Non-canonical Notch Signaling Research Articles

Aberrant activation of Notch1 signaling in the mouse uterine epithelium promotes hyper-proliferation by increasing estrogen sensitivity.

In mammals, the endometrium undergoes dynamic changes in response to estrogen and progesterone to prepare for blastocyst implantation. Two distinct types of endometrial epithelial cells, the luminal (LE) and glandular (GE) epithelial cells play different functional roles during this physiological process. Previously, we have reported that Notch signaling plays multiple roles in embryo implantation, decidualization, and postpartum repair. Here, using the uterine epithelial-specific Ltf-iCre, we showed that Notch1 signaling over-activation in the endometrial epithelium caused dysfunction of the epithelium during the estrous cycle, resulting in hyper-proliferation. During pregnancy, it further led to dysregulation of estrogen and progesterone signaling, resulting in infertility in these animals. Using 3D organoids, we showed that over-activation of Notch1 signaling increased the proliferative potential of both LE and GE cells and reduced the difference in transcription profiles between them, suggesting disrupted differentiation of the uterine epithelium. In addition, we demonstrated that both canonical and non-canonical Notch signaling contributed to the hyper-proliferation of GE cells, but only the non-canonical pathway was involved with estrogen sensitivity in the GE cells. These findings provided insights into the effects of Notch1 signaling on the proliferation, differentiation, and function of the uterine epithelium. This study demonstrated the important roles of Notch1 signaling in regulating hormone response and differentiation of endometrial epithelial cells and provides an opportunity for future studies in estrogen-dependent diseases, such as endometriosis.

Notch signaling functions in noncanonical juxtacrine manner in platelets to amplify thrombogenicity.

Notch signaling dictates cell fate decisions in mammalian cells including megakaryocytes. Existence of functional Notch signaling in enucleate platelets remains elusive. Transcripts/peptides of Notch1 and Delta-like ligand (DLL)-4 were detected in platelets isolated from human blood by RT-qPCR, Western analysis and flow cytometry. Platelet aggregation, granule secretion and platelet-leukocyte interaction were analyzed by lumi-aggregometry and flow cytometry. Platelet-derived extracellular vesicles were documented with Nanoparticle Tracking Analyzer. Platelet thrombus on immobilized collagen was quantified using microfluidics platform. Intracellular calcium was monitored by fluorescence spectrophotometry. Whole blood coagulation was studied by thromboelastography. Ferric chloride-induced mouse mesenteric arteriolar thrombosis was imaged by intravital microscopy. We demonstrate expression of Notch1, its ligand DLL-4 and their respective transcripts in human platelets. Synthesis and surface translocation of Notch1 and DLL-4 were upregulated by thrombin. DLL-4, in turn, instigated neighbouring platelets to switch to 'activated' phenotype through cleavage of Notch receptor and release of its intracellular domain (NICD), which was averted by inhibition of γ-secretase and phosphatidylinositol-3-kinase (PI3K). Inhibition of Notch signaling, too, restrained agonist-induced platelet activation, and significantly impaired arterial thrombosis in mice. Strikingly, prevention of DLL-4-Notch1 interaction by a blocking antibody abolished platelet aggregation and extracellular vesicle shedding induced by thrombin. Our study presents compelling evidence in support of non-canonical juxtacrine Notch signaling within platelet aggregates that synergizes with physiological agonists to generate occlusive intramural thrombi. Thus, Notch pathway can be a potential anti-platelet/anti-thrombotic therapeutic target. Research was supported by grants received by DD from JC Bose Fellowship (JCB/2017/000029), ICMR (71/4/2018-BMS/CAR), DBT (BT/PR-20645/BRB/10/1541/2016) and SERB (EMR/2015/000583). SNC, ME and VS are recipients of ICMR-Scientist-C, CSIR-SRF and UGC-SRF support, respectively. Funders had no role in design, analysis and reporting of study.

VEGF in regulation of non-canonical Notch signaling pathway in endothelial cells

<h3></h3> Формирование сосудистой сети (ангиогенез и последующая васкуляризация) в зоне повреждения происходит при взаимодействии эндотелиальных и муральных клеток. Межклеточная коммуникация осуществляется путем паракринного воздействия, в частности, за счет секреции муральными клетками VEGF (фактор роста эндотелия сосудов) — ключевого фактора, стимулирующего ангиогенез, а также прямыми межклеточными контактами. Одним из основных механизмов, регулирующих межклеточные взаимодействия, является сигнальный путь Notch, определяющий выбор направления развития контактирующих клеток и регулирующий их способность к самообновлению, росту, выживанию, дифференцировке и апоптозу. <h3>ЦЕЛЬ ИССЛЕДОВАНИЯ</h3> Изучение возможности пересечения сигнальных путей, запускаемых VEGF, и неканонического Notch. Мы оценили степень влияния VEGF и Notch на способность клеток эндотелия пупочной вены (HUVEC) формировать сосудистые отростки на 3D-модели ангиогенеза в фибриновом геле <i>in vitro</i>. <h3></h3> Мы обнаружили, что ингибитор VEGF (DMH4) значительно снижал количество и длину эндотелиальных отростков. Ингибиторы сигнализации Notch также подавляли формирование отростков, но в меньшей степени. Анализ изменения протеомного профиля фосфоформ ряда киназ в эндотелиальных клетках после добавления VEGF и/или Jagged1 (лиганд системы Notch) показал, что VEGF усиливает фосфорилирование фактора транскрипции CREB, в то время как Jagged1 активирует такие компоненты неканонической сигнализации Notch, как HCK, усиливает экспрессию Hsp60 и b-катенина и подавляет фосфорилирование киназы p70S6. Совместное действие VEGF и Jagged1 приводит к усилению активации HCK и подавлению p70S6.

Noncanonical Notch signals have opposing roles during cardiac development

The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stage-specific roles during cardiac development.

Deficiency of Rbpj Leads to Defective Stress-Induced Hematopoietic Stem Cell Functions and Hif Mediated Activation of Non-canonical Notch Signaling Pathways.

Deregulated notch signaling has been associated with human pathobiology. However, functions of notch pathways in hematopoiesis remain incompletely understood. Here, we ablated canonical notch pathways, through genetic deletion of Rbpj, in hematopoietic stem cells (HSCs). Our data identified that loss of canonical notch results in normal adult HSC pool, at steady state conditions. However, HSC maintenance and functions in response to radiation-, chemotherapy-, and cytokine- induced stress were compromised in the absence of canonical notch. Rbpj deficient HSCs exhibit decreased proliferation rates and elevated expression of p57Kip2. Surprisingly, loss of Rbpj resulted in upregulation of key notch target genes and augmented binding of Hes1 to p57 and Gata2 promoters. Further molecular analyses identified an increase in notch activity, elevated expression and nuclear translocation of Hif proteins, and augmented binding of Hif1α to Hes1 promoter in the absence of Rbpj. These studies, for the first time, identify a previously unknown role for non-canonical notch signaling and establish a functional link between Hif and Notch pathways in hematopoiesis.

Abstract IA01: Strategies for targeted and immune therapy in melanoma

Abstract Melanoma cells activate pathways that are also activated in proliferating fibroblasts, endothelial cells, or even inflammatory and immune cells. The tumor-secreted proteins and expressed cell surface receptors are associated with growth, migration, invasion, and antiapoptosis. Then there is a group of molecules that is not associated with the phenotype of differentiated cells in skin or tumor stroma but that represents re-expressed developmental genes. Several developmental pathway genes, such as those associated with noncanonical Wnt or Notch signaling, are commonly found on stem cells from the dermis that can give rise to melanocytes and on melanoma cells, but they are absent from or not activated in epidermal melanocytes. We have recently identified a G protein-coupled receptor, LPAR1, that is highly activated in neural crest-like stem cells and melanoma cells and absent from melanocytes. Expression is associated with resistance to BRAFV600E inhibition. When other subpopulations of melanoma cells were characterized that were associated with resistance to BRAF, MEK, or ERK small-molecule inhibitors, their major common property was very slow proliferation, which was in contrast to the bulk of the tumor cells. These “label-retaining cells,” characterized by expression of the H3K4 demethylase JARID1B and the protease SERPIN E2, and the EGF and NGF receptors, are not only drug resistant but also highly invasive in vitro and in experimental animal model. Nonproliferating, drug-resistant malignant cells may pre-exist in tumors. As the majority of melanoma cells are killed by kinase inhibitors, multikinase receptor-expressing cells begin proliferating to repopulate the tumor to its original heterogeneity. Thus, melanomas contain small subpopulations representing only 0.1-5% of all malignant cells that are critical for growth, invasion, and therapy resistance. To achieve total elimination of all malignant cells, we have to develop strategies to target not only the bulk of the tumor cells but also those with intrinsic resistance to common drugs. To closely mimic the conditions in patients’ tumors, we have developed &amp;gt;500 patient-derived xenografts. Together with the &amp;gt;300 permanent cell lines, they represent all clinical, genetic, and biologic groups of the disease. Since the human tumors only proliferate in immune-deficient mice, we have developed two humanized models that recapitulate the infiltration by human leukocytes into the human tumors. Inhibition of checkpoints with clinical-grade inhibitors stimulated the immune response, leading to focal or general tumor regression; thus, the models allow investigating the mechanisms of both stimulation and suppression of the immune system. Citation Format: Meenhard Herlyn. Strategies for targeted and immune therapy in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA01.

Quality-control mechanisms targeting translationally stalled and C-terminally extended poly(GR) associated with ALS/FTD

Maintaining the fidelity of nascent peptide chain (NP) synthesis is essential for proteome integrity and cellular health. Ribosome-associated quality control (RQC) serves to resolve stalled translation, during which untemplated Ala/Thr residues are added C terminally to stalled peptide, as shown during C-terminal Ala and Thr addition (CAT-tailing) in yeast. The mechanism and biological effects of CAT-tailing-like activity in metazoans remain unclear. Here we show that CAT-tailing-like modification of poly(GR), a dipeptide repeat derived from amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD)-associated GGGGCC (G4C2) repeat expansion in C9ORF72, contributes to disease. We find that poly(GR) can act as a mitochondria-targeting signal, causing some poly(GR) to be cotranslationally imported into mitochondria. However, poly(GR) translation on mitochondrial surface is frequently stalled, triggering RQC and CAT-tailing-like C-terminal extension (CTE). CTE promotes poly(GR) stabilization, aggregation, and toxicity. Our genetic studies in Drosophila uncovered an important role of the mitochondrial protease YME1L in clearing poly(GR), revealing mitochondria as major sites of poly(GR) metabolism. Moreover, the mitochondria-associated noncanonical Notch signaling pathway impinges on the RQC machinery to restrain poly(GR) accumulation, at least in part through the AKT/VCP axis. The conserved actions of YME1L and noncanonical Notch signaling in animal models and patient cells support their fundamental involvement in ALS/FTD.

Covid-19: Pandemic is having "severe" impact on non-communicable disease care, WHO survey finds.

<h3>Abstract</h3> Current treatment of triple negative breast cancer patients is hindered by a high incidence of chemoresistance (30-50%). The prevailing theory is that resistance and subsequent recurrence is driven by cancer stem cells. Unfortunately, the functional characterization of cancer stem cells at the molecular level is still incomplete. We show here, that within the canonical breast cancer stem cell population, a subset of cells characterized by high Notch1 expression possesses the tumor-initiating property associated with cancer stem cells. Moreover, the tumor initiating property of these high Notch1-expressing breast cancer stem cells is mediated by a cleavage independent Notch signaling pathway culminating in the repression of SIRT1. Of note, the Notch1-mediated repression of SIRT1 is required not only for tumor initiation, but also for chemoresistance in breast cancer stem cells. Strikingly, inhibition of SIRT1 obviates the requirement for Notch1, marking the first example of conferring cancer stem cell function by inhibiting the activity of a single protein. We also demonstrate that progenitor-like mammary epithelial cells, which possess both luminal and basal properties, are also characterized by high Notch1 expression and repression of SIRT1 via the non-canonical pathway. These findings provide the first functional mechanistic requirements for tumor initiation by breast cancer stem cells and suggest that activation of the non-canonical Notch1 pathway is hardwired into tumor-initiating progenitor cells and thus a prerequisite for tumor initiation. <h3>Statement of Significance</h3> We demonstrate that chemoresistant and tumor-initiating properties of breast cancer stem cells are driven by repression of SIRT1 via non-canonical Notch signaling, suggesting a novel therapeutic strategy for triple negative breast cancer.

Rab11 plays a key role in stellate cell differentiation via non-canonical Notch pathway in Malpighian tubules of Drosophila melanogaster

Rab11, a member of Rab-GTPase family, and a marker of recycling endosomes has been reported to be involved in the differentiation of various tissues in Drosophila. Here we report a novel role of Rab11 in the differentiation of stellate cells via the non-canonical Notch pathway in Malpighian tubules. During Malpighian tubule development caudal visceral mesodermal cells intercalate into the epithelial tubule of ectodermal origin consisting of principal cells, undergo mesenchymal to epithelial transition and differentiate into star shaped stellate cells in adult Malpighian tubule. Two transcription factors, Teashirt and Cut (antagonistic to each other) are known to be expressed in stellate cells and principal cells, respectively, from early stages of development and serve as markers for these cells. Inhibition of Rab11 function or over-expression of activated Notch in stellate cells resulted in the expression of Cut that leads to down-regulation of Teashirt or vice-versa that leads to hampered differentiation of stellate cells. The stellate cells do not transform to star/bar shaped and remain in mesenchymal state in adult Malpighian tubule. Over-expression of Deltex, which plays important role in non-canonical Notch signaling pathway, shows similar phenotype of stellate cells as seen in individuals with down-regulated Rab11, while down-regulation of Deltex in genetic background of Rab11RNAi rescues Teashirt expression and shape of stellate cells. Our experiments suggest that an inhibition or reduction of Rab11 function in stellate cells results in the faulty recycling of Notch receptors to plasma membrane as they accumulate in early and late endosomes, leading to Deltex mediated non-canonical Notch activation.

Phosphorylation and Proteolytic Cleavage of Notch in Canonical and Noncanonical Notch Signaling.

The Notch signaling pathway seems deceptively simple, with its key feature being a direct connection between extracellular signal and transcriptional output without the need for an extended chain of protein intermediaries as required by so many other signaling paradigms. However, this apparent simplicity hides considerable complexity. Consistent with its central role in many aspects of development, Notch signaling has an extensive collection of mechanisms that it employs alongside of its core transcriptional machinery. These so-called noncanonical Notch pathways diversify the potential outputs of Notch, and allow it to coordinate regulation of many aspects of the biology of cells. Here we will review noncanonical Notch signaling with special attention to the role of posttranslational modifications of Notch. We will also consider the importance of coordinating the activity of gene expression with regulation of cell morphology in biological processes, including axon guidance and other morphological events during embryogenesis.

Abnormally localized DLK1 interacts with NCOR1 in non-small cell lung cancer cell nuclear.

Delta-like homolog 1 (DLK1) regulates noncanonical Notch signaling pathway as ligand. DLK1 was abnormally expressed in a variety of tumors, affecting tumorigenesis and developments. The biological function of DLK1 toward cell proliferation and signaling activation was controversial across different cell types. Two currently known isoforms of DLK1, which are membrane-tethered isoform and soluble isoform, are believed to be the key of DLK1 dual behaviors. While these isoforms are not enough to explain the phenomena, our observations offer the possibility of a third isoform of DLK1. In the present study, we verified the nuclear localization of DLK1 in lung cancer cells. The nuclear localized DLK1 was observed in 107 of 351 non-small cell lung cancer (NSCLC) samples and was associated with tissue differentiation and tumor size. Through co-immunoprecipitation (co-IP) combined mass spectrometry (MS), we identified nuclear receptor corepressor 1 (NCOR1) as DLK1’s novel interaction protein and confirmed their interaction in nuclear. We analyzed the expression of NCOR1 in two independent cohorts and demonstrated that NCOR1 is a tumor suppressor and has prognosis potential in lung squamous carcinomas. At last, we analyzed the colocalization of DLK1 and NCOR1 in 147 NSCLC samples by immunohistochemistry (IHC). The result indicated NCOR1 might participate with nuclear localized DLK1 in regulating cell differentiation.

Non-canonical Notch Signaling Regulates Actin Remodeling in Cell Migration by Activating PI3K/AKT/Cdc42 Pathway.

Tumor cell migration is a critical step in cancer metastasis. Over-activated Notch pathway can promote the migration of cancer cells, especially in the breast cancer. However, the underlying mechanism of non-canonical Notch signaling in modulating the migration has not yet been clearly characterized. Here we demonstrated that DAPT, a gamma secretase inhibitor, inhibited protrusion formation and cell motility, and then reduced the migration of triple-negative breast cancer cells, through increasing the activity of Cdc42 by non-canonical Notch pathway. Phosphorylation of AKT on S473 was surprisingly increased when Notch signaling was inhibited by DAPT. Inhibition of PI3K and AKT by LY294002 and MK2206, respectively, or knockdown of AKT expression by siRNA blocked DAPT-induced activation of Cdc42. Moreover, immunofluorescence staining further showed that DAPT treatment reduced the formation of lamellipodia and induced actin cytoskeleton remodeling. Taken together, these results indicated that DAPT inhibited Notch signaling and consequently activated PI3K/AKT/Cdc42 signaling by non-canonical pathway, facilitated the formation of filopodia and inhibited the assembly of lamellipodia, and finally resulted in the decrease of migration activity of breast cancer cells.

Abstract 157: Targeting cancer stem-like cells in triple negative breast cancer

Abstract Triple Negative Breast Cancer (TNBC), an aggressive, heterogeneous subtype of breast cancer, and immunohistochemically negative for estrogen receptor α (ER-), progesterone receptor (PR-), and lacking amplification of the human epidermal growth factor receptor 2 locus (HER2-). TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore CSC-targeted therapies are keenly sought. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlates with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Moreover the role of non-canonical Notch signaling in TNBC remains unknown. Here, we demonstrate that Notch1 promotes cell survival in MDA-MB-231 cells, representative of MSL TNBC, in part by activating NF-κB. Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment of NF-κB subunits, IKKα and Notch1 to the cIAP-2 promoter. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by dual mTORC1/2 inhibitors, AKT inhibitors and IKKα inhibitors but not Everolimus (mTORC1-selective inhibitor). These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1-activated pathway. We also demonstrate that recombinant Jagged1 increases the cellular metabolism of TNBC cells and knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. We have found that CSCs derived from MDA-MB-231 cells have increased Notch1, p-AKT, and mitochondrial respiration. AKT inhibition or NF-κB inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF-03084014) in combination with AKT inhibitor (MK-2206) or NF-κB inhibitor (Bay11-7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. These data suggest that combination treatments affecting Notch, NF-kB or AKT pathways have potential therapeutic importance in targeting CSCs of TNBC. Citation Format: Fokhrul Hossain, Ayse D Ucar Bilyeu, Claudia Sorrentino, Judy Crabtree, Antonio Pannuti, Margarite Matossian, Matthew Burow, Todd Golde, Barbara Osborne, Lucio Miele. Targeting cancer stem-like cells in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 157.

The non-canonical Notch signaling is essential for the control of fertility in Aedes aegypti.

The Notch signaling pathway is a highly evolutionarily-conserved cell-cell signaling pathway that regulates many events during development. It plays a pivotal role in the regulation of fundamental cellular processes, such as cell proliferation, stem cell maintenance, and differentiation during embryonic and adult development. However, functions of Notch signaling in Aedes aegypti, the major mosquito vector for dengue, are largely unknown. In this study, we identified a unique feature of A. aegypti Notch (AaNotch) in the control of the sterile-like phenotype in female mosquitoes. Silencing AaNotch with a reverse genetic approach significantly reduced the fecundity and fertility of the mosquito. Silencing AaNotch also resulted in the prevention of micropyle formation, which led to impaired fertilization. In addition, JNK phosphorylation (a signaling molecule in the non-canonical Notch signaling pathway) was inhibited in the absence of AaNotch. Furthermore, treatment with a JNK inhibitor in the mosquito resulted in impaired fecundity and fertility. Taken together, our results demonstrate that non-canonical Notch signaling is essential for controlling fertility in the A. aegypti mosquito.

Abstract P6-07-06: Targeting cancer stem-like cells metabolism via non-canonical notch signaling pathways in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore CSC-targeted therapies are keenly sought. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment to the cIAP-2 promoter of NF-κB subunits, IKKα and Notch1. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by AKT inhibitors, IKK inhibitors, and dual mTORC1/2 inhibitors but not an mTORC1-selective inhibitor. These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF-κB activation. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1-activated pathway. Importantly, we find co-localization of Notch1 with Mitochondria in MDA-MB-231 cells by confocal microscopy and Western blot of isolated mitochondrial fractions. We demonstrate that recombinant Jagged1 increases metabolism of TNBC cells. Knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. CSCs derived from MDA-MB-231 cells have increased Notch1, p-AKT, and oxidative metabolism compared to non-stem cells. AKT inhibition or IKK inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF-03084014) in combination with AKT inhibitor (MK-2206) or IKK inhibitor (Bay11-7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. Notably, we find similar results in TNBC patient derived xenograft (PDX) models. These data suggest that combination treatments affecting the intersection of Notch, NF-kB and AKT pathways have potential therapeutic importance in targeting CSCs in TNBC cases with high Notch1 expression. Citation Format: Hossain F, Sorrentino C, Ucar Bilyeu AD, Matossian M, Crabtree J, Pannuti A, Burow M, Golde T, Osborne B, Miele L. Targeting cancer stem-like cells metabolism via non-canonical notch signaling pathways in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-06.

Notching a New Pathway in Vascular Flow Sensing.

Vascular barrier function is controlled at cell-cell junctions in response to blood flow, but how vascular endothelial cells sense and respond to flow remains to be understood. A recent study describes a flow-sensing pathway involving non-canonical Notch and cadherin signaling that sheds new light on mechanisms controlling the endothelial barrier.

Mechanisms of Non-canonical Signaling in Health and Disease: Diversity to Take Therapy up a Notch?

Non-canonical Notch signaling encompasses a wide range of cellular processes, diverging considerably from the established paradigm. It can dispense of ligand, proteolytic or nuclear activity. Non-canonical Notch signaling events have been studied mostly in the fruit fly Drosophila melanogaster, the organism in which Notch was identified first and a powerful model for understanding signaling outcomes. However, non-canonical events are ill-defined and their involvement in human physiology is not clear, hampering our understanding of diseases arising from Notch signaling alterations. At a time in which therapies based on specific targeting of Notch signaling are still an unfulfilled promise, detailed understanding of non-canonical Notch events might be key to devising more specific and less toxic pharmacologic options. Based on the blueprint of non-canonical signaling in Drosophila, here, we review and rationalize current evidence about non-canonical Notch signaling. Our effort might inform Notch biologists developing new research avenues and clinicians seeking future treatment of Notch-dependent diseases.

Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling

ARMMs (arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles) are extracellular vesicles that bud directly at the plasma membrane; however, little is known about the molecular composition and physiological function of these vesicles. Here we report that ARMMs contain active NOTCH receptors and mediate a non-canonical intercellular NOTCH signaling. We identify over 100 proteins that are significantly enriched in ARMMs, including ARRDC1, TSG101 and multiple ESCRT complex proteins. About a third of ARMMs-enriched proteins are plasma membrane proteins, including the NOTCH2 receptor. The incorporation of NOTCH2 into ARMMs is facilitated by the ITCH E3 ligase and the metalloprotease ADAM10, both of which are also secreted into ARMMs. NOTCH2 in ARMMs can be delivered into recipient cells, and upon activation by γ-secretase cleavage, induces NOTCH-specific gene expression. Together, our findings reveal a role for ARMMs in a novel NOTCH signaling pathway that acts in distance and is independent of direct cell–cell contact.

Abstract 2904: Targeting cancer stem-like cells in triple negative breast cancer cells through non-canonical notch signaling

Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore CSC-targeted therapies are keenly sought. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch1 promotes cell survival in MDA-MB-231 cells, representative of MSL TNBC, in part by activating NF-κB. Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment to the cIAP-2 promoter of NF-κB subunits, IKKα and Notch1. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by dual mTORC1/2 inhibitors, AKT inhibitors and IKKα inhibitors but not Everolimus (mTORC1-selective inhibitor). These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF-κB activation. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1-activated pathway. We demonstrate that recombinant Jagged1 increases the cellular metabolism of TNBC cells and knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. We have found that CSCs derived from MDA-MB-231 cells have increased Notch1, p-AKT, and oxidative metabolism. AKT inhibition or IKKα inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF-03084014) in combination with AKT inhibitor (MK-2206) or NF-κB inhibitor (Bay11-7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. These data suggest that combination treatments affecting Notch, NF-kB and AKT pathways have potential therapeutic importance in targeting CSCs of TNBC cases with wild type PTEN and high Notch1 expression. Note: This abstract was not presented at the meeting. Citation Format: Fokhrul Hossain, Claudia Sorrentino, Ayse Bilyeu, Judy Crabtree, Antonio Pannuti, Todd Golde, Barbara Osborne, Lucio Miele. Targeting cancer stem-like cells in triple negative breast cancer cells through non-canonical notch signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2904. doi:10.1158/1538-7445.AM2017-2904

The expression levels of Notch-related signaling molecules in pulmonary microvascular endothelial cells in bleomycin-induced rat pulmonary fibrosis.

Previous studies have suggested that the Notch signaling pathway plays a very important role in the proliferation and differentiation of pulmonary microvascular endothelial cells (PMVECs). Therefore, we aimed to investigate the expression level of Notch-related signaling molecules in PMVECs in bleomycin (BLM)-induced rat pulmonary fibrosis. Immunohistochemistry, immunofluorescence, Western blotting, and real-time PCR were used to analyze the differences in protein and mRNA expression levels of Notch-related signaling molecules, i.e. Notch1, Jagged1, Delta-like ligand 4 (Dll4), and hairy and enhancer of split homolog 1 (Hes1), between a control group treated with intratracheal instillation of saline and a study group treated with intratracheal instillation of BLM solution. Expression levels of the receptor Notch1 and one of its ligands, Jagged1, were upregulated, while the expression levels of the ligand Dll4 and the target molecule of the Notch signaling pathway, Hes1, were downregulated. The differences in protein and mRNA expression levels between the control and study groups were significant (p<0.001). The Jagged1/Notch1 signaling pathway is activated in the pathogenesis of BLM-induced rat pulmonary fibrosis, while the Dll4/Notch1 signaling pathway is inhibited, which inhibits the suppressive effect of Dll4/Notch1 signaling on PMVEC overproliferation, further causing PMVEC dysfunction in cell sprouting and maturation as well as abnormal differentiation of the cell phenotype. Conversely, the down-expression of Hes1 indicates that the Jagged1/Notch1 signaling pathway could be a non-canonical Notch signaling pathway independent of Hes1 activation, which differs from the canonical Dll4/Notch1 signaling pathway.