IκBα inhibits apoptosis at the outer mitochondrial membrane through a novel, NF-κB–independent, interaction with VDAC1

The inducible transcription factor NF-κB is tightly regulated by the inhibitory IκB-family of proteins that associate with the transcription factor and act in response to stress stimuli. The best studied inhibitory protein is IκBα which resides in the cytosol where it retains NF-κB. Our study shows that IκBα also associates with the outer mitochondrial membrane (OMM) and exerts an unexpected novel anti-apoptotic function, independent of NF-κB inhibition. IκBα-/- cells become refractory to apoptosis when IκBα is specifically reconstituted at the OMM. We found that cancer cells with constitutively active NF-κB accumulate IκBα at the OMM and when its expression is down-regulated these cells are sensitised to apoptosis. At the OMM IκBα associates with VDAC1 and hexokinase II (HKII). Our findings show that IκBα inhibits the dissociation of HKII from VDAC1 and prevents Bax-mediated cytochrome c release. Deletion mutants of IκBα reveal a domain necessary for apoptosis inhibition that is different from the domain for NF-κB retention, thereby separating the two functions. These results reveal an unexpected activity of IκΒα in guarding the integrity of the OMM against apoptosis induction and open possibilities for more specific interference in diseases involving deregulated NF-κB

Cell demise inhibited: Unexpected liaisons between mitochondria and IκΒα

IκΒα (the protein product of NFKBIA gene) has widely been considered a pro- apoptotic factor due to its ability to inhibit the anti-apoptotic transcription factor NFκB. Our findings indicate that IκΒα also exerts a strong anti-apoptotic activity at the outer mitochondria membrane (OMM). This function we uncovered is distinct from its ability to sequester and inhibit NFκB. IκΒα instead binds to voltage dependent anion channel 1 (VDAC1) and Hexokinase 2 (HK2), stabilizes this complex and prevents mitochondria outer membrane permeabilisation (MOMP) and apoptosis

The transporter and permeability interactions of asymmetric dimethylarginine (ADMA) with the human blood–brain barrier in vitro

AbstractThe blood–brain barrier (BBB) is a biological firewall that carefully regulates the cerebral microenvironment by acting as a physical, metabolic and transport barrier. This selectively permeable interface was modelled using the immortalised human cerebral microvascular endothelial cell line (hCMEC/D3) to investigate interactions with the cationic amino acid (CAA) L-arginine, the precursor for nitric oxide (NO), and with asymmetric dimethylarginine (ADMA), an endogenously derived analogue of L-arginine that potently inhibits NO production. The transport mechanisms utilised by L-arginine are known but they are not fully understood for ADMA, particularly at the BBB. This is of clinical significance giving the emerging role of ADMA in many brain and cerebrovascular diseases and its potential as a therapeutic target. We discovered that high concentrations of ADMA could induce endothelial dysfunction in the hCMEC/D3s BBB permeability model, leading to an increase in paracellular permeability to the paracellular marker FITC-dextran (40kDa). We also investigated interactions of ADMA with a variety of transport mechanisms, comparing the data with L-arginine interactions. Both molecules are able to utilise the CAA transport system y+. Furthermore, the expression of CAT-1, the best known protein from this group, was confirmed in the hCMEC/D3s. It is likely that influx systems, such as y+L and b0,+, have an important physiological role in ADMA transport at the BBB. These data are not only important with regards to the brain, but apply to other microvascular endothelia where ADMA is a major area of investigation

Dose-dependent neuroprotection of VEGF165 in Huntington's disease striatum

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by abnormal polyglutamine expansion in the huntingtin protein (Exp-Htt). Currently, there are no effective treatments for HD. We used bidirectional lentiviral transfer vectors to generate in vitro and in vivo models of HD and to test the therapeutic potential of vascular endothelial growth factor 165 (VEGF165). Lentiviral-mediated expression of Exp-Htt caused cell death and aggregate formation in human neuroblastoma SH-SY5Y and rat primary striatal cultures. Lentiviral-mediated VEGF165 expression was found to be neuroprotective in both of these models. Unilateral stereotaxic vector delivery of Exp-Htt vector in adult rat striatum led to progressive inclusion formation and striatal neuron loss at 10 weeks post-transduction. Coinjection of a lower dose VEGF165 significantly attenuated DARPP-32 + neuronal loss, enhanced NeuN staining and reduced Exp-Htt aggregation. A tenfold higher dose VEGF165 led to overt neuronal toxicity marked by tissue damage, neovascularization, extensive astrogliosis, vascular leakage, chronic inflammation and distal neuronal loss. No overt behavioral phenotype was observed in these animals. Expression of VEGF165 at this higher dose in the brain of wild-type rats led to early mortality with global neuronal loss. This report raises important safety concerns about unregulated VEGF 165 CNS applications. © The American Society of Gene & Cell Therapy

Novel computational method for predicting polytherapy switching strategies to overcome tumor heterogeneity and evolution.

The success of targeted cancer therapy is limited by drug resistance that can result from tumor genetic heterogeneity. The current approach to address resistance typically involves initiating a new treatment after clinical/radiographic disease progression, ultimately resulting in futility in most patients. Towards a potential alternative solution, we developed a novel computational framework that uses human cancer profiling data to systematically identify dynamic, pre-emptive, and sometimes non-intuitive treatment strategies that can better control tumors in real-time. By studying lung adenocarcinoma clinical specimens and preclinical models, our computational analyses revealed that the best anti-cancer strategies addressed existing resistant subpopulations as they emerged dynamically during treatment. In some cases, the best computed treatment strategy used unconventional therapy switching while the bulk tumor was responding, a prediction we confirmed in vitro. The new framework presented here could guide the principled implementation of dynamic molecular monitoring and treatment strategies to improve cancer control

NF-κB-Activating Complex Engaged in Response to EGFR Oncogene Inhibition Drives Tumor Cell Survival and Residual Disease in Lung Cancer

Although oncogene-targeted therapy often elicits profound initial tumor responses in patients, responses are generally incomplete because some tumor cells survive initial therapy as residual disease that enables eventual acquired resistance. The mechanisms underlying tumor cell adaptation and survival during initial therapy are incompletely understood. Here, through the study of EGFR mutant lung adenocarcinoma, we show that NF-κB signaling is rapidly engaged upon initial EGFR inhibitor treatment to promote tumor cell survival and residual disease. EGFR oncogene inhibition induced an EGFR-TRAF2-RIP1-IKK complex that stimulated an NF-κB-mediated transcriptional survival program. The direct NF-κB inhibitor PBS-1086 suppressed this adaptive survival program and increased the magnitude and duration of initial EGFR inhibitor response in multiple NSCLC models, including a patient-derived xenograft. These findings unveil NF-κB activation as a critical adaptive survival mechanism engaged by EGFR oncogene inhibition and provide rationale for EGFR and NF-κB co-inhibition to eliminate residual disease and enhance patient responses

AXL Mediates Resistance to PI3Kα Inhibition by Activating the EGFR/PKC/mTOR Axis in Head and Neck and Esophageal Squamous Cell Carcinomas

Phosphoinositide-3-kinase (PI3K)-α inhibitors have shown clinical activity in squamous cell carcinomas (SCCs) of head and neck (H&N) bearing PIK3CA mutations or amplification. Studying models of therapeutic resistance, we have observed that SCC cells that become refractory to PI3Kα inhibition maintain PI3K-independent activation of the mammalian target of rapamycin (mTOR). This persistent mTOR activation is mediated by the tyrosine kinase receptor AXL. AXL is overexpressed in resistant tumors from both laboratory models and patients treated with the PI3Kα inhibitor BYL719. AXL dimerizes with and phosphorylates epidermal growth factor receptor (EGFR), resulting in activation of phospholipase Cγ (PLCγ)-protein kinase C (PKC), which, in turn, activates mTOR. Combined treatment with PI3Kα and either EGFR, AXL, or PKC inhibitors reverts this resistance

Synthetic Essentiality of Metabolic Regulator PDHK1 in PTEN-Deficient Cells and Cancers.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor and bi-functional lipid and protein phosphatase. We report that the metabolic regulator pyruvate dehydrogenase kinase1 (PDHK1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The PTEN protein phosphatase dephosphorylates nuclear factor κB (NF-κB)-activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NF-κB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to induce aerobic glycolysis and PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, a biomarker of decreased patient survival. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers