FBP1 inhibits NSCLC stemness by promoting ubiquitination of Notch1 intracellular domain and accelerating degradation.

The existence of cancer stem cells is widely acknowledged as the underlying cause for the challenging curability and high relapse rates observed in various tumor types, including non-small cell lung cancer (NSCLC). Despite extensive research on numerous therapeutic targets for NSCLC treatment, the strategies to effectively combat NSCLC stemness and achieve a definitive cure are still not well defined. The primary objective of this study was to examine the underlying mechanism through which Fructose-1,6-bisphosphatase 1 (FBP1), a gluconeogenic enzyme, functions as a tumor suppressor to regulate the stemness of NSCLC. Herein, we showed that overexpression of FBP1 led to a decrease in the proportion of CD133-positive cells, weakened tumorigenicity, and decreased expression of stemness factors. FBP1 inhibited the activation of Notch signaling, while it had no impact on the transcription level of Notch 1 intracellular domain (NICD1). Instead, FBP1 interacted with NICD1 and the E3 ubiquitin ligase FBXW7 to facilitate the degradation of NICD1 through the ubiquitin-proteasome pathway, which is independent of the metabolic enzymatic activity of FBP1. The aforementioned studies suggest that targeting the FBP1-FBXW7-NICD1 axis holds promise as a therapeutic approach for addressing the challenges of NSCLC recurrence and drug resistance.

Alcohol drinking inhibits NOTCH-PAX9 signaling in esophageal squamous epithelial cells.

Alcohol drinking has been established as a major risk factor for esophageal diseases. Our previous study showed that ethanol exposure inhibited PAX9 expression in human esophageal squamous epithelial cells in vitro and in vivo. In this study, we aimed to investigate the molecular pathways through which alcohol drinking suppresses PAX9 in esophageal squamous epithelial cells. We first demonstrated the inhibition of NOTCH by ethanol exposure in vitro. NOTCH regulated PAX9 expression in KYSE510 and KYSE410 cells in vitro and in vivo. RBPJ and NOTCH intracellular domain (NIC) D1 ChIP-PCR confirmed Pax9 as a direct downstream target of NOTCH signaling in mouse esophagus. NOTCH inhibition by alcohol drinking was further validated in mouse esophagus and human tissue samples. In conclusion, ethanol exposure inhibited NOTCH signaling and thus suppressed PAX9 expression in esophageal squamous epithelial cells in vitro and in vivo. Our data support a novel mechanism of alcohol-induced esophageal injury through the inhibition of NOTCH-PAX9 signaling. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Getting a Notch closer to renal dysfunction: activated Notch suppresses expression of the adaptor protein Disabled-2 in tubular epithelial cells.

Reactivation of Notch signaling in kidneys of animal models and patients with chronic kidney disease (CKD) has been shown to contribute to epithelial injury and fibrosis development. Here, we investigated the mechanisms of Notch-induced injury in renal epithelial cells. We performed genome-wide transcriptome analysis to identify Notch target genes using an in vitro system of cultured tubular epithelial cells expressing the intracellular domain of Notch1. One of the top downregulated genes was Disabled-2 ( Dab2). With the use of Drosophila nephrocytes as a model system, we found that Dab (the Drosophila homolog of Dab2) knockdown resulted in a significant filtration defect, indicating that loss of Dab2 plays a functional role in kidney disease development. We showed that Dab2 expression in cultured tubular epithelial cells is involved in endocytic regulation and that it also protects cells from TGF-ß-induced epithelial-to-mesenchymal transition. In vivo correlation studies indicated its additional role in renal ischemia-induced injury. Together, these data suggest that Dab2 plays a versatile role in the kidney and may impact on acute and CKDs.-Schütte-Nütgen, K., Edeling, M., Mendl, G., Krahn, M. P., Edemir, B., Weide, T., Kremerskothen, J., Michgehl, U., Pavenstädt, H. Getting a Notch closer to renal dysfunction: activated Notch suppresses expression of the adaptor protein Disabled-2 in tubular epithelial cells.

ASH2L-mediated H3K4me3 drives diabetic nephropathy through HIPK2 and Notch1 pathway.

Diabetic nephropathy (DN) is one of the complications of diabetes. Long-term hyperglycemia in the kidney results in renal insufficiency, and eventually leads to end-stage renal disease. Epigenetic factor ASH2L has long been identified as a transcriptional activator, and we previously indicated that ASH2L aggravated fibrosis and inflammation in high glucose-induced glomerular mesangial cells, but the pathophysiological relevance and the mechanism of ASH2L-mediated H3K4me3 in DN is not well understood. Here we demonstrated that ASH2L is upregulated in glomeruli isolated from db/db mice. Loss of ASH2L protected glomerular injury caused by hyperglycemia, as evidenced by reduced albuminuria, preserved structure, decreased glomerular extracellular matrix deposition, and lowered renal glomerular expression of proinflammatory and profibrotic markers in db/db mice. Furthermore, we demonstrated that enrichment of ASH2L-mediated H3K4me3 on the promoter regions of ADAM17 and HIPK2 triggered their transcription, leading to aberrant activation of Notch1 signaling pathway, thereby contributing to fibrosis and inflammation in DN. The findings of this study provide compelling evidence for targeting ASH2L as a potential therapeutic strategy to prevent or slow down the progression of DN.

Prognostic impact of Notch1 receptor and clinicopathological High-Risk Predictors in lacrimal gland adenoid cystic carcinoma.

PURPOSE: Adenoid Cystic Carcinoma (ACC) is an aggressive malignant lacrimal gland tumour associated with poor prognosis. Aberrant Notch signalling has been investigated in various tumours. However, very few studies on Notch signalling in lacrimal gland ACC are reported. The aim of the present study was to evaluate the status of Notch1 receptor and activated Notch1 (NICD1) in lacrimal gland ACC and to correlate it with high-risk clinicopathological features. METHODS: A total of 23 cases of histopathologically proven lacrimal gland ACC, who underwent surgical treatment, were included in this study. Expression of Notch1 receptor and NICD1 was evaluated by immunohistochemistry on formalin fixed paraffin embedded tissues. The results obtained were correlated with clinicopathological high-risk features and survival of the patients. Kaplan-Meier survival and multivariate analysis was performed to determine the prognostic significance. RESULTS: Overexpression of Notch1 receptor and NICD1 was observed in 65% and 39% of lacrimal gland ACC cases, respectively. On Kaplan-Meier survival analysis, patients with Notch1 receptor overexpression had reduced disease free survival. On univariate analysis, male gender, bone erosion, perineural invasion, solid histologic pattern, intracranial extension and advanced tumour stage were also indicators of poor prognosis. On multivariate analysis bone erosion was the most significant poor prognostic indicator. CONCLUSION: Our study demonstrates that overexpression of Notch1 receptor plays a critical role in the biology and aggressive behaviour of lacrimal gland ACC. Bone erosion, solid histologic pattern, advanced T stage, perineural invasion and intracranial extension are other high-risk clinicopathological predictors of lacrimal gland ACC.

Notch-1 Signaling Activation and Progesterone Receptor Expression in Ectopic Lesions of Women With Endometriosis.

CONTEXT: Progesterone (P) resistance is a hallmark of endometriosis, but the underlying mechanism(s) for loss of P sensitivity leading to lesion establishment remains poorly understood. OBJECTIVE: To evaluate the association between Notch-1 signaling activation and P resistance in the progression of endometriosis. DESIGN: Case control study; archived formalin-fixed, paraffin-embedded tissues. SETTING: University hospitals (United States, Taiwan). PATIENTS: Women with endometriosis; human endometrial stromal cell line (HESC). INTERVENTION: Eutopic endometria (EU) and ectopic lesions (ECs) were collected from surgically diagnosed patients. Archived tissue sections of EU and ECs were identified. HESCs were treated with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) and valproic acid (VPA) to, respectively, suppress and induce Notch-1 activation. OUTCOME MEASURES: Tissues were analyzed for Notch Intra-Cellular Domain 1 (NICD1) and progesterone receptor (PGR) protein expression by immunohistochemistry and for transcript levels of NICD1 target genes HES1, PGR, and PGR-B by quantitative reverse transcription polymerase chain reaction. DAPT- or VPA-treated HESCs with and without P cotreatment were evaluated for cell numbers and for PGR, HES1, and PGR target gene DKK1 transcript levels. RESULTS: Nuclear-localized stromal NICD1 protein levels were inversely associated with those of total PGR in EU and ECs. Stromal ECs displayed higher HES1 and lower total PGR and PGR-B transcript levels than EU. In HESCs, DAPT reduction of NICD1 decreased cell numbers and increased PGR transcript and nuclear PGR protein levels and, with P cotreatment, maintained P sensitivity. Conversely, VPA induction of NICD1 decreased PGR transcript levels and, with P cotreatment, abrogated P-induced DKK1 and maintained HES1 transcript levels. CONCLUSIONS: Aberrant Notch-1 activation is associated with decreased PGR that contributes to P resistance in endometriosis.

NOTCH1 activates the Wnt/ß-catenin signaling pathway in colon cancer.

PURPOSE AND METHODS: The translocation of ß-catenin/CTNNB1 to the nucleus activates Wnt signaling and cell proliferation; however, the precise mechanism underlying this phenomenon remains unknown. Previous reports have provided evidence that NOTCH1 is involved in the Wnt signaling pathway. Therefore, we sought to determine the mechanism by which NOTCH1 influences the Wnt/ß-catenin pathway. We constructed a vector expressing the NOTCH1 intracellular domain (NICD1) and transfected the vector into HCT116 which has low expression of NICD1. Furthermore, inhibition of NOTCH signal pathway in SW480 which has abundant NICD1 expression, was performed by transfection of siNICD1 or DAPT, gamma secretase inhibitor, treatment. In addition, we evaluated NICD1 and ß-catenin localization in colon cancer cell lines and in 189 colon cancer tissue samples and analyzed the correlation between the nuclear localization of NICD1 and the clinicopathological features of colon cancer patients. RESULTS: Immunohistochemical assays demonstrated that NICD1 and ß-catenin exhibited a similar localization pattern in colon cancer tissues. In addition, we found that NICD1 induced the translocation of ß-catenin to the nucleus and that NICD1 and ß-catenin co-localized in the nucleus. Overexpression of NICD1 increased luciferase activity of Wnt signal pathway. On the other hand, reduction of NICD1 reduced luciferase activity of Wnt signaling pathway. In the 189 analyzed colon cancer cases, multivariate COX regression analysis demonstrated the independent prognostic impact of nuclear localization of NICD1(p=0.0376). CONCLUSION: NOTCH1 plays a key role in the Wnt pathway and activation of NOTCH1 is associated with the translocation of ß-catenin to the nucleus.

Notch Signaling Rescues Loss of Satellite Cells Lacking Pax7 and Promotes Brown Adipogenic Differentiation.

Pax7 is a nodal transcription factor that is essential for regulating the maintenance, expansion, and myogenic identity of satellite cells during both neonatal and adult myogenesis. Deletion of Pax7 results in loss of satellite cells and impaired muscle regeneration. Here, we show that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) rescues the loss of Pax7-deficient satellite cells and restores their proliferative potential. Strikingly NICD1-expressing satellite cells do not undergo myogenic differentiation and instead acquire a brown adipogenic fate both in vivo and in vitro. NICD-expressing Pax7(-/-) satellite cells fail to upregulate MyoD and instead express the brown adipogenic marker PRDM16. Overall, these results show that Notch1 activation compensates for the loss of Pax7 in the quiescent state and acts as a molecular switch to promote brown adipogenesis in adult skeletal muscle.

KSHV viral cyclin interferes with T-cell development and induces lymphoma through Cdk6 and Notch activation in vivo.

Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that in vivo expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis.