Iron-responsive element of Divalent metal transporter 1 (Dmt1) controls Notch-mediated cell fates.

Notch receptor activation is regulated by the intramembrane protease γ-secretase, which cleaves and liberates the Notch intracellular domain (Nicd) that regulates gene transcription. While γ-secretase cleavage is necessary, we demonstrate it is insufficient for Notch activation and requires vesicular trafficking. Here, we report Divalent metal transporter 1 (Dmt1, Slc11A2) as a novel and essential regulator of Notch signalling. Dmt1-deficient cells are defective in Notch signalling and have perturbed endolysosomal trafficking and function. Dmt1 encodes for two isoforms, with and without an iron response element (ire). We show that isoform-specific silencing of Dmt1-ire and Dmt1+ire has opposite consequences on Notch-dependent cell fates in cell lines and intestinal organoids. Loss of Dmt1-ire suppresses Notch activation and promotes differentiation, whereas loss of Dmt1+ire causes Notch activation and maintains stem-progenitor cell fates. Dmt1 isoform expression correlates with Notch and Wnt signalling in Apc-deficient intestinal organoids and human colorectal cancers. Consistently, Dmt1-ire silencing induces Notch-dependent differentiation in colorectal cancer cells. These data identify Dmt1 isoforms as binary switches controlling Notch cell fate decisions in normal and tumour cells.

A lineage-tracing tool to map the fate of hypoxic tumour cells.

Intratumoural hypoxia is a common characteristic of malignant treatment-resistant cancers. However, hypoxia-modification strategies for the clinic remain elusive. To date, little is known on the behaviour of individual hypoxic tumour cells in their microenvironment. To explore this issue in a spatial and temporally controlled manner, we developed a genetically encoded sensor by fusing the O2-labile hypoxia-inducible factor 1α (HIF-1α) protein to eGFP and a tamoxifen-regulated Cre recombinase. Under normoxic conditions, HIF-1α is degraded but, under hypoxia, the HIF-1α-GFP-Cre-ERT2 fusion protein is stabilised and in the presence of tamoxifen activates a tdTomato reporter gene that is constitutively expressed in hypoxic progeny. We visualise the random distribution of hypoxic tumour cells from hypoxic or necrotic regions and vascularised areas using immunofluorescence and intravital microscopy. Once tdTomato expression is induced, it is stable for at least 4 weeks. Using this system, we could show in vivo that the post-hypoxic cells were more proliferative than non-labelled cells. Our results demonstrate that single-cell lineage tracing of hypoxic tumour cells can allow visualisation of their behaviour in living tumours using intravital microscopy. This tool should prove valuable for the study of dissemination and treatment response of post-hypoxic tumour cells in vivo at single-cell resolution.This article has an associated First Person interview with the joint first authors of the paper.

Rosuvastatin Enhances VSV-G Lentiviral Transduction of NK Cells via Upregulation of the Low-Density Lipoprotein Receptor.

Adoptive natural killer (NK) cell therapy is attaining promising clinical outcomes in recent years, but improvements are needed. Genetic modification of NK cells with a tumor antigen-specific receptor on their surface coupled to intracellular signaling domains may lead to enhanced cytotoxicity against malignant cells. One of the most common approaches is by lentivirus-mediated transduction. However, NK cells are difficult to transduce and various methods have been attempted with different success rates. Because the low-density lipoprotein-receptor (LDLR) is the receptor of vesicular stomatitis virus (VSV) and is expressed only at low levels on NK cells, we tested the potential of 5 statins and 5 non-statin compounds to increase the LDLR expression, thereby facilitating viral transduction. We found that the transduction efficiency of VSV-G pseudotyped lentivirus is augmented by statins that induced higher LDLR expression. In both NK-92 cells and primary NK cells, the transduction efficiency increased after treatment with statins. Furthermore, statins have been reported to suppress NK cell cytotoxicity; however, we showed that this can be completely reversed by adding geranylgeranyl-pyrophosphate (GGPP). Among the statins tested, we found that the combination of rosuvastatin with GGPP most potently improved viral transduction without affecting the cytotoxic properties of the NK cells.

NOTCH inhibition promotes bronchial stem cell renewal and epithelial barrier integrity after irradiation.

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.

NOTCH signaling promotes the survival of irradiated basal airway stem cells.

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.

The role of ENPP1/PC-1 in osteoinduction by calcium phosphate ceramics.

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 anti-malarial drug chloroquine sensitizes oncogenic NOTCH1 driven human T-ALL to γ-secretase inhibition.

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.

HIF-1α and HIF-2α Differently Regulate the Radiation Sensitivity of NSCLC Cells.

The hypoxia-inducible transcription factors (HIF)-1/2α 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-1α and HIF-2α, we use CRISPR gene-editing to generate isogenic H1299 non-small cell lung carcinoma cells lacking HIF-1α, HIF-2α or both. We found that in HIF1 knockout cells, HIF-2α was strongly induced by hypoxia compared to wild type but the reverse was not seen in HIF2 knockout cells. Cells lacking HIF-1α were more radiation resistant than HIF2 knockout and wildtype cells upon hypoxia, which was associated with a reduced recruitment of γH2AX foci directly after irradiation and not due to differences in proliferation. Conversely, double-HIF1/2 knockout cells were most radiation sensitive and had increased γH2AX recruitment and cell cycle delay. Compensatory HIF-2α 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-α 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.

An orthotopic non-small cell lung cancer model for image-guided small animal radiotherapy platforms.

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.

Synergistic Effects of NOTCH/γ-Secretase Inhibition and Standard of Care Treatment Modalities in Non-small Cell Lung Cancer Cells.

Background: Lung cancer is the leading cause of cancer death worldwide. More effective treatments are needed to increase durable responses and prolong patient survival. Standard of care treatment for patients with non-operable stage III-IV NSCLC is concurrent chemotherapy and radiation. An activated NOTCH signaling pathway is associated with poor outcome and treatment resistance in non-small cell lung cancer (NSCLC). NOTCH/γ-secretase inhibitors have been effective in controlling tumor growth in preclinical models but the therapeutic benefit of these inhibitors as monotherapy in patients has been limited so far. Because NOTCH signaling has been implicated in treatment resistance, we hypothesized that by combining NOTCH inhibitors with chemotherapy and radiotherapy this could result in an increased therapeutic effect. A direct comparison of the effects of NOTCH inhibition when combined with current treatment combinations for NSCLC is lacking. Methods: Using monolayer growth assays, we screened 101 FDA-approved drugs from the Cancer Therapy Evaluation Program alone, or combined with radiation, in the H1299 and H460 NSCLC cell lines to identify potent treatment interactions. Subsequently, using multicellular three-dimensional tumor spheroid assays, we tested a selection of drugs used in clinical practice for NSCLC patients, and combined these with a small molecule inhibitor, currently being tested in clinical trials, of the NOTCH pathway (BMS-906024) alone, or in combination with radiation, and measured specific spheroid growth delay (SSGD). Statistical significance was determined by one-way ANOVA with post-hoc Bonferroni correction, and synergism was assessed using two-way ANOVA. Results: Monolayer assays in H1299 and H460 suggest that 21 vs. 5% were synergistic, and 17 vs. 11% were additive chemoradiation interactions, respectively. In H1299 tumor spheroids, significant SSGD was obtained for cisplatin, etoposide, and crizotinib, which increased significantly after the addition of the NOTCH inhibitor BMS-906024 (but not for paclitaxel and pemetrexed), and especially in triple combination with radiation. Synergistic interactions were observed when BMS-906024 was combined with chemoradiation (cisplatin, paclitaxel, docetaxel, and crizotinib). Similar results were observed for H460 spheroids using paclitaxel or crizotinib in dual combination treatment with NOTCH inhibition and triple with radiation. Conclusions: Our findings point to novel synergistic combinations of NOTCH inhibition and chemoradiation that should be tested in NSCLC in vivo models for their ability to achieve an improved therapeutic ratio.

Prognostic Role of Hypoxia-Inducible Factor-2α Tumor Cell Expression in Cancer Patients: A Meta-Analysis.

Hypoxia-inducible factor-2α (HIF-2α) plays an important role in tumor progression and metastasis. A number of studies have evaluated the correlation between 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 with solid tumors, 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 February 2018. Forty-nine studies with 6,052 patients were included in this study. The pooled hazard ratios (HRs) with corresponding confidence intervals were calculated to assess the prognostic value of HIF-2α protein expression in tumor cells. 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). Subgroup analyses revealed similar associations in the majority of tumor sites. Overall, these data demonstrate a negative prognostic role of HIF-2α in patients suffering from different types of solid tumors.

Blocking 17ß-hydroxysteroid dehydrogenase type 1 in endometrial cancer: a potential novel endocrine therapeutic approach.

The enzyme type 1 17ß-hydroxysteroid dehydrogenase (17ß-HSD-1), responsible for generating active 17ß-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 17ß-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 17ß-HSD-1 similar to human tissues. In these cells, HPLC analysis showed that 17ß-HSD-1 activity could be blocked by a specific 17ß-HSD-1 inhibitor. In vitro, E1 administration elicited colony formation similar to E2, and this was impaired by 17ß-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 17ß-HSD-1 inhibition. Neither in vitro nor in vivo effects of E1 were observed using 17ß-HSD-1-negative cells (negative control). Using a patient cohort of 52 primary ECs, we demonstrated the presence of 17ß-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 17ß-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, 17ß-HSD-1, in metastatic lesions using a second cohort of 37 EC patients. In conclusion, 17ß-HSD-1 inhibition efficiently blocks the generation of E2 from E1 using various EC models. Further preclinical investigations and 17ß-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.

Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aß Pool.

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 Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß 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 Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

NOTCH blockade combined with radiation therapy and temozolomide prolongs survival of orthotopic glioblastoma.

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.

Insights into Autoregulation of Notch3 from Structural and Functional Studies of Its Negative Regulatory Region.

Notch 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.

Human NOTCH2 Is Resistant to Ligand-independent Activation by Metalloprotease Adam17.

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 γ-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.

Regulated proteolysis of NOTCH2 and NOTCH3 receptors by ADAM10 and presenilins.

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 γ-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 γ-secretase complex, leading to transcriptional activation.

High NOTCH activity induces radiation resistance in non small cell lung cancer.

BACKGROUND AND PURPOSE: Patients with advanced NSCLC have survival rates <15%. The NOTCH pathway plays an important role during lung development and physiology but is often deregulated in lung cancer, making it a potential therapeutic target. We investigated NOTCH signaling in NSCLC and hypothesized that high NOTCH activity contributes to radiation resistance. MATERIALS AND METHODS: NOTCH signaling in NSCLC patient samples was investigated using quantitative RT-PCR. H460 NSCLC cells with either high or blocked NOTCH activity were generated and their radiation sensitivity monitored using clonogenic assays. In vivo, xenograft tumors were irradiated and response assessed using growth delay. Microenvironmental parameters were analyzed by immunohistochemistry. RESULTS: Patients with high NOTCH activity in tumors showed significantly worse disease-free survival. In vitro, NOTCH activity did not affect the proliferation or intrinsic radiosensitivity of NSCLC cells. In contrast, xenografts with blocked NOTCH activity grew slower than wild type tumors. Tumors with high NOTCH activity grew significantly faster, were more hypoxic and showed a radioresistant phenotype. CONCLUSIONS: We demonstrate an important role for NOTCH in tumor growth and correlate high NOTCH activity with poor prognosis and radioresistance. Blocking NOTCH activity in NSCLC might be a promising intervention to improve outcome after radiotherapy.

The role of Adams in Notch signaling.

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.