.

Experience Modulates the Effects of Histone Deacetylase Inhibitors on Gene and Protein Expression in the Hippocampus: Impaired Plasticity in Aging.

The therapeutic potential of histone deacetylase inhibitor (HDACi) treatment has attracted considerable attention in the emerging area of cognitive neuroepigenetics. The possibility that ongoing cognitive experience importantly regulates the cell biological effects of HDACi administration, however, has not been systematically examined. In an initial experiment addressing this issue, we tested whether water maze training influences the gene expression response to acute systemic HDACi administration in the young adult rat hippocampus. Training powerfully modulated the response to HDACi treatment, increasing the total number of genes regulated to nearly 3000, including many not typically linked to neural plasticity, compared with <300 following HDACi administration alone. Although water maze training itself also regulated nearly 1800 genes, the specific mRNAs, gene networks, and biological pathways involved were largely distinct when the same experience was provided together with HDACi administration. Next, we tested whether the synaptic protein response to HDACi treatment is similarly dependent on recent cognitive experience, and whether this plasticity is altered in aged rats with memory impairment. Whereas synaptic protein labeling in the young hippocampus was selectively increased when HDACi administration was provided in conjunction with water maze training, combined treatment had no effect on synaptic proteins in the aged hippocampus. Our findings indicate that ongoing experience potently regulates the molecular consequences of HDACi treatment and that the interaction of recent cognitive experience with histone acetylation dynamics is disrupted in the aged hippocampus. SIGNIFICANCE STATEMENT: The possibility that interventions targeting epigenetic regulation could be effective in treating a range of neurodegenerative disorders has attracted considerable interest. Here we demonstrate in the rat hippocampus that ongoing experience powerfully modifies the molecular response to one such intervention, histone deacetylase inhibitor (HDACi) administration. A single learning episode dramatically shifts the gene expression profile induced by acute HDACi treatment, yielding a qualitatively distinct hippocampal transcriptome compared with the influence of behavioral training alone. The downstream synaptic protein response to HDACi administration is similarly experience-dependent, and we report that this plasticity is disrupted in the aged hippocampus. The findings highlight that accommodating the modulatory influence of ongoing experience represents a challenge for therapeutic development in the area of cognitive neuroepigenetics.

RPTOR, a novel target of miR-155, elicits a fibrotic phenotype of cystic fibrosis lung epithelium by upregulating CTGF.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the most frequent of which is F508del-CFTR. CF is characterized by excessive secretion of pro-inflammatory mediators into the airway lumen, inducing a highly inflammatory cellular phenotype. This process triggers fibrosis, causing airway destruction and leading to high morbidity and mortality. We previously reported that miR-155 is upregulated in CF lung epithelial cells, but the molecular mechanisms by which miR-155 affects the disease phenotype is not understood. Here we report that RPTOR (regulatory associated protein of mTOR, complex 1) is a novel target of miR-155 in CF lung epithelial cells. The suppression of RPTOR expression and subsequent activation of TGF-ß signaling resulted in the induction of fibrosis by elevating connective tissue growth factor (CTGF) abundance in CF lung epithelial cells. Thus, we propose that miR-155 might regulate fibrosis of CF lungs through the increased CTGF expression, highlighting its potential value in CF therapy.

Blast traumatic brain injury-induced cognitive deficits are attenuated by preinjury or postinjury treatment with the glucagon-like peptide-1 receptor agonist, exendin-4.

INTRODUCTION: Blast traumatic brain injury (B-TBI) affects military and civilian personnel. Presently, there are no approved drugs for blast brain injury. METHODS: Exendin-4 (Ex-4), administered subcutaneously, was evaluated as a pretreatment (48 hours) and postinjury treatment (2 hours) on neurodegeneration, behaviors, and gene expressions in a murine open field model of blast injury. RESULTS: B-TBI induced neurodegeneration, changes in cognition, and genes expressions linked to dementia disorders. Ex-4, administered preinjury or postinjury, ameliorated B-TBI-induced neurodegeneration at 72 hours, memory deficits from days 7-14, and attenuated genes regulated by blast at day 14 postinjury. DISCUSSION: The present data suggest shared pathologic processes between concussive and B-TBI, with end points amenable to beneficial therapeutic manipulation by Ex-4. B-TBI-induced dementia-related gene pathways and cognitive deficits in mice somewhat parallel epidemiologic studies of Barnes et al. who identified a greater risk in US military veterans who experienced diverse TBIs, for dementia in later life.

Delineation of a conserved arrestin-biased signaling repertoire in vivo.

Biased G protein-coupled receptor agonists engender a restricted repertoire of downstream events from their cognate receptors, permitting them to produce mixed agonist-antagonist effects in vivo. While this opens the possibility of novel therapeutics, it complicates rational drug design, since the in vivo response to a biased agonist cannot be reliably predicted from its in cellula efficacy. We have employed novel informatic approaches to characterize the in vivo transcriptomic signature of the arrestin pathway-selective parathyroid hormone analog [d-Trp(12), Tyr(34)]bovine PTH(7-34) in six different murine tissues after chronic drug exposure. We find that [d-Trp(12), Tyr(34)]bovine PTH(7-34) elicits a distinctive arrestin-signaling focused transcriptomic response that is more coherently regulated across tissues than that of the pluripotent agonist, human PTH(1-34). This arrestin-focused network is closely associated with transcriptional control of cell growth and development. Our demonstration of a conserved arrestin-dependent transcriptomic signature suggests a framework within which the in vivo outcomes of arrestin-biased signaling may be generalized.

Global dissociation of HuR-mRNA complexes promotes cell survival after ionizing radiation.

Ionizing radiation (IR) triggers adaptive changes in gene expression. Here, we show that survival after IR strongly depends on the checkpoint kinase Chk2 acting upon its substrate HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. Microarray analysis showed that in human HCT116 colorectal carcinoma cells (WT), IR-activated Chk2 triggered the dissociation of virtually all of HuR-bound mRNAs, since IR did not dissociate HuR target mRNAs in Chk2-null (CHK2-/-) HCT116 cells. Accordingly, several HuR-interacting mRNAs encoding apoptosis- and proliferation-related proteins (TJP1, Mdm2, TP53BP2, Bax, K-Ras) dissociated from HuR in WT cells, but remained bound and showed altered post-transcriptional regulation in CHK2-/- cells. Use of HuR mutants that were not phosphorylatable by Chk2 (HuR(3A)) and HuR mutants mimicking constitutive phosphorylation by Chk2 (HuR(3D)) revealed that dissociation of HuR target transcripts enhanced cell survival. We propose that the release of HuR-bound mRNAs via an IR-Chk2-HuR regulatory axis improves cell outcome following IR.

Distinct inhibitory effects on mTOR signaling by ethanol and INK128 in diffuse large B-cell lymphoma.

BACKGROUND: The mechanistic target of rapamycin, (mTOR) kinase plays a pivotal role in controlling critical cellular growth and survival pathways, and its aberrant induction is implicated in cancer pathogenesis. Therefore, suppression of active mTOR signaling has been of great interest to researchers; several mTOR inhibitors have been discovered to date. Ethanol (EtOH), similar to pharmacologic mTOR inhibitors, has been shown to suppress the mTOR signaling pathway, though in a non-catalytic manner. Despite population studies showing that the consumption of EtOH has a protective effect against hematological malignancies, the mechanisms behind EtOH's modulation of mTOR activity in cells and its downstream consequences are largely unknown. Here we evaluated the effects of EtOH on the mTOR pathway, in comparison to the active-site mTOR inhibitor INK128, and compared translatome analysis of their downstream effects in diffuse large B-cell lymphoma (DLBCL). RESULTS: Treatment of DLBCL cells with EtOH suppressed mTORC1 complex formation while increasing AKT phosphorylation and mTORC2 complex assembly. INK128 completely abrogated AKT phosphorylation without affecting the structure of mTORC1/2 complexes. Accordingly, EtOH less profoundly suppressed cap-dependent translation and global protein synthesis, compared to a remarkable inhibitory effect of INK128 treatment. Importantly, EtOH treatment induced the formation of stress granules, while INK128 suppressed their formation. Microarray analysis of polysomal RNA revealed that although both agents primarily affected cell growth and survival, EtOH and INK128 regulated the synthesis of mostly distinct genes involved in these processes. Though both EtOH and INK128 inhibited cell cycle, proliferation and autophagy, EtOH, in contrast to INK128, did not induce cell apoptosis. CONCLUSION: Given that EtOH, similar to pharmacologic mTOR inhibitors, inhibits mTOR signaling, we systematically explored the effect of EtOH and INK128 on mTOR signal transduction, components of the mTORC1/2 interaction and their downstream effectors in DLBCL malignancy. We found that EtOH partially inhibits mTOR signaling and protein translation, compared to INK128's complete mTOR inhibition. Translatome analysis of mTOR downstream target genes established that differential inhibition of mTOR by EtOH and INK128 distinctly modulates translation of specific subsets of mRNAs involved in cell growth and survival, leading to differential cellular response and survival.

A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high-grade serous carcinoma development.

Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter. Histological analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal-appearing p53-positive epithelium that are similar to 'p53 signatures' in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these non-invasive precursor lesions, we also identified invasive adenocarcinoma in the ovaries of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and wild-type (WT) C57BL/6. One of these genes, Top2a, which encodes topoisomerase IIα, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and was specifically elevated in mouse STICs but not in the surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumourigenesis, as well as for developing novel approaches for the prevention, diagnosis and therapy of this disease.