Inhibition of autophagy sensitizes malignant pleural mesothelioma cells to dual PI3K/mTOR inhibitors

Malignant pleural mesothelioma (MPM) originates in most of the cases from chronic inflammation of the mesothelium due to exposure to asbestos fibers. Given the limited effect of chemotherapy, a big effort is being made to find new treatment options. The PI3K/mTOR pathway was reported to be upregulated in MPM. We tested the cell growth inhibition properties of two dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 on 19 MPM cell lines. We could identify resistant and sensitive lines; however, there was no correlation to the downregulation of PI3K/mTOR activity markers. As a result of mTOR inhibition, both drugs efficiently induced long-term autophagy but not cell death. Autophagy blockade by chloroquine in combination with the dual PI3K/mTOR inhibitors significantly induced caspase-independent cell death involving RIP1 in the sensitive cell line SPC212. Cell death in the resistant cell line Mero-82 was less pronounced, and it was not induced via RIP1-dependent mechanism, suggesting the involvement of RIP1 downstream effectors. Cell death induction was confirmed in 3D systems. Based on these results, we identify autophagy as one of the main mechanisms of cell death resistance against dual PI3K/mTOR inhibitors in MPM. As PI3K/mTOR inhibitors are under investigation in clinical trials, these results may help interpreting their outcome and suggest ways for intervention.Malignant pleural mesothelioma (MPM) is sensitive to phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling inhibitors due to the activation of PI3K/mTOR signaling.^{1, 2} The activation may result from inactivation of INP4A phosphatase, which is downregulated in 44% of MPM (presented at IMIG2014), or alterations in PI3K signaling components, which are mutated in 9% of MPM,³ while receptor tyrosine kinase mutations/amplifications have not been identified in two recent high-throughput studies.^{4, 5}One of the tumor-suppressor genes frequently mutated in MPM is NF2 and NF2-null cells were shown to be sensitive to growth-inhibitory effects of rapamycin⁶ via mechanisms involving PI3K signaling-independent mTORC1 activation. However, the mTOR inhibitor, everolimus, showed no therapeutic benefit in unselected MPM patients.⁷ As mTORC1 inhibitors often lead to a feedback activation of PI3K activation in cancers,^{8, 9} we postulated that dual PI3K–mTOR inhibitors may yield greater therapeutic benefit. Furthermore, NF2 was also shown to inhibit PI3K activity by binding to PI3K enhancer-L (PIKE-L), which disrupts binding of PIKE-L to PI3K¹⁰ and loss of NF2 in schwannoma was shown to sensitize to PI3K inhibitors.¹¹In a screen on the dual PI3K/mTOR inhibitor NVP-BEZ235, within the Sanger Institute/MGH''s ‘Genomics of Drug Sensitivity'' screening panel,¹² CDKN2A deletion was shown to be associated with increased sensitivity. Because NF2 and CDKN2A are indeed the genes most frequently mutated in MPM, blocking PI3K/mTOR signaling might be a valid approach to circumvent the difficulty of applying targeted therapy in the absence of an identified oncogene. The rationale for targeting the PI3K/mTOR pathway is also supported by the association of increased activity with a worse clinical outcome.^{13, 14}NVP-BEZ235^(ref15) and GDC-0980^(ref16) are small-molecule inhibitors of class I PI3K and mTOR (mTORC1 and mTORC2). GDC-0980 has been tested in phase I studies where the phase I extension cohort showed two objective responses among 26 patients with mesothelioma.¹⁷ Despite these encouraging results, this drug will not be explored further because of side effects observed in another clinical trial.¹⁸ This, however, should not deter us for trying to find means to improve the antitumor effect of this class of agents. We have previously shown that PI3K/mTOR signaling inhibition sensitizes mesothelioma cells to drugs that are effluxed via ABCG2 transporter by inhibiting the function of ABCG2.¹⁹ In this study, we aimed at identifying the underlying mechanisms responsible for sensitivity versus resistance towards PI3K/mTOR inhibition in a large panel of mesothelioma cell lines. We observed that PI3K/mTOR inhibition increases autophagic rate, which constitutes an efficient mechanism of resistance by inducing growth arrest and survival. However, blocking autophagy, which per se affects cell growth, is synthetically lethal when combined with PI3K/mTOR inhibitors by a mechanism involving receptor-interacting protein kinase 1 (RIP1)-dependent cell death.     

Altered sense of Agency in children with spastic cerebral palsy

Background  

Children diagnosed with spastic Cerebral Palsy (CP) often show perceptual and cognitive problems, which may contribute to their functional deficit. Here we investigated if altered ability to determine whether an observed movement is performed by themselves (sense of agency) contributes to the motor deficit in children with CP.     

Differential Effects of Lovastatin on Cisplatin Responses in Normal Human Mesothelial Cells versus Cancer Cells: Implication for Therapy

The cancer killing efficacy of standard chemotherapeutic agents such as cisplatin (CDDP) is limited by their side effects to normal tissues. Therefore, research efforts optimizing the safety and efficacy of those agents are clinically relevant. We did screen for agents that specifically protect normal human mesothelial cells against CDDP without reducing the cancer cell killing efficacy. Lovastatin was identified from the screen. Lovastatin at a pharmacologically relevant concentration strongly arrested the proliferation of normal cells, whereas cancer cells were less affected. CDDP-induced DNA damage response was not activated and normal cells showed enhanced tolerance to CDDP when normal cells were treated with the combination of CDDP and lovastatin. We demonstrate that interfering with protein geranylgeranylation is involved in the lovastatin-mediated CDDP protective effect in normal cells. In contrast to normal cells, in cancer cells lovastatin did not change the CDDP-induced response, and cancer cells were not protected by lovastatin. Furthermore, lovastatin at the pharmacological relevant concentration per se induced DNA damage, oxidative stress and autophagy in cancer cells but not in normal mesothelial cells. Therefore, our data suggest that lovastatin has a potential to improve the therapeutic index of cisplatin-based therapy.     

The Cytochrome b5 Tail Anchors and Stabilizes Subdomains of Human DNA Topoisomerase IIα in the Cytoplasm of Retrovirally Infected Mammalian Cells

DNA topoisomerase II (topo II) is the target of many anticancer drugs and is often altered in drug-resistant cell lines. In some tumor cell lines truncated isoforms of topo IIα are localized to the cytoplasm. To study the localization and function of individual enzyme domains, we have epitope-tagged several fragments of human topo IIα and expressed them by retroviral infection of rodent and human cells. We find that fusion of the topo II fragments to the hydrophobic tail of human liver cytochrome b₅ anchors the fusion protein to the outer face of cytoplasmic membranes, as determined by colocalization with calnexin and selective detergent permeabilization. Moreover, whereas the minimal ATPase domain (aa 1–266) is weakly and diffusely expressed, addition of the cytb₅ anchor (1–266-b₅) increases its steady-state level 16-fold with no apparent toxicity. Similar results are obtained with the complete ATPase domain (aa 1–426). A C-terminal domain (aa 1030–1504) of human topo IIα containing an intact dimerization motif is stably expressed and accumulates in the nucleus. Fusion to the cytb₅ anchor counteracts the nuclear localization signal and relocalizes the protein to cytoplasmic membranes. In conclusion, we describe a technique that stabilizes and targets retrovirally expressed proteins such that they are exposed on the cytoplasmic surface of cellular membranes. This approach may be of general use for regulating the nuclear accumulation of drugs or proteins in living cells.     

Identification of candidate biomarkers of brain damage in a mouse model of closed head injury: a metabolomic pilot study

We aim to identify candidate brain biomarkers for, and to elucidate the pathophysiology of closed traumatic brain injury (TBI). Nuclear magnetic resonance (NMR) based metabolomic analysis was performed on the whole brain of mice undergoing TBI using a validated technique. There were 10 TBI mice compared to 8 sham operated controls. A total of 45 metabolites were evaluated. There was a statistically significant alteration in concentrations of 29 metabolites in TBI brains as compared to controls (FDR <0.05). Profound disturbances of several metabolic pathways (FDR <1E-07), including pathways associated with purine, alanine, aspartate and glutamine and glutathione metabolism were observed. Also, a significant elevation in glutamate (the main excitatory neurotransmitter) and depression of GABA (the main inhibitory neurotransmitter) was observed. Four metabolites, ADP, AMP, NAD+, and IMP were the most important indicators of TBI, relative to normal controls. All were elevated in the TBI mice. A combination of these 4 biomarkers produced a perfect predictor of TBI status, AUC (95 % CI) = 1.0 (1.0, 1.0). We also detected significant disturbances in mitochondrial function, energy metabolism, neurotransmitter metabolism and other important biochemical pathways in TBI mouse brains. Further studies to assess the utility of metabolomics to detect and classify the severity of and assess the prognosis of TBI is warranted.     

Serum metabolomic markers for traumatic brain injury: a mouse model

Introduction

Traumatic brain injury (TBI) is physical injury to brain tissue that temporarily or permanently impairs brain function.

Objectives

Evaluate the use of metabolomics for the development of biomarkers of TBI for the diagnosis and timing of injury onset.

Methods

A validated model of closed injury TBI was employed using 10 TBI mice and 8 sham operated controls. Quantitative LC–MS/MS metabolomic analysis was performed on the serum.

Results

Thirty-six (24.0 %) of 150 metabolites were altered with TBI. Principal component analysis (PCA) and Partial least squares discriminant analysis (PLS-DA) analyses revealed clear segregation between TBI versus control sera. The combination of methionine sulfoxide and the lipid PC aa C34:4 accurately diagnosed TBI, AUC (95 % CI) 0.85 (0.644–1.0). A combination of metabolite markers were highly accurate in distinguishing early (4 h post TBI) from late (24 h) TBI: AUC (95 % CI) 1.0 (1.0–1.0). Spermidine, which is known to have an antioxidant effect and which is known to be metabolically disrupted in TBI, was the most discriminating biomarker based on the variable importance ranking in projection (VIP) plot. Several important metabolic pathways were found to be disrupted including: pathways for arginine, proline, glutathione, cysteine, and sphingolipid metabolism.

Conclusion

Using serum metabolomic analysis we were able to identify novel putative serum biomarkers of TBI. They were accurate for detecting and determining the timing of TBI. In addition, pathway analysis provided important insights into the biochemical mechanisms of brain injury. Potential clinical implications for diagnosis, timing, and monitoring brain injury are discussed.

     

Vorkommen vonAstropecten aranciacus (Asteroidea,Echinodermata) in Abhängigkeit von Umweltbedingungen

Zusammenfassung 1.Astropecten aranciacus L. ist die größte von sechs Arten der Gattung, die im Mittelmeer nachgewiesen sind. Dieser Seestern lebt auf Sedimentböden; die obere Grenze des Vorkommens findet sich auf etwa drei Meter Tiefe.2. Auf 70 Stichprobenfeldern von je 25 m² Fläche wurde die Individuenzahl vonA. aranciacus zusammen mit biotischen und abiotischen Bedingungen registriert. Das Untersuchungsgebiet lag zwischen 3 und 10 m Tiefe.3. Die statistische Datenauswertung ergab, daß die Abundanz vonA. aranciacus mit zunehmender Tiefe und steigendem mittlerem Sandkorndurchmesser zunimmt. Von den biotischen Faktoren war die Anzahl Molluskenarten positiv, die Anzahl Echinocardien negativ mit der Abundanz vonA. aranciacus korreliert.4. Die Bestandesdichte vonA. aranciacus schwankte in dem heterogenen, mosaikartig gegliederten Biotop zwischen 0 und 12 Individuen je 100 m². Im Mittel entfielen auf 100 m² 4 Seesterne. Der Radius R der Tiere variierte zwischen 7 und 15 cm.5. Die Makro-Infauna des Gebiets fiel durch geringe Abundanz auf. Bei den Mollusken betrug die durchschnittliche Bestandesdichte 36 Tiere pro m². Dagegen war die Arten-Diversität groß: In Infaunenproben von gesamthaft 17,5 m² Fläche fanden sich 37 Molluskenarten.

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Environmental conditions affecting the occurrence ofAstropecten aranciacus (Asteroidea, Echinodermata)

In shallow water off the north coast of Sardinia (western Mediterranean) the frequency of occurrence of the starfishAstropecten aranciacus was recorded in 70 sample areas of 25 m² each. Four environmental factors, namely depth, average sand grain diameter, number of mollusc species, and number of individuals of the genusEchinocardium proved to be related to the abundance ofA. aranciacus. The sediment contained many species, each, as a rule, being present at low abundance. The molluscs found and their sizes are listed.