Phospho-Akt expression is high in a subset of triple negative breast cancer patients

The most commonly used biomarkers to predict the response of breast cancer patients to therapy are oestrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Patients positive for these biomarkers are eligible for specific therapies such as anti-oestrogen therapy in the event of ER and PgR positivity, and trastuzumab, a monoclonal antibody, in the case of HER2 positive patients. Patients who are negative for all these three biomarkers, the so-called triple negatives, however, derive little benefit from such therapies. The PI3K/Akt pathway is activated in triple negative breast cancer cases, providing a possible target for therapy. The activation of Akt was investigated in Maltese triple negative breast cancer cases using an antibody detecting Akt phosphorylated at serine 473 (anti-Akt pS473). The study showed that 26\% of triple negative breast cancer patients had an elevated level of Akt (pS473). Furthermore, FTY720, a pharmacological activator of the phosphatase PP2A, was shown to block Akt activation at a concentration of 1\textmu M, in HCC1937 cells subjected to insulin-like growth factor 1 (IGF-1). Our data defined a subset of triple negative breast cancer patients based on high activity of AKT (pS473). This subset would be eligible for treatment using therapies which target the PI3K/Akt pathway, such as kinase inhibitors or phosphatase activators. In support of this, the BRCA1 mutant cells (HCC1937) were sensitive to the PP2a activator, FTY720. This suggests that FTY720 is a potential drug for use in adjuvant therapy in breast cancer cases having a high Akt (pS473).peer-reviewe

Deregulation of the phosphatase, PP2A is a common event in breast cancer, predicting sensitivity to FTY720

We would like to thank Professor Andrew Hanby, Professor Valerie Speirs and Dr Thomas A Hughes for the breast cancer cell lines used in this study. This research was supported by the Faculty of Medicine and Surgery, University of Malta.Background: The most commonly used biomarkers to predict the response of breast cancer patients to therapy are the oestrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Patients positive for these biomarkers are eligible for specific therapies such as endocrine treatment in the event of ER and PgR positivity, and the monoclonal antibody, trastuzumab, in the case of HER2-positive patients. Patients who are negative for these three biomarkers, the so-called triple negatives, however, derive little benefit from such therapies and are associated with a worse prognosis. Deregulation of the protein serine/threonine phosphatase type 2A (PP2A) and its regulatory subunits is a common event in breast cancer, providing a possible target for therapy. Methods: The data portal, cBioPortal for Cancer Genomics was used to investigate the incidence of conditions that are associated with low phosphatase activity. Four (4) adherent human breast cancer cell lines, MDA-MB-468, MDA-MB-436, Hs578T and BT-20 were cultured to assess their viability when exposed to various dosages of rapamycin or FTY720. In addition, RNA was extracted and cDNA was synthesised to amplify the coding sequence of PPP2CA. Amplification was followed by high-resolution melting to identify variations. Results and conclusion: The sequence of PPP2CA was found to be conserved across a diverse panel of solid tumour and haematological cell lines, suggesting that low expression of PPP2CA and differential binding of inhibitory PPP2CA regulators are the main mechanisms of PP2A deregulation. Interestingly, the cBioPortal for Cancer Genomics shows that PP2A is deregulated in 59.6% of basal breast tumours. Viability assays performed to determine the sensitivity of a panel of breast cancer cell lines to FTY720, a PP2A activator, indicated that cell lines associated with ER loss are sensitive to lower doses of FTY720. The subset of patients with suppressed PP2A activity is potentially eligible for treatment using therapies which target the PI3K/AKT/mTOR pathway, such as phosphatase activators.peer-reviewe

Electric fields can control the transport of water in carbon nanotubes

The properties of water confined inside nanotubes are of considerable scientific and technological interest. We use molecular dynamics to investigate the structure and average orientation of water flowing within a carbon nanotube. We find that water exhibits biaxial paranematic liquid crystal ordering both within the nanotube and close to its ends. This preferred molecular ordering is enhanced when an axial electric field is applied, affecting the water flow rate through the nanotube. A spatially patterned electric field can minimize nanotube entrance effects and significantly increase the flow rate

IGBP1 protein as a regulator of mTOR pathway and its role in haematopoiesis

Aims: Haematopoiesis is the life-long process of blood cell formation derived from haematopoietic stem cells (HSC). In haematopoietic precursor cells, the PI3K/mTOR pathway plays an important role in controlling the balance between proliferation and differentiation. Grech et al., (2008) found that the mTOR regulatory protein Igbp1 is potent in arresting erythroblast differentiation to mature erythrocytes. Progenitor cells constitutively expressing Igbp1 in fact proliferate exponentially for at least 72 hours without evidence of differentiation. PI3K activates mTOR through a cascade of phosphorylation events following the activation of PKB. The mTOR targets S6 kinase and 4E-BP1 enhance translation initiation of specific transcripts leading to increased protein synthesis and cell growth.In this study we specifically aim to investigate (1) the activation kinetics of mTOR targets by growth factors that regulate erythroid proliferation and (2) the role of protein phosphatase 2A (PP2A) on mTOR signaling with particular interest in the balance of haematopoietic cell proliferation versus differentiation. Methods: The methodology mainly focuses on the investigation of the effects of PP2A inhibitor Igbp1 and PP2A activator FTY720 on the control of mTOR signaling in a murine cell culture model developed by Von Lindern et al., (2001). This immortal murine factor-sensitive erythroid progenitor clones are cultured in a serum-free culture medium (Stem Pro 34TM), and can be factorprogrammed for renewal or terminal differentiation. Western blotting is the technique of choice in ultimately establishing and targeting the protein interplay at the levels of phorphorylated p70-S6 kinase and unphosphorylated p70-S6 kinase. This will enable us to track the action of Igbp1 on PP2A, since sequestration of PP2A by Igbp1 will inhibit dephosphorylation of S6 kinase. Results: Interestingly the synergistic action of erythropoietin and stem cell factor is required to phosphorylate S6K, giving a peak of S6K phosphorylation approximately 30 minutes after initial stimulation. Also, an experiment has been carried out to investigate FTY720 effects on growth of progenitor cells, were it was found that FTY720 attenuates cell growth in a dose-dependant fashion. Evidence of haemoglobin formation in cells dosed with FTY720 has been shown through haemoglobin assays, and thus we can anticipate that the reduced blast cell population was due to progenitor cell differentiation and not apoptosis. This shows that the block of differentiation empirically constituted by erythropoietin and stem cell factor is released by FTY720. Conclusions: Characterization of the mTOR signaling pathway regulators open up additional possibilities for the development of anticancer drugs. The importance of growth regulation in carcinogenesis has been further supported by the discovery that for example rapamycin, rapamycin analogues and FTY720 modulate mTOR. Therefore, the outcome of this study will eventually lead to understanding more the fundamental regulatory mechanisms involved in such an important pathway with potential identification of prognostic factors and therapeutic targets.peer-reviewe

P-Akt as a biomarker of a subset of triple negative breast cancer patients potentially sensitive to the pp2a activator, FTY720

Introduction: The most commonly used biomarkers to predict the response of breast cancer patients to therapy are oestrogen receptor (ER), progesterone receptor (PgR), and HER2. Patients positive for these biomarkers are eligible for specific therapies including anti-oestrogen therapy for ER and PgR positive patients, and trastuzumab, a monoclonal antibody, in the case of HER2 positive patients. Patients who are negative for all three biomarkers, the so-called triple negatives, however, derive little benefit from such therapies and are associated with a worse prognosis. The PI3K/Akt pathway has been found to be activated in triple negative breast cancer cases, providing a possible target for therapy. Patients having an elevated activation of the PI3K/Akt pathway could benefit from therapies targeting this pathway. Possibilities include using inhibitors of the PI3K/Akt pathway, or drugs which activate phosphatases involved in the pathway such as FTY720 which activates the phosphatase PP2A. Aim: The purpose of this study was to investigate the incidence of increased Akt activation in triple negative breast cancers in Malta by immunohistochemical staining, and the effect of FTY720 on the activation of Akt in two human breast cancer cell lines. Methodology: A serine-473 Akt1 antibody (p-Akt (S473)) was used to investigate the activation of Akt in triple negative breast cancer cases in the Maltese population. Scoring of stained sections was performed on the basis of intensity. Furthermore, the effect of FTY720, a pharmacological activator of the phosphatase PP2A which negatively regulates Akt activity, on the activity of Akt in two human breast cancer cell lines: MCF-7 and HCC1937, was investigated. This was tested under conditions of starvation, and also Akt stimulation by IGF-1 using In-Cell Western blotting. HCC1937 was of particular interest since it is also negative for ER, PgR, and HER2, and is known to have enhanced Akt activity. Results: 27% of triple negative breast cancer patients had an elevated level of p-Akt (S473). FTY720 at a concentration of 1μM, which did not affect cell viability, was shown to suppress Akt activation in MCF-7 and HCC1937 cells subjected to IGF- 1, an activator of Akt. Conclusion: The subset of triple negative having elevated Akt activation (27%) would be eligible for treatment using therapies which target the PI3K/Akt pathway, such as kinase inhibitors or phosphatase activators. The in vitro experiment using FTY720 suggests that it is a potential drug for use in adjuvant therapy in breast cancer cases having a high p-Akt (S473).peer-reviewe

High dietary fat intake increases fat oxidation and reduces skeletal muscle mitochondrial respiration in trained humans.

High-fat, low-carbohydrate (CHO) diets increase whole-body rates of fat oxidation and down-regulate CHO metabolism. We measured substrate utilization and skeletal muscle mitochondrial respiration to determine whether these adaptations are driven by high fat or low CHO availability. In a randomized crossover design, 8 male cyclists consumed 5 d of a high-CHO diet [>70% energy intake (EI)], followed by 5 d of either an isoenergetic high-fat (HFAT; >65% EI) or high-protein diet (HPRO; >65% EI) with CHO intake clamped at <20% EI. During the intervention, participants undertook daily exercise training. On d 6, participants consumed a high-CHO diet before performing 100 min of submaximal steady-state cycling plus an ∼30-min time trial. After 5 d of HFAT, skeletal muscle mitochondrial respiration supported by octanoylcarnitine and pyruvate, as well as uncoupled respiration, was decreased at rest, and rates of whole-body fat oxidation were higher during exercise compared with HPRO. After 1 d of high-CHO diet intake, mitochondrial respiration returned to baseline values in HFAT, whereas rates of substrate oxidation returned toward baseline in both conditions. These findings demonstrate that high dietary fat intake, rather than low-CHO intake, contributes to reductions in mitochondrial respiration and increases in whole-body rates of fat oxidation after a consuming a high-fat, low-CHO diet.-Leckey, J. J., Hoffman, N. J., Parr, E. B., Devlin, B. L., Trewin, A. J., Stepto, N. K., Morton, J. P., Burke, L. M., Hawley, J. A. High dietary fat intake increases fat oxidation and reduces skeletal muscle mitochondrial respiration in trained humans

Typical features of Parkinson disease and diagnostic challenges with microdeletion 22q11.2

Objective: To delineate the natural history, diagnosis, and treatment response of Parkinson disease (PD) in individuals with 22q11.2 deletion syndrome (22q11.2DS), and to determine if these patients differ from those with idiopathic PD. Methods: In this international observational study, we characterized the clinical and neuroimaging features of 45 individuals with 22q11.2DS and PD (mean follow-up 7.5 ± 4.1 years). Results: 22q11.2DS PD had a typical male excess (32 male, 71.1%), presentation and progression of hallmark motor symptoms, reduced striatal dopamine transporter binding with molecular imaging, and initial positive response to levodopa (93.3%). Mean age at motor symptom onset was relatively young (39.5 ± 8.5 years); 71.4% of cases had early-onset PD (<45 years). Despite having a similar age at onset, the diagnosis of PD was delayed in patients with a history of antipsychotic treatment compared with antipsychotic-naive patients (median 5 vs 1 year, p = 0.001). Preexisting psychotic disorders (24.5%) and mood or anxiety disorders (31.1%) were common, as were early dystonia (19.4%) and a history of seizures (33.3%). Conclusions: Major clinical characteristics and response to standard treatments appear comparable in 22q11.2DS-associated PD to those in idiopathic PD, although the average age at onset is earlier. Importantly, treatment of preexisting psychotic illness may delay diagnosis of PD in 22q11.DS patients. An index of suspicion and vigilance for complex comorbidity may assist in identifying patients to prioritize for genetic testing

Postexercise High-Fat Feeding Supresses p70S6K1 Activity in Human Skeletal Muscle.

PURPOSE: To examine the effects of reduced CHO but high post-exercise fat availability on cell signalling and expression of genes with putative roles in regulation of mitochondrial biogenesis, lipid metabolism and muscle protein synthesis (MPS). METHODS: Ten males completed a twice per day exercise model (3.5 h between sessions) comprising morning high-intensity interval (HIT) (8 x 5-min at 85% VO2peak) and afternoon steady-state (SS) running (60 min at 70% VO2peak). In a repeated measures design, runners exercised under different isoenergetic dietary conditions consisting of high CHO (HCHO: 10 CHO, 2.5 Protein and 0.8 Fat g.kg per whole trial period) or reduced CHO but high fat availability in the post-exercise recovery periods (HFAT: 2.5 CHO, 2.5 Protein and 3.5 Fat g.kg per whole trial period). RESULTS: Muscle glycogen was lower (P<0.05) at 3 (251 vs 301 mmol.kgdw) and 15 h (182 vs 312 mmol.kgdw) post-SS exercise in HFAT compared to HCHO. AMPK-α2 activity was not increased post-SS in either condition (P=0.41) though comparable increases (all P<0.05) in PGC-1α, p53, CS, Tfam, PPAR and ERRα mRNA were observed in HCHO and HFAT. In contrast, PDK4 (P=0.003), CD36 (P=0.05) and CPT1 (P=0.03) mRNA were greater in HFAT in the recovery period from SS exercise compared with HCHO. p70S6K activity was higher (P=0.08) at 3 h post-SS exercise in HCHO versus HFAT (72.7 ± 51.9 vs 44.7 ± 27 fmol.min mg). CONCLUSION: Post-exercise high fat feeding does not augment mRNA expression of genes associated with regulatory roles in mitochondrial biogenesis though it does increase lipid gene expression. However, post-exercise p70S6K1 activity is reduced under conditions of high fat feeding thus potentially impairing skeletal muscle remodelling processes

The Val158Met COMT polymorphism is a modifier of the age at onset in Parkinson's disease with a sexual dimorphism

The catechol-O-methyltranferase (COMT) is one of the main enzymes that metabolise dopamine in the brain. The Val158Met polymorphism in the COMT gene (rs4680) causes a trimodal distribution of high (Val/Val), intermediate (Val/Met) and low (Met/Met) enzyme activity. We tested whether the Val158Met polymorphism is a modifier of the age at onset (AAO) in Parkinson's disease (PD). The rs4680 was genotyped in a total of 16 609 subjects from five independent cohorts of European and North American origin (5886 patients with PD and 10 723 healthy controls). The multivariate analysis for comparing PD and control groups was based on a stepwise logistic regression, with gender, age and cohort origin included in the initial model. The multivariate analysis of the AAO was a mixed linear model, with COMT genotype and gender considered as fixed effects and cohort and cohort-gender interaction as random effects. COMT genotype was coded as a quantitative variable, assuming a codominant genetic effect. The distribution of the COMT polymorphism was not significantly different in patients and controls (p=0.22). The Val allele had a significant effect on the AAO with a younger AAO in patients with the Val/Val (57.1±13.9, p=0.03) than the Val/Met (57.4±13.9) and the Met/Met genotypes (58.3±13.5). The difference was greater in men (1.9 years between Val/Val and Met/Met, p=0.007) than in women (0.2 years, p=0.81). Thus, the Val158Met COMT polymorphism is not associated with PD in the Caucasian population but acts as a modifier of the AAO in PD with a sexual dimorphism: the Val allele is associated with a younger AAO in men with idiopathic PD