Notch Proteins Research Articles

C-fos, ERK1/2, MAP2, NOTCH1 Proteins Expression Patterns in Human Cerebral Cortex Neurons after Ischemic Stroke

Background. The search for protein (these include c-fos, ERK1/2, MAP2, NOTCH1) expression that provide neuroplasticity mechanisms of the cerebral cortex after ischemic stroke (IS) patterns is an urgent task.
 Aims to reveal c-fos, ERK1/2, MAP2, NOTCH1 proteins expression patterns in human cerebral cortex neurons after IS.
 Materials and methods. We studied 9 left middle cerebral artery (LMCA) IS patients cerebral cortex samples from 3 zones: 1 the zone adjacent to the necrotic tissue focus; 2 zone remote from the previous one by 47 cm; 3 zone of the contralateral hemisphere, symmetric to the IS focus. Control samples were obtained from 3 accident died people. Identification of targeted proteins NSE, c-fos, ERK1/2, MAP2, NOTCH1 was performed by indirect immunoperoxidase immunohistochemical method.
 Results. Moving away from the ischemic focus, there is an increase in the density of neurons and a decrease in the damaged neurons proportion, the largest share of c-fos protein positive neurons in zone 2, NOTCH1 positive neurons in zone 1, smaller fractions of ERK1/2 and MAP2 positive neurons compared to the control only in samples of zone 1.
 Conclusions. With the IS development, the contralateral hemisphere is intact tissue increased activation zone, while the zones 1 and 2 have pathological activation signs. In zone 1 of the range, the adaptive response of the tissue decreases, and in zone 2 it expands. Therefore, a key target for therapeutic intervention is zone 2.

Membrane dynamics of γ-secretase with the anterior pharynx-defective 1B subunit.

The four-subunit protease complex γ-secretase cleaves many single-pass transmembrane (TM) substrates, including Notch and β-amyloid precursor protein to generate amyloid-β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx-defective 1 (APH-1) and presenilin (PS) exist in two homologous forms APH1-A and APH1-B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ-secretase medicine. Here, we developed the first complete structural model of the APH-1B subunit using the published cryo-electron microscopy (cryo-EM) structures of APH1-A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all-atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH-1B alone and in γ-secretase without and with substrate C83-bound. We show that APH-1B adopts a 7TM topology with a water channel topology similar to APH-1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo-EM structures with APH-1A, however with subtle differences: The substrate-bound APH-1B γ-secretase was quite stable, but some TM helices of PS1 and APH-1B rearranged in the membrane consistent with the disorder seen in the cryo-EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH-1B, that is, it represents a more closed state, due to interactions with the C-terminal fragment of PS1. Our structural-dynamic model of APH-1B alone and in γ-secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.

Abstract 5218: Inhibition of γ-secretase decreases Notch signaling and cell migration in uterine carcinosarcoma

Abstract Carcinosarcoma (CS) is a rare and aggressive type II endometrial adenocarcinoma (EmCa) accounting for less than 5% of all uterine malignancies, but accounting for a disproportionately high disease-specific mortality rate. Notch signaling is an evolutionarily conserved pathway with oncogenic roles in several cancers. Notch proteins are expressed in Type I and Type II EmCa, cell lines, and human tissue, but Notch function has not been characterized in CS. To determine whether inhibiting Notch signaling may be a therapeutic target in CS we assessed expression of Notch family genes and the impact of γ-secretase inhibition on cellular migration in the CS cell line, CS-99, and compared to lower risk Type I EmCa cell line (HEC-1-A). Expression of Notch receptors, ligands and effectors was determined using RT-PCR. We determined the impact of exposure to DAPT, a γ-secretase inhibitor, vs DMSO (vehicle control) on cell viability with a MTT assay. The ability of DAPT to inhibit Notch signaling was assessed by quantitative (q) RT-PCR for the Notch effector, HES1. The relative expression level of each target gene was normalized to 18s rRNA. Cellular migration after exposure to DAPT was assessed using a scratch assay for CS-99 and HEC-1-A. The extent of cell migration was measured using ImageJ (NIH Bethesda, MD). CS-99 and HEC-1-A, expressed receptors NOTCH1-4, ligands DLL4, JAG1, JAG2, and effectors HES1, HEY1, and NRARP. The MTT assay revealed >88% cellular viability after 24 hr exposure to 10µM in CS-99 and 50µM DAPT in HEC-1-A. Exposure to DAPT significantly decreased expression HES1 in CS-99 and HEC-1-A. Migration was decreased in CS-99 and unchanged in HEC-1-A with 10µM and 50 µM of DAPT, respectively. Inhibition of Notch signaling activity decreased cellular migration in CS-99 cell line suggesting that Notch signaling may impact disease growth in CS. Unchanged HEC-1-A cellular migration suggests that Notch signaling might not impact disease growth in Type I EmCa. Inhibition of Notch signaling may be a promising therapeutic target in CS. Inhibition of Notch signaling in CS-99 and HEC-1-A with a γ-secretase inhibitor (median + IQR)*HEC-1-A + DMSOHEC-1-A + DAPT 50μMP-valueCS-99 + DMSOCS-99 + DAPT 10μMP-valueHES1 (normalized fold change)0.94 (0.83-1.3)0.35 (0.25-0.41)0.0220.99 (0.84-1.2)0.19 (0.16-0.23)0.002% wound closure (0-6h)12.5 (8.5-15.5)24.1 (10.5-33.0)0.1311.4 (9.7-11.5)11.3 (9.1-11.4)0.7% wound closure (0-24h)38.1 (31.4-46.1)63.0 (33.6-100.0)0.2473.3 (66.6-73.9)61.7 (52.6-64.7)0.05 Citation Format: Sofia D. Gabrilovich, Yasmin Abedin, Sreevidya Santha, Tracy Wu, Mark Einstein, Nataki C. Douglas, Jenna Z. Marcus. Inhibition of γ-secretase decreases Notch signaling and cell migration in uterine carcinosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5218.

Abstract LB-047: CHMP5 dependency is a therapeutic vulnerability of T cell acute lymphoblastic leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a very aggressive hematologic malignancy that results from the transformation of immature T-cell progenitors. Aberrant cell growth and proliferation is sustained by strong oncogenic drivers, notably NOTCH1 and MYC, whose activation is present in more than half of all T-ALL cases. However, although it is known that post-translational modifications, such as ubiquitination by the E3-ligase FBXW7, regulate NOTCH1 and MYC protein stability and activity, regulatory proteins involved in their stability remain unestablished. The Charged multivesicular body protein 5 (CHMP5), an accessory protein of the ESCRT (endosomal sorting complex required for transport) pathway, has been shown to regulate the stability of client proteins in thymocytes through the recruitment of de-ubiquitinating enzymes. Here, we report that CHMP5 protein is not only highly expressed in T-ALL cells, but also promotes the stability of transactivating intracellular NOTCH1 (ICN1) and MYC. We found that depletion of CHMP5 significantly reduced the abundance of ICN1 and MYC, and the transcription of their target genes in vitro, as well as suppressed the development of T-ALL in vivo in a murine model of NOTCH1 driven T-ALL. Additionally, loss of CHMP5 restored T-ALL cell susceptibility to chemotherapeutic agents, including glucocorticoids and gamma secretase inhibitors, resistance to which contributes to disease relapse in patients. We further reveal CHMP5 as an adaptor protein required to orchestrate T-ALL client protein deubiquitination and stability. Altogether, our data suggest that CHMP5 is a non-oncogenic dependency of T-ALL and its depletion may be an effective therapeutic strategy for T-ALL. Citation Format: Katharine Umphred-Wilson, Stanley Adoro. CHMP5 dependency is a therapeutic vulnerability of T cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-047.

Abstract 6405: Fosciclopirox suppresses growth of high-grade urothelial cancer by targeting Notch signaling

Abstract Ciclopirox (CPX) is a FDA-approved topical antifungal agent that has demonstrated preclinical anticancer activity in solid and hematologic malignancies. It's clinical utility as an anticancer agent, however, is limited by poor oral bioavailability, gastrointestinal toxicity, and poor water solubility. Fosciclopirox, the phosphoryloxymethyl ester of CPX (Ciclopirox Prodrug, CPX-POM), is rapidly and completely metabolized to CPX, the active metabolite, which subsequently undergoes renal elimination resulting in urine concentrations of CPX that exceed in vitro IC50's several-fold. We characterized the activity of CPX-POM and its major metabolites in vitro utilizing authenticated human T24, HT-1376, and UM-UC-3 high-grade urothelial cancer cell lines. CPX inhibited cell proliferation, clonogenicity, and spheroid formation, and increased cell cycle arrest at S and G0/G1 phases. Mechanistically, CPX suppressed activation of Notch signaling, which was partially rescued by ectopic expression of the intracellular domain of Notch1. Molecular modeling and cellular thermal shift assays demonstrated CPX binding to γ-secretase complex proteins Presenilin1 and Nicastrin, which are essential for Notch activation. Interrogation of The Cancer Genome Atlas (TCGA) database demonstrated that both proteins were upregulated in bladder tumor tissue, and that higher levels of Presenilin1 and Nicastrin were significantly associated with lower overall survival in muscle invasive bladder cancer (MIBC) patients. To establish in vivo preclinical proof of principle, we tested fosciclopirox in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) mouse bladder cancer model in two separate studies. Intraperitoneal (IP) administration of CPX-POM once daily for four weeks at doses ranging from 25 to 200 mg/kg significantly decreased bladder weight and resulted in a migration to lower stage tumors in CPX-POM treated animals compared to untreated animals. This was coupled with a reduction in proliferation index, as well as reductions in Presenilin1 and Hey1 expression in bladder tumor tissues in CPX-POM treated animals. A similar anti-tumor response was observed following once daily versus three times weekly IP CPX-POM in this chemical carcinogen mouse model of bladder cancer. The safety, dose tolerance, pharmacokinetics and pharmacodynamics of intravenous (IV) CPX-POM were characterized in a US multi-center, First-in-Human, Phase 1, open-label, dose escalation study (NCT03348514). Eight cohorts of 19 patients received IV CPX-POM doses ranging from 30 to 1200 mg/m2 for as many as six 21-day treatment cycles. Adequate systemic and urinary tract CPX exposures were achieved at the maximum tolerated dose of 900 mg/m2 with evidence of Notch inhibition. An expansion cohort study in 12 cisplatin-ineligible MIBC patients receiving two treatment cycles of CPX-POM prior to radical cystectomy (RC) is underway. Evidence of pharmacologic activity is being characterized in bladder tumor tissues obtained at RC. Citation Format: Scott James Weir, Prasad Dandawate, Prabhu Ramamoorthy, Parthasarathy Ranjarajan, Robyn Wood, Amanda Brinker, Benjamin Woolbright, Mehmet Tanol, Tammy Ham, William McCulloch, Michael Dalton, Michael J. Baltezor, Roy A. Jensen, John A. Taylor, Shrikant Anant. Fosciclopirox suppresses growth of high-grade urothelial cancer by targeting Notch signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6405.

Abstract 5858: Multiplatform analyses identify upregulated hematopoietic cell kinase activity in poorly prognostic high-grade serous ovarian cancer and its role in regulating tumor aggressiveness

Abstract High-grade serous ovarian cancer (HGSOC) is the most predominant and lethal subtype of ovarian cancer. While most patients initially respond to standard-of-care treatment, the majority will eventually relapse. Despite improved treatment strategies, the prognosis has not improved significantly and the overall 5-year survival rate remains ~31%. To understand the underlying mechanisms that govern the distinctions between tumors with good and poor prognoses, we implemented a proteogenomic approach to analyze gene expression, proteomic and phosphoproteomic profiles of HGSOC tumors available in the TCGA and the CPTAC studies. Our analyses identified upregulated hematopoietic cell kinase (HCK) signaling along with innate immunity related and ERBB activity in poor prognostic tumors. These results were confirmed using independent datasets to demonstrate HCK up-regulation in tumor samples compared to borderline tumors and/or normal samples. Further analyses of micro-dissected HGSOC tumor tissue determined that HCK was specifically upregulated in tumor epithelial cells. Importantly, differential analysis of CPTAC phospho-proteomic data identified significant enrichment of phosphorylated HCK in the primary tumors of patients with tumor recurrence within 1 year compared to tumor-free patients. Interestingly, in-silico analyses suggest that HCK may play a dichotomous tumorigenic role as HCK was strongly associated with pro-survival signaling pathways as well as enrichment of pro-tumorigenic immune signaling. Consistent with these results, in vitro studies in NIH-OVCAR3 and CAOV3 cell lines with high endogenous HCK expression demonstrated that HCK silencing decreased cell proliferation, invasion, and colony formation. Conversely, HCK overexpression significantly induced proliferation and clonogenic capacity in OVSAHO cells (low HCK). These results suggest that HCK is essential for HGSOC proliferation and survival. Reverse phase protein array (RPPA) analysis was next used to identify potential mechanisms by which HCK modulates HGSOC tumorigenesis. Our analyses identified a number of candidate proteins that are significantly downregulated following shRNA-mediated silencing of HCK including PD-L1, CD44, and NOTCH3. Further in silico analyses demonstrated significant enrichment of PD-L1 protein expression in HGSOC tumors with high HCK activity suggesting a potential role for HCK in regulating the tumor microenvironment. These latter analyses also identified a significant correlation between HCK and CD44 mRNA and protein expression in human tumors. Importantly, experimental studies in CAOV3 cells confirmed that shRNA silencing of HCK inhibited PD-L1, CD44 and NOTCH3 protein expression; these results were confirmed by lentiviral overexpression of HCK which resulted in increased CD44 and NOTCH3 expression in OVSAHO cells. Given that CD44 and NOTCH3 signaling have been shown to mediate tumor cell proliferation and survival, our data, which indicate that CD44 and NOTCH3 expression are regulated by HCK activity, suggest that these genes may contribute to HCK mediated HGSOC tumorigenesis. Our data collectively implicate HCK as a novel driver of HGSOC development and progression and may represent a novel therapeutic opportunity in this subset of patients. Citation Format: Christen A. Khella, Michael L. Gatza. Multiplatform analyses identify upregulated hematopoietic cell kinase activity in poorly prognostic high-grade serous ovarian cancer and its role in regulating tumor aggressiveness [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5858.

Overexpression of AMPD2 indicates poor prognosis in colorectal cancer patients via the Notch3 signaling pathway.

BACKGROUNDAMPD2 is a critical enzyme catalyzing smooth muscle energy supply and metabolism; however, its cellular biological function and clinical implication in colorectal cancer (CRC) are largely unknown.AIMTo clarify the role of AMPD2 in CRC and study the pathway and prognostic value of its role.METHODSAMPD2 expression was analyzed by integrated bioinformatics analysis based on TCGA data sets and immunohistochemistry in tissue microarrays, and the correlation between AMPD2 expression and clinicopathological parameters, Notch3 expression, and prognostic features was assessed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were then performed to investigate the regulatory pathway involved. The effects of AMPD2 expression on CRC cells and Notch3 protein expression were investigated by downregulation and overexpression of AMPD2.RESULTSAMPD2 mRNA was significantly overexpressed in tumor tissue when compared with normal tissue in a cohort of the TCGA-COAD data set. Biological function enrichment analysis indicated that the Notch pathway strongly correlated with AMPD2 expression, and that the expression of Notch3 and JAG2 mRNA was positively associated with AMPD2 in CRC tissues. In vitro, AMPD2 overexpression markedly reduced Notch3 protein expression in CRC cells, while knockdown of AMPD2 showed the opposite findings. In addition, protein expression was significantly up-regulated in our CRC cohort as indicated by tissue microarray analysis. High expression of AMPD2 protein correlated with advanced depth of tumor and poor differentiation. Furthermore, high AMPD2 expression in CRC tissues was an indicator of poor outcome for CRC patients.CONCLUSIONAMPD2 is commonly overexpressed in CRC, and acts as a metabolism oncogene to induce CRC progression through the Notch signaling pathway. Thus, AMPD2 may be a novel prognostic biomarker for CRC.

NOTCH pathway activation in infantile hemangiomas

ObjectiveThe objective of this study was to characterize the role of NOTCH signaling cascade activation in the angiogenic drive of infantile hemangioma (IH) tissue. MethodsBoth IH tissue and normal skin were collected from 55 surgical patients. Of these, 14 were proliferating hemangiomas, 14 were stationary, 14 were involuted, and 13 had been treated with propranolol. Protein and RNA were extracted from all tissues and subjected to Western blotting and reverse transcription-polymerase chain reaction, respectively, for components of the NOTCH signaling pathway. ResultsCompared with normal skin from the same patients, proliferating IHs contained higher levels of messenger RNA and protein for the majority of NOTCH receptors and ligands as well as the downstream coactivator MAML1. Expression of NOTCH receptor ligand messenger RNA and protein was significantly lower in involuting and propranolol-treated IHs. ConclusionsPerturbations in NOTCH signaling follow the natural course and treatment outcome of IHs, suggesting a critical role in their pathogenesis.

Hydroxysafflor yellow A improves diabetes-induced renal fibrosis

IntroductionThis study aimed to discuss the use of hydroxysafflor yellow A (HSYA) to improve renal fibrosis induced by diabetes and its relative mechanisms, as well as to evaluate the level of renal histopathology and fibrosis in different rat groups.Material and methodsSprague-Dawley (SD) rats (n = 27) were divided into the normal control (NC), diabetes mellitus (DM), and HSYA groups. The miRNA-140-5p mRNA, blood glucose (BG), 24-h urine protein (UP), total cholesterol (TC), triglyceride (TG), total anti-oxidant capacity (T-AOC), malondialdehyde (MDA), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α) were measured in different groups. Moreover, the relative protein expression was measured via immunohistochemistry (IHC) assay. In the in vitro cell experiment, we discussed the effects of miRNA-140-5p on renal fibrosis induced by diabetes. Toll-like receptor 4 (TLR4), nuclear factor B (p65) (NF-κB(p65)), NOD-like receptor protein 3 (NLRP3), Notch2, and collagen IV (Col-IV) protein expression were evaluated using western blotting in the different cell groups. We evaluated the important protein expressions and NF-κB(p65) nuclear volume by cellular immunofluorescence. A correlation between miRNA-140-5p and TLR4 was found by dual-luciferase reporter assay.ResultsIn the in vivo study, HSYA improved diabetes-induced renal fibrosis. The severity of fibrosis significantly decreased after treatment with HSYA. In the HSYA group, the miRNA-140-5p mRNA, BG, 24-h UP, TC, TG, T-AOC, MDA, IL-6, and TNF-α significantly improved, as well as the TLR4, NF-κB(p65), NLRP3, Notch2, and Col-IV proteins. In the in vitro experiment, miRNA-140-5p was significantly decreased after treatment with HSYA in diabetes-induced renal fibrosis.ConclusionsHSYA improved diabetes-induced renal fibrosis by regulating miRNA-140-5p.

Cholesterol and Mevalonate: Two Metabolites Involved in Breast Cancer Progression and Drug Resistance through the ERRα Pathway.

Breast cancer is the second greatest cause of cancer-related death in women. Resistance to endocrine treatments or chemotherapy is a limiting drawback. In this context, this work aims to evaluate the effects of cholesterol and mevalonate during tumor progression and their contribution in the onset of resistance to clinical treatments in use today. In this study, we demonstrated that cholesterol and mevalonate treatments were able to activate the estrogen-related receptor alpha (ERRα) pathway, increasing the expression levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), ERbB2/human epithelial receptor (HER2), tumor protein D52 (TPD52), and NOTCH2 proteins in breast cancer cells. The activation of this pathway is shown to be responsible for intense metabolic switching, higher proliferation rates, sustained motility, the propagation of cancer stem-like cells (CSCs), and lipid droplet formation. All of these events are related to greater tumor propagation, aggressiveness, and drug resistance. Furthermore, the activation and expression of proteins induced by the treatment with cholesterol or mevalonate are consistent with those obtained from the MCF-7/TAMr cell line, which is largely used as a breast cancer model of acquired endocrine therapy resistance. Altogether, our data indicate that cholesterol and mevalonate are two metabolites implicated in breast cancer progression, aggressiveness, and drug resistance, through the activation of the ERRα pathway. Our findings enable us to identify the ERRα receptor as a poor prognostic marker in patients with breast carcinoma, suggesting the correlation between cholesterol/mevalonate and ERRα as a new possible target in breast cancer treatment.

Broad phenotype of cysteine-altering NOTCH3 variants in UK Biobank: CADASIL to nonpenetrance.

ObjectiveTo determine the small vessel disease spectrum associated with cysteine-altering NOTCH3 variants in community-dwelling individuals by analyzing the clinical and neuroimaging features of UK Biobank participants harboring such variants.MethodsThe exome and genome sequencing datasets of the UK Biobank (n = 50,000) and cohorts of cognitively healthy elderly (n = 751) were queried for cysteine-altering NOTCH3 variants. Brain MRIs of individuals harboring such variants were scored according to Standards for Reporting Vascular Changes on Neuroimaging criteria, and clinical information was extracted with ICD-10 codes. Clinical and neuroimaging data were compared to age- and sex-matched UK Biobank controls and clinically diagnosed patients from the Dutch cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) registry.ResultsWe identified 108 individuals harboring a cysteine-altering NOTCH3 variant (2.2 of 1,000), of whom 75% have a variant that has previously been reported in CADASIL pedigrees. Almost all variants were located in 1 of the NOTCH3 protein epidermal growth factor–like repeat domains 7 to 34. White matter hyperintensity lesion load was higher in individuals with NOTCH3 variants than in controls (p = 0.006) but lower than in patients with CADASIL with the same variants (p < 0.001). Almost half of the 24 individuals with brain MRI had a Fazekas score of 0 or 1 up to age 70 years. There was no increased risk of stroke.ConclusionsAlthough community-dwelling individuals harboring a cysteine-altering NOTCH3 variant have a higher small vessel disease MRI burden than controls, almost half have no MRI abnormalities up to age 70 years. This shows that NOTCH3 cysteine altering variants are associated with an extremely broad phenotypic spectrum, ranging from CADASIL to nonpenetrance.

USP28 and USP25 are downregulated by Vismodegib in vitro and in colorectal cancer cell lines.

Deubiquitinase USP28 plays a crucial role in tumorigenesis by enhancing the stabilities of multiple cancer-related proteins including c-Myc, Notch1, and LSD1, and has become an attractive target for anticancer drug development. However, to date, only a few of USP28-targeted active compounds have been developed, and the active compound-binding pocket in USP28 has not been experimentally revealed yet. In this study, bioassay-based high-throughput screening was applied to discover USP28-targeted inhibitors from the commercially available drug library. Vismodegib, an inhibitor of Hedgehog signaling pathway and FDA-approved drug for the treatment of basal cell carcinoma, was found to exhibit inhibition activity against USP28 (IC50 : 4.41±1.08μm). Multiple biophysical and biochemical techniques including NMR, ITC, thermal shift assay, HDX-MS, and site-directed mutagenesis analysis were then used to characterize the interaction between Vismodegib and USP28. The binding pocket in USP28 for Vismodegib, which is mainly composed of two helical structures spanning D255-N278 and N286-Y293, was revealed. According to the possible binding pose generated by HDX-MS data-defined molecular docking, the binding cavity occupied by Vismodegib in USP28 aligns well with one of the reported-binding pockets in USP7 for its inhibitors. Furthermore, cellular assays were conducted to confirm that Vismodegib could interact with the evolutionarily related deubiquitinases USP28 and USP25 and downregulate the levels of the two enzymes' substrate proteins c-Myc, Notch1, and Tankyrase-1/2.

Expression of NANOG and Its Regulation in Oral Squamous Cell Carcinoma.

Background Results of previous studies suggest that NANOG may be an important prognostic biomarker in oral squamous cell carcinoma (OSCC), but there are contradictory results regarding NANOG expression patterns on mRNA and protein levels, and the mechanisms of its regulation are poorly understood. Our aim was to analyze the expression and diagnostic significance of NANOG in OSCC, and the possible mechanisms of its regulation, i.e., protein regulators on mRNA level (OCT4, SOX2, KLF4, AGR2, and NOTCH1), methylation status, copy number variation, and regulatory miRNAs, miR-145, miR-335, miR-150, miR-34a, miR-128, and miR-27a. Methods Our study included 120 patients with OSCC. Expression of NANOG protein and mRNA was analyzed using immunohistochemistry and qPCR. Expression of regulatory factors, miRNAs, and copy number variation was performed using qPCR. Methylation status of NANOG promoter was determined using PCR and Sanger sequencing. Results We detected upregulation of NANOG and OCT4 and downregulation of NOTCH1 and AGR2 mRNA in OSCC with lymph node metastases compared to OSCC without lymph node metastases. We observed a strong positive correlation between mRNAs of NANOG and those of its protein regulators OCT4, SOX2, NOTCH1, AGR2, and KLF4. The expression of NANOG was in positive correlation with the expression of miR-34a. There was also a correlation between T status of OSCC and the expression of miR-335 and miR-150 and a correlation of miR-150 with the N status of T2 OSCC. NANOG promoter methylation and copy number variation were only observed in a small proportion of samples. Conclusions Our findings confirm the diagnostic significance of NANOG in OSCC and provide information on NANOG expression patterns on both mRNA and protein levels. They also suggest that protein regulators and microRNAs might play a crucial role, whereas methylation of its promoter and copy number variation probably have a minor role in the regulation of NANOG expression in OSCC.

Hepatocyte Notch Signaling Deregulation Related to Lipid Metabolism in Women with Obesity and Nonalcoholic Fatty Liver.

This cohort study aimed to explore the relationship between the Notch signaling pathway and the degree of nonalcoholic fatty liver disease (NAFLD). Moreover, this study intended to investigate whether this pathway is related to hepatic lipid metabolism and Toll-like receptors (TLRs). This study used real-time polymerase chain reaction analysis to evaluate the hepatic expression level of all genes studied (Notch receptors NOTCH1, NOTCH2, NOTCH3, and NOTCH4, transcription factors HES1 and HES5, and Hes-related repressor proteins HEY1 and HEY2) in hepatic tissue from two cohorts: women with severe obesity (n = 57) and normal liver structure (n = 20) or NAFLD (n = 37). In women with severe obesity and NAFLD, this study found downregulation of hepatic HES5 expression. This expression correlated positively with the hepatic expression of HES1, HEY1, and NOTCH3. This study also found a positive correlation between HES5 expression and sterol regulatory element-binding protein 1c (SREBP1c) and between NOTCH3 and several genes related to hepatic lipid metabolism (encoding liver X nuclear receptor α variant 1, farnesoid X nuclear receptor, SREBP1c, acetyl-CoA carboxylase 1, fatty acid synthase, peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1, carnitine O-octanoyltransferase, ATP-binding cassette subfamily A member 1, and ATP-binding cassette subfamily G member 1). Finally, this study found a positive correlation between NOTCH2 and TLR2, TLR4, and TLR9 and a positive relationship between NOTCH1 and TLR9. Taken together, these findings suggest that hepatic expression of Notch proteins and ligands in relation to lipid metabolism pathways in the liver could have a role in NAFLD pathogenesis.

Isolation and evaluation of cardenolides from Lansium domesticum as Notch inhibitors.

Since Notch signaling plays important roles in cell proliferation and differentiation, aberrant activation of this signaling contributes to cancer progression. In neural stem cells, Notch signaling inhibits differentiation by activating HES1 expression. Therefore, Notch signaling inhibitors may be candidates for new anticancer drugs or have applications in neural regenerative medicine. In this study, six naturally occurring Notch inhibitors were isolated from the methanol (MeOH) extract of Lansium domesticum using our novel cell-based assay. Hongherin (2), a cardiac glycoside, demonstrated potent Notch inhibitory activity with an IC50 of 0.62μM and was found to be cytotoxic in HPB-ALL human T cell acute lymphoblastic leukemia cells. Hongherin (2) also induced the differentiation of C17.2 neural stem cells to neurons, causing a 65% increase in differentiation compared to the control. Mechanistically, hongherin (2) reduced the amount of Notch1 (full length) and mastermind-like protein (MAML). This indicates that hongherin (2) inhibits Notch signaling through a dual mechanism involving the reduction of both Notch1 and MAML protein levels.

Cathepsin K Deficiency Impaired Ischemia-Induced Neovascularization in Aged Mice.

Background Aging is a major risk factor for cardiovascular disease. Cysteine protease cathepsin K (CatK) has been implicated in the process of angiogenesis, but the exact roles of individual CatK in vessel formation during aging are poorly understood. Methods and Results To study the putative role of CatK in ischemia-induced angiogenesis, we applied a hindlimb ischemia model to aged wild-type (CatK+/+) and CatK-deficient (CatK−/−) mice. A serial laser Doppler blood-flow analysis revealed that the recovery of the ischemic/normal blood-flow ratio in the aged CatK−/−mice was impaired throughout the follow-up period. On postoperative day 14, CatK deficiency had also impaired capillary formation. CatK deficiency reduced the levels of cleaved Notch1, phospho-Akt, and/or vascular endothelial growth factor (VEGF) proteins in the ischemic muscles and bone marrow-derived c-Kit+ cells. A flow cytometry analysis revealed that CatK deficiency reduced the numbers of endothelial progenitor cell (EPC)-like CD31+/c-Kit+ cells in the peripheral blood as well as the ischemic vasculature. In vitro experiments, CatK−/− impaired bone-derived c-Kit+ cellular functions (migration, invasion, proliferation, and tubulogenesis) in aged mice. Our findings demonstrated that aging impaired the ischemia-induced angiogenesis associated with the reductions of the production and mobilization of CD31+/c-Kit+ cells in mice. Conclusions These findings established that the impairment of ischemia-induced neovascularization in aged CatK−/− mice is due, at least in part, to the reduction of EPC mobilization and the homing of the cells into vasculature that is associated with the impairment of Notch1 signaling activation at advanced ages.

In situ evidence of collagen V and signaling pathway of found inflammatory zone 1 (FIZZ1) is associated with silicotic granuloma in lung mice

Inhalation of silica particles causes silicosis: an occupational lung disease characterized by persistent inflammation with granuloma formation that leads to tissue remodeling and impairment of lung function. Although silicosis has been studied intensely, little is known about the crucial cellular mechanisms that initiate and drive the process of inflammation and fibrosis. Recently, found in inflammatory zone 1 (FIZZ1) protein, produced by alveolar macrophages and fibroblasts have been shown to induce the proliferation of myofibroblasts and their transdifferentiation, causing tissue fibrosis. Moreover, autoimmunogenic collagen V, produced by alveolar epithelial cells and fibroblasts, is involved in the pathophysiology of interstitial pulmonary fibrosis and bleomycin-induced lung fibrosis. Based on the aforementioned we hypothesized that FIZZ1 and collagen V may be involved in the silicotic granuloma process in mice lungs. Male C57BL/6 mice (N = 20) received intratracheal administration of silica particles (Silica; 20 mg in 50 μL saline) or saline (Control; 50 μL). After 15 days, the lung histology was performed through immunohistochemistry and morphometric analysis. Within silicotic granulomas, collagen V and FIZZ1 increased, while peroxisome proliferator-activated receptor gamma (PPARγ) positive cells decreased. In addition, the expression of proteins Notch-1, alpha smooth muscle actin (α-SMA) and macrophages163 (CD163) were higher in silicotic granulomas than control lungs. A significant positive correlation was found between collagen V and FIZZ1 (r = 0.70; p < 0.05), collagen V and Notch-1 (r = 0.72; p < 0.05), whereas Collagen V was inversely associated with peroxisome proliferator-activated receptor gamma (r=-0.69; p < 0.05). These findings suggested that collagen V association with FIZZ1, Notch-1 and PPARγ might be a key pathogenic mechanism for silicotic granulomas in mice lungs.

Evaluation of Notch1 gene expression in prostate carcinoma

Aim: Prostate cancer is one of the most common cancers in the world and its incidence is rapidly increasing. Currently, several new treatment strategies that target specific signaling pathways as well as immunotherapy are being explored for prostate cancer. In the current study, we aimed to investigate the frequency and clinicopathologic features associated with Notch1 gene expression using immunohistochemistry and real-time-polymerase chain reaction in patients with prostate cancer. Methods: This research was planned as a cohort study. Tissue samples of 60 patients with prostate cancer and 20 patients with benign prostatic hyperplasia were selected from the archives. Twenty patients with benign prostatic hyperplasia were used as the control group. The relative expression levels of Notch1 gene were determined by real-time-polymerase chain reaction while Notch1 protein expression was analyzed using immunohistochemistry. Results: We found Notch1 gene expression at a significant level in 17 (28.3%) of 60 patients with prostate carcinoma. We found a significant correlation between high Gleason scores (P=0.007), high ISUP (International Society of Urologic Pathologists) grade group scores (P=0.008) and high prostate-specific antigen levels (P=0.018) for Notch1 gene expression. Conclusions: We conclude that Notch1 is important in prostate carcinogenesis. Therefore, determining Notch1 will be an essential step in targeted therapies in patients with prostate carcinoma.

MicroRNA-128-3p Enhances the Chemosensitivity of Temozolomide in Glioblastoma by Targeting c-Met and EMT

Temozolomide is a first line anti-tumor drug used for the treatment of patients with Glioblastoma multiforme (GBM). However, the drug resistance to temozolomide limits its clinical application. Therefore, novel strategies to overcome chemoresistance are desperately needed for improved treatment of human GBM. Recent studies have demonstrated that miRNAs are closely related to resistance to cancer chemotherapy. This study aimed to further validate the biological role of miR-128-3p and to investigate whether miR-128-3p can enhance the chemosensitivity of glioblastoma to temozolomide (TMZ) and the underlying mechanisms. The effects of miR-128-3p and TMZ on the proliferation of glioblastoma cells were investigated by cell counting kit-8 (cck8). Transwell and intracerebral invasion assays were applied to determine the effects of the combination of miR-128-3p and TMZ on the invasion and migration of glioblastoma in vitro and in vivo. Flow cytometry was used to detect apoptosis in each group, and immunofluorescence was used to determine the expression levels of EMT-related proteins. RT-PCR and Western-blot were applied to detect EMT-transformed proteins (c-Met, PDGFRα, Notch1, and Slug) and EMT phenotype-associated proteins (Vim, CD44, and E-cadherin) at both mRNA and protein levels. Based on the microRNA.org database, we predicted the target genes of miR-128-3p. The target-relationship between miR-128-3p and c-Met and PDGFRα was verified by dual luciferase reporter gene. The tumor volume, weight and the expression levels of the proteins described above were measured in subcutaneously transplanted tumor model in nude mice. We found that the expression of miR-128-3p was down-regulated in glioblastoma tissue samples and cell lines. miR-128-3p suppressed the proliferation, migration, and invasion of GBM both in vitro and in vivo; miR-128-3p enhanced the therapeutic effect of TMZ via inhibition of proliferation, invasion and migration of glioblastoma cells and induction of apoptosis. Overexpression of miR-128-3p down-regulated the expression levels of EMT-transformed proteins (c-Met, PDGFRα, Notch1 and Slug) to enhance the effect of TMZ. In addition, we found that miR-128-3p targeted and bound c-Met. More importantly, the upregulation of c‐Met significantly prompted U87 and U251 cell proliferation. This effect could be abolished when c‐Met was silenced. The investigation in tumor bearing nude mice showed that miR-128-3p in combination with TMZ reduced tumor volume and the invasion extent, and increased the sensitivity of glioblastoma to TMZ. miR-128-3p is capable of enhancing the sensitivity of glioblastoma to TMZ through regulating c-Met/EMT.

Therapeutic Effects of Focused Ultrasound on Expression of Notch1, C-Fos and Transforming Growth Factor-β3 in Vulvar Skin of SD Rats with Vulvar Lichen Simplex Chronicus

The purpose of this study was to investigate the therapeutic effects of focused ultrasound on the expression of notch1, c-fos and transforming growth factor-β3 (TGF-β3) in genital skin of SD rats with vulvar lichen simplex chronicus (LSC). Fifty-six female SD rats with LSC were randomly divided into therapy and sham groups. The therapy group was exposed to focused ultrasound. The sham group received the same therapy with an instrument that had no power output. Four wk after a singly focused ultrasound therapy, histologic analyses revealed that recovered SD rats accounted for 75% of SD rats in the therapy group and 10.7% in the sham group. Total collagen fiber density in the superficial layer of dermis in the therapy group was significantly lower than that in the sham group. Notch1 and c-fos protein expression in the therapy group was significantly lower than that in the sham group, with the opposite effect present for TGF-β3. Focused ultrasound therapy may inhibit superficial collagen fibrosis in the dermis by affecting expression of notch1, c-fos and TGF-β3 in vulvar skin tissue and consequently reduce the recurrence rate of LSC.