Evaluation of the combination of the dual m-TORC1/2 inhibitor vistusertib (AZD2014) and paclitaxel in ovarian cancer models.

Activation of the PI3K/mTOR pathway has been shown to be correlated with resistance to chemotherapy in ovarian cancer. We aimed to investigate the effects of combining inhibition of mTORC1 and 2 using the mTOR kinase inhibitor vistusertib (AZD2014) with paclitaxel in in vitro and in vivo ovarian cancer models. The combination of vistusertib and paclitaxel on cell growth was additive in a majority of cell lines in the panel (n = 12) studied. A cisplatin- resistant model (A2780Cis) was studied in vitro and in vivo. We demonstrated inhibition of mTORC1 and mTORC2 by vistusertib and the combination by showing reduction in p-S6 and p-AKT levels, respectively. In the A2780CisR xenograft model compared to control, there was a significant reduction in tumor volumes (p = 0.03) caused by the combination and not paclitaxel or vistusertib alone. In vivo, we observed a significant increase in apoptosis (cleaved PARP measured by immunohistochemistry; p = 0.0003). Decreases in phospholipid and bioenergetic metabolites were studied using magnetic resonance spectroscopy and significant changes in phosphocholine (p = 0.01), and ATP (p = 0.04) were seen in tumors treated with the combination when compared to vehicle-control. Based on this data, a clinical trial evaluating the combination of paclitaxel and vistusertib has been initiated (NCT02193633). Interestingly, treatment of ovarian cancer patients with paclitaxel caused an increase in p-AKT levels in platelet-rich plasma and it was possible to abrogate this increase with the co-treatment with vistusertib in 4/5 patients: we believe this combination will benefit patients with ovarian cancer

Combination of mTORC1/2 inhibitor vistusertib plus fulvestrant in vitro and in vivo targets oestrogen receptor-positive endocrine-resistant breast cancer.

BACKGROUND:Endocrine therapies are still the main strategy for the treatment of oestrogen receptor-positive (ER+) breast cancers (BC), but resistance remains problematic. Cross-talk between ER and PI3K/AKT/mTORC has been associated with ligand-independent transcription of ER. We have previously reported the anti-proliferative effects of the combination of everolimus (an mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape via AKT signalling can lead to resistance; therefore, the use of dual mTORC1/2 inhibitors has met with significant interest. METHODS:To address this, we tested the effect of vistusertib, a dual mTORC1 and mTORC2 inhibitor, in a panel of endocrine-resistant and endocrine-sensitive ER+ BC cell lines, with varying PTEN, PIK3CA and ESR1 mutation status. End-points included proliferation, cell signalling, cell cycle and effect on ER-mediated transcription. Two patient-derived xenografts (PDX) modelling endocrine resistance were used to assess the efficacy of vistusertib, fulvestrant or the combination on tumour progression, and biomarker studies were conducted using immunohistochemistry and RNA-seq technologies. RESULTS:Vistusertib caused a dose-dependent decrease in proliferation of all the cell lines tested and reduced abundance of mTORC1, mTORC2 and cell cycle markers, but caused an increase in abundance of EGFR, IGF1R and ERBB3 in a context-dependent manner. ER-mediated transcription showed minimal effect of vistusertib. Combined therapy of vistusertib with fulvestrant showed synergy in two ER+ PDX models of resistance to endocrine therapy and delayed tumour progression after cessation of therapy. CONCLUSIONS:These data support the notion that models of acquired endocrine resistance may have a different sensitivity to mTOR inhibitor/endocrine therapy combinations

Peroxisome proliferators-activated alpha agonist treatment ameliorates hepatic damage in rats with obstructive jaundice: an experimental study

<p>Abstract</p> <p>Background</p> <p>Peroxisome proliferators-activated receptor alpha (PPARα) activation modulates cholesterol metabolism and suppresses bile acid synthesis. This study aims to evaluate the effect of short-term administration of fenofibrate, a PPARα agonist, on proinflammatory cytokines, apoptosis, and hepatocellular damage in cholestasis.</p> <p>Methods</p> <p>Forty male Wistar rats were randomly divided into four groups: I = sham operated, II = bile duct ligation (BDL), III = BDL + vehicle (gum Arabic), IV = BDL + fenofibrate (100 mg/kg/day). All rats were sacrificed on 7^thday after obtaining blood samples and liver tissue. Total bilirubin, aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), gamma-glutamyl transferase, (GGT), tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1 β), and total bile acid (TBA) in serum, and liver damage scores; portal inflammation, necrosis, bile duct number, in liver tissue were evaluated. Apoptosis in liver was also assessed by immunohistochemical staining.</p> <p>Results</p> <p>Fenofibrate administration significantly reduced serum total bilirubin, AST, ALT, ALP, and GGT, TNF-α, IL-1 β levels, and TBA (<it>P </it>< 0.01). Hepatic portal inflammation, hepatic necrosis, number of the bile ducts and apoptosis in rats with BDL were more prominent than the sham-operated animals (<it>P </it>< 0.01). PPARα induction improved all histopathologic parameters (<it>P </it>< 0.01), except for the number of the bile duct, which was markedly increased by fenofibrate therapy (<it>P </it>< 0.01).</p> <p>Conclusion</p> <p>Short-term administration of fenofibrate to the BDL rats exerts beneficial effects on hepatocellular damage and apoptosis.</p

Caspase cleavage of the Golgi stacking factor GRASP65 is required for Fas/CD95-mediated apoptosis

GRASP65 (Golgi reassembly and stacking protein of 65 KDa) is a cis-Golgi protein with roles in Golgi structure, membrane trafficking and cell signalling. It is cleaved by caspase-3 early in apoptosis, promoting Golgi fragmentation. We now show that cleavage is needed for Fas-mediated apoptosis: expression of caspase-resistant GRASP65 protects cells, whereas expression of membrane proximal caspase-cleaved GRASP65 fragments dramatically sensitises cells. GRASP65 coordinates passage through the Golgi apparatus of proteins containing C-terminal hydrophobic motifs, via its tandem PDZ type ‘GRASP' domains. Fas/CD95 contains a C-terminal leucine–valine pairing so its trafficking might be coordinated by GRASP65. Mutagenesis of the Fas/CD95 LV motif reduces the number of cells with Golgi-associated Fas/CD95, and generates a receptor that is more effective at inducing apoptosis; however, siRNA-mediated silencing or expression of mutant GRASP65 constructs do not alter the steady state distribution of Fas/CD95. We also find no evidence for a GRASP65–Fas/CD95 interaction at the molecular level. Instead, we find that the C-terminal fragments of GRASP65 produced following caspase cleavage are targeted to mitochondria, and ectopic expression of these sensitises HeLa cells to Fas ligand. Our data suggest that GRASP65 cleavage promotes Fas/CD95-mediated apoptosis via release of C-terminal fragments that act at the mitochondria, and we identify Bcl-XL as a candidate apoptotic binding partner for GRASP65

Kinase-substrate enrichment analysis provides insights into the heterogeneity of signaling pathway activation in leukemia cells

Kinases determine the phenotypes of many cancer cells, but the frequency with which individual kinases are activated in primary tumors remains largely unknown. We used a computational approach, termed kinase-substrate enrichment analysis (KSEA), to systematically infer the activation of given kinase pathways from mass spectrometry-based phosphoproteomic analysis of acute myeloid leukemia (AML) cells. Experiments conducted in cell lines validated the approach and, furthermore, revealed that DNAdependent protein kinase (DNA-PK) was activated as a result of inhibiting the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling pathway. Application of KSEA to primary AML cells identified PI3K, casein kinases (CKs), cyclin-dependent kinases (CDKs), and p21-activated kinases (PAKs) as the kinase substrate groups most frequently enriched in this cancer type. Substrates phosphorylated by extracellular signal-regulated kinase (ERK) and cell division cycle 7 (CDC7) were enriched in primary AML cells that were resistant to inhibition of PI3K-mTOR signaling, whereas substrates of the kinases Abl, Lck, Src, and CDK1 were increased in abundance in inhibitor-sensitive cells. Modeling based on the abundances of these substrate groups accurately predicted sensitivity to a dual PI3K and mTOR inhibitor in two independent sets of primary AML cells isolated from patients. Thus, our study demonstrates KSEA as an untargeted method for the systematic profiling of kinase pathway activities and for increasing our understanding of diseases caused by the dysregulation of signaling pathways. Copyright 2008 by the American Association for the Advancement of Scienc