Hairy And Enhancer Of Split-1 Research Articles

HES1 potentiates high salt stress response as an enhancer of NFAT5-DNA binding

High salt conditions and subsequent hyperosmolarity are injurious cellular stresses that can activate immune signaling. Nuclear factor of activated T-cells 5 (NFAT5) is an essential transcription factor that induces osmoprotective genes such as aldose reductase (AR) and betaine-GABA transporter 1 (BGT1). High salt stress-mediated NFAT5 activation is also reported to accelerate the inflammatory response and autoimmune diseases. However, the systemic regulation of NFAT5 remains unclear. Here, we performed a genome-wide siRNA screen to comprehensively identify the regulators of NFAT5. We monitored NFAT5 nuclear translocation and identified one of the Notch signaling effectors, Hairy and enhancer of split-1 (HES1), as a positive regulator of NFAT5. HES1 was induced by high salinity via ERK signaling and facilitated NFAT5 recruitment to its target promoter region, resulting in the proper induction of osmoprotective genes and cytoprotection under high salt stress. These findings suggest that, though HES1 is well known as a transcriptional repressor, it positively regulates NFAT5-dependent transcription in the context of a high salinity/hyperosmotic response.

A role for sirtuin 1 in FGF23 activation following β-glycerophosphate treatment

Phosphate homeostasis is vital for many biological processes and disruptions in circulating levels can be detrimental. While the mechanisms behind FGF23 regulation have been regularly studied, the role of extracellular phosphate sensing and its impact on fibroblast growth factor 23 (FGF23) expression remains unclear. This study aimed to investigate the involvement of reactive oxygen species (ROS), silent information regulator 1 (SIRT1), and Hairy and Enhancer of Split-1 (HES1) in regulating FGF23 in FGF23 expressing MC3T3-E1 cells. MC3T3-E1 cells treated with β-glycerophosphate (BGP) resulted in increased Fgf23 expression. Inhibition of ROS formation by inhibition of NADPH oxidase, which is essential for ROS production, did not affect this response to BGP, suggesting ROS is not involved in this process. Moreover, treatment with tert-butyl hydroperoxide (TBHP), a ROS-inducing agent, did not increase Fgf23 expression. This suggests that ROS machinery is not involved in FGF23 stimulation as previously suggested. Nonetheless, inhibition of SIRT1 using Ex527 eliminated the Fgf23 response to BGP, indicating its involvement in FGF23 regulation after BGP treatment. Indeed, activation of SIRT1 using SRT1720 increased Fgf23 expression. Moreover, transcription factor Hes1 was upregulated by BGP treatment, which was diminished when cells were treated with Ex527 implying it is also regulated through SIRT1. These findings suggest the existence of an upstream SIRT1-HES1 axis in the regulation of FGF23 by phosphate, though we were unable to find a role for ROS in this process. Further research should provide insights into phosphate homeostasis and potential therapeutic targets for phosphate-related disorders.

Hypoxia preconditioning increases Notch1 activity by regulating DNA methylation in vitro and in vivo.

Our previous research has demonstrated that hypoxic preconditioning (HPC) can improve spatial learning and memory abilities in adult mice. Adult hippocampal neurogenesis has been associated with learning and memory. The Neurogenic locus notch homolog protein (Notch) was involved in adult hippocampal neurogenesis, as well as in learning and memory. It is currently unclear whether the Notch pathway regulates hippocampal neuroregeneration by modifying the DNA methylation status of the Notch gene following HPC. The HPC animal model and cell model were established through repeated hypoxia exposure using mice and the mouse hippocampal neuronal cell line HT22. Step-down test was conducted on HPC mice. Real-time PCR and Western blot analysis were used to assess the mRNA and protein expression levels of Notch1 and hairy and enhancer of split1 (HES1). The presence of BrdU-positive cells and Notch1 expression in the hippocampal dental gyrus (DG) were examined with confocal microscopy. The methylation status of the Notch1 was analyzed using methylation-specific PCR (MS-PCR). HT22 cells were employed to elucidate the impact of HPC on Notch1 in vitro. HPC significantly improved the step-down test performance of mice with elevated levels of mRNA and protein expression of Notch1 and HES1 (P < 0.05). The intensities of the Notch1 signal in the control group, the H group and the HPC group were 2.62 ± 0.57 × 107, 2.87 ± 0.84 × 107, and 3.32 ± 0.14 × 107, respectively, and the number of BrdU (+) cells in the hippocampal DG were 1.83 ± 0.54, 3.71 ± 0.64, and 7.29 ± 0.68 respectively. Compared with that in C and H group, the intensity of the Notch1 signal and the number of BrdU (+) cells increased significantly in HPC group (P < 0.05). The methylation levels of the Notch1 promoter 0.82 ± 0.03, 0.65 ± 0.03, and 0.60 ± 0.02 in the C, H, and HPC groups, respectively. The methylation levels of Notch1 decreased significantly (P < 0.05). The effect of HPC on HT22 cells exhibited similarities to that observed in the hippocampus. HPC may confer neuroprotection by activating the Notch1 signaling pathway and regulating its methylation level, resulting in the regeneration of hippocampal neurons.

HES1 induces ITPR1-mediated autophagy to exert anti-metastatic effects in pituitary adenomas.

Pituitary adenomas (PAs) are prevalent intracranial tumors necessitating a comprehensive exploration of their molecular intricacies. This study delved into the molecular interactions among HES1 (hairy and enhancer of split 1), ITPR1 (inositol 1,4,5-trisphosphate receptor, type 1), and autophagy to elucidate their contributions to PA progression. Our in-depth bioinformatics analysis identified ITPR1 as a central hub gene in the PA-associated dataset. It exhibited reduced expression in PA and held significant clinical diagnostic relevance. Motivated by this discovery, we investigated the consequences of ITPR1 overexpression, as well as the use of autophagy inhibitors 3-Methyladenine (3-MA) and Baf A1, while considering the transcriptional influence of HES1. In vitro experiments utilizing PA cell lines revealed that ITPR1 overexpression significantly hindered tumorigenic activities. In contrast, both 3-MA and Baf A1 exacerbated these tumorigenic properties, confirmed by a decreased LC3 II/LC3 I ratio, indicative of autophagy inhibition. Intriguingly, the concurrent introduction of ITPR1 and these inhibitors mitigated these intensified effects, implying a tumor-suppressive role for ITPR1. Further investigations pinpoint HES1 as a potential upstream regulator of ITPR1 transcription. Silencing HES1 lead to reduced ITPR1 promoter activity, weakening the impact of ITPR1 overexpression on autophagy. This neutralized the ITPR1-mediated suppressions on PA cell activities, including proliferation, invasion, and migration. In summary, our research uncovered a complex regulatory interplay among HES1, ITPR1, and autophagy in the context of PA progression. These findings opened up promising avenues for novel therapeutic interventions targeting this intricate network to enhance PA treatment.

Carvedilol attenuates inflammatory reactions of lipopolysaccharide-stimulated BV2 cells and modulates M1/M2 polarization of microglia via regulating NLRP3, Notch, and PPAR-γ signaling pathways.

Microglial cells coordinate immune responses in the central nervous system. Carvedilol (CVL) is a non-selective β-blocker with anti-inflammatory and anti-oxidant effects. This study aims to investigate the anti-inflammatory effects and the underlying mechanisms of CVL on lipopolysaccharide (LPS)-induced inflammation in microglial BV2 cells. BV2 cells were stimulated with LPS, and the protective effects of CVL were investigated via measurement of cell viability, reactive oxygen species (ROS), and interleukin (IL)-1β liberation. The protein levels of some inflammatory cascade, Notch, and peroxisome proliferator-activated receptor (PPAR)-γ pathways and relative markers of M1/M2 microglial phenotypes were assessed. Neuroblastoma SH-SY5Y cells were cultured with a BV2-conditioned medium (CM), and the capacity of CVL to protect cell viability was evaluated. CVL displayed a protective effect against LPS stress through reducing ROS and down-regulating of nuclear factor kappa B (NF-κB) p65, NLR family pyrin domain containing-3 (NLRP3), and IL-1β proteins. LPS treatment significantly increased the levels of the M1 microglial marker inducible nitric oxide synthase (iNOS) and M1-associated cleaved-NOTCH1 and hairy and enhancer of split-1 (HES1) proteins. Conversely, LPS treatment reduced the levels of the M2 marker arginase-1 (Arg-1) and PPAR-γ proteins. CVL pre-treatment reduced the protein levels of iNOS, cleaved-NOTCH1, and HES1, while increased Arg-1 and PPAR-γ. CM of CVL-primed BV2 cells significantly improved SH-SY5Y cell viability as compared with the LPS-induced cells. CVL suppressed ROS production and alleviated the expression of inflammatory markers in LPS-stimulated BV2 cells. Our results demonstrated that targeting Notch and PPAR-γ pathways as well as directing BV2 cell polarization toward the M2 phenotype may provide a therapeutic strategy to suppress neuroinflammation by CVL.

Tibetan medicine Qi-Sai-Er-Sang-Dang-Song Decoction inhibits TNF-α-induced rheumatoid arthritis in human fibroblast-like synoviocytes via regulating NOTCH1/NF-κB/NLRP3 pathway.

Qi-Sai-Er-Sang-Dang-Song Decoction (QSD, ཆུ་སེར་སེང་ལྡེང་སུམ་ཐང་།), a Tibetan classical herbal formula, is commonly used in Tibetan hospital preparation for the treatment of rheumatoid arthritis (RA). Its efficacy is to relieve inflammation, dispel cold, remove dampness, and alleviate pain. However, its anti-RA mechanism is still unclear. This study aimed to investigate the effect of QSD on rheumatoid arthritis and explore its anti-inflammatory mechanism against human fibroblast-like synoviocytes (HFLSs) by regulating the notch family of receptors (NOTCH1)/Nuclear factor-κB (NF-κB)/nucleotide-binding (NLRP3) pathway. We used ultra-performance liquid chromatography coupled with Q-TOF mass spectrometry (UPLC-Q-TOF-MS) to identify the chemical composition of QSD. Then, HFLSs were exposed to drug-containing serum. The effect of QSD drug-containing serum on HFLS viability was detected using the cell counting kit-8 (CCK-8) assay. Next, we explored the anti-inflammatory effect of QSD using enzyme-linked immunosorbent assay (ELISA) for inflammatory factors, such as interleukin-18 (IL-18), interleukin-1β (IL-1β), and interleukin-6 (IL-6). The expression of NOTCH-related proteins, a member of the NOTCH1, Cleaved NOTCH1, hairy and enhancer of split-1 (HES-1), NF-κB p65, NF-κB pp65, NLRP3, and delta-like 1 (DLL-1), was examined using western blotting. Furthermore, the relative mRNA expression levels of NOTCH1, NF-κB p65, NLRP3, DLL-1, and HES-1 were detected using real-time quantitative (RT-qPCR). To explore the mechanism underlying the anti-RA effect of QSD, we the used the NOTCH signaling pathway inhibitor LY411575 and transfection with a NOTCH1 siRNA. In addition, we employed immunofluorescence to determine the expression of HES-1 and NF-κB p65 in vitro. Our results revealed that QSD ameliorated inflammation in HFLSs. Compared with the model group, the QSD drug-containing serum group had obviously down-regulated levels of IL-18, IL-1β, and IL-6. Consistently, the CCK-8 results showed that the QSD drug-containing serum had no obvious toxicity towards HFLSs. Moreover, both LY411575 and siNOTCH1, QSD could reduce NOTCH1, NLRP3, and HES-1 protein expression levels, and LY411575 could significantly inhibit the expression levels of NF-κB p65, NF-κB pp65, and Cleaved NOTCH1 (p<0.05). siNOTCH1 could also suppress the expression of DLL-1. The RT-qPCR results indicated that QSD could downregulate the relative mRNA expression levels of NOTCH1, NF-κB p65, NLRP3, DLL-1, and HES-1 in HFLSs (p<0.05). In the immunofluorescence experiment, the fluorescence intensities of HES-1 and NF-κB p65 in HFLSs were found to decrease after exposure to QSD drug-containing serum (p<0.05). Ultimately, 44 chemical components were detected in QSD using UPLC-Q-TOF-MS. This study reveals that the QSD can markedly ameliorate inflammation induced by TNF-α on HFLS. The effect of QSD on HFLS may be exerted by inhibition of the NOTCH1/NF-κB/NLRP3 signaling pathway.

Neuritin affects the activity of neuralized-like 1 by promoting degradation and weakening its affinity for substrate.

Neuritin plays a key role in neural development and regeneration by promoting neurite outgrowth and synapse maturation. Our previous research revealed the mechanism by which neuritin inhibits Notch signaling through interaction with neuralized-like 1 (Neurl1) to promote neurite growth. However, how neuritin regulates Notch signaling through Neurl1 has not been elucidated. Here, we first confirm that neuritin is an upstream regulator of Neurl1 and inhibits Notch signaling through Neurl1. Neurl1 is an E3 ubiquitin ligase that can promote ubiquitination and endocytosis of the Notch1 ligand Jagged1. Therefore, we observe the effect of neuritin on the ligase activity of Neurl1. The results indicate that neuritin inhibits Neurl1 activity by reducing the ubiquitination level and endocytosis of the target protein Jagged1. Moreover, we find that decreased activity of Neurl1 results in reduced expression of Notch receptor Notch intracellular domain (NICD) and downstream target gene hairy and enhancer of split-1 ( HES1). Furthermore, we investigate how neuritin affects Neurl1 enzyme activity. The results show that neuritin not only weakens the affinity between Neurl1 and Jagged1 but also promotes the degradation of Neurl1 by the 26S proteasome pathway. Taken together, our results suggest that neuritin negatively regulates Notch signaling by inhibiting the activity of Neurl1, promoting the degradation of Neurl1 and weakening the affinity of Neurl1 for Jagged1. Our study clarifies the molecular mechanisms of neuritin in regulating the Notch signaling pathway and provides new clues about how neuritin mediates neural regeneration and plasticity.

Abstract 3928: Therapeutic targeting of NOTCH1 and neddylation pathway in T cell acute lymphoblastic leukemia

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is hematologic tumor characterized by the diffuse infiltration of the bone marrow by malignant hematopoietic cells expressing immature T cell markers. Although T-ALL currently has better cure rates primarily due to multiagent or intensified chemotherapy, the prognosis for patients who are resistant or develop relapse to therapy remains very poor. Aberrant Notch homolog-1 (NOTCH1) signaling is a major driver of T-ALL pathogenesis as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. Gamma Secretase Inhibitors (GSIs) which effectively block the activation of oncogenic protein NOTCH1 are potential candidates for the treatment of T-ALL. However, the clinical application of GSIs is hampered by severe gastrointestinal toxicity due to the inhibition of NOTCH1 signaling in the gut. Here we demonstrate that combination therapy of GSIs and a small molecule inhibitor of the neddylation pathway circumvents the GSI-induced gut toxicity in vitro and in vivo. Genome-wide CRISPR loss-of-function screen in LS174T adenocarcinoma cells revealed neddylation pathway as a main regulator of massive goblet cell differentiation upon NOTCH1 inhibition. Genetic and pharmacologic inhibition of the neddylation pathway in LS174T cells rescued GSI-induced differentiation and cell death. Mechanistically, neddylation inhibition increases the protein stability of Hairy and enhancer of split-1 (HES1), a known regulator of absorptive and secretory cell fate decisions. Combination treatment of GSI and neddylation inhibitor in C57/BL6 mice showed a profound decrease in the number of goblet cells and maintained HES1 protein levels in the intestinal epithelium compared to GSI treatment alone. Remarkably, combined treatment of GSI and neddylation inhibitor in NOTCH1-induced T-ALL mice showed leukemia regression and improved overall survival without any associated gut toxicity. Overall, these results substantiate the potential of targeting NOTCH1 and neddylation pathway in the treatment of NOTCH1-induced T-ALL. Citation Format: Carla Bertulfo, Pablo Perez-Duran, Teresa Palomero, Adolfo Ferrando. Therapeutic targeting of NOTCH1 and neddylation pathway in T cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3928.

Low HES-1 and positive DLL4 expression predicts poor prognosis of colorectal cancers

Hairy and enhancer of split-1 (HES-1) is a downstream transcription factor and delta-like 4 (DLL4) is a ligand of the Notch signalling pathway. HES-1 and DLL4 expression are known to have an association with the progression and metastasis of cancers. We evaluated HES-1 and DLL4 expression and assessed their correlation with biological behaviour and prognostic significance of 327 colorectal cancers. Low HES-1 expression was identified in 210 (64.2%) cases and was significantly correlated with large tumour size, lymphovascular invasion, and distant metastasis. DLL4 was positive in 132 (40.4%) cases and significantly correlated with perineural invasion, distant metastasis, and involved resection margin. Patients with low HES-1 expression showed significantly worse overall survival than patients with high HES-1 expression [hazard ratio (HR)=3.017; 95% confidence interval (CI) 1.880-4.841; p<0.001]. The overall survival of patients with positive DLL4 expression was significantly worse than that of patients with negative DLL4 expression (HR=2.922; 95% CI 1.976-4.322; p<0.001). Furthermore, the combined HES-1lowDLL4positive expression group showed the worst overall survival compared to other groups (p<0.001) and was an independent poor prognostic factor of colorectal cancer patients. Thus, low HES-1 and positive DLL4 expression are associated with aggressive biological behaviour and can be used as prognostic factors in colorectal cancer patients.

Context-Dependent Roles of Hes1 in the Adult Pancreas and Pancreatic Tumor Formation

The Notch signaling pathway is an important pathway in the adult pancreas and in pancreatic ductal adenocarcinoma (PDAC), with hairy and enhancer of split-1 (HES1) as the core molecule in this pathway. However, the roles of HES1 in the adult pancreas and PDAC formation remain controversial. We used genetically engineered dual-recombinase mouse models for inducing Hes1 deletion under various conditions. The loss of Hes1 expression in the adult pancreas did not induce phenotypic alterations. However, regeneration was impaired after caerulein-induced acute pancreatitis. In a pancreatic intraepithelial neoplasia (PanIN) mouse model, PanINs rarely formed when Hes1 deletion preceded PanIN formation, whereas more PanINs were formed when Hes1 deletion succeeded PanIN formation. In a PDAC mouse model, PDAC formation was also enhanced by Hes1 deletion after PanIN/PDAC development; therefore, Hes1 promotes PanIN initiation but inhibits PanIN/PDAC progression. RNA sequencing and chromatin immunoprecipitation-quantitative polymerase chain reaction revealed that Hes1 deletion enhanced epithelial-to-mesenchymal transition via Muc5ac up-regulation in PDAC progression. The results indicated that HES1 is not required for maintaining the adult pancreas under normal conditions, but is important for regeneration during recovery from pancreatitis; moreover, Hes1 plays different roles, depending on the tumor condition. Our findings highlight the context-dependent roles of HES1 in the adult pancreas and pancreatic cancer.

CircSKIL promotes osteoblastic differentiation of periodontal ligament cells by sponging miR-532-5p to activate Notch signaling.

Periodontal ligament cells (PDLCs) possess the capacity to differentiate into a variety of cell types to benefit periodontal regeneration. In this study, we examined the circSKIL/miR-532-5p/Notch1 axis in controlling the osteoblastic differentiation of PDLCs. Primary human PDLCs (hPDLCs) were isolated and induced to differentiate into osteoblasts. Osteogenic responses were assessed for the expressions of osteoblast-related marker proteins (including alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein-2 (BMP2), and runt-related transcription factor 2 (RUNX2) by RT-PCR. The formation of mineralized nodules was examined by Alizarin Red S (ARS) staining and ALP activity. Expressions of circSKIL, miR-532-5p, and Notch1 were measured by RT-PCR and western blotting, and their regulations by combining bioinformatic analysis and luciferase reporter assay. Notch signaling was assessed for the expressions of hairy and enhancer of split-1 (HES1) and Notch intracellular domain (NICD). During osteoblastic differentiation of hPDLCs, circSKIL, and Notch1 were up-regulated, while miR-532-5p down-regulated. By sponging miR-532-5p, circSKIL activated Notch signaling, increasing levels of Notch1, HES1, and NICD. Functionally, knocking down circSKIL or overexpressing miR-532-5p inhibited osteoblastic differentiation of PDLCs, down-regulating ALP, OCN, BMP2, and RUNX2, and reducing ARS staining or ALP activity. The impacts of circSKIL knockdown were rescued by miR-532-5p inhibitor or overexpressing Notch1, while those caused by up-regulating miR-532-5p were reversed by overexpressing Notch1. By targeting miR-532-5p and up-regulating Notch1, circSKIL critically controls osteoblastic differentiation of hPDLCs. Therefore, modulating this axis may maximize the differentiation of PDLCs into osteoblasts and benefit periodontal regeneration.

Conditional overexpression of neuritin in supporting cells (SCs) mitigates hair cell (HC) damage and induces HC regeneration in the adult mouse cochlea after drug-induced ototoxicity

Hearing loss due to the loss of auditory hair cells is normally irreversible because mammalian hair cells do not regenerate. Using neurotrophic factors to induce the regeneration of hair cells (HCs) from adjacent nonsensory supporting cells (SCs) may be a promising strategy to treat hearing loss. Here, we demonstrate that overexpression of neuritin in SCs could mitigate drug-induced HC damage and directly induce HC regeneration from SCs by inhibiting the Notch signaling pathway. Using neuritin conditional knock-in mice, we found that upregulation of neuritin in SCs results in preserved HCs and partial recovery of hearing, inducing the regeneration of HCs from the transdifferentiation of SCs in ears damaged by kanamycin. Furthermore, neuritin overexpression in SCs downregulates the expression levels of Notch receptor Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) protein, two core components of the Notch signaling pathway. These observations confirmed in vitro that in cultured neonatal mouse cochleae, neuritin overexpression in SCs significantly inhibited gentamicin-induced HC damage and induced regeneration of HCs from the transdifferentiation of SCs, and that these effects were eliminated by adding the Notch ligand Jagged-1. These findings may provide a new avenue to stimulate HC regeneration after HC loss and highlight the therapeutic potential of neuritin for sensorineural hearing loss.

Dual targeting of Notch and Wnt/β-catenin pathways: Potential approach in triple-negative breast cancer treatment.

Despite the continuously growing repertoire of new and improved anti-cancer therapies, triple-negative breast cancer (TNBC) remains a clinical challenge to treat. In this sense, targeting signaling pathways such as Notch and Wnt/β-catenin have attracted growing attention. This work aimed at investigating the possible antitumor effects of IMR-1 as a Notch inhibitor, PRI-724 as a Wnt/β-catenin inhibitor, as well as their combination and to explore the possible crosstalk between Notch and Wnt/β-catenin signaling pathways in MDA-MB-231 TNBC cell line. Microculture tetrazolium test (MTT) was used to determine the drug growth inhibition (GI50), and the results were analyzed using CompuSyn 3.0.1 software. MDA-MB-231 cells were divided into four treatment groups including positive control, IMR-1-treated, PRI-724-treated, and combination-treated groups. Sandwich enzyme-linked immunosorbent assay (ELISA) was used for the determination of the protein levels of hairy and enhancer of split-1 (HES-1), Notch-1, β-catenin, cyclin-D1, and vascular endothelial growth factor (VEGF1). HES-1 gene expression was assessed by quantitative real-time polymerase chain reaction. Statistical analyses were performed using GraphPad Prism Software. The GI50 for IMR-1 and PRI-724 were 15.3μM and 0.69μM, respectively. Upon treatment of MDA-MB-231 cells with these drugs, HES-1 gene expression was up-regulated due to single and combined treatments. Moreover, the protein levels of cyclin-D1, VEGF1, HES-1, and Notch-1 were reduced, while those of active β-catenin and active caspase-3 were elevated. IMR-1/PRI-724 combination augmented IMR-1- and PRI-724-mediated effects on MDA-MB-231 cells by initiating apoptotic cell death. Further in vitro and in vivo studies are warranted to support our findings.

MicroRNA-216b enhances cisplatin-induced apoptosis in osteosarcoma MG63 and SaOS-2 cells by binding to JMJD2C and regulating the HIF1\u03b1/HES1 signaling axis

BackgroundAlthough cisplatin-based chemotherapy represents the standard regimen for osteosarcoma (OS), OS patients often exhibit treatment failure and poor prognosis due to chemoresistance to cisplatin. Emerging research has highlighted the tumor suppressive properties of microRNAs (miRNAs or miRs) in various human cancers via the inhibition of the histone demethylase jumonji domain containing protein 2C (JMJD2C). As a coactivator for hypoxia-inducible factor 1α (HIF1α), JMJD2C targets hairy and enhancer of split-1 (HES1) gene. Hence, the current study aimed to elucidate the role of miR-216b in OS cell cisplatin resistance to identify the underlying mechanism of miR-216b regulating the JMJD2C//HIF1α/HES1 signaling.MethodsTumor and paracancerous tissues were collected from OS patients to determine the expression patterns of miR-216b and JMJD2C. After ectopic expression and knockdown experiments in the OS cells, CCK-8 assay and flow cytometry were employed to determine cell viability and apoptosis. The interaction of miR-216b, JMJD2C, HIF1α and HES1 was subsequently determined by dual luciferase reporter, co-immunoprecipitation (IP) and ChIP-qPCR assays. In vivo experiments were conducted to further verify the role of the miR-216b in the resistance of OS cells to cisplatin.ResultsmiR-216b expression was reduced in the OS tissues, as well as the MG63 and SaOS-2 cells. Heightened miR-216b expression was found to be positively correlated with patient survival, and miR-216b further enhanced cisplatin-induced apoptosis of MG63 and SaOS-2 cells. Mechanistically, miR-216b inhibited JMJD2C expression by binding to its 3’UTR. Through interaction with HIF1α, JMJD2C removed the H3K9 methylation modification at the HES1 promoter region, leading to upregulation of HES1 in vitro. Furthermore, miR-216b was observed to increase the tumor growth in nude mice in the presence of cisplatin treatment. HES1 overexpression weakened the effects of miR-216b in MG63 and SaOS-2 cells and in nude mouse xenografts.ConclusionOverall, miR-216b enhanced the sensitivity of OS cells to cisplatin via downregulation of the JMJD2C/HIF1α/HES1 signaling axis, highlighting the capacity of miR-216b as an adjunct to cisplatin chemotherapy in the treatment of OS.

Loss of HES-1 Expression Predicts a Poor Prognosis for Small Intestinal Adenocarcinoma Patients.

Objective: Hairy and enhancer of split-1 (HES-1), which is a downstream target of the Notch signaling pathway, has been linked to KRAS mutations. HES-1 has been proposed as harboring oncogenic activity in colorectal cancer but has not been investigated in adenocarcinoma of the small intestine, where the drivers of oncogenesis are not as well-understood.Materials and Methods: To investigate the clinicopathologic and prognostic implications of HES-1, HES-1 immunohistochemical expression was analyzed in digital images along with clinicopathological variables, including survival and KRAS genotype, in 185 small intestinal adenocarcinomas.Results: The loss of HES-1 expression (HES-1Loss) was observed in 38.4% (71/185) of the patients, and was associated with higher pT category (P = 0.018), pancreatic invasion (P = 0.005), high grade (P = 0.043), and non-tubular histology (P = 0.004). Specifically, in tumors with mutant KRAS (KRASMT), HES-1Loss was related to proximal location (P = 0.024), high T and N categories (P = 0.005 and 0.047, respectively), and pancreatic invasion (P = 0.004). Patients with HES-1Loss showed worse overall survival compared to those with intact HES-1 (HES-1Intact) (P = 0.013). Patients with HES-1Loss/KRASMT (median, 17.3 months) had significantly worse outcomes than those with HES-1Intact/KRASWT (39.9 months), HES-1Intact/KRASMT (47.6 month), and HES-1Loss/KRASWT (36.2 months; P = 0.010). By multivariate analysis, HES-1Loss (hazard ratio = 1.55, 95% confidence interval (CI), 1.07–2.26; P = 0.022) remained an independent prognostic factor.Conclusion: HES-1expression can be used as a potential prognostic marker and may aid in the management of patients with small intestinal adenocarcinomas.

Analysis of changes in the expression of Notch1 and HES1 and the prognosis of osteosarcoma patients following surgery.

The present study aimed to analyze the changes in the expression of Notch1 and hairy and enhancer of split-1 (HES1) and the prognosis of patients with osteosarcoma following surgery. Samples from 62 patients with osteosarcoma treated at Shandong Cancer hospital from April, 2011 to June, 2013 were collected as the research group, and those from 52 healthy individuals undergoing physical examination were collected as the control group. The expression levels of Notch1 and HES1 in the serum of patients with osteosarcoma were measured by ELISA before and after surgery. Pearson's correlation analysis was used to analyze the correlation between Notch1 expression and HES1 expression in the osteosarcoma patients. According to the expression levels of Notch1 and HES1, the patients were divided into the high expression group and the low expression group, and the 5-year survival rate of the patients was observed. The expression levels of Notch1 and HES1 in the osteosarcoma patients before surgery were higher than those after surgery (P<0.05). The sensitivity, specificity and AUC of Notch1 for osteosarcoma were 93.55%, 58.06% and 0.732 respectively, and those of HES1 were 82.26%, 61.29% and 0.766, respectively. The expression level of Notch1 positively correlated with the expression level of HES1 in the osteosarcoma patients (r=0.795, P<0.001). According to the expression levels of Notch1 and HES1, the patients were divided into the high and low expression groups. The survival rate of the low expression group was significantly higher than that of the high expression groups (P=0.045). Finally, multiple factors were analyzed by logistic regression, and it was found that tumor location, chemotherapy response, tumor size, Notch1 and HES1 were independent risk factors for prognosis. Notch1 and HES1 exhibited a low expression in patients following surgery. ROC curve analysis revealed that the two indicators had good diagnostic efficacy and were expected to become markers for diagnosis and prognosis of osteosarcoma.

Effects of per- and poly-fluorinated alkyl substances on pancreatic and endocrine differentiation of human pluripotent stem cells

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are typical per- and poly-fluorinated alkyl substances (PFASs) that epidemiological studies have already associated with diabetes. However, insufficient data on their toxicity have been reported to explain any mechanism of action, which could justify such an association. Meanwhile, short-chain PFASs designed to substitute PFOA and PFOS, have already raised increasing concerns for their biosafety. Here, we evaluated whether common PFASs affected pancreatic and endocrine cell development using a human pluripotent stem cell pancreatic induction model and human pancreatic progenitor cell (hPP) endocrine induction model. The short-chain PFASs, pentafluorobenzoic acid, perfluorohexanoic acid, perfluorobutanesulfonic acid, and perfluorohexanesulfonic acid, homologous to PFOA or PFOS, did not significantly disrupt hPP generation, unlike PFOA and PFOS, based on pancreatic and duodenal homeobox 1 (PDX1) expression. However, SRY box 9 (SOX9) expression was suppressed in PDX1+ hPPs. All six PFASs did not disrupt SOX9 expression or hPP proliferation. However, endocrine differentiation of hPPs was affected, as indicated by neurogenin-3 (NGN3) downregulation, owing to abnormal increases in SOX9 and hairy and enhancer of split-1 (HES1) expressions. Thus, hyperactivation of NOTCH signaling was repressed after hPPs committed to the endocrine lineage. In conclusion, our study demonstrates how powerful human pluripotent stem cell-based pancreatic differentiation models can be in developmental toxicity evaluations, compared to traditional toxicity assays, mostly based on live animals. Moreover, our findings suggest that PFASs may alter pancreatic development after the pancreatic domain emerges from the gut tube, and provide insights into their toxicity mechanisms.

Proliferation of poorly differentiated endometrial cancer cells through autocrine activation of FGF receptor and HES1 expression.

Patients with poorly differentiated endometrial cancer show poor prognosis, and effective molecular target-based therapies are needed. Endometrial cancer cells proliferate depending on the activation of HES1 (hairy and enhancer of split-1), which is induced by several pathways, such as the Notch and fibroblast growth factor receptor (FGFR) signaling pathways. In addition, aberrant, ligand-free activation of the FGFR signaling pathway resulting from mutations in FGFR2 was also reported in endometrial cancer. However, a clinical trial showed that there was no difference in the effectiveness of FGFR inhibitors between patients with and without the FGFR2 mutation, suggesting a presence of another signaling pathway for the FGFR activation. Here, we investigated the signaling pathway regulating the expression of HES1 and proliferation of poorly and well-differentiated endometrial cancer cell lines Ishikawa and HEC-50B, respectively. Whereas Ishikawa cells proliferated and expressed HES1 in a Notch signaling-dependent manner, Notch signaling was not involved in HES1 and proliferation of HEC-50B cells. The FGFR inhibitor, NVP-BGJ398, decreased HES1 expression and proliferation of HEC-50B cells; however, HEC50B cells had no mutations in the FGFR2 gene. Instead, HEC-50B cells highly expressed ligands for FGFR2, suggesting that FGFR2 is activated by an autocrine manner, not by ligand-free activation. This autocrine pathway activated Akt downstream of FGFR for cell proliferation. Our findings suggest the usefulness of HES1 as a marker for the proliferation signaling and that FGFR inhibitor may be effective for poorly differentiated endometrial cancers that harbor wild-type FGFR.

O-GlcNAc Transferase Inhibition Differentially Affects Breast Cancer Subtypes

Post-translational modification of intracellular proteins with a single N-acetylglucosamine sugar (O-GlcNAcylation) regulates signaling, proliferation, metabolism and protein stability. In breast cancer, expression of the enzyme that catalyzes O-GlcNAcylation – O-GlcNAc-transferase (OGT), and the extent of protein O-GlcNAcylation, are upregulated in tumor tissue, and correlate with cancer progression. Here we compare the significance of O-GlcNAcylation in a panel of breast cancer cells of different phenotypes. We find a greater dependency on OGT among triple-negative breast cancer (TNBC) cell lines, which respond to OGT inhibition by undergoing cell cycle arrest and apoptosis. Searching for the cause of this response, we evaluate the changes in the proteome that occur after OGT inhibition or knock-down, employing a reverse-phase protein array (RPPA). We identify transcriptional repressor - hairy and enhancer of split-1 (HES1) - as a mediator of the OGT inhibition response in the TNBC cells. Inhibition of OGT as well as the loss of HES1 results in potent cytotoxicity and apoptosis. The study raises a possibility of using OGT inhibition to potentiate DNA damage in the TNBC cells.

M1-Polarized Macrophages Promote Self-Renewing Phenotype of Hepatic Progenitor Cells with Jagged1-Notch Signalling Involved: Relevance in Primary Sclerosing Cholangitis.

The immunologic interaction between parenchyma cells and encircling inflammatory cells is thought to be the most important mechanism of biliary damage and repair in primary sclerosing cholangitis (PSC). Monocytes/macrophages as master regulators of hepatic inflammation have been demonstrated to contribute to PSC pathogenesis. Macrophages coordinate with liver regeneration, and multiple phenotypes have been identified with diverse expressions of surface proteins and cytokine productions. We analyzed the expression of Notch ligand Jagged1 in polarized macrophages and investigated the relevance of Notch signalling activation in liver regeneration. M1 or M2 macrophages were generated from mouse bone marrow-derived macrophages (BMDMs) by classical or alternative activation, respectively. Then, the expression levels of Jagged1 (Jag1) of each phenotype were measured. The effects of polarized BMDMs on the expression of hepatic progenitor cell- (HPC-) specific markers and hairy and enhancer of split-1 (HES1) in HPCs in coculture were also analyzed. Monocyte-macrophage and Notch signalling-associated gene signatures were evaluated in the GEO database (access ID: GSE61260) by gene set enrichment analysis (GSEA). M1 macrophages were found associated with elevated Jag1 expression, which increased the fraction of HPC with self-renewing phenotypes (CD326+CD44+ or CD324+CD44+) and HES1 expression level in cocultured HPC. Blocking Jagged1 by siRNA or antibody in the coculture system attenuates HPC self-renewing phenotypes as well as HES1 expression in HPC. GSEA data show that macrophage activation and Notch signalling-associated gene signatures are enriched in PSC patients. These findings suggest that M1 macrophages promote an HPC self-renewing phenotype which is associated with Notch signalling activation within HPC. In the liver of PSC patients, the prevalence of activated macrophages, with M1 polarized accounting for the main part, is associated with increment of Notch signalling and enhancement of HPC self-renewal.