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Hyperactivity of the NOTCH pathway is associated with tumor growth and radiotherapy resistance in lung cancer, and NOTCH/γ-secretase inhibitors (GSIs) are a potential therapeutic target. The therapeutic outcome, however, is often restricted by the dose-limiting toxicity of combined treatments on the surrounding healthy tissue. The NOTCH signaling pathway is also crucial for homeostasis and repair of the normal airway epithelium. The effects of NOTCH/γ-secretase inhibition on the irradiation of normal lung epithelium are unknown and may counteract antitumor activity. Here we, therefore, investigated whether normal tissue toxicity to radiation is altered upon NOTCH pathway inhibition. We established air-liquid interface pseudostratified and polarized cultures from primary human bronchial epithelial cells and blocked NOTCH signaling alone or after irradiation with small-molecule NOTCH inhibitor/GSI. We found that the reduction in proliferation and viability of bronchial stem cells (TP63+) in response to irradiation is rescued with concomitant NOTCH inhibition. This correlated with reduced activation of the DNA damage response and accelerated repair by 24 hours and 3 days postirradiation. The increase in basal cell proliferation and viability in GSI-treated and irradiated cultures resulted in an improved epithelial barrier function. Comparable results were obtained after in vivo irradiation, where the combination of NOTCH inhibition and irradiation increased the percentage of stem cells and ciliated cells ex vivo. These encourage further use of normal patient tissue for toxicity screening of combination treatments and disclose novel interactions between NOTCH inhibition and radiotherapy and opportunities for tissue repair after radiotherapy. Significance statement Radiation-induced lung injury is a dose-limiting toxicity that limits the effective dose that should be administered and forces the interruption of the treatment. The NOTCH signaling pathway is a potential therapeutic target for lung cancer because its inhibition reduces tumor growth and synergizes with radiotherapy and chemotherapy in preclinical models. However, the effect of inhibiting NOTCH in irradiated normal lung tissue is not known and could impact the therapeutic benefit of combination treatments. This study demonstrates that small-molecule inhibitors of the NOTCH pathway enhance the survival of irradiated primary human and murine bronchial epithelial lung stem cells. This finding may be beneficial in lung cancer treatment with radiotherapy and NOTCH inhibitors by protecting normal lung tissue while increasing tumor control.

Radiation-induced lung injury to normal airway epithelium is a frequent side-effect and dose-limiting factor in radiotherapy of tumors in the thoracic cavity. NOTCH signaling plays key roles in self-renewal and differentiation of upper airway basal lung stem cells during development, and the NOTCH pathway is frequently deregulated in lung cancer. In preclinical lung cancer models, NOTCH inhibition was shown to improve the radiotherapy response by targeting tumor stem cells, but the effects in combination with irradiation on normal lung stem cells are unknown. NOTCH/γ-secretase inhibitors are potent clinical candidates to block NOTCH function in tumors, but their clinical implementation has been hampered by normal tissue side-effects. Here we show that NOTCH signaling is active in primary human- and murine-derived airway epithelial stem cell models and when combined with radiation NOTCH inhibition provokes a decrease in S-phase and increase in G1-phase arrest. We show that NOTCH inhibition in irradiated lung basal stem cells leads to a more potent activation of the DNA damage checkpoint kinases pATM and pCHK2 and results in an increased level of residual 53BP1 foci in irradiated lung basal stem cells reducing their capacity for self-renewal. The effects are recapitulated in ex vivo cultured lung basal stem cells after in vivo whole thorax irradiation and NOTCH inhibition. These results highlight the importance of studying normal tissue effects that may counteract the therapeutic benefit in the use of NOTCH/γ-secretase inhibitors in combination with radiation for antitumor treatment.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from T-cell progenitors. Although current treatments, including chemotherapy and glucocorticoids, have significantly improved survival, T-ALL remains a fatal disease and new treatment options are needed. Since more than 60% of T-ALL cases bear oncogenic NOTCH1 mutations, small molecule inhibitors of NOTCH1 signalling; gamma-secretase inhibitors (GSI), are being actively investigated for the treatment of T-ALL. Unfortunately, GSI have shown limited clinical efficacy and dose-limiting toxicities. We hypothesized that by combining known drugs, blocking NOTCH activity through another mechanism, may synergize with GSI enabling equal efficacy at a lower concentration. Here, we show that the clinically used anti-malarial drug chloroquine (CQ), an inhibitor of lysosomal function and autophagy, decreases T-ALL cell viability and proliferation. This effect of CQ was not observed in GSI-resistant T-ALL cell lines. Mechanistically, CQ impairs the redox balance, induces ds DNA breaks and activates the DNA damage response. CQ also interferes with intracellular trafficking and processing of oncogenic NOTCH1. Interestingly, we show for the first time that the addition of CQ to gamma-secretase inhibition has a synergistic therapeutic effect on T-ALL and reduces the concentration of GSI required to obtain a reduction in cell viability and a block of proliferation. Overall, our results suggest that CQ may be a promising repurposed drug in the treatment of T-ALL, as a single treatment or in combination with GSI, increasing the therapeutic ratio.

In the past decade, calcium phosphate (CaP) ceramics have emerged as alternatives to autologous bone grafts for the treatment of large, critical-sized bone defects. In order to be effective in the regeneration of such defects, ceramics must show osteoinductive behaviour, defined as the ability to induce de novo heterotopic bone formation. While a set of osteoinductive CaP ceramics has been developed, the exact processes underlying osteoinduction, and the role of the physical and chemical properties of the ceramics, remain largely unknown. Previous studies have focused on the role of the transcriptome to shed light on the mechanism of osteoinduction at the mRNA level. To complement these studies, a proteomic analysis was performed to study the behaviour of hMSCs on osteoinductive and non-osteoinductive CaPs. The results of this analysis suggest that plasma cell glycoprotein 1 (PC-1), encoded by the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene, plays a key role in the process of osteoinduction by CaP ceramics. Validation experiments have confirmed that indeed, the mRNA expression of ENPP1 and the production of PC-1 are higher on osteoinductive than on non-osteoinductive CaP ceramics, a trend that was also observed for other osteogenic markers such as bone morphogenetic protein 2 (BMP2) and osteopontin (OPN), but not for alkaline phosphatase (ALP). Our results also showed that the expression of PC-1 is restricted to those cells which are in direct contact with the CaP ceramic surface, plausibly due to the localised depletion of calcium and inorganic phosphate ions from the supersaturated cell culture medium as CaP crystallises on the ceramic surface. Replicating the surface of the osteoinductive ceramic in polystyrene resulted in a significant decrease in ENPP1 expression, suggesting that surface structural properties alone are not sufficient to induce ENPP1 expression. Finally, knocking down ENPP1 expression in hMSCs resulted in increased BMP2 expression, both at the mRNA and protein level, suggesting that ENPP1 is a negative regulator of BMP-2 signalling. Taken together, this study shows, for the first time, that ENPP1/PC-1 plays an important role in CaP-induced osteogenic differentiation of hMSCs and thus possibly osteoinduction by CaP ceramics. Furthermore, we have identified a crucial role for the interfacial (chemical) events occurring on the CaP ceramic surface in the process of osteoinduction. This knowledge can contribute to the development of new bone graft substitutes, with improved osteoinductive potential.

The enzyme type 1 17β-hydroxysteroid dehydrogenase (17β-HSD-1), responsible for generating active 17β-estradiol (E2) from low-active estrone (E1), is overexpressed in endometrial cancer (EC), thus implicating an increased intra-tissue generation of E2 in this estrogen-dependent condition. In this study, we explored the possibility of inhibiting 17β-HSD-1 and impairing the generation of E2 from E1 in EC using in vitro, in vivo, and ex vivo models. We generated EC cell lines derived from the well-differentiated endometrial adenocarcinoma Ishikawa cell line and expressing levels of 17β-HSD-1 similar to human tissues. In these cells, HPLC analysis showed that 17β-HSD-1 activity could be blocked by a specific 17β-HSD-1 inhibitor. In vitro, E1 administration elicited colony formation similar to E2, and this was impaired by 17β-HSD-1 inhibition. In vivo, tumors grafted on the chicken chorioallantoic membrane (CAM) demonstrated that E1 upregulated the expression of the estrogen responsive cyclin A similar to E2, which was impaired by 17β-HSD-1 inhibition. Neither in vitro nor in vivo effects of E1 were observed using 17β-HSD-1-negative cells (negative control). Using a patient cohort of 52 primary ECs, we demonstrated the presence of 17β-HSD-1 enzyme activity (ex vivo in tumor tissues, as measured by HPLC), which was inhibited by over 90% in more than 45% of ECs using the 17β-HSD-1 inhibitor. Since drug treatment is generally indicated for metastatic/recurrent and not primary tumor, we next demonstrated the mRNA expression of the potential drug target, 17β-HSD-1, in metastatic lesions using a second cohort of 37 EC patients. In conclusion, 17β-HSD-1 inhibition efficiently blocks the generation of E2 from E1 using various EC models. Further preclinical investigations and 17β-HSD-1 inhibitor development to make candidate compounds suitable for the first human studies are awaited. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Introduction/Background The enzyme type 1 17β-hydroxysteroid dehydrogenase (HSD17B1), responsible for generating active 17β-estradiol (E2) from low-active estrone (E1), is over-expressed in endometrial cancer (EC) thus implicating an increased intra-tissue generation of E2 in this estrogen-dependent condition. In this study we explored the possibility of inhibiting HSD17B1 and impairing the generation of E2 from E1 in EC using in vitro, in vivo and ex vivo models. Methodology We generated EC cell lines derived from the well-differentiated endometrial adenocarcinoma Ishikawa cell line and expressing levels of HSD17B1 similar to human tissues. In these cells, HPLC analysis showed that HSD17B1 activity could be blocked by a specific HSD17B1 inhibitor. In vitro, E1 administration elicited colony formation similar to E2, and this was impaired by HSD17B1 inhibition. In vivo, tumours grafted on the chicken chorioallantoic membrane (CAM) demonstrated that E1 upregulated the expression of the estrogen responsive cyclin A similar to E2, which was impaired by HSD17B1 inhibition. Neither in vitro nor in vivo effects of E1 were observed using HSD17B1 negative cells (negative control). Results Using a patient cohort of 52 primary ECs, we demonstrated the presence of HSD17B1 enzyme activity (ex vivo in tumour tissues, as measured by HPLC), which was inhibited by over 90% in more than 45% of ECs using the HSDβB1 inhibitor. Since drug treatment is generally indicated for metastatic/recurrent and not primary tumour, we next demonstrated the mRNA expression of the potential drug target, HSD17B1, in metastatic lesions using a second cohort of 37 EC patients. Conclusion In conclusion, HSD17B1 inhibition efficiently blocks the generation of E2 from E1 using various EC models. Further preclinical investigations and HSD17B1 inhibitor development to make candidate compounds suitable for the first human studies are awaited. Disclosure Nothing to disclose.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. The current standard of care includes surgery followed by radiotherapy (RT) and chemotherapy with temozolomide (TMZ). Treatment often fails due to the radiation resistance and intrinsic or acquired TMZ resistance of a small percentage of cells with stem cell-like behavior (CSC). The NOTCH signaling pathway is expressed and active in human glioblastoma and NOTCH inhibitors attenuate tumor growth in vivo in xenograft models. Here we show using an image guided micro-CT and precision radiotherapy platform that a combination of the clinically approved NOTCH/γ-secretase inhibitor (GSI) RO4929097 with standard of care (TMZ + RT) reduces tumor growth and prolongs survival compared to dual combinations. We show that GSI in combination with RT and TMZ attenuates proliferation, decreases 3D spheroid growth and results into a marked reduction in clonogenic survival in primary and established glioma cell lines. We found that the glioma stem cell marker CD133, SOX2 and Nestin were reduced following combination treatments and NOTCH inhibitors albeit in a different manner. These findings indicate that NOTCH inhibition combined with standard of care treatment has an anti-glioma stem cell effect which provides an improved survival benefit for GBM and encourages further translational and clinical studies.

The hypoxia-inducible transcription factors (HIF)-1/2&alpha, are the main oxygen sensors which regulate the adaptation to intratumoral hypoxia. The aim of this study was to assess the role of the HIF proteins in regulating the radiation response of a non-small cell lung cancer (NSCLC) in vitro model. To directly assess the unique and overlapping functions of HIF-1&alpha, and HIF-2&alpha, we use CRISPR gene-editing to generate isogenic H1299 non-small cell lung carcinoma cells lacking HIF-1&alpha, HIF-2&alpha, or both. We found that in HIF1 knockout cells, HIF-2&alpha, was strongly induced by hypoxia compared to wild type but the reverse was not seen in HIF2 knockout cells. Cells lacking HIF-1&alpha, were more radiation resistant than HIF2 knockout and wildtype cells upon hypoxia, which was associated with a reduced recruitment of &gamma, H2AX foci directly after irradiation and not due to differences in proliferation. Conversely, double-HIF1/2 knockout cells were most radiation sensitive and had increased &gamma, H2AX recruitment and cell cycle delay. Compensatory HIF-2&alpha, activity in HIF1 knockout cells is the main cause of this radioprotective effect. Under hypoxia, HIF1 knockout cells uniquely had a strong increase in lactate production and decrease in extracellular pH. Using genetically identical HIF-&alpha, isoform-deficient cells we identified a strong radiosensitizing of HIF1, but not of HIF2, which was associated with a reduced extracellular pH and reduced glycolysis.

Objective: Lung cancer is the deadliest cancer worldwide. To increase treatment potential for lung cancer, pre-clinical models that allow testing and follow up of clinically relevant treatment modalities are essential. Therefore, we developed a single-nodule-based orthotopic non-small cell lung cancer tumor model which can be monitored using multimodal non-invasive imaging to select the optimal image-guided radiation treatment plan.Methods: An orthotopic non-small cell lung cancer model in NMRI-nude mice was established to investigate the complementary information acquired from 80 kVp microcone-beam CT (micro-CBCT) and bioluminescence imaging (BLI) using different angles and filter settings. Different micro-CBCT-based radiation-delivery plans were evaluated based on their dose-volume histogram metrics of tumor and organs at risk to select the optimal treatment plan.Results: H1299 cell suspensions injected directly into the lung render exponentially growing single tumor nodules whose CBCT-based volume quantification strongly correlated with BLI-integrated intensity. Parallel-opposed single angle beam plans through a single lung are preferred for smaller tumors, whereas for larger tumors, plans that spread the radiation dose across healthy tissues are favored.Conclusions: Closely mimicking a clinical setting for lung cancer with highly advanced preclinical radiation treatment planning is possible in mice developing orthotopic lung tumors.Advances in knowledge: BLI and CBCT imaging of orthotopic lung tumors provide complementary information in a temporal manner. The optimal radiotherapy plan is tumor volume-dependent.

Hypoxia-inducible factor-2α (HIF-2α) plays an important role in tumor progression and metastasis. A number of studies have evaluated the correlation between hypoxia-inducible factor-2α (HIF-2α) overexpression and clinical outcome in cancer patients but yielded inconsistent results. To comprehensively and quantitatively summarize the evidence on the capability of HIF-2α to predict the prognosis of cancer patients, a meta-analysis was carried out. Renal cell carcinoma (CC-RCC) was separately analyzed due to an alternative mechanism of regulation. Systematic literature searches were performed in PubMed and Embase databases for relevant original articles until Feb 2018. Forty nine studies with 6,052 patients were included in the present study. The pooled hazard ratios (HRs) with corresponding confidence intervals (CIs) were calculated to assess the prognostic value of HIF-2α protein expression. The meta-analysis revealed strong significant negative associations between HIF-2α expression and five endpoints: overall survival (HR = 1.69, 95% confidence interval (95%CI) 1.39–2.06), disease-free survival (HR = 1.87, 95%CI 1.2–2.92), disease-specific survival (HR = 1.57, 95%CI 1.06–2.34), metastasis-free survival (HR = 2.67, 95%CI 1.32–5.38) and progression-free survival (HR = 2.18, 95%CI 1.25–3.78). No association was observed with locoregional control. Subgroup analyses revealed similar associations in the majority of tumor sites. A positive (but not significant) association between good prognosis and HIF-2α expression was seen for kidney and bladder cancer. This meta-analysis indicated that the HIF-2α may serve as a useful prognostic biomarker in various cancers.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from T-cell progenitors. Although current treatments, including chemotherapy and glucocorticoids, have significantly improved survival, T-ALL remains a fatal disease and new treatment options are needed. Since more than 60% of T-ALL cases bear oncogenic NOTCH1 mutations, small molecule inhibitors of NOTCH1 signalling; γ-secretase inhibitors (GSI), are being actively investigated for the treatment of T-ALL. Unfortunately, GSI have shown limited clinical efficacy and dose-limiting toxicities. We hypothesized that by combining known drugs, blocking NOTCH activity through another mechanism, may synergize with GSI enabling equal efficacy at a lower concentration. Here, we show that the clinically used anti-malarial drug chloroquine (CQ), an inhibitor of lysosomal function and autophagy, decreases T-ALL cell viability and proliferation. This effect of CQ was not observed in GSI-resistant T-ALL cell lines. Mechanistically, CQ impairs the redox balance, induces ds DNA breaks and activates the DNA damage response. CQ also interferes with intracellular trafficking and processing of oncogenic NOTCH1. Interestingly, we show for the first time that the addition of CQ to γ-secretase inhibition has a synergistic therapeutic effect on T-ALL and reduces the concentration of GSI required to obtain a reduction in cell viability and a block of proliferation. Overall, our results suggest that CQ may be a promising repurposed drug in the treatment of T-ALL, as a single treatment or in combination with GSI, increasing the therapeutic ratio.

SummaryNotch receptors are transmembrane proteins that undergo activating proteolysis in response to ligand stimulation. A negative regulatory region (NRR) maintains receptor quiescence by preventing protease cleavage prior to ligand binding. We report here the X-ray structure of the NRR of autoinhibited human Notch3, and compare it with the Notch1 and Notch2 NRRs. The overall architecture of the autoinhibited conformation, in which three LIN12-Notch repeat (LNR) modules wrap around a heterodimerization domain, is preserved in Notch3, but the autoinhibited conformation of the Notch3 NRR is less stable. The Notch3 NRR uses a highly conserved surface on the third LNR module to form a dimer in the crystal. Similar homotypic interfaces exist in Notch1 and Notch2. Together, these studies reveal distinguishing structural features associated with increased basal activity of Notch3, demonstrate increased ligand-independent signaling for disease-associated mutations that map to the Notch3 NRR, and identify a conserved dimerization interface present in multiple Notch receptors.

Cell surface receptors of the NOTCH family of proteins are activated by ligand induced intramembrane proteolysis. Unfolding of the extracellular negative regulatory region (NRR), enabling successive proteolysis by the enzymes Adam10 and gamma-secretase, is rate-limiting in NOTCH activation. Mutations in the NOTCH1 NRR are associated with ligand-independent activation and frequently found in human T-cell malignancies. In mammals four NOTCH receptors and five Delta/Jagged ligands exist, but mutations in the NRR are only rarely reported for receptors other than NOTCH1. Using biochemical and functional assays, we compared the molecular mechanisms of ligand-independent signaling in NOTCH1 and the highly related NOTCH2 receptor. Both murine Notch1 and Notch2 require the metalloprotease protease Adam17, but not Adam10 during ligand-independent activation. Interestingly, the human NOTCH2 receptor is resistant to ligand-independent activation compared with its human homologs or murine orthologs. Taken together, our data reveal subtle but functionally important differences for the NRR among NOTCH paralogs and homologs.

The enzyme type 1 17β-hydroxysteroid dehydrogenase (17β-HSD-1), responsible for generating active 17β-estradiol (E2) from low-active estrone (E1), is overexpressed in endometrial cancer (EC), thus implicating an increased intra-tissue generation of E2 in this estrogen-dependent condition. In this study, we explored the possibility of inhibiting 17β-HSD-1 and impairing the generation of E2 from E1 in EC using in vitro, in vivo, and ex vivo models. We generated EC cell lines derived from the well-differentiated endometrial adenocarcinoma Ishikawa cell line and expressing levels of 17β-HSD-1 similar to human tissues. In these cells, HPLC analysis showed that 17β-HSD-1 activity could be blocked by a specific 17β-HSD-1 inhibitor. In vitro, E1 administration elicited colony formation similar to E2, and this was impaired by 17β-HSD-1 inhibition. In vivo, tumors grafted on the chicken chorioallantoic membrane (CAM) demonstrated that E1 upregulated the expression of the estrogen responsive cyclin A similar to E2, which was impaired by 17β-HSD-1 inhibition. Neither in vitro nor in vivo effects of E1 were observed using 17β-HSD-1-negative cells (negative control). Using a patient cohort of 52 primary ECs, we demonstrated the presence of 17β-HSD-1 enzyme activity (ex vivo in tumor tissues, as measured by HPLC), which was inhibited by over 90% in more than 45% of ECs using the 17β-HSD-1 inhibitor. Since drug treatment is generally indicated for metastatic/recurrent and not primary tumor, we next demonstrated the mRNA expression of the potential drug target, 17β-HSD-1, in metastatic lesions using a second cohort of 37 EC patients. In conclusion, 17β-HSD-1 inhibition efficiently blocks the generation of E2 from E1 using various EC models. Further preclinical investigations and 17β-HSD-1 inhibitor development to make candidate compounds suitable for the first human studies are awaited. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

Introduction Hypoxia plays a role in many types of cancer, and is linked to metastasis, genetic instability, resistance to therapy and poor prognosis. Hypoxic modification has been tested in clinical trials, but results have been inconclusive, in part due to a knowledge gap into the characterisation and behaviour of hypoxic tumour cells. In this project, hypoxic cells will be lineage traced to investigate their dissemination within the primary tumour and for their presence in circulating tumour cells and distant metastasis. To investigate the contribution of the hypoxic fraction to the tumour resistance, hypoxic cells will be specifically ablated, and the growth and response to treatment of tumours with and without this hypoxic population investigated. Material and methods To accomplish these aims, two complementary systems will be generated in the 4 T1 mouse mammary gland cell line, a model for stage IV metastatic human breast cancer. The first consists of a HIF-1α-CRE-ER TAM ‘hypoxia sensing’ construct coupled with a loxp DsRed loxp DTR-EGFP cassette (Diphtheria Toxin Receptor: DTR). Under normal conditions the HIF-1α-CRE-ER TAM fusion protein is expressed, but immediately degraded, but under hypoxia HIF-1α degradation is inhibited leading to accumulation. 4-OHT Administration translocates HIF-1α-CRE-ER TAM to the nucleus mediating loxp recombination in the coupled cassette. This allows lineage tracing of hypoxic cells and their progeny and selective ablation throught the DTR using Diphthteria toxin. Secondly, a DTR-UnaG-ODDD-HA construct (Oxygen-dependant degradation domain: ODDD), driven by the hypoxia dependent 5XHRE promoter will be used. This system allows ablation of hypoxic cells through the DTR and identification of hypoxic cells through the UnaG fluorescent protein. Only cells that are hypoxic at the time of administration/analysis will be labelled. Both cell lines will be transduced with luciferase to allow identification of distant metastasis. Results and discussions The two systems for this project have been generated and transduced into the 4 T1 cell line. Lineage tracing and selective ablation have been shown to work in vitro and clones are being characterised. In vitro experiments have shown the HIF-1α-CRE-ER TAM system to be extremely tightly regulated in an oxygen and tamoxifen dependant manner. Conclusion Once optimised, both systems will be used in an in vivo orthotopic mouse model to investigate the role of the hypoxic cells in breast cancer, metastasis and response to radiotherapy.

Introduction T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive cancer arising from T-cell progenitors. Although current treatments, including chemotherapy and glucocorticoids, have significantly improved survival, T-ALL remains a fatal disease and new treatment options are needed. Since more than 60% of T-ALL cases bear oncogenic NOTCH1 mutations, inhibitors of NOTCH1 signalling are being actively investigated for the treatment of T-ALL. However, they have limited efficacy in tumours due to dose-limiting toxicities. Recent evidence indicates that γ-secretase activity not only occurs at the cell surface, but also in the acidic environment of lysosomes and endosomes. In addition, endosomes have been shown to play a key role in the NOTCH signal relay. Therefore, we hypothesised that chloroquine (CQ), a clinically used antimalarial drug that inhibits lysosomal function and autophagy, may inhibit T-ALL growth and survival. Material and methods T-ALL cell lines were treated with γ-secretase inhibitor (GSI), CQ or a combination treatment. Cell viability was determined by trypan blue exclusion. Propidium Iodide (PI) staining was combined with Annexin V staining or Edu incorporation to measure apoptosis and perform cell cycle analysis, respectively. Flow cytometry was used to determine cytoplasmic ROS levels and yH2AX expression. To address the effect of CQ on NOTCH1 signalling, cleaved NOTCH1 was assessed by immunoblotting, NOTCH1 target gene expression by qPCR and NOTCH1 expression at the cell surface by flow cytometry. In vivo T-ALL xenografts were grown and treated with different doses of GSI, CQ or a combination treatment. Tumour growth and gastro-intestinal toxicity were assessed. Results and discussions CQ decreases T-ALL cell viability and proliferation and reduces GSI sensitive T-ALL xenograft growth, which was not observed in GSI-resistant T-ALL cell lines or xenografts. Mechanistically, CQ impairs the redox balance and induces DNA damage with a subsequent DNA damage response. In addition, we observed that CQ interferes with intracellular trafficking and processing of oncogenic NOTCH1. Interestingly, we show for the first time that the addition of CQ to γ-secretase inhibition has an additive therapeutic effect on T-ALL and reduces the concentration of GSI required to obtain tumour control. Conclusion Overall, our results suggest that CQ may be a promising alternative as a repurposed drug in the treatment of T-ALL, either as a single treatment or in combination with NOTCH1 inhibition, increasing the therapeutic ratio.

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aβ that contains longer Aβ; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aβ further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aβ42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis. publisher: Elsevier articletitle: Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aβ Pool journaltitle: Cell articlelink: http://dx.doi.org/10.1016/j.cell.2016.05.020 content_type: article copyright: © 2016 Elsevier Inc. ispartof: Cell vol:166 issue:1 pages:193-208 ispartof: location:United States status: published

Background and purpose: Hypoxia is a hallmark of solid cancers and associated with metastases and treatment failure. During tumor progression epithelial cells often acquire mesenchymal features, a phenomenon known as epithelial-to-mesenchymal transition (EMT). Intratumoral hypoxia has been linked to EMT induction. We hypothesized that signals from the tumor microenvironment such as growth factors and tumor oxygenation collaborate to promote EMT and thereby contribute to radioresistance. Materials and methods: Gene expression changes under hypoxia were analyzed using microarray and validated by qRT-PCR. Conversion of epithelial phenotype upon hypoxic exposure, TGF beta addition or oncogene activation was investigated by Western blot and immunofluorescence. Cell survival following ionizing radiation was assayed using clonogenic survival. Results: Upon hypoxia, TGF beta addition or EGFRvIII expression, MCF7, A549 and NMuMG epithelial cells acquired a spindle shape and lost cell-cell contacts. Expression of epithelial markers such as E-cadherin decreased, whereas mesenchymal markers such as vimentin and N-cadherin increased. Combining hypoxia with TGF beta or EGFRvIII expression, lead to more rapid and pronounced EMT-like phenotype. Interestingly, E-cadherin expression and the mesenchymal appearance were reversible upon reoxygenation. Mesenchymal conversion and E-cadherin loss were associated with radioresistance. Conclusions: Our findings describe a mechanism by which the tumor microenvironment may contribute to tumor radioresistance via E-cadherin loss and EMT.

A unique feature of the retina is the presence of photoreceptors, which require an enormous amount of oxygen for the conversion of light to an electrical signal. Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a transcription factor that is the master regulator of cellular adaptation to low oxygen tension. Only in hypoxic conditions is HIF-1 alpha protein stabilized and translocated to the nucleus, where it induces transcription of target genes involved in oxygen delivery and energy metabolism. We hypothesized that HIF-1 alpha is constitutively stabilized and active in the normal human retina. We investigated the cellular distribution of HIF-1 alpha and the expression of its downstream targets, vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT-1), and carbonic anhydrase IX (CAIX), by immunohistochemistry and immunoblotting in the retina of normal rats and human donor eyes. Both human and rat retinas displayed prominent staining of HIF-1 alpha. in nuclei of most cell types in inner and outer nuclear layers and the ganglion cell layer, a cellular distribution pattern which was confirmed in human retina by immunoblotting of nuclear extracts. A negative correlation was found between HIF-1 alpha protein levels and postmortem times. In human retina, staining of VEGF, GLUT-1, and CAIX was found. Our observations indicate that active HIF-1 signaling occurs constitutively in the normal human and rat retina, suggesting that HIF-1 has a physiological role in the retina. (J Histochem Cytochem 58:247-254, 2010)

Mast cells play a crucial role at the early stages of immune response against bacteria and parasites where their functionality is based on their capability of releasing highly bioactive compounds, among them TNF. Mast cells are considered the only cells storing preformed TNF, which allows for the immediate release of this cytokine upon contact with pathogens. We approached the question of mechanisms and amino acid motifs directing newly synthesized TNF for storage in cytoplasmic granules by analyzing the trafficking of a series of TNF-enhanced GFP fusion proteins in human mast cell lines HMC-1 and LAD2. Protein covering the full TNF sequence was successfully sorted into secretory granules in a process involving transient exposure on the outer membrane and re-endocytosis. In human cells, contrary to results previously obtained in a rodent model, TNF seems not to be glycosylated and, thus, trafficking is carbohydrate independent. In an effort to localize the amino acid motif responsible for granule targeting, we constructed additional fusion proteins and analyzed their trafficking, concluding that granule-targeting sequences are localized in the mature chain of TNF and that the cytoplasmic tail is expendable for endocytotic sorting of this cytokine, thus excluding direct interactions with intracellular adaptor proteins.

With the completion of the sequence of the human genome, emphasis is now switching to the human proteome. However, the number of proteins is not only larger than mRNAs in the transcriptome, proteins need often to be in complex with other proteins to be functional. A favourable option to study proteins in their natural context is with a combination of biochemical and microscopic techniques using specific antibodies. Therefore, we designed a fast, reliable and controllable selection and screening of single-domain antibody fragments (VHH) from a Camelid non-immune library. We isolated VHH for four muscle disease related proteins; emerin, actin, tropomyosin-1, and nuclear poly(A)-binding protein. Important features of antibodies for target validation studies are recognition of the antigen in natural conformations and biologically relevant complexes. We show that selected antibody fragments are functional in various immunological techniques and prove useful in diagnostic applications. Our selection strategy is amenable to automation and to the establishment of proteomics platforms. It opens the way to quickly and cost-effectively obtain multiple antibody fragments for many antigens that can detect changes in their localization, level, and modification as well as subtle changes in supramolecular structures, which often associate with disease.

The t(9;11) has been described in patients with acute myeloid leukemia (AML), and two genes [AF9 (at 9p21) and FBP17 (at 9q34)] have been cloned as fusion partners of the MLL gene. From an AML-M5 with a t(9;11)(q34;q23), we identified a novel MLL fusion partner, AF9Q34. The AF9Q34 protein shows high homology with nGAP, a RAS GTPase-activating protein (RASGAP), and contains the highly conserved GRD and FLR motifs characteristic of RASGAPs. Recently, the rat homologue (DAB2IP) also was identified and reported to act as a RASGAP both in vivo and in vitro. RASGAPs negatively regulate the activity of RAS proteins that modulate diverse cellular processes by cycling between an inactive GDP-bound and an active GTP-bound state. In addition, the NH2 terminus harbors an amino acid stretch with homology to the pleckstrin homology (PH) domain implicated in regulating the interaction between RAS and the catalytic domain of RASGAP. As a result of the breakpoint in the AF9Q34–MLL fusion protein, this PH domain is disrupted. This suggests that because of the translocation, the normal function of the AF9Q34 gene is aborted. Thus, AF9Q34 encodes a novel RASGAP gene that appears to be deregulated as a result of the translocation. The identification of this RASGAP protein in a novel MLL fusion implies that an indirect RAS-deregulating mechanism could be involved in leukemic transformation. © 2004 Wiley-Liss, Inc.

Item does not contain fulltext Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics. To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein. OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle. An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli. The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library. This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis. Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease.

In mammals, there are four NOTCH receptors and five Delta-Jagged-type ligands regulating many aspects of embryonic development and adult tissue homeostasis. NOTCH proteins are type I transmembrane receptors that interact with ligands on adjacent cells and are activated by regulated intramembrane proteolysis (RIP). The activation mechanism of NOTCH1 receptors upon ligand binding is well understood and requires cleavage by ADAM10 metalloproteases prior to intramembranous cleavage by gamma-secretase. How the other human NOTCH receptor homologues are activated upon ligand binding is not known. Here, we dissect the proteolytic activation mechanism of the NOTCH2 and NOTCH3 receptors. We show that NOTCH2 and NOTCH3 signaling can be triggered by both Delta-Jagged-type ligands and requires ADAM10 and presenilin-1 or -2. Importantly, we did not find any role for the highly related ADAM17/TACE (tumor necrosis factor alpha-converting enzyme) protease in ligand-induced NOTCH2 or NOTCH3 signaling. These results demonstrate that canonical ligand-induced proteolysis of the NOTCH1, -2, and -3 receptors strictly depends on consecutive cleavage of these receptors by ADAM10 and the presenilin-containing gamma-secretase complex, leading to transcriptional activation.

Item does not contain fulltext BACKGROUND AND PURPOSE: Patients with advanced NSCLC have survival rates

Regulated intramembrane proteolysis (RIP) is a highly conserved signaling paradigm whereby membrane-bound signaling proteins are cleaved in their transmembrane region and then released into the cytoplasm to act as signaling molecules. In most if not all cases intramembrane cleavage is preceded and regulated by a membrane proximal cleavage step called ‘ectodomain shedding’. Here we will review the role of ectodomain shedding in RIP of the NOTCH signaling pathway, a highly conserved cell-cell communication pathway that mediates cell fate decisions during development and in adult tissues.

The gene encoding COMM domain-containing 1 (COMMD1) is a prototypical member of the COMMD gene family that has been shown to inhibit both NF-kappa B- and HIF-mediated gene expression. NF-kappa B and HIF are transcription factors that have been shown to play a role in promoting tumor growth, survival, and invasion. In this study, we demonstrate that COMMD1 expression is frequently suppressed in human cancer and that decreased COMMD1 expression correlates with a more invasive tumor phenotype. We found that direct repression of COMMD1 in human cell lines led to increased tumor invasion in a chick xenograft model, while increased COMMD1 expression in mouse melanoma cells led to decreased lung metastasis in a mouse model. Decreased COMMD1 expression also correlated with increased expression of genes known to promote cancer cell invasiveness, including direct targets of HIF. Mechanistically, our studies show that COMMD1 inhibits HIF-mediated gene expression by binding directly to the amino terminus of HIF-1 alpha, preventing its dimerization with HIF-1 beta and subsequent DNA binding and transcriptional activation. Altogether, our findings demonstrate a role for COMMD1 in tumor invasion and provide a detailed mechanism of how this factor regulates the HIF pathway in cancer cells.

Copper metabolism MURR1 domain1 (COMMD1) is a novel inhibitor of the transcription factors NF-kappa B and HIF-1, which play important roles in inflammation and tumor growth, respectively. In this study, we identified two highly conserved nuclear export signals (NESs) in COMMD1 and revealed that these NESs were essential and sufficient to induce maximal nuclear export of COMMD1. Inhibition of CRM1-mediated nuclear export by Leptomycin B resulted in nuclear accumulation of COMMD1. In addition, low oxygen concentrations induced the active export of COMMD1 from the nucleus in a CRM1-dependent manner. Disruption of the NESs in COMMD1 increased the repression of COMMD1 in transcriptional activity of NF-kappa B and HIF-1. In conclusion, these data indicate that COMMD1 undergoes constitutive nucleocytoplasmic transport as a novel mechanism to regulate NF-kappa B and HIF-1 signaling.

Therapeutic approaches aimed at targeting tumor surface markers using monoclonal antibodies provide a powerful strategy in cancer treatment. Here we report selection of single variable domains (VHH) of llama heavy chain antibodies, using a VHH-phage-display library. A reverse proteomic approach was used to identify the cognate proteins recognized by enriched VHH on HeLa cells. One of these VHH bound the integrin α3β1 (VLA-3) and was further characterized. Most interestingly, this VHH could inhibit VLA-3 mediated cell-matrix adhesion. Our approach provides a fast and efficient method to screen for novel cell surface markers on normal and tumor cells that may find diagnostic or therapeutic application in disease management or treatment.

Metastases formation is a major factor in disease progression and accounts for the majority of cancer deaths. The molecular mechanisms controlling invasion, dissemination to blood or lymphatic systems and spread of tumor cells to distant organs are still poorly understood. Recent observations indicate that the meta-static phenotype may already be present during the angiogenic switch of tumors. Intratumoral hypoxia correlates with poor prognosis and enhanced metastases formation. The Hypoxia Inducible Factors (HIFs) are key molecules in the hypoxic response and play critical roles during tumor cell expansion by regulating energy metabolism and the induction of angiogenesis. Increasing evidence implicates HIF function in metastatic cell characteristics, like epithelial to mesenchymal transition, cell detachment, invasion and tumor cell seeding. Here, we review the link between tumor cell hypoxia and the acquisition of metastatic behavior. We hypothesize that polyclonal tumor selection by hypoxia enhances metastatic capacity by transcriptional control of key regulators of metastasis. This polyclonal hypoxic gene profile potentially develops into a metastatic profile, driving metastasis formation. The hypoxic gene profile in primary tumors may therefore provide a prognostic indicator in clinical decision-making.

Hypoxia is a hallmark of solid cancer and characterized by regions of low oxygen and necrosis due to insufficient blood perfusion. Intratumoral hypoxia triggers the transcription of genes responsible for cell survival. The transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha) is a key regulator of this response. HIF activation is associated with resistance to radio- and chemotherapy and poor clinical outcome, and may therefore provide an attractive therapeutic target. Clostridium-based oncolysis is a promising therapeutic strategy for the treatment of hypoxic tumors where these microorganisms naturally home. Here, we report for the first time the isolation of transconjugants of two excellent tumor colonizing Clostridium strains, C. novyi-NT and C. sporogenes, expressing single chain antibodies specific for human HIF-1alpha. This is a first step towards Clostridium-directed antibody therapy (CDAT) that holds promise as a carrier of cancer therapeutics targeting the most resistant regions in human solid cancer.

COMMD1 is a protein which is associated with multiple cellular pathways, including NFkappaB signaling, copper homeostasis and sodium transport. Recently we found that COMMD1 is also essential for normal mouse embryogenesis. Embryos deficient for Commd1 are retarded and die between 9.5 and 10.5 dpc. Increased HIF-1 activity and elevated HIF-1alpha protein expression were observed in 9.5 dpc Commd1-deficient embryos. In line with these in vivo data, in vitro studies showed that reduced COMMD1 expression caused increased HIF-1alpha protein stability and HIF-1 activity. Functional characterization of COMMD1 in NFkappaB signaling and ATP7B-dependent biliary copper excretion suggested that COMMD1 also has a role in regulating the protein degradation of RelA (p65) and ATP7B. The exact function of COMMD1 in these pathways remains elusive but these recent studies suggest that COMMD1 is associated with the ubiquitin-proteasomal system for regulating protein stability.

OMMD1 (previously known as MURR1) belongs to a novel family of proteins termed the copper metabolism gene MURR1 domain (COMMD) family. The 10 COMMD family members are well conserved between vertebrates, but the functions of most of the COMMD proteins are unknown. We recently established that COMMD1 is associated with the hepatic copper overload disorder copper toxicosis in Bedlington terriers. Recent in vitro studies indicate that COMMD1 has multiple functions, including sodium transport and NF-KB signaling. To elucidate the function of Commd1 in vivo, we generated homozygous Commd1 null (Commd1(-/-)) mice. Commd1(-/-) embryos died in utero between 9.5 and 10.5 days postcoiturn. (dpc), their development was generally retarded, and placenta vascularization was absent. Microarray analysis identified transcriptional upregulation of hypoxia-inducible factor 1 (HIF-1) target genes in 9.5-dpc Commd1(-/-) embryos compared to normal embryos, a feature that was associated with increased Hif-1 alpha stability. Consistent with these observations, COMMD1 physically associates with HIF-1 alpha and inhibits HIF-1 alpha stability and HIF-1 transactivation in vitro. Thus, this study identifies COMMD1 as a novel regulator of HIF-1 activity and shows that Commd1 deficiency in mice leads to embryonic lethality associated with dysregulated placenta vascularization.

Hypoxia triggers the transcription of genes responsible for cell survival via the key player transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). Overexpression of this protein has been implicated in cardiovascular disorders, carcinogenesis and cancer progression. For functional and diagnostic studies on the HIF-1alpha protein, we have identified single-domain antibody fragments directed against this protein by using a llama-derived nonimmune phage display library. This library displays the variable domains of the heavy-chain antibody subclass, found in these animals. Phage display selection with six recombinant HIF-1alpha proteins yielded five different antibody fragments. By epitope-mapping, we show that all five antibody fragments bind within the functionally important oxygen-dependent degradation domain of the HIF-1alpha protein. Two of these antibody fragments were engineered into bivalent antibodies that were able to detect human HIF-1alpha by immunohistochemistry, Western blotting and immunoprecipitation, and mouse HIF-1alpha by immunofluorescence and immunoprecipitation. These are the first single-domain antibody fragments that may be used in exploration of HIF-1alpha as a possible therapeutic target through molecular applications.

Hypoxia-inducible factor-1 alpha (HIF-1alpha) is the regulatory subunit of the heterodimeric transcription factor HIF-1 and the key factor in cellular response to low oxygen tension. Expression of HIF-1alpha protein is associated with poor patient survival and therapy resistance in many types of solid tumors. Insight into HIF-1alpha regulation in solid tumors is important for therapeutic strategies. In this study, we determined the pathophysiological relevance of HIF-1alpha regulation by the oncogenic phosphatidylinositol 3'-kinase (PI 3-kinase)/Akt signaling pathway. We modeled the physiology of hypoxic tumor regions by culturing carcinoma cells under low oxygen tension in the absence of serum. We observed that hypoxic induction of HIF-1alpha protein was decreased by serum deprivation. Overexpression of dominant-active Akt1 restored HIF-1alpha expression, whereas inhibition of PI 3-kinase activity reduced hypoxic HIF-1alpha protein levels to a similar extent as serum deprivation. Immunohistochemical analysis of 95 human breast cancers revealed that lack of Akt1 phosphorylation correlates with low HIF-1alpha levels. To our knowledge, this is the first reported comparison between HIF-1alpha expression and Akt phosphorylation in human carcinomas. We conclude that Akt activity is physiologically relevant for HIF-1alpha expression in breast cancer. This implies that HIF-1alpha function might be therapeutically targeted by inhibition of the PI 3-kinase/Akt pathway.

Background: The Copper Metabolism MURR1 Domain containing 1 protein COMMD1 has been associated with copper homeostasis, NF-kappa B signaling, and sodium transport. Recently, we identified COMMD1 as a novel protein in HIF-1 signaling. Mouse embryos deficient for Commd1 have increased expression of hypoxia/HIF-regulated genes i.e. VEGF, PGK and Bnip3. Hypoxia-inducible factors (HIFs) are master regulators of oxygen homeostasis, which control angiogenesis, erythropoiesis, glycolysis and cell survival/proliferation under normal and pathologic conditions. Although HIF activity is mainly controlled by ubiquitination and protein degradation by the von Hippel Lindau (pVHL) tumor suppressor gene other mechanisms have recently been identified that regulate HIF signaling independently of pVHL.Principal Findings: Here we characterized the mechanism by which COMMD1 regulates HIF-1 alpha protein degradation. We show that COMMD1 competes with the chaperone heat shock protein HSP90b for binding to the NH2-terminal DNA-binding and heterodimerization domain of HIF-1 alpha to regulate HIF-1 alpha stability together with HSP70. Inhibition of HSP90 activity with 17-Allylamino-17-demethoxygeldanamycin (17-AAG) increased COMMD1-mediated HIF-1 alpha degradation independent of ubiquitin and pVHL.Conclusion/Significance: These data reveal a novel role for COMMD1 in conjunction with HSP90 beta/HSP70 in the ubiquitin and O-2-independent regulation of HIF-1 alpha.