Notch1 Cleavage Research Articles

Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells

Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.

Abstract 3439: Sulindac sulfide as a gamma secretase modifier to target triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is defined as pathologically negative for estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 amplification (HER2-). TNBCs are a heterogeneous group of clinically aggressive breast cancers with high risk of recurrence and metastasis, but the current treatment options remain limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, Including Gamma Secretase Inhibitors (GSIs) are quite effective in preclinical models of TNBC. However the success of GSIs in clinical trials is limited by their intestinal toxicity and adverse immunological effects. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects. We identified Sulindac Sulfide (SS), the active metabolite of FDA-approved NSAID Sulindac, as a potential candidate to replace GSIs. SS has documented Gamma Secretase Modifier (GSM) activity, in addition to cyclo-oxygenase (COX) inhibition. We confirmed that SS inhibits Notch1 cleavage in TNBC cells, but not in murine T-cells. SS significantly inhibited mammospheres growth in all human and murine TNBC models we tested: 1) human MDA-MB-231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe (LFng-/-); and 3) Two TNBC patient-derived xenograft models, 2K1 and 4IC. In C0321 tumors, we found that SS had remarkable single-agent anti-tumor activity and virtually eliminated Notch1 expression in tumors. SS caused an increase in intra-tumoral CD11c+ dendritic cells, but decreased CD4 cells, which in this model are largely PD-1 positive (exhausted). CD8 cells were modestly increased. SS did not affect the numbers of tumor infiltrating macrophages or myeloid-derived suppressor cells (MDSC). However, SS blocked the immunosuppressive function of bone marrow-derived MDSC. RNA-Sequencing of SS-treated tumors revealed significant reduction of CXCL14, EGR1, HOXC6, MAGI2, NCAM1, APOE, CLU (a Wnt target), DTX4 (an E3-ligase positive regulator of Notch activation), and TGFB3 genes and upregulation of CCL17, EPCAM, FABP4, C4A, LTF, ZBTB16, INADL, and FGFR2 genes. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be significantly easier and more cost-effective than developing unproven investigational agents. Citation Format: Fokhrul Hossain, Deniz A Ucar, Samarpan Majumder, Margarite Matossian, Keli Xu, Yong Ran, Lisa Minter, Yaguang Xi, Matthew Burow, Todd Golde, Barbara Osborne, Lucio Miele. Sulindac sulfide as a gamma secretase modifier to target triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3439.

γ‐secretase inhibitor DAPT mitigates cisplatin‐induced acute kidney injury by suppressing Notch1 signaling

Organ toxicity, including kidney injury, limits the use of cisplatin for the treatment of multiple human cancers. Hence, interventions to alleviate cisplatin‐induced nephropathy are of benefit to cancer patients. Recent studies have demonstrated that pharmacological inhibition of the Notch signaling pathway enhances cisplatin efficacy against several cancer cells. However, whether augmentation of the anti‐cancer effect of cisplatin by Notch inhibition comes at the cost of increased kidney injury is unclear. We show here that treatment of mice with cisplatin resulted in a significant increase in Notch ligand Delta‐like 1 (Dll1) and Notch1 intracellular domain (N1ICD) protein expression levels in the kidneys. N‐ [N‐ (3,5‐difluorophenacetyl) ‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT), a γ‐secretase inhibitor reversed cisplatin‐induced increase in renal N1ICD expression and plasma or urinary levels of predictive biomarkers of acute kidney injury (AKI). DAPT also mitigated cisplatin‐induced tubular injury and reduction in glomerular filtration rate. Real‐time multiphoton microscopy revealed marked necrosis and peritubular vascular dysfunction in the kidneys of cisplatin‐treated mice which were abrogated by DAPT. Cisplatin‐induced Dll1/Notch1 signaling was recapitulated in a human proximal tubule epithelial cell line (HK‐2). siRNA‐mediated Dll1 knockdown and DAPT attenuated cisplatin‐induced Notch1 cleavage and cytotoxicity in HK‐2 cells. These data suggest that Dll1‐mediated Notch1 signaling contributes to cisplatin‐induced AKI. Hence, the Notch signaling pathway could be a potential therapeutic target to alleviate renal complications associated with cisplatin chemotherapy.Support or Funding InformationWe acknowledge a fellowship (to A. Adebiyi) from the O'Brien Center for Advanced Renal Microscopic Analysis (NIH‐NIDDKP30DK079312).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GSAP modulates γ-secretase specificity by inducing conformational change in PS1

The mechanism by which γ-secretase activating protein (GSAP) regulates γ-secretase activity has not yet been elucidated. Here, we show that knockout of GSAP in cultured cells directly reduces γ-secretase activity for Aβ production, but not for Notch1 cleavage, suggesting that GSAP may induce a conformational change contributing to the specificity of γ-secretase. Furthermore, using an active-site-directed photoprobe with double cross-linking moieties, we demonstrate that GSAP modifies the orientation and/or distance of the PS1 N-terminal fragment and the PS1 C-terminal fragment, a region containing the active site of γ-secretase. This work offers insight into how GSAP regulates γ-secretase specificity.

Cigarette smoke extracts and cadmium induce COX-2 expression through γ-secretase-mediated p38 MAPK activation in C6 astroglia cells.

Exposure to cigarette smoke has been implicated in the progression of cerebrovascular and neurological disorders like stroke through inflammation and blood-brain barrier disruption. In this study, we investigated the signaling cascade activated by cigarette smoke extracts (CSE) and cadmium (Cd) resulting in the COX-2 induction in C6 rat astroglia cells. CSE or Cd induced Notch1 cleavage and activated p38 MAPK and CREB signaling pathways in C6 astroglia cells. Knockdown of nicastrin using siRNA or γ-secretase inhibitors, DAPT and L-685,486, reduced Notch1 cleavage and phosphorylation of p38 MAPK and CREB, while phosphorylation of ERK and JNK remained unaffected. Additionally, the blockage of γ-secretase activity did not show any effect on the phosphorylation of AKT, another upstream activator of CREB, indicating that γ-secretase-mediated CREB activation occurs via p38 MAPK. γ-secretase inhibitor also inhibited the CSE and Cd-mediated increase in the expression of COX-2. Furthermore, recombinant overexpression of Notch1 intracellular domain resulted in an increase in the expression of COX-2. Notch signaling induced by CSE and Cd induced apoptosis in C6 cells. Our results demonstrate that CSE exposure activated the p38 MAPK and CREB-mediated induction in COX-2 expression in astrocytes via γ-secretase-mediated Notch1 signaling. Our data provides novel insights into the potential mechanism of pro-inflammatory response activated by exposure to cigarette smoke.

Abstract P6-22-01: Repurposing sulindac sulfide as a notch inhibitor to target cancer stem-like cells in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive breast cancers. TNBC patients have a high risk of recurrence and metastasis, and current treatment options remain limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Emerging evidence suggests that cancer stem-like cells (CSCs) that escape chemo or radiation therapy in TNBC are often Notch-dependent. At the same time, there is evidence that active tumor immunity predicts good response to neo-adjuvant chemotherapy in TNBC. Notch inhibitors, including Gamma Secretase Inhibitors (GSIs) are quite effective in preclinical models of TNBC, where they eliminate CSCs resistant to chemotherapy. However, the success of GSIs in clinical trials is limited by their intestinal toxicity and adverse immunological effects. CD4 and CD8 T-cells, necessary to adaptive tumor immunity, require Notch1 for activation. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects. We identified Sulindac Sulfide (SS), the active metabolite of FDA-approved NSAID Sulindac, as a potential candidate to replace GSI. SS has Gamma Secretase Modifier (GSM) activity. We confirmed that SS inhibits Notch1 cleavage in TNBC cells. SS significantly inhibited mammosphere growth in all human and murine TNBC models we tested: 1) human MDA-MB-231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe (LFng-/-); and 3) Two TNBC patient-derived xenograft models, 2K1 and 4IC. In contrast, SS did not inhibit Notch expression or cleavage in murine T cells. In C0321 tumors, which recapitulate human mesenchymal TNBC, we found that SS had remarkable single-agent anti-tumor activity and virtually eliminated Notch1 expression in tumors. SS caused an increase in intra-tumoral CD11c+ dendritic cells, but decreased CD4 cells, which in this model are largely PD-1 positive (exhausted). CD8 cells were modestly increased. SS did not affect the number of tumor infiltrating macrophages or myeloid-derived suppressor cells (MDSC). However, SS blocked the immunosuppressive function of bone marrow-derived MDSC. We are currently investigating the mechanisms of this anti-tumor activity. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy agents. Repurposing an FDA-approved, safe agent for the treatment of TNBC would be significantly easier and more cost-effective than developing unproven investigational agents. Citation Format: Hossain F, Ucar D, Majumder S, Xu K, Ran Y, Minter L, Xi Y, Burow M, Golde T, Osborne B, Miele L. Repurposing sulindac sulfide as a notch inhibitor to target cancer stem-like cells in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-22-01.

Adenosine A2A Receptor Stimulation Inhibits TCR-Induced Notch1 Activation in CD8+T-Cells.

Notch receptors signaling is required for optimal T-cell activation and function. T-cell receptor (TCR) engagement can activate Notch receptors in T-cells in a ligand-independent fashion. In this study, we examined the role of adenosine A2A receptor (A2AR) signaling pathway in regulating the activity of Notch1 induced by TCR stimulation in CD8+T-cells. A selective A2AR agonist decreased Notch1 protein expression and Notch1 cleavage, and reduced transcripts of Notch1-target genes Hes1 and Myc in activated CD8+T-cells. Inhibition of TCR-induced Notch1 expression by an A2AR agonist was accompanied by increased cAMP concentration and mimicked by forskolin. This effect was associated with reduced IFN-γ and granzyme B production. The effect of an A2AR agonist was abrogated by a selective A2AR antagonist and absent in CD8+T-cells harvested from A2AR−/− mice. Stimulation of A2AR reduced Notch1 receptor levels by inhibiting upstream TCR signals, including ZAP70 phosphorylation, in turn impairing the generation of the active Notch1 intracellular domain (N1ICD). Direct activation of PKC with PMA and ionomycin bypassed A2AR-induced Notch1 inhibition. Overexpression of N1ICD in CD8+T-cells prevented the suppressive effects of an A2AR agonist on proliferation and cytokine release during activation. Our results identify the A2AR signaling pathway as an important regulator of TCR-induced Notch1 receptor activation in CD8+T-cells, and Notch as an important target of the immune suppressive effects of A2AR. We propose a mechanism whereby A2AR impairs CD8 T-cells function through inhibition of Notch1 receptor activation.

γ‐secretase inhibitor DAPT mitigates cisplatin‐induced acute kidney injury by suppressing Notch1 signaling

Organ toxicity, including kidney injury, limits the use of cisplatin for the treatment of multiple human cancers. Hence, interventions to alleviate cisplatin‐induced nephropathy are of benefit to cancer patients. Recent studies have demonstrated that pharmacological inhibition of the Notch signaling pathway enhances cisplatin efficacy against several cancer cells. However, whether augmentation of the anti‐cancer effect of cisplatin by Notch inhibition comes at the cost of increased kidney injury is unclear. We show here that treatment of mice with cisplatin resulted in a significant increase in Notch ligand Delta‐like 1 (Dll1) and Notch1 intracellular domain (N1ICD) protein expression levels in the kidneys. N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT), a γ‐secretase inhibitor reversed cisplatin‐induced increase in renal N1ICD expression and plasma or urinary levels of predictive biomarkers of acute kidney injury (AKI). DAPT also mitigated cisplatin‐induced tubular injury and reduction in glomerular filtration rate. Real‐time multiphoton microscopy revealed marked necrosis and peritubular vascular dysfunction in the kidneys of cisplatin‐treated mice which were abrogated by DAPT. Cisplatin‐induced Dll1/Notch1 signaling was recapitulated in a human proximal tubule epithelial cell line (HK‐2). siRNA‐mediated Dll1 knockdown and DAPT attenuated cisplatin‐induced Notch1 cleavage and cytotoxicity in HK‐2 cells. These data suggest that Dll1‐mediated Notch1 signaling contributes to cisplatin‐induced AKI. Hence, the Notch signaling pathway could be a potential therapeutic target to alleviate renal complications associated with cisplatin chemotherapy.

A presenilin-1 mutation causes Alzheimer disease without affecting Notch signaling.

Presenilin-1 (PSEN1) is the catalytic subunit of the γ-secretase complex, and pathogenic mutations in the PSEN1 gene account for the majority cases of familial AD (FAD). FAD-associated mutant PSEN1 proteins have been shown to affect APP processing and Aβ generation and inhibit Notch1 cleavage and Notch signaling. In this report, we found that a PSEN1 mutation (S169del) altered APP processing and Aβ generation, and promoted neuritic plaque formation as well as learning and memory deficits in AD model mice. However, this mutation did not affect Notch1 cleavage and Notch signaling in vitro and in vivo. Taken together, we demonstrated that PSEN1S169del has distinct effects on APP processing and Notch1 cleavage, suggesting that Notch signaling may not be critical for AD pathogenesis and serine169 could be a critical site as a potential target for the development of novel γ-secretase modulators without affecting Notch1 cleavage to treat AD.

Bexarotene Binds to the Amyloid Precursor Protein Transmembrane Domain, Alters Its α-Helical Conformation, and Inhibits γ-Secretase Nonselectively in Liposomes.

Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-β (Aβ)-lowering drug for the treatment of Alzheimer's disease (AD). It acts by upregulation of an apolipoprotein E (apoE)-mediated Aβ clearance mechanism. However, whether bexarotene induces removal of Aβ plaques in mouse models of AD has been controversial. Here, we show by NMR and CD spectroscopy that bexarotene directly interacts with and stabilizes the transmembrane domain α-helix of the amyloid precursor protein (APP) in a region where cholesterol binds. This effect is not mediated by changes in membrane lipid packing, as bexarotene does not share with cholesterol the property of inducing phospholipid condensation. Bexarotene inhibited the intramembrane cleavage by γ-secretase of the APP C-terminal fragment C99 to release Aβ in cell-free assays of the reconstituted enzyme in liposomes, but not in cells, and only at very high micromolar concentrations. Surprisingly, in vitro, bexarotene also inhibited the cleavage of Notch1, another major γ-secretase substrate, demonstrating that its inhibition of γ-secretase is not substrate specific and not mediated by acting via the cholesterol binding site of C99. Our data suggest that bexarotene is a pleiotropic molecule that interfere with Aβ metabolism through multiple mechanisms.

Endothelial Cell Caveolin‐1 Depletion Modulates TGF and Notch Signaling Associated with Pulmonary Arterial Hypertension

Reduced expression of caveolin‐1 (Cav1) in endothelial cells (ECs) is associated with pulmonary vascular remodeling. Pulmonary vascular pathology in global Cav1−/− mice is prevented when eNOS is also deleted. Thus, we hypothesized that eNOS‐derived oxidants in absence of Cav‐1 result in dysfunctional ECs and pathological vascular remodeling. Hypoxia exposure following VEGFRII inhibition in Sprague‐Dawley rats produced a significant reduction in pulmonary Cav‐1 expression. Moreover, C57BL6 WT (Tie2.Cre−;Cav1lox/lox) mice exposed for 1 month to hypoxia exhibited an increase in the right ventricular systolic pressure (RVSP) from 25.33 to 31.68 ± 1.75 mmHg which was even higher in EC‐Cav1−/− (Tie2.Cre+;Cav1lox/lox) mice (27.95 to *38.48 ± 1.76 mmHg) when compared to normoxia. The increase in pulmonary arterial pressure induced by hypoxia was associated mild cardiac hypertrophy, increased plasma TGF‐β levels, and increased microvascular area/thickness indicative of vascular remodeling. Interestingly, under normoxic conditions, we observed reduced VEGF level in the lungs of EC‐Cav1−/− mice (8998 ± 1062 and 4927 ± 505.4 pg/mL, n=3–7 mice) suggesting Cav‐1 expression may be required to maintain normal VEGF signaling. Since VEGFRII inhibition may promote EC injury, we hypothesized that in the absence of endothelial Cav‐1, a defect in VEGF‐dependent vascular repair after injury may be the mechanism underlying pathological vascular remodeling. Then, mouse lung ECs (MLECs) isolated from WT, Cav1−/−, and Cav1−/−;eNOS−/− mice were used to assess gene and protein expression, and to quantify indices of angiogenesis. We have observed a defect in tube formation in Cav1−/− MLECs which was abo‐lished with genetic ablation of eNOS−/−. Moreover, in support of these findings, we observed increased Notch‐2 and ‐3 gene expression, and reduced Notch‐1 cleavage in Cav1−/− but not Cav1−/−;eNOS−/− MLECs. Finally, our data suggest the absence of endothelial Cav‐1 may promote pathological vascular remodeling by modulating TGF‐β and Notch signaling.Support or Funding InformationCNPq Fellowship‐CsF/Brazil; HL125356; DOD W911NF‐15‐R‐002.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SUMOylation Negatively Regulates Angiogenesis by Targeting Endothelial NOTCH Signaling.

The highly conserved NOTCH (neurogenic locus notch homolog protein) signaling pathway functions as a key cell-cell interaction mechanism controlling cell fate and tissue patterning, whereas its dysregulation is implicated in a variety of developmental disorders and cancers. The pivotal role of endothelial NOTCH in regulation of angiogenesis is widely appreciated; however, little is known about what controls its signal transduction. Our previous study indicated the potential role of post-translational SUMO (small ubiquitin-like modifier) modification (SUMOylation) in vascular disorders. The aim of this study was to investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. Endothelial SENP1 (sentrin-specific protease 1) deletion, in newly generated endothelial SENP1 (the major protease of the SUMO system)-deficient mice, significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling, as confirmed in cultured endothelial cells. An in vitro SUMOylation assay and immunoprecipitation revealed that when SENP1 associated with N1ICD (NOTCH1 intracellular domain), it functions as a deSUMOylase of N1ICD SUMOylation on conserved lysines. Immunoblot and immunoprecipitation analyses and dual-luciferase assays of natural and SUMO-conjugated/nonconjugated NOTCH1 forms demonstrated that SUMO conjugation facilitated NOTCH1 cleavage. This released N1ICD from the membrane and stabilized it for translocation to the nucleus where it functions as a cotranscriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 (Delta-like 4) stimulation. This in turn suppressed VEGF (vascular endothelial growth factor) receptor signaling and angiogenesis, as evidenced by immunoblotted signaling molecules and in vitro angiogenesis assays. These results establish reversible NOTCH1 SUMOylation as a regulatory mechanism in coordinating endothelial angiogenic signaling; SENP1 acts as a critical intrinsic mediator of this process. These findings may apply to NOTCH-regulated biological events in nonvascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.

Abstract 791: Inhibitory effects of theaflavin-3, 3′-digallate (TF3) on ovarian cancer cells

Abstract Theaflavin-3, 3'-digallate (TF3) is a black tea polyphenol produced from polymerization and oxidization of green tea ployphenols epicatechin gallate and (−)-epigallocatechin-3-gallate (EGCG) during fermentation of fresh tea leaves. TF3 has been reported to have anti-cancer properties. However, the effect of TF3 on tumor angiogenesis and the underlying mechanisms are not clear. In the current research, TF3 was verified to inhibit tumor angiogenesis. Compared with EGCG, TF3 was more potent. TF3 inhibited human ovarian carcinoma OVCAR-3 cells induced angiogenesis in human umbilical vein endothelial cell model and in chick chorioallantoic membrane model. TF3 reduced tumor angiogenesis by down-regulating HIF-1α and VEGF. One of the mechanisms was TF3 inactivated Akt/ mTOR/ p70S6K/ 4E-BP1 pathway and Akt/ c-Myc pathway. Besides, TF3 suppressed the cleavage of Notch-1, subsequently decreased the expression of c-Myc, HIF-1α and VEGF, and finally impaired cancer cells induced angiogenesis. Nevertheless, TF3 didn’t have any influence on MAPK pathways. Taken together, these findings suggest that TF3 might serve as a potential anti-angiogenic agent for cancer treatment. Citation Format: Yi C. Chen, Gary O. Rankin. Inhibitory effects of theaflavin-3, 3′-digallate (TF3) on ovarian cancer cells [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 791. doi:10.1158/1538-7445.AM2017-791

γ‐Secretase inhibitors in cancer clinical trials are pharmacologically and functionally distinct

γ‐Secretase inhibitors (GSIs) are being actively repurposed as cancer therapeutics based on the premise that inhibition of NOTCH1 signaling in select cancers is therapeutic. Using novel assays to probe effects of GSIs against a broader panel of substrates, we demonstrate that clinical GSIs are pharmacologically distinct. GSIs show differential profiles of inhibition of the various NOTCH substrates, with some enhancing cleavage of other NOTCH substrates at concentrations where NOTCH1 cleavage is inhibited. Several GSIs are also potent inhibitors of select signal peptide peptidase (SPP/SPPL) family members. Extending these findings to mammosphere inhibition assays in triple‐negative breast cancer lines, we establish that these GSIs have different functional effects. We also demonstrate that the processive γ‐secretase cleavage pattern established for amyloid precursor protein (APP) occurs in multiple substrates and that potentiation of γ‐secretase cleavage is attributable to a direct action of low concentrations of GSIs on γ‐secretase. Such data definitively demonstrate that the clinical GSIs are not biological equivalents, and provide an important framework to evaluate results from ongoing and completed human trials with these compounds.

A Presenilin/Notch1 pathway regulated by miR-375, miR-30a, and miR-34a mediates glucotoxicity induced-pancreatic beta cell apoptosis.

The presenilin-mediated Notch1 cleavage pathway plays a critical role in controlling pancreatic beta cell fate and survival. The aim of the present study was to investigate the role of Notch1 activation in glucotoxicity-induced beta cell impairment and the contributions of miR-375, miR-30a, and miR-34a to this pathway. We found that the protein levels of presenilins (PSEN1 and PSEN2), and NOTCH1 were decreased in INS-1 cells after treatment with increased concentrations of glucose, whereas no significant alteration of mRNA level of Notch1 was observed. Targeting of miR-375, miR-30a, and miR-34a to the 3′utr of Psen1, Psen2, and Notch1, respectively, reduced the amounts of relevant proteins, thereby reducing NICD1 amounts and causing beta cell apoptosis. Overexpression of NICD1 blocked the effects of glucotoxicity as well as miRNA overabundance. Downregulating the expression of miR-375, miR-30a, and miR-34a restored PSEN1, PSEN2, and NICD1 production and prevented glucotoxicity-induced impairment of the beta cells. These patterns of miRNA regulation of the Notch1 cleavage pathway were reproduced in GK rats as well as in aged rats. Our findings demonstrated that miRNA-mediated suppression of NICD1 links the presenilin/Notch1 pathway to glucotoxicity in mature pancreatic beta cells.

Sensitizing effect of juglone is mediated by down regulation of Notch1 signaling pathway in trastuzumab-resistant SKBR3 cells.

Trastuzumab (Herceptin) monoclonal antibody directed against HER2 receptor has been administered as a treatment for metastatic HER2 positive breast cancer. The problematic issue in treatment of HER2 positive breast cancer cells is commonly the induction of resistance to trastuzumab which might be due to modulation of some vital signaling elements such as Notch1 and Pin1. In this study, we were aimed to investigate whether the cross talk between pin1 and Notch1 has a role in this event. Our results indicated that the expression level of Pin1 in resistant SKBR3 cells increased by about twofold relative to sensitive SKBR3 cells. Besides, Pin1 inhibition via juglone reduced the extent of proliferation, colony formation and migration capacity of resistant SKBR3 cells. In addition, despite a feed forward loop between Notch1 and Pin1 in sensitive SKBR3 cells, inhibition of Notch1 cleavage in resistant SKBR3 cells did not affect pin1 level whereas pin1 inhibition by juglone reduced the level of Hes1, p-Akt and increased the cellular content of Numb. Therefore, we concluded that pin1 inhibition could be considered as a promising sensitizing strategy to weaken trastuzumab resistance.

Valproic acid exhibits different cell growth arrest effect in three HPV-positive/negative cervical cancer cells and possibly via inducing Notch1 cleavage and E6 downregulation.

We investigated the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, and the mechanism of VPA-induced growth inhibition on three cervical cancer cell lines with different molecular and genetic background. We found that VPA induced proliferation suppression, cell apoptosis and cell cycle arrest in all tested cell lines, with an increase of Notch1 active form ICN1 as a tumor suppressor and its target gene HES1. Noteworthy, blocking of Notch signaling with DAPT resulted in growth inhibition in ICN1-overexpressing CaSki and HT-3 cells. Thus, endogenous Notch signaling may be necessary for survival of ICN1-overexpressing cervical cancer cell lines. Furthermore, G1 phase arrest was induced in HeLa and CaSki cells by VPA while G2 phase arrest was induced in HT-3 cells, suggesting different mechanism in this cycle arrest. We also found VPA suppressed oncogene E6 in a Notch-independent manner, and induced significant apoptosis in E6-overexpressing HPV positive CaSki cells. Cell morphological change was also observed in HeLa and HT-3 cell lines after VPA treatment with an upregulation of EMT transcription factor Snail1. Notch signaling inhibitor DAPT partly reversed VPA-induced Snail1 upregulation in HeLa cells. This discovery supports that VPA may induce EMT at least partly via Notch activation.

Hepatitis B Virus HBx Activates Notch Signaling via Delta-Like 4/Notch1 in Hepatocellular Carcinoma.

Hepatitis virus B (HBV) infection is one of the major causes of hepatocellular carcinomas (HCC). HBx protein encoded in HBV genome is one of the key viral factors leading to malignant transformation of infected cells. HBx functions by interfering with cellular functions, causing aberration in cellular behaviour and transformation. Notch signalling is a well-conserved pathway involved in cellular differentiation, cell survival and cell death operating in various types of cells. Aberration in the Notch signalling pathways is linked to various tumors, including HCC. The role of HBx on the Notch signalling in HCC, however, is still controversial. In this study, we reported that HBV genome-containing HCC cell line HepG2 (HepG2.2.15) expressed higher Notch1 and Delta-like 4 (Dll4), compared to the control HepG2 without HBV genome. This upregulation coincided with increased appearance of the cleavage of Notch1, indicating constitutively activated Notch signalling. Silencing of HBx specifically reduced the level of Dll4 and cleaved Notch1. The increase in Dll4 level was confirmed in clinical specimens of HCC lesion, in comparison with non-tumor lesions. Using specific signalling pathway inhibitors, we found that MEK1/2, PI3K/AKT and NF-κB pathways are critical for HBx-mediated Dll4 upregulation. Silencing of HBx clearly decreased the level of phosphorylation of Akt and Erk1/2. Upon silencing of Dll4 in HepG2.2.15, decreased cleaved Notch1, increased apoptosis and cell cycle arrest were observed, suggesting a critical role of HBx-Dll4-Notch1 axis in regulating cell survival in HCC. Furthermore, clonogenic assay confirmed the important role of Dll4 in regulating cell survival of HBV-genome containing HCC cell line. Taken together, we reported a link between HBx and the Notch signalling in HCC that affects cell survival of HCC, which can be a potential target for therapy.

Theaflavin-3, 3'-digallate decreases human ovarian carcinoma OVCAR-3 cell-induced angiogenesis via Akt and Notch-1 pathways, not via MAPK pathways.

Theaflavin-3, 3′-digallate (TF3) is a black tea poly-phenol produced from polymerization and oxidization of the green tea ployphenols epicatechin gallate and (−)-epigallocatechin-3-gallate (EGCG) during fermentation of fresh tea leaves. TF3 has been reported to have anticancer properties. However, the effect of TF3 on tumor angiogenesis and the underlying mechanisms are not clear. In the present study, TF3 was verified to inhibit tumor angiogenesis. Compared with EGCG, TF3 was more potent. TF3 inhibited human ovarian carcinoma OVCAR-3 cell-induced angiogenesis in human umbilical vein endothelial cell model and in chick chorioallantoic membrane model. TF3 reduced tumor angiogenesis by downregulating HIF-1α and VEGF. One of the mechanisms was TF3 inactivated Akt/mTOR/p70S6K/4E-BP1 pathway and Akt/c-Myc pathway. Besides, TF3 suppressed the cleavage of Notch-1, subsequently decreased the expression of c-Myc, HIF-1α and VEGF, and finally the impaired cancer cells induced angiogenesis. Nevertheless, TF3 did not have any influence on the MAPK pathways. Taken together, these findings suggest that TF3 might serve as a potential anti-angiogenic agent for cancer treatment.

Notch1 Autoactivation via Transcriptional Regulation of Furin, Which Sustains Notch1 Signaling by Processing Notch1-Activating Proteases ADAM10 and Membrane Type 1 Matrix Metalloproteinase.

Notch1 is an evolutionarily conserved transmembrane receptor involved in melanoma growth. Notch1 is first cleaved by furin in the Golgi apparatus to produce the biologically active heterodimer. Following ligand binding, Notch1 is cleaved at the cell membrane by proteases such as ADAM10 and -17 and membrane type 1 matrix metalloproteinase (MT1-MMP), the latter of which we recently identified as a novel protease involved in Notch1 processing. The final cleavage is γ-secretase dependent and releases the active Notch intracellular domain (NIC). We now demonstrate that Notch1 directly regulates furin expression. Aside from activating Notch1, furin cleaves and activates several proteases, including MT1-MMP, ADAM10, and ADAM17. By chromatin immunoprecipitation and a reporter assay, we demonstrate that Notch1 binds at position -1236 of the furin promoter and drives furin expression. The Notch1-dependent enhancement of furin expression increases the activities of MT1-MMP and ADAM10 but not that of ADAM17, as demonstrated by short hairpin RNA (shRNA) knockdown of furin, and promotes the cleavage of Notch1 itself. These data highlight a novel positive-feedback loop whereby Notch1-dependent furin expression can induce Notch1 signaling by increasing Notch1 processing and by potentiating the activity of the proteases responsible for Notch1 activation. This leads to Notch1 signal amplification, which can promote melanoma tumor growth and progression, as demonstrated by the inhibition of cell migration and invasion upon furin inhibition downstream of Notch1. Disruption of such feedback signaling might represent an avenue for the treatment of melanoma.