LncRNA PCAT6 promotes the occurrence of laryngeal squamous cell carcinoma via modulation of the miR-4731-5p/NOTCH3 axis.

Laryngeal squamous cell carcinoma (LSCC) is one of the most aggressive cancers that affect the head and neck region. Recent researches have confirmed that long non-coding RNAs (lncRNAs) present an emerging role in diversiform diseases including cancers. Prostate cancer-associated ncRNA transcript 6 (PCAT6) is an oncogene in lung cancer, cervical cancer, colon cancer and gastric cancer, but its role in LSCC is still unknown. In the current study, we attempted to figure out the role of PCAT6 in LSCC. RT-qPCR was to analyze PCAT6 expression in LSCC cells. Functional assays were to uncover the role of PCAT6 in LSCC. Mechanism assays were to explore the regulatory mechanism behind PCAT6 in LSCC. PCAT6 exhibited higher expression in LSCC cells and PCAT6 strengthened cell proliferation and inhibited cell apoptosis. Furthermore, lncRNA PCAT6 modulated notch receptor 3 expression and activated NOTCH signaling pathway via serving as a sponge for miR-4731-5p. Taken together, lncRNA PCAT6 was identified as an oncogene in LSCC, which revealed that PCAT6 might be used as potential therapeutic target for LSCC.

NOTCH3 as a prognostic biomarker and its correlation with immune infiltration in gastrointestinal cancers.

NOTCH receptor 3 (NOTCH3) is known to regulate the transcription of oncogenes or tumor suppressor genes, thereby playing a crucial role in tumor development, invasion, maintenance, and chemotherapy resistance. However, the specific mechanism of how NOTCH3 drives immune infiltration in gastrointestinal cancer remains uncertain. The expression of NOTCH3 was analyzed through Western blot, PCR, Oncomine database, and the Tumor Immune Estimation Resource (TIMER) site. Kaplan-Meier plotter, PrognoScan database, and gene expression profile interactive analysis (GEPIA) were used to assess the impact of NOTCH3 on clinical prognosis. The correlation between NOTCH3 expression and immune infiltration gene markers was investigated using TIMER and GEPIA. NOTCH3 was found to be commonly overexpressed in various types of gastrointestinal tumors and was significantly associated with poor prognosis. Furthermore, the expression level of NOTCH3 showed a significant correlation with the tumor purity of gastrointestinal tumors and the extent of immune infiltration by different immune cells. Our findings suggest that NOTCH3 may act as a crucial regulator of tumor immune cell infiltration and can serve as a valuable prognostic biomarker in gastrointestinal cancers.

NRN1 interacts with Notch to increase oncogenic STAT3 signaling in melanoma.

BACKGROUND: Melanoma is a highly heterogeneous cancer, in which frequent changes in activation of signaling pathways lead to a high adaptability to ever changing tumor microenvironments. The elucidation of cancer specific signaling pathways is of great importance, as demonstrated by the inhibitor of the common BrafV600E mutation PLX4032 in melanoma treatment. We therefore investigated signaling pathways that were influenced by neurotrophin NRN1, which has been shown to be upregulated in melanoma. METHODS: Using a cell culture model system with an NRN1 overexpression, we investigated the influence of NRN1 on melanoma cells' functionality and signaling. We employed real time cell analysis and spheroid formation assays, while for investigation of molecular mechanisms we used a kinase phosphorylation kit as well as promotor activity analysis followed by mRNA and protein analysis. RESULTS: We revealed that NRN1 interacts directly with the cleaved intracellular domain (NICD) of Notch1 and Notch3, causing a potential retention of NICD in the cytoplasm and thereby reducing the expression of its direct downstream target Hes1. This leads to decreased sequestration of JAK and STAT3 in a Hes1-driven phosphorylation complex. Consequently, our data shows less phosphorylation of STAT3 while presenting an accumulation of total protein levels of STAT3 in association with NRN1 overexpression. The potential of the STAT3 signaling pathway to act in both a tumor suppressive and oncogenic manner led us to investigate specific downstream targets - namely Vegf A, Mdr1, cMet - which were found to be upregulated under oncogenic levels of NRN1. CONCLUSIONS: In summary, we were able to show that NRN1 links oncogenic signaling events between Notch and STAT3 in melanoma. We also suggest that in future research more attention should be payed to cellular regulation of signaling molecules outside of the classically known phosphorylation events.

SNP and Structural Study of the Notch Superfamily Provides Insights and Novel Pharmacological Targets against the CADASIL Syndrome and Neurodegenerative Diseases.

The evolutionary conserved Notch signaling pathway functions as a mediator of direct cell-cell communication between neighboring cells during development. Notch plays a crucial role in various fundamental biological processes in a wide range of tissues. Accordingly, the aberrant signaling of this pathway underlies multiple genetic pathologies such as developmental syndromes, congenital disorders, neurodegenerative diseases, and cancer. Over the last two decades, significant data have shown that the Notch signaling pathway displays a significant function in the mature brains of vertebrates and invertebrates beyond neuronal development and specification during embryonic development. Neuronal connection, synaptic plasticity, learning, and memory appear to be regulated by this pathway. Specific mutations in human Notch family proteins have been linked to several neurodegenerative diseases including Alzheimer's disease, CADASIL, and ischemic injury. Neurodegenerative diseases are incurable disorders of the central nervous system that cause the progressive degeneration and/or death of brain nerve cells, affecting both mental function and movement (ataxia). There is currently a lot of study being conducted to better understand the molecular mechanisms by which Notch plays an essential role in the mature brain. In this study, an in silico analysis of polymorphisms and mutations in human Notch family members that lead to neurodegenerative diseases was performed in order to investigate the correlations among Notch family proteins and neurodegenerative diseases. Particular emphasis was placed on the study of mutations in the Notch3 protein and the structure analysis of the mutant Notch3 protein that leads to the manifestation of the CADASIL syndrome in order to spot possible conserved mutations and interpret the effect of these mutations in the Notch3 protein structure. Conserved mutations of cysteine residues may be candidate pharmacological targets for the potential therapy of CADASIL syndrome.

Stromal cartilage oligomeric matrix protein as a tumorigenic driver in ovarian cancer via Notch3 signaling and epithelial-to-mesenchymal transition.

BACKGROUND: Cartilage oligomeric matrix protein (COMP), an extracellular matrix glycoprotein, is vital in preserving cartilage integrity. Further, its overexpression is associated with the aggressiveness of several types of solid cancers. This study investigated COMP's role in ovarian cancer, exploring clinicopathological links and mechanistic insights. METHODS: To study the association of COMP expression in cancer cells and stroma with clinicopathological features of ovarian tumor patients, we analyzed an epithelial ovarian tumor cohort by immunohistochemical analysis. Subsequently, to study the functional mechanisms played by COMP, an in vivo xenograft mouse model and several molecular biology techniques such as transwell migration and invasion assay, tumorsphere formation assay, proximity ligation assay, and RT-qPCR array were performed. RESULTS: Based on immunohistochemical analysis of epithelial ovarian tumor tissues, COMP expression in the stroma, but not in cancer cells, was linked to worse overall survival (OS) of ovarian cancer patients. A xenograft mouse model showed that carcinoma-associated fibroblasts (CAFs) expressing COMP stimulate the growth and metastasis of ovarian tumors through the secretion of COMP. The expression of COMP was upregulated in CAFs stimulated with TGF-ß. Functionally, secreted COMP by CAFs enhanced the migratory capacity of ovarian cancer cells. Mechanistically, COMP activated the Notch3 receptor by enhancing the Notch3-Jagged1 interaction. The dependency of the COMP effect on Notch was confirmed when the migration and tumorsphere formation of COMP-treated ovarian cancer cells were inhibited upon incubation with Notch inhibitors. Moreover, COMP treatment induced epithelial-to-mesenchymal transition and upregulation of active ß-catenin in ovarian cancer cells. CONCLUSION: This study suggests that COMP secretion by CAFs drives ovarian cancer progression through the induction of the Notch pathway and epithelial-to-mesenchymal transition.

Identification of MUC1-C as a Target for Suppressing Progression of Head and Neck Squamous Cell Carcinomas.

The MUC1-C protein is aberrantly expressed in adenocarcinomas of epithelial barrier tissues and contributes to their progression. Less is known about involvement of MUC1-C in the pathogenesis of squamous cell carcinomas (SCC). Here, we report that the MUC1 gene is upregulated in advanced head and neck SCCs (HNSCC). Studies of HNSCC cell lines demonstrate that the MUC1-C subunit regulates expression of (i) RIG-I and MDA5 pattern recognition receptors, (ii) STAT1 and IFN regulatory factors, and (iii) downstream IFN-stimulated genes. MUC1-C integrates chronic activation of the STAT1 inflammatory pathway with induction of the ∆Np63 and SOX2 genes that are aberrantly expressed in HNSCCs. In extending those dependencies, we demonstrate that MUC1-C is necessary for NOTCH3 expression, self-renewal capacity, and tumorigenicity. The findings that MUC1 associates with ∆Np63, SOX2 and NOTCH3 expression by single-cell RNA sequencing analysis further indicate that MUC1-C drives the HNSCC stem cell state and is a target for suppressing HNSCC progression. SIGNIFICANCE: This work reports a previously unrecognized role for MUC1-C in driving STAT1-mediated chronic inflammation with the progression of HNSCC and identifies MUC1-C as a druggable target for advanced HNSCC treatment.

FPR3 reprograms glycolytic metabolism and stemness in gastric cancer via calcium-NFATc1 pathway.

Aerobic glycolysis accelerates tumor proliferation and progression, and inhibitors or drugs targeting abnormal cancer metabolism have been developing. Cancer stem-like cells (CSCs) significantly contribute to tumor initiation, metastasis, therapy resistance, and recurrence. Formyl peptide receptor 3 (FPR3), a member of FPR family, involves in inflammation, tissue repair, and angiogenesis. However, studies in exploring the regulatory mechanisms of aerobic glycolysis and CSCs by FPR3 in gastric cancer (GC) remain unknown. Here, we demonstrated that overexpressed FPR3 suppressed glycolytic capacity and stemness of tumor cells, then inhibited GC cells proliferation. Mechanistically, FPR3 impeded cytoplasmic calcium ion flux and hindered nuclear factor of activated T cells 1 (NFATc1) nuclear translocation, leading to the transcriptional inactivation of NFATc1-binding neurogenic locus notch homolog protein 3 (NOTCH3) promoter, subsequently obstructing NOTCH3 expression and the AKT/mTORC1 signaling pathway, and ultimately downregulating glycolysis. Additionally, NFATc1 directly binds to the sex determining region Y-box 2 (SOX2) promoter and modifies stemness in GC. In conclusion, our work illustrated that FPR3 played a negative role in GC progression by modulating NFATc1-mediated glycolysis and stemness in a calcium-dependent manner, providing potential insights into cancer therapy.

Notch3 as a novel therapeutic target for the treatment of ADPKD by regulating cell proliferation and renal cyst development.

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic kidney disease. Emerging research indicates that the Notch signaling pathway plays an indispensable role in the pathogenesis of numerous kidney diseases, including ADPKD. Herein, we identified that Notch3 but not other Notch receptors was overexpressed in renal tissues from mice with ADPKD and ADPKD patients. Inhibiting Notch3 with γ-secretase inhibitors, which block a proteolytic cleavage required for Notch3 activation, or shRNA knockdown of Notch3 significantly delayed renal cyst growth in vitro and in vivo. Subsequent mechanistic study elucidated that the cleaved intracellular domain of Notch3 (N3ICD) and Hes1 could bind to the PTEN promoter, leading to transcriptional inhibition of PTEN. This further activated the downstream PI3K-AKT-mTOR pathway and promoted renal epithelial cell proliferation. Overall, Notch3 was identified as a novel contributor to renal epithelial cell proliferation and cystogenesis in ADPKD. We envision that Notch3 represents a promising target for ADPKD treatment.

Pro-Hemorrhagic Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy Associated with NOTCH3 p.R75P Mutation with Low Vascular NOTCH3 Aggregation Property.

OBJECTIVES: Intracerebral hemorrhage (ICH) and cerebral microbleeds (CMB) in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy are more common in East Asian populations than in people of white European ancestry. We hypothesized that the ethnic difference is explained by the East Asian-specific NOTCH3 p.R75P mutation. METHODS: This retrospective observational study included 118 patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy in Japanese and Korean cohorts. We investigated whether the p.R75P mutation is associated with symptomatic ICH and multiple CMB (>5) using quasi-Poisson regression models. We predicted the NOTCH3 extracellular domain protein structures in silico and graded NOTCH3 extracellular domain immunostaining in skin vessels of some patients, with subsequent comparisons between p.R75P and other conventional mutations. RESULTS: Among 63 Japanese patients (median age 55 years; 56% men), 15 had a p.R75P mutation, significantly associated with symptomatic ICH (adjusted relative risk 9.56, 95% CI 2.45-37.31), multiple CMB (3.00, 1.34-6.71), and absence of temporopolar lesions (4.91, 2.29-10.52) after adjustment for age, sex, hypertension, and antithrombotics. In the Korean cohort (n = 55; median age 55 years; 51% men), the p.R75P mutation (n = 13) was also associated with symptomatic ICH (8.11, 1.83-35.89), multiple CMB (1.90, 1.01-3.56), and absence of temporopolar lesions (2.32, 1.08-4.97). Structural analysis revealed solvent-exposed free cysteine thiols in conventional mutations, directly causing aggregation, whereas a stereochemically incompatible proline residue structure in p.R75P lowers correct disulfide bond formation probability, indirectly causing aggregation. Pathologically, the p.R75P mutation resulted in less vascular NOTCH3 extracellular domain accumulation than the other conventional mutations. INTERPRETATION: NOTCH3 p.R75P mutation is associated with hemorrhagic presentations, milder temporopolar lesions, and distinct mutant protein structure properties. ANN NEUROL 2024;95:1040-1054.

CYP7B1-mediated 25-hydroxycholesterol degradation maintains quiescence-activation balance and improves therapeutic potential of mesenchymal stem cells.

Stem cells remain quiescent in vivo and become activated in response to external stimuli. However, the mechanism regulating the quiescence-activation balance of bone-marrow-derived mesenchymal stem cells (BM-MSCs) is still unclear. Herein, we demonstrated that CYP7B1 was the common critical molecule that promoted activation and impeded quiescence of BM-MSCs under inflammatory stimulation. Mechanistically, CYP7B1 degrades 25-hydroxycholesterol (25-HC) into 7α,25-dihydroxycholesterol (7α,25-OHC), which alleviates the quiescence maintenance effect of 25-HC through Notch3 signaling pathway activation. CYP7B1 expression in BM-MSCs was regulated by NF-κB p65 under inflammatory conditions. BM-MSCs from CYP7B1 conditional knockout (CKO) mice had impaired activation abilities, relating to the delayed healing of bone defects. Intravenous infusion of BM-MSCs overexpressing CYP7B1 could improve the pathological scores of mice with collagen-induced arthritis. These results clarified the quiescence-activation regulatory mechanism of BM-MSCs through the NF-κB p65-CYP7B1-Notch3 axis and provided insight into enhancing BM-MSCs biological function as well as the subsequent therapeutic effect.

Association of Rare NOTCH3 Variants With Prevalent and Incident Stroke and Dementia in the General Population.

BACKGROUND: It is uncertain whether rare NOTCH3 variants are associated with stroke and dementia in the general population and whether they lead to alterations in cognitive function. This study aims to determine the associations of rare NOTCH3 variants with prevalent and incident stroke and dementia, as well as cognitive function changes. METHODS AND RESULTS: In the prospective community-based Shunyi Study, a total of 1007 participants were included in the baseline analysis. For the follow-up analysis, 1007 participants were included in the stroke analysis, and 870 participants in the dementia analysis. All participants underwent baseline brain magnetic resonance imaging, carotid ultrasound, and whole exome sequencing. Rare NOTCH3 variants were defined as variants with minor allele frequency <1%. A total of 137 rare NOTCH3 carriers were enrolled in the baseline study. At baseline, rare NOTCH3 variant carriers had higher rates of stroke (8.8% versus 5.6%) and dementia (2.9% versus 0.8%) compared with noncarriers. After adjustment for associated risk factors, the epidermal growth factor-like repeats (EGFr)-involving rare NOTCH3 variants were associated with a higher risk of prevalent stroke (odds ratio [OR], 2.697 [95% CI, 1.266-5.745]; P=0.040) and dementia (OR, 8.498 [95% CI, 1.727-41.812]; P=0.032). After 5 years of follow-up, we did not find that the rare NOTCH3 variants increased the risk of incident stroke and dementia. There was no statistical difference in the change in longitudinal cognitive scale scores. CONCLUSIONS: Rare NOTCH3 EGFr-involving variants are genetic risk factors for stroke and dementia in the general Chinese population.

Notch signaling and targeted therapy in non-small cell lung cancer.

The Notch signaling pathway plays pivotal roles in cell proliferation, stemness and invasion of non-small cell lung cancer (NSCLC). The human Notch family consists of four receptors, namely Notch1, Notch2, Notch3, and Notch4. These receptors are transmembrane proteins that play crucial roles in various cellular processes. Notch1 mostly acts as a pro-carcinogenic factor in NSCLC but sometimes acts as a suppressor. Notch2 has been demonstrated to inhibit the growth and progression of NSCLC, whereas Notch3 facilitates these biological behaviors of NSCLC. The role of Notch4 in NSCLC has not been fully elucidated, but it is evident that Notch4 promotes tumor progression. At present, drugs targeting the Notch pathway are being explored for NSCLC therapy, a majority of which are already in the stage of preclinical research and clinical trials, with bright prospects in the clinical treatment of NSCLC.

Single-Cell Analysis Identifies NOTCH3-Mediated Interactions between Stromal Cells That Promote Microenvironment Remodeling and Invasion in Lung Adenocarcinoma.

Cancer immunotherapy has revolutionized the treatment of lung adenocarcinoma (LUAD); however, a significant proportion of patients do not respond. Recent transcriptomic studies to understand determinants of immunotherapy response have pinpointed stromal-mediated resistance mechanisms. To gain a better understanding of stromal biology at the cellular and molecular level in LUAD, we performed single-cell RNA sequencing of 256,379 cells, including 13,857 mesenchymal cells, from 9 treatment-naïve patients. Among the mesenchymal cell subsets, FAP+PDPN+ cancer-associated fibroblasts (CAF) and ACTA2+MCAM+ pericytes were enriched in tumors and differentiated from lung-resident fibroblasts. Imaging mass cytometry revealed that both subsets were topographically adjacent to the perivascular niche and had close spatial interactions with endothelial cells (EC). Modeling of ligand and receptor interactomes between mesenchymal and ECs identified that NOTCH signaling drives these cell-to-cell interactions in tumors, with pericytes and CAFs as the signal receivers and arterial and PLVAPhigh immature neovascular ECs as the signal senders. Either pharmacologically blocking NOTCH signaling or genetically depleting NOTCH3 levels in mesenchymal cells significantly reduced collagen production and suppressed cell invasion. Bulk RNA sequencing data demonstrated that NOTCH3 expression correlated with poor survival in stroma-rich patients and that a T cell-inflamed gene signature only predicted survival in patients with low NOTCH3. Collectively, this study provides valuable insights into the role of NOTCH3 in regulating tumor stroma biology, warranting further studies to elucidate the clinical implications of targeting NOTCH3 signaling. SIGNIFICANCE: NOTCH3 signaling activates tumor-associated mesenchymal cells, increases collagen production, and augments cell invasion in lung adenocarcinoma, suggesting its critical role in remodeling tumor stroma.

NOTCH3 promotes docetaxel resistance of prostate cancer cells through regulating TUBB3 and MAPK signaling pathway.

Docetaxel is the preferred chemotherapeutic agent in patients with castrate-resistant prostate cancer (CRPC). However, patients eventually develop docetaxel resistance and in the absence of effective treatment options. Consequently, it is essential to investigate the mechanisms generating docetaxel resistance and develop novel alternative therapeutic targets. RNA sequencing was undertaken on docetaxel-sensitive and docetaxel-resistant prostate cancer (PCa) cells. Subsequently, chemoresistance, cancer stemness, and lipid metabolism were investigated. To obtain insight into the precise activities and action mechanisms of NOTCH3 in docetaxel-resistant PCa, immunoprecipitation, mass spectrometry, ChIP, luciferase reporter assay, cell metabolism, and animal experiments were performed. Through RNA sequencing analysis, we found that NOTCH3 expression was markedly higher in docetaxel-resistant cells relative to parental cells, and that this trend was continued in docetaxel-resistant PCa tissues. Experiments in vitro and in vivo revealed that NOTCH3 enhanced stemness, lipid metabolism, and docetaxel resistance in PCa. Mechanistically, NOTCH3 is bound to TUBB3 and activates the MAPK signaling pathway. Moreover, NOTCH3 was directly regulated by MEF2A in docetaxel-resistant cells. Notably, targeting NOTCH3 and the MEF2A/TUBB3 signaling axis was related to docetaxel chemoresistance in PCa. Overall, these results demonstrated that NOTCH3 fostered stemness, lipid metabolism, and docetaxel resistance in PCa via the TUBB3 and MAPK signaling pathways. Therefore, NOTCH3 may be employed as a prognostic biomarker in PCa patients. NOTCH3 could be a therapeutic target for PCa patients, particularly those who have developed docetaxel resistance.

Notch3 restricts metastasis of breast cancers through regulation of the JAK/STAT5A signaling pathway.

PURPOSE: To explore the potential role of signal transducer and activator of transcription 5A (STAT5A) in the metastasis of breast cancer, and its mechanism of regulation underlying. METHODS AND RESULTS: TCGA datasets were used to evaluate the expression of STAT5A in normal and different cancerous tissues through TIMER2.0, indicating that STAT5A level was decreased in breast cancer tissues compared with normal ones. Gene Set Enrichment Analysis predicted that STAT5A was associated with the activation of immune cells and cell cycle process. We further demonstrated that the infiltration of immune cells was positively associated with STAT5A level. Influorescence staining revealed the expression and distribution of F-actin was regulated by STAT5A, while colony formation assay, wound healing and transwell assays predicted the inhibitory role of STAT5A in the colony formation, migratory and invasive abilities in breast cancer cells. In addition, overexpression of the Notch3 intracellular domain (N3ICD), the active form of Notch3, resulted in the increased expression of STAT5A. Conversely, silencing of Notch3 expression by siNotch3 decreased STAT5A expression, supporting that STAT5A expression is positively associated with Notch3 in human breast cancer cell lines and breast cancer tissues. Mechanistically, chromatin immunoprecipitation showed that Notch3 was directly bound to the STAT5A promoter and induced the expression of STAT5A. Moreover, overexpressing STAT5A partially reversed the enhanced mobility of breast cancer cells following Notch3 silencing. Low expression of Notch3 and STAT5A predicted poorer prognosis of patients with breast cancer. CONCLUSION: The present study demonstrates that Notch3 inhibits metastasis in breast cancer through inducing transcriptionally STAT5A, which was associated with tumor-infiltrating immune cells, providing a novel strategy to treat breast cancer.

The m6A reader IGF2BP3 preserves NOTCH3 mRNA stability to sustain Notch3 signaling and promote tumor metastasis in nasopharyngeal carcinoma.

Metastasis remains the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC), in which sustained activation of the Notch signaling plays a critical role. N6-Methyladenosine (m6A)-mediated post-transcriptional regulation is involved in fine-tuning the Notch signaling output; however, the post-transcriptional mechanisms underlying NPC metastasis remain poorly understood. In the present study, we report that insulin-like growth factor 2 mRNA-binding proteins 3 (IGF2BP3) serves as a key m6A reader in NPC. IGF2BP3 expression was significantly upregulated in metastatic NPC and correlated with poor prognosis in patients with NPC. IGF2BP3 overexpression promoted, while IGF2BP3 downregulation inhibited tumor metastasis and the stemness phenotype of NPC cells in vitro and in vivo. Mechanistically, IGF2BP3 maintains NOTCH3 mRNA stability via suppression of CCR4-NOT complex-mediated deadenylation in an m6A-dependent manner, which sustains Notch3 signaling activation and increases the transcription of stemness-associated downstream genes, eventually promoting tumor metastasis. Our findings highlight the pro-metastatic function of the IGF2BP3/Notch3 axis and revealed the precise role of IGF2BP3 in post-transcriptional regulation of NOTCH3, suggesting IGF2BP3 as a novel prognostic biomarker and potential therapeutic target in NPC metastasis.

NOTCH3 C201R variant causes cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) that can be confused with early-onset Alzheimer's disease.

BACKGROUND: NOTCH3 is the causative gene for autosomal dominant cerebral arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL) which is associated with both stroke and dementia. When CADASIL presents primarily as dementia it can be difficult to distinguish from Alzheimer's disease (AD) at both the clinical and neuropathological levels. METHODS: We performed exome sequencing of several affected individuals from a large family affected with AD. PCR amplification and direct Sanger sequencing were used to verify variants detected by exome analysis and to screen family members at-risk to carry those variants. Neuropathologic brain evaluation by immunohistochemistry and MRI were performed for the carriers of the NOTCH3 variant. RESULTS: In a three-generation family with AD, we found a c.601 T > C p.Cys201Arg variant in the NOTCH3 gene that caused clinical and neuropathological manifestations of CADASIL. These features included earlier onset of dementia accompanied by behavioral abnormalities in the father and son and white matter abnormalities in the asymptomatic grandson. The family is one branch of a large pedigree studied by the Alzheimer's Disease Sequencing Project (ADSP). As part of the ADSP linkage analysis and whole genome sequencing endeavor, an ABCA1 variant, p.Ala937Val, was previously found associated with AD in this pedigree. CONCLUSIONS: Our findings, together with other reported pathogenic missense variants of the C201 codon in NOTCH3, support the role of cysteine 201 as a mutation hotspot for CADASIL and highlight the genetic complexity both clinically and pathologically of AD and related dementia.

Enhancer remodeling activates NOTCH3 signaling to confer chemoresistance in advanced nasopharyngeal carcinoma.

Acquired resistance to chemotherapy is one of the major causes of mortality in advanced nasopharyngeal carcinoma (NPC). However, effective strategies are limited and the underlying molecular mechanisms remain elusive. In this study, through transcriptomic profiling analysis of 23 tumor tissues, we found that NOTCH3 was aberrantly highly expressed in chemoresistance NPC patients, with NOTCH3 overexpression being positively associated with poor clinical outcome. Mechanistically, using an established NPC cellular model, we demonstrated that enhancer remodeling driven aberrant hyperactivation of NOTCH3 in chemoresistance NPC. We further showed that NOTCH3 upregulates SLUG to induce chemo-resistance of NPC cells and higher expression of SLUG have poorer prognosis. Genetic or pharmacological perturbation of NOTCH3 conferred chemosensitivity of NPC in vitro and overexpression of NOTCH3 enhanced chemoresistance of NPC in vivo. Together, these data indicated that genome-wide enhancer reprogramming activates NOTCH3 to confer chemoresistance of NPC, suggesting that targeting NOTCH3 may provide a potential therapeutic strategy to effectively treat advanced chemoresistant NPC.

The cysteine-altering p.R544C variant in the NOTCH3 gene is a probable candidate for blood pressure and relevant traits in the Taiwan Biobank.

BACKGROUND: The cysteine-altering variants in NOTCH3 have been suggested to be associated with stroke, dementia, and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), where aberrant blood pressure levels represent the characteristics of these diseases. We aimed to assess whether the cysteine-altering p.Arg544Cys (p.R544C; rs201118034) variant and common single nucleotide variants (SNVs) in NOTCH3 could contribute to systolic and diastolic blood pressure and related phenotypes in the Taiwan Biobank. METHODS: We employed a discovery sample of 68,925 individuals, an independent replication sample of 45,676 individuals, and a combined/total sample of 114,601 individuals; all from the Taiwan Biobank. Blood pressure, such as systolic and diastolic blood pressure, was measured for all participants. Association was evaluated using a general linear model, where results were considered statistically significant if the P value < 0.05 divided by the number of independent tests per model. RESULTS: From our analysis, we identified and replicated three novel candidates for blood pressure that have not previously been reported: the cysteine-altering p.R544C variant for systolic blood pressure, the common SNV rs11669950 for diastolic blood pressure, and the common SNV rs4808235 for diastolic blood pressure. We also generalized two previously identified SNVs (i.e., rs10418305 and rs7408868) in NOTCH3 for blood pressure in European and non-Taiwanese East Asian populations to the Taiwanese population. Moreover, the participants with NOTCH3 p.R544C had an increased stroke frequency (P < 1.0 × 10-5) and a higher dementia frequency (P = 2.0 × 10-4) compared with the whole Taiwan Biobank population in the combined/total sample. CONCLUSION: NOTCH3 is a strong candidate for a role in stroke, dementia, and CADASIL, which has previously been linked to blood pressure changes. While our preliminary study suggests that NOTCH3 p.R544C may influence blood pressure, stroke, and dementia in the Taiwan Biobank, replication in a well-powered external sample is required. This study also underlines considerable prospects of detecting novel genetic biomarkers in underrepresented worldwide populations.

Preferential rabbit antibody responses to C-termini of NOTCH3 peptide immunogens.

Antibodies raised in peptide-immunized rabbits have been used in biological research for decades. Although there has been wide implementation of this approach, specific proteins are occasionally difficult to target for multiple reasons. One consideration that was noted in mice is that humoral responses may preferentially target the carboxyl terminus of the peptide sequence which is not present in the intact protein. To shed light on the frequency of preferential rabbit antibody responses to C-termini of peptide immunogens, we present our experience with generation of rabbit antibodies to human NOTCH3. A total of 23 antibodies were raised against 10 peptide sequences of human NOTCH3. Over 70% (16 of 23) of these polyclonal antibodies were determined to be C-terminal preferring: NOTCH3 peptide-reactive antibodies largely targeted the terminating free carboxyl group of the immunizing peptide. The antibodies that preferred C-terminal epitopes reacted weakly or not at all with recombinant target sequences with extension the C-terminus that eliminated the free carboxyl group of the immunogen structure; furthermore, each of these antisera revealed no antibody reactivity to proteins truncated before the C-terminus of the immunogen. In immunocytochemical applications of these anti-peptide antibodies, we similarly found reactivity to recombinant targets that best binding to cells expressing the free C-terminus of the immunizing sequence. In aggregate, our experience demonstrates a strong propensity for rabbits to mount antibody responses to C-terminal epitopes of NOTCH3-derived peptides which is predicted to limit their use against the native protein. We discuss some potential approaches to overcome this bias that could improve the efficiency of generation of antibodies in this commonly utilized experimental paradigm.