IGF1 regulates PKM2 function through Akt phosphorylation

Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1α, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1α expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells.     

Next generation sequencing demonstrates association between tumor suppressor gene aberrations and poor outcome in patients with cancer

Next generation sequencing is transforming patient care by allowing physicians to customize and match treatment to their patients’ tumor alterations. Our goal was to study the association between key molecular alterations and outcome parameters. We evaluated the characteristics and outcomes (overall survival (OS), time to metastasis/recurrence, and best progression-free survival (PFS)) of 392 patients for whom next generation sequencing (182 or 236 genes) had been performed. The Kaplan-Meier method and Cox regression models were used for our analysis, and results were subjected to internal validation using a resampling method (bootstrap analysis). In a multivariable analysis (Cox regression model), the parameters that were statistically associated with a poorer overall survival were the presence of metastases at diagnosis (P = 0.014), gastrointestinal histology (P < 0.0001), PTEN (P < 0.0001), and CDKN2A alterations (P = 0.0001). The variables associated with a shorter time to metastases/recurrence were gastrointestinal histology (P = 0.004), APC (P = 0.008), PTEN (P = 0.026) and TP53 (P = 0.044) alterations. TP53 (P = 0.003) and PTEN (P = 0.034) alterations were independent predictors of a shorter best PFS. A personalized treatment approach (matching the molecular aberration with a cognate targeted drug) also correlated with a longer best PFS (P = 0.046). Our study demonstrated that, across diverse cancers, anomalies in specific tumor suppressor genes (PTEN, CDKN2A, APC, and/or TP53) were independently associated with a worse outcome, as reflected by time to metastases/recurrence, best PFS on treatment, and/or overall survival. These observations suggest that molecular diagnostic tests may provide important prognostic information in patients with cancer.     

A Model of Drosophila Larva Chemotaxis

Detailed observations of larval Drosophila chemotaxis have characterised the relationship between the odour gradient and the runs, head casts and turns made by the animal. We use a computational model to test whether hypothesised sensorimotor control mechanisms are sufficient to account for larval behaviour. The model combines three mechanisms based on simple transformations of the recent history of odour intensity at the head location. The first is an increased probability of terminating runs in response to gradually decreasing concentration, the second an increased probability of terminating head casts in response to rapidly increasing concentration, and the third a biasing of run directions up concentration gradients through modulation of small head casts. We show that this model can be tuned to produce behavioural statistics comparable to those reported for the larva, and that this tuning results in similar chemotaxis performance to the larva. We demonstrate that each mechanism can enable odour approach but the combination of mechanisms is most effective, and investigate how these low-level control mechanisms relate to behavioural measures such as the preference indices used to investigate larval learning behaviour in group assays.     

Model-Based Evaluation of Spontaneous Tumor Regression in Pilocytic Astrocytoma

Pilocytic astrocytoma (PA) is the most common brain tumor in children. This tumor is usually benign and has a good prognosis. Total resection is the treatment of choice and will cure the majority of patients. However, often only partial resection is possible due to the location of the tumor. In that case, spontaneous regression, regrowth, or progression to a more aggressive form have been observed. The dependency between the residual tumor size and spontaneous regression is not understood yet. Therefore, the prognosis is largely unpredictable and there is controversy regarding the management of patients for whom complete resection cannot be achieved. Strategies span from pure observation (wait and see) to combinations of surgery, adjuvant chemotherapy, and radiotherapy. Here, we introduce a mathematical model to investigate the growth and progression behavior of PA. In particular, we propose a Markov chain model incorporating cell proliferation and death as well as mutations. Our model analysis shows that the tumor behavior after partial resection is essentially determined by a risk coefficient γ, which can be deduced from epidemiological data about PA. Our results quantitatively predict the regression probability of a partially resected benign PA given the residual tumor size and lead to the hypothesis that this dependency is linear, implying that removing any amount of tumor mass will improve prognosis. This finding stands in contrast to diffuse malignant glioma where an extent of resection threshold has been experimentally shown, below which no benefit for survival is expected. These results have important implications for future therapeutic studies in PA that should include residual tumor volume as a prognostic factor.     

Super-Resolution Imaging of ESCRT-Proteins at HIV-1 Assembly Sites

The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley’s L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.     

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.     

Independent Neuronal Origin of Seizures and Behavioral Comorbidities in an Animal Model of a Severe Childhood Genetic Epileptic Encephalopathy

The childhood epileptic encephalopathies (EE’s) are seizure disorders that broadly impact development including cognitive, sensory and motor progress with severe consequences and comorbidities. Recently, mutations in DNM1 (dynamin 1) have been implicated in two EE syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Dnm1 encodes dynamin 1, a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Dnm1^Ftfl or “fitful” mice carry a spontaneous mutation in the mouse ortholog of DNM1 and recapitulate many of the disease features associated with human DNM1 patients, providing a relevant disease model of human EE’s. In order to examine the cellular etiology of seizures and behavioral and neurological comorbidities, we engineered a conditional Dnm1^Ftfl mouse model of DNM1 EE. Observations of Dnm1 ^Ftfl/flox mice in combination with various neuronal subpopulation specific cre strains demonstrate unique seizure phenotypes and clear separation of major neurobehavioral comorbidities from severe seizures associated with the germline model. This demonstration of pleiotropy suggests that treating seizures per se may not prevent severe comorbidity observed in EE associated with dynamin-1 mutations, and is likely to have implications for other genetic forms of EE.     

The regulation of connective tissue growth factor expression influences the viability of human trabecular meshwork cells

Connective tissue growth factor (CTGF) induces extracellular matrix (ECM) synthesis and contractility in human trabecular meshwork (HTM) cells. Both processes are involved in the pathogenesis of primary open‐angle glaucoma. To date, little is known about regulation and function of CTGF expression in the trabecular meshwork (TM). Therefore, we analysed the effects of different aqueous humour proteins and stressors on CTGF expression in HTM cells. HTM cells from three different donors were treated with endothelin‐1, insulin‐like growth factor (IGF)‐1, angiotensin‐II, H₂O₂ and heat shock and were analysed by immunohistochemistry, real‐time RT‐PCR and Western blotting. Viability after H₂O₂ treatment was measured in CTGF silenced HTM‐N cells and their controls. Latrunculin A reduced expression of CTGF by about 50% compared to untreated HTM cells, whereas endothelin‐1, IGF‐1, angiotensin‐II, heat shock and oxidative stress led to a significant increase. Silencing of CTGF resulted in a delayed expression of αB‐crystallin and in reduced cell viability in comparison to the controls after oxidative stress. Conversely, CTGF treatment led to a higher cell viability rate after H₂O₂ treatment. CTGF expression is induced by factors that have been linked to glaucoma. An increased level of CTGF appears to protect TM cells against damage induced by stress. The beneficial effect of CTGF for viability of TM cells is likely associated with the effects on increased ECM synthesis and higher contractility of the TM, thereby contributing to reduced aqueous humour outflow facility causing increased intraocular pressure.