Hes1 Expression Research Articles

Loss of p53 enhances the tumor-initiating potential and drug resistance of clonogenic multiple myeloma cells.

Tumor relapse and drug resistance are major factors that limit the curability of multiple myeloma (MM). New regimens have improved overall MM survival rates, but patients with high-risk features continue to have inferior outcomes. Chromosome 17p13 deletion (del17p) that includes the loss of the TP53 gene is a high-risk cytogenetic abnormality and is associated with poor clinical outcomes owing to relatively short remissions and the development of pan-drug resistant disease. Increased relapse rates suggest that del17p enhances clonogenic growth, and we found that the loss of p53 increased both the frequency and drug resistance of tumor-initiating MM cells (TICs). Subsequent RNA sequencing (RNA-seq) studies demonstrated significant activation of the Notch signaling pathway and upregulation of inhibitor of DNA binding (ID1/ID2) genes in p53-knock out (p53-KO) cells. We found that the loss of ID1 or HES-1 expression or treatment with a gamma-secretase inhibitor (GSI) significantly decreased the clonogenic growth of p53-KO but not p53 wild-type cells. GSI treatment in a small set of MM specimens also reduced the clonogenic growth in del17p samples but not in non-del17p samples. This effect was specific as overexpression of the Notch intracellular domain (NICD) rescued the effects of GSI treatment. Our study demonstrates that the Notch signaling and ID1 expression are required for TIC expansion in p53-KO MM cells. These findings also suggest that GSI may be specifically active in patients with p53 mutant MM.

REST, RCOR1 and RCOR2 expression is reduced in osteoarthritic chondrocytes and contributes to increasing MMP13 and ADAMTS5 expression through upregulating HES1

Expression of key transcriptional regulators is altered in chondrocytes in osteoarthritis (OA). This contributes to an increase in production of cartilage-catabolizing enzymes such as MMP13 and ADAMTS5. RCOR1 and RCOR2, binding partners for the transcriptional repressor REST, have previously been found to be downregulated in OA chondrocytes although their function in chondrocytes is unclear. HES1 is a known REST/RCOR1 target gene and HES1 has been shown to promote MMP13 and ADAMTS5 expression in murine OA chondrocytes. The purpose of this study was to determine whether reduced REST/RCOR levels leads to increased HES1 expression in human OA chondrocytes and whether HES1 also promotes ADAMTS5 and MMP13 expression in these cells.Chondrocytes were isolated from osteoarthritic and adjacent macroscopically normal cartilage obtained from patients undergoing total knee arthroplasty. RNA and protein levels of REST, RCOR1 and RCOR2 were lower, but levels of HES1 higher, in chondrocytes isolated from osteoarthritic compared to macroscopically normal cartilage. Over-expression of either REST, RCOR1 or RCOR2 resulted in reduced HES1 levels in OA chondrocytes whereas knockdown of REST, RCOR1 or RCOR2 led to increased HES1 expression in chondrocytes from macroscopically normal cartilage. In OA chondrocytes, ADAMTS5 and MMP13 expression were reduced following HES1 knockdown, but further enhanced following HES1 over-expression. Levels of phosphorylated CaMKII were higher in chondrocytes from OA cartilage consistent with previous findings that HES1 only promotes gene transcription in the presence of active CaMKII.These findings identify the REST/RCOR/HES1 pathway as a contributing factor leading to increased ADAMTS5 and MMP13 expression in OA chondrocytes.

JAG-1/Notch signaling axis in the spinal cord contributes to bone cancer pain in rats.

Notch signal plays an important role in regulating cell-cell interactions with the adjacent cells. However, it remains unknown whether Jagged1 (JAG-1) mediated Notch signaling regulates bone cancer pain (BCP) via the spinal cell interactions mechanism. Here, we showed that intramedullary injection of Walker 256 breast cancer cells increased the expression of JAG-1 in spinal astrocytes and knockdown of JAG-1 reduced BCP. The supplementation of exogenous JAG-1 to the spinal cord induced BCP-like behavior and promoted expression of c-Fos and hairy and enhancer of split homolog-1 (Hes-1) in the spinal cord of the naïve rats. These effects were reversed when the rats were administered intrathecal injections of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). The intrathecal injection of DAPT reduced BCP and inhibited Hes-1 and c-Fos expression in the spinal cord. Furthermore, our results showed that JAG-1 up-regulated Hes-1 expression by inducing the recruitment of Notch intracellular domain (NICD) to the RBP-J/CSL-binding site located within the Hes-1 promoter sequence. Finally, the intrathecal injection of c-Fos-antisense oligonucleotides (c-Fos-ASO) and administration of sh-Hes-1 to the spinal dorsal horn also alleviated BCP. The study indicates that inhibition of the JAG-1/Notch signaling axis may be a potential strategy for the treatment of BCP.

Beta cell dysfunction induced by bone morphogenetic protein (BMP)-2 is associated with histone modifications and decreased NeuroD1 chromatin binding

SummaryInsufficient insulin secretion is a hallmark of type 2 diabetes and has been attributed to beta cell identity loss characterized by decreased expression of several key beta cell genes. The pro-inflammatory factor BMP-2 is upregulated in islets of Langerhans from individuals with diabetes and acts as an inhibitor of beta cell function and proliferation. Exposure to BMP-2 induces expression of Id1-4, Hes-1, and Hey-1 which are transcriptional regulators associated with loss of differentiation. The aim of this study was to investigate the mechanism by which BMP-2 induces beta cell dysfunction and loss of cell maturity. Mouse islets exposed to BMP-2 for 10 days showed impaired glucose-stimulated insulin secretion and beta cell proliferation. BMP-2-induced beta cell dysfunction was associated with decreased expression of cell maturity and proliferation markers specific to the beta cell such as Ins1, Ucn3, and Ki67 and increased expression of Id1-4, Hes-1, and Hey-1. The top 30 most regulated proteins significantly correlated with corresponding mRNA expression. BMP-2-induced gene expression changes were associated with a predominant reduction in acetylation of H3K27 and a decrease in NeuroD1 chromatin binding activity. These results show that BMP-2 induces loss of beta cell maturity and suggest that remodeling of H3K27ac and decreased NeuroD1 DNA binding activity participate in the effect of BMP-2 on beta cell dysfunction.

An investigation into the role of Notch signaling, altered angiogenesis, and inflammatory-induced preterm delivery and related complications in Northeast Indian patients

IntroductionNotch signaling is crucial during pregnancy with ability to regulate angiogenesis and inflammatory response. Considering the enigmatic importance of Notch signaling in pregnancy including placenta development, gestational disorders and adverse pregnancy; we performed experimental analysis to identify the Notch receptor-ligands association with Preterm delivery (PTD) and linked complication. MethodA total of 245 cases [Term n = 135 and Preterm n = 110] were enrolled for the study from Northeast Indian Population. The differential mRNA expression of Notch receptors , ligands, its downstream target Hes1 and Immune markers (IL-10, IL-12 and TNF-α) was studied by real time polymerase chain reaction. Further the protein study of Notch1 and 4, Hes1, VEGF and TNF-α was performed by immunofluorescence. ResultsPlacental mRNA expression of all the four notch receptors [Notch1 = 2.15 ± 1.02 fold, Notch2 = 6.85 ± 2.70 fold, and Notch3 = 1.74 ± 0.90 fold and Notch4 = 14.15 ± 6.72 fold]; ligands [JAG1 = 2.71 ± 1.22, JAG2 = 4.41 ± 2.31, DLL1 = 3.55 ± 1.38, DLL3 = 4.31 ± 2.82 and DLL4 = 3.07 ± 1.30 folds] and downstream target [Hes1 = 6.09 ± 2.89 folds] was elevated in PTD cases compared to Term delivery (TD) cases. The mRNA expression of pro-inflammatory marker (IL-12 = 3.99 ± 1.02 fold and TNF-α = 16.83 ± 2.97), was upregulated. The upregulated expression of Notch1(p < 0.001), JAG1 (p = 0.006), JAG2 (p = 0.009), DLL1 (p = 0.001), DLL4 (p < 0.001) Hes1 (p < 0.001), TNF-α (p < 0.001) and IL-12 (p = 0.006) were associated with the baby death; and Notch4 significantly inversely correlated with low birth weight (LBW). Consistently higher protein level expression of Notch1, Hes1, VEGFA and TNF-α was observed in preterm with highest expression in negative outcome cases. DiscussionTo conclude, the increased Notch1 expression and angiogenesis linked inflammation holds key in understanding the pathogenesis of PTD and linked complications and underlines its potential as therapeutic target for PTD interventions.

Immunohistochemical expression of Hairy and Enhancer of Split 1 (HES1) protein in breast carcinoma

Background Hairy and Enhancer of Split 1 (HES1) is a transcription factor involved in cell differentiation, proliferation, and in various cancer progression. Recently, HES1 was addressed as a potential biomarker that induces stem cell features in breast cancer and stimulates epithelial-mesenchymal transition in triple-negative breast cancer (TNBC). Aim This study aimed to assess HES1 immunohistochemical (IHC) expression in invasive breast carcinoma and correlate it with the clinicopathological variables and different molecular subtypes of the cases. Patients and methods IHC was conducted on 76 paraffin blocks with an anti-HES1 monoclonal antibody, and a final IHC score was calculated based on staining intensity and percentage of positively stained cells. A final IHC of greater than or equal to 4 was considered an HES1 positive expression. Results HES1 expression was positive in 18 (23.7%) cases and was significantly associated with breast cancer HER2-enriched molecular subtype, followed by triple-negative breast cancer (P&lt;0.05). HES1 expression was also significantly associated with estrogen and progesterone receptor negativity and HER2 overexpression (P&lt;0.05). Positive HES1 expression was associated with high tumor grade, advanced stage, high Ki-67 proliferation index, and presence of tumor vascular emboli, although it did not reach statistical significance (P&gt;0.05). Conclusion Our study demonstrated that the HES1 protein showed a significantly higher expression in nonluminal breast cancer subtypes. In addition, it could have a potential role in the growth and spread of breast cancer cells. These findings suggest that HES1 is a possible promising target for therapeutic interventions.

Porcine Deltacoronavirus Infection Disrupts the Intestinal Mucosal Barrier and Inhibits Intestinal Stem Cell Differentiation to Goblet Cells via the Notch Signaling Pathway.

Goblet cells and their secreted mucus are important elements of the intestinal mucosal barrier, which allows host cells to resist invasion by intestinal pathogens. Porcine deltacoronavirus (PDCoV) is an emerging swine enteric virus that causes severe diarrhea in pigs and causes large economic losses to pork producers worldwide. To date, the molecular mechanisms by which PDCoV regulates the function and differentiation of goblet cells and disrupts the intestinal mucosal barrier remain to be determined. Here, we report that in newborn piglets, PDCoV infection disrupts the intestinal barrier: specifically, there is intestinal villus atrophy, crypt depth increases, and tight junctions are disrupted. There is also a significant reduction in the number of goblet cells and the expression of MUC-2. In vitro, using intestinal monolayer organoids, we found that PDCoV infection activates the Notch signaling pathway, resulting in upregulated expression of HES-1 and downregulated expression of ATOH-1 and thereby inhibiting the differentiation of intestinal stem cells into goblet cells. Our study shows that PDCoV infection activates the Notch signaling pathway to inhibit the differentiation of goblet cells and their mucus secretion, resulting in disruption of the intestinal mucosal barrier. IMPORTANCE The intestinal mucosal barrier, mainly secreted by the intestinal goblet cells, is a crucial first line of defense against pathogenic microorganisms. PDCoV regulates the function and differentiation of goblet cells, thereby disrupting the mucosal barrier; however, the mechanism by which PDCoV disrupts the barrier is not known. Here, we report that in vivo, PDCoV infection decreases villus length, increases crypt depth, and disrupts tight junctions. Moreover, PDCoV activates the Notch signaling pathway, inhibiting goblet cell differentiation and mucus secretion in vivo and in vitro. Thus, our results provide a novel insight into the mechanism underlying intestinal mucosal barrier dysfunction caused by coronavirus infection.

Jagged 1 Regulates The Proliferation and Metastasis of Human MDA-T68 Thyroid Cancer Cells.

The current study evaluated the expression profile and explored the therapeutic implications of Jagged 1 in human thyroid cancer. This experimental study was conducted in 60 paired specimens of papillary thyroid and adjacent normal tissues. Gene expression was determined by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. Transfection of cancer cells was performed by using Lipofectamine 2000. The cell proliferation of PTC cells was estimated by MTT assay. Clonogenic assay was performed for analysis of colony forming potential of cancer cells. The apoptosis of PTC cells was studied by using AO/EB and Annexin V-FITC/PI staining methods. Flow cytometry was done to analyze the cell cycle phase distribution of cancer cells. Migration and invasion PTC cells were determined respectively with the wound-healing and transwell assays. The impact of Jagged 1 silencing was investigated in vivo in a xenografted mice model followed by Immunohistochemistry (IHC) analysis. We found significant (P<0.05) upregulation of Jagged 1 in human thyroid cancer. Silencing of Jagged 1 caused significant (P<0.05) reduction in proliferation and colony formation of MDA-T68 cells. The inhibitory effects of Jagged 1 silencing were found to be due to the induction of apoptosis. We also found enhancement of Bax and repression of Bcl-2 protein levels in MDA-T68 cells. Wound healing assay indicated significant (P<0.05) inhibition of cell migration of MDA-T68 thyroid cancer cells. Additionally, we found that invasion of the thyroid cancer cells was reduced by 55% upon silencing of Jagged 1. Moreover, Jagged 1 silencing was found to cause inhibition of the Notch intracellular domain (NICD) and Notch target gene, Hes-1 expression. Finally, Jagged 1 silencing inhibited the xenografted tumors in vivo. The findings suggest that Jagged 1 regulates the development of thyroid cancer that may act as a therapeutic target for managing thyroid cancer.

KDM6B Negatively Regulates the Neurogenesis Potential of Apical Papilla Stem Cells via HES1.

Stem cells from the apical papilla (SCAPs) are used to regulate the microenvironment of nerve defects. KDM6B, which functions as an H3K27me3 demethylase, is known to play a crucial role in neurogenesis. However, the mechanism by which KDM6B influences the neurogenesis potential of SCAPs remains unclear. We evaluated the expression of neural markers in SCAPs by using real-time RT-PCR and immunofluorescence staining. To assess the effectiveness of SCAP transplantation in the SCI model, we used the BBB scale to evaluate motor function. Additionally, toluidine blue staining and Immunofluorescence staining of NCAM, NEFM, β-III-tubulin, and Nestin were used to assess nerve tissue remodeling. Further analysis was conducted through Microarray analysis and ChIP assay to study the molecular mechanisms. Our results show that KDM6B inhibits the expression of NeuroD, TH, β-III tubulin, and Nestin. In vivo studies indicate that the SCAP-KDM6Bsh group is highly effective in restoring spinal cord structure and motor function in rats suffering from SCI. Our findings suggest that KDM6B directly binds to the HES1 promoter via regulating H3K27me3 and HES1 expression. In conclusion, our study can help understand the regulatory role of KDM6B in neurogenesis and provide more effective treatments for nerve injury.

NOTCH4ΔL12_16 sensitizes lung adenocarcinomas to EGFR-TKIs through transcriptional down-regulation of HES1

Resistance to epidermal growth factor tyrosine kinase inhibitors (EGFR-TKI) remains one of the major challenges in lung adenocarcinoma (LUAD) therapy. Here, we find an increased frequency of the L12_16 amino acid deletion mutation in the signal peptide region of NOTCH4 (NOTCH4ΔL12_16) in EGFR-TKI-sensitive patients. Functionally, exogenous induction of NOTCH4ΔL12_16 in EGFR-TKI -resistant LUAD cells sensitizes them to EGFR-TKIs. This process is mainly mediated by the reduction of the intracellular domain of NOTCH4 (NICD4) caused by the NOTCH4ΔL12_16 mutation, which results in a lower localization of NOTCH4 in the plasma membrane. Mechanistically, NICD4 transcriptionally upregulates the expression of HES1 by competitively binding to the gene promoter relative to p-STAT3. Because p-STAT3 can downregulate the expression of HES1 in EGFR-TKI-resistant LUAD cells, the reduction of NICD4 induced by NOTCH4ΔL12_16 mutation leads to a decrease in HES1. Moreover, inhibition of the NOTCH4-HES1 pathway using inhibitors and siRNAs abolishes the resistance of EGFR-TKI. Overall, we report that the NOTCH4ΔL12_16 mutation sensitizes LUAD patients to EGFR-TKIs through transcriptional down-regulation of HES1 and that targeted blockade of this signaling cohort could reverse EGFR-TKI -resistance in LUAD, providing a potential approach to overcome resistance to EGFR-TKI -therapy.

Expressions and Clinical Significance of Notch1 and Hes1 in Diffuse Large B-Cell Lymphoma

To investigate the expressions of Notch1 and Hes1 in diffuse large B-cell lymphoma (DLBCL), and their correlations with clinical features. Immunohistochemistry (IHC) was performed on DLBCL samples (54 cases) and lymphadenitis tissues (20 cases) to evaluate the expressions of Notch1 and Hes1, and analyze their correlations with clinical characteristics of patients. Based on Oncomine database, the expressions of Notch1 and Hes1 mRNA and DNA were also explored. IHC result showed that the positive expression rates of Notch1 and Hes1 in DLBCL patients were significantly higher than those in the control group (P <0.05). In DLBCL patients, the expression of Notch1 was closely associated with B symptoms, Ann Arbor stage, lymphocyte count and the level of lactate dehydrogenase (P <0.05), while the expression level of Hes1 was significantly higher in patients with B symptoms (P <0.05). Notch+/Hes1+ expression was found in 21 DLBCL tissues (38.9%), and there was a correlation between Notch1 and Hes1 expression (r =0.296, P <0.05). Bioinformatics analysis (Oncomine database) showed that the mRNA expressions of Notch1 and Hes1 in the Brune dataset were significantly higher than those in the control tissues (P <0.05). The expressions of Notch1 and Hes1 in DLBCL are significantly higher than those in lymphadenitis, and correlated with B symptoms and Ann Arbor stage, suggesting that Notch1 and Hes1 play important roles in the occurrence and development of DLBCL.

N-acetylcysteine improves autism-like behavior by recovering autophagic deficiency and decreasing Notch-1/Hes-1 pathway activity.

N-acetylcysteine (NAC) has been reported to improve social interaction behavior, irritability, self-injury, and anxiety-like behavior in autism. However, the molecular mechanism underlying the therapeutic roles of NAC in autism remains unknown. This study mainly aimed to investigate the therapeutic effect of NAC on valproic acid (VPA)-induced autism model and the underlying mechanisms. Our results showed that NAC ameliorated the deficits in sociability and the anxiety- and repetitive-like behaviors displayed by VPA-exposed rats. In addition, VPA exposure induced autophagic deficiency and enhanced Notch-1/Hes-1 pathway activity based on lowered Beclin-1 and LC3B levels, while increased expression of p62, Notch-1, and Hes-1 expression at the protein level. However, NAC recovered VPA-induced autophagic deficiency and reduced Notch-1/Hes-1 pathway activity in a VPA-exposed autism rat model and SH-SY5Y neural cells. The present results demonstrated that NAC improves autism-like behavioral abnormalities by inactivating Notch-1/Hes-1 signaling pathway and recovering autophagic deficiency. Taken together, this study helps to elucidate a novel molecular mechanism that underlies the therapeutic actions of NAC in autism and suggests its potential to ameliorate behavioral abnormalities in neurodevelopmental disorders.

Notch signaling pathway involved in Echinococcus granulosus infection regulates dendritic cell development and differentiation.

The Notch signaling pathway is involved in the development of many diseases; it regulates the development of dendritic cells (DCs), and affects the immune response of DC-mediated T cells. We previously found that ferritin and malate dehydrogenase (mMDH) in Echinococcus granulosus (E.granulosus) induced different immune responses through sensitized DCs. Therefore, in the study we explored whether the Notch signaling pathway affects the development and differentiation of DCs, causing changes in the immune response of DCs sensitized with E. granulosus antigens, and clarified whether it is involved in E.granulosus infection. We used the Notch signaling pathway inhibitor [N-[3,5-difluorophenace-tyl] -L-alanyl]-S-phenylglycinet-butyl ester (DAPT) or activator Jagged1 to construct in vitro cell models with blocked or activated Notch signaling respectively. We analyzed the effect of Notch signaling on the development and differentiation of DCs by detecting their morphology, migration function, capacity to promote T cell proliferation, and cytokine secretion. We observed the changes in DC response to E. granulosus antigens and the mediated immune response. DAPT inhibited the development and maturation of DCs, which were in a non-responsive or incompetent state, reduced the sensitization of DCs to Eg.ferritin, weakened the migration ability of DCs, disrupted their ability to mediate T-cell proliferation, reduced DC expression of MHCII, CD80, CD60, and CD40 co-stimulatory molecules, prevented the secretion of cytokines and attenuated the expression of Notch1, Notch2, Notch3 receptors, Jagged1, Delta-like 4 (Delta4), and Hes1. Following Jagged1 addition, the function of DCs was restored to some extent, and the expression of Notch1, Delta4 and Hes1 was activated in response to the stimulation of Eg.ferritin. However, Eg.mMDH stimulated DCs to produce an immune response showing weak interference by DAPT and Jagged1. The study suggests that the Notc h signaling pathway is involved in the Eg.ferritin-sensitized DC-mediated immune response, which may become a new target for treating E.granulosus infection.

Differential cell-cycle control by oscillatory versus sustained Hes1 expression via p21

Oscillatory Hes1 expression activates cell proliferation, while high and sustained Hes1 expression induces quiescence, but the mechanism by which Hes1 differentially controls cell proliferation depending on its expression dynamics is unclear. Here, we show that oscillatory Hes1 expression down-regulates the expression of the cyclin-dependent kinase inhibitor p21 (Cdkn1a), which delays cell-cycle progression, and thereby activates the proliferation of mouse neural stem cells (NSCs). By contrast, sustained Hes1 overexpression up-regulates p21 expression and inhibits NSC proliferation, although it initially down-regulates p21 expression. Compared with Hes1 oscillation, sustained Hes1 overexpression represses Dusp7, a phosphatase for phosphorylated Erk (p-Erk), and increases the levels of p-Erk, which can up-regulate p21 expression. These results indicate that p21 expression is directly repressed by oscillatory Hes1 expression, but indirectly up-regulated by sustained Hes1 overexpression, suggesting that depending on its expression dynamics, Hes1 differentially controls NSC proliferation via p21.

Effect of the Lipoxin Receptor Agonist BML-111 on Cigarette Smoke Extract-Induced Macrophage Polarization and Inflammation in RAW264.7 Cells.

Macrophages are known to play a crucial role in the chronic inflammation associated with Chronic Obstructive Pulmonary Disease (COPD). BML-111, acting as a lipoxin A4 (LXA4) receptor agonist, has shown to be effective in protecting against COPD. However, the precise mechanism by which BML-111 exerts its protective effect remains unclear. In order to establish a cell model of inflammation, cigarette smoke extract (CSE) was used on the RAW264.7 cell line. Afterwards, an Enzyme-linked immunosorbent assay (ELISA) kit was employed to measure concentrations of tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1β), interleukin-18 (IL-18), and interleukin-10 (IL-10) in the cell supernatants of the RAW264.7 cells.In this study, we examined the markers of macrophage polarization using two methods: quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Additionally, we detected the expression of Notch-1 and Hes-1 through Western blotting. BML-111 effectively suppressed the expression of pro-inflammatory cytokines TNF-α, IL-1β, and IL-18, as well as inflammasome factors NLRP3 and Caspase-1, while simultaneously up-regulating the expression of the anti-inflammatory cytokine IL-10 induced by CSE. Moreover, BML-111 reduced the expression of iNOS, which is associated with M1 macrophage polarization, and increased the expression of Arg-1, which is associated with M2 phenotype. Additionally, BML-111 downregulated the expression of Hes-1 and the ratio of activated Notch-1 to Notch-1 induced by CSE. The effect of BML-111 on inflammation and macrophage polarization was reversed upon administration of the Notch-1 signaling pathway agonist Jagged1. BML-111 has the potential to suppress inflammation and modulate M1/M2 macrophage polarization in RAW264.7 cells. The underlying mechanism may involve the Notch-1 signaling pathway.

The Notch pathway regulates autophagy after hypoxic-ischemic injury and affects synaptic plasticity.

Following neonatal hypoxic-ischemia (HI) injury, it is crucial factor to reconstruct neural circuit and maintain neural network homeostasis for neurological recovery. A dynamic balance between the synthesis and degradation of synaptic protein is required for maintaining synaptic plasticity. Protein degradation is facilitated by autophagy. This study aimed to investigate the regulation of synaptic structural plasticity by the Notch pathway, by assessing changes in Notch pathway activation and their effects on synaptic proteins and autophagy after HI injury. The study involved 48 male newborn Yorkshire piglets, each weighing 1.0-1.5kg and 3days old. They were randomly assigned to two groups: the HI group and the Notch pathway inhibitor + HI group (n = 24 per group). Each group was further divided into six subgroups according to HI duration (n = 4 per group): a control subgroup, and 0-6, 6-12, 12-24, 24-48, and 48-72h subgroups. The expression of Notch pathway-related proteins, including Notch1, Hes1, and Notch intracellular domains, increased following HI injury. The expression of autophagy proteins increased at 0-6h and 6-12h post-HI. The expression of synaptic proteins, such as postsynaptic density protein 95 (PSD95) and synaptophysin, increased 6-12h and 12-24h after HI, respectively. Notably, the increased expression of these proteins was reversed by a Notch pathway inhibitor. Transmission electron microscopy revealed the presence of autophagosome structures in synapses. These findings shed light on the underlying mechanisms of neurological recovery after HI injury and may provide insights into potential therapeutic targets for promoting neural circuit reconstruction and maintaining neural network homeostasis.

The HDAC inhibitor domatinostat induces type I interferon α in Merkel cell carcinoma by HES1 repression

BackgroundClass I selective histone deacetylase inhibitors (HDACi) have been previously demonstrated to not only increase major histocompatibility complex class I surface expression in Merkel cell carcinoma (MCC) cells by restoring the antigen processing and presentation machinery, but also exert anti-tumoral effect by inducing apoptosis. Both phenomena could be due to induction of type I interferons (IFN), as has been described for HDACi. However, the mechanism of IFN induction under HDACi is not fully understood because the expression of IFNs is regulated by both activating and inhibitory signaling pathways. Our own preliminary observations suggest that this may be caused by suppression of HES1.MethodsThe effect of the class I selective HDACi domatinostat and IFNα on cell viability and the apoptosis of MCPyV-positive (WaGa, MKL-1) and -negative (UM-MCC 34) MCC cell lines, as well as, primary fibroblasts were assessed by colorimetric methods or measuring mitochondrial membrane potential and intracellular caspase-3/7, respectively. Next, the impact of domatinostat on IFNA and HES1 mRNA expression was measured by RT-qPCR; intracellular IFNα production was detected by flow cytometry. To confirm that the expression of IFNα induced by HDACi was due to the suppression of HES1, it was silenced by RNA interference and then mRNA expression of IFNA and IFN-stimulated genes was assessed.ResultsOur studies show that the previously reported reduction in viability of MCC cell lines after inhibition of HDAC by domatinostat is accompanied by an increase in IFNα expression, both of mRNA and at the protein level. We confirmed that treatment of MCC cells with external IFNα inhibited their proliferation and induced apoptosis. Re-analysis of existing single-cell RNA sequencing data indicated that induction of IFNα by domatinostat occurs through repression of HES1, a transcriptional inhibitor of IFNA; this was confirmed by RT-qPCR. Finally, siRNA-mediated silencing of HES1 in the MCC cell line WaGa not only increased mRNA expression of IFNA and IFN-stimulated genes but also decreased cell viability.ConclusionOur results demonstrate that the direct anti-tumor effect of HDACi domatinostat on MCC cells is at least in part mediated via decreased HES1 expression allowing the induction of IFNα, which in turn causes apoptosis.

750 Epidermal expression of Hes1 controls immune response

750 Epidermal expression of Hes1 controls immune response

NDRG2 regulates the formation of reactive astrocyte-derived progenitor cells via Notch signaling pathway after brain traumatic injury in rats.

In response to traumatic brain injury, a subpopulation of cortical astrocytes is activated, resulting in acquisition of stem cell properties, known as reactive astrocytes-derived progenitor cells (Rad-PCs). However, the underlying mechanisms remain largely unknown during this process. In this study, we examined the role of N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, in Rad-PCs after cortical stab injury in adult rats. Immunohistochemical analysis showed that in the cerebral cortex of normal adult rats, NDRG2 was exclusively expressed in astrocytes. After liu cortical injury, the expression of NDRG2 was significantly elevated around the wound and most cells expressing NDRG2 also expressed GFAP, a reactive astrocyte marker. Importantly, NDRG2-expressing cells were co-labeled with Nestin, a marker for neural stem cells, some of which also expressed cell proliferation marker Ki67. Overexpression of NDRG2 further increased the number of NDRG2/Nestin double-labeling cells around the lesion. In contrast, shRNA knockdown of NDRG2 decreased the number of NDRG2+/Nestin+ cells. Intracerebroventricular administration of stab-injured rats with a Notch antagonist, DAPT, led to a significant decrease in Nestin+/NDRG2+ cells around the injured boundary, but did not affect NDRG2+ cells. Moreover, overexpression or knockdown of NDRG2 led to up- and down-regulation of the expression of Notch intracellular domain NICD and Notch target gene Hes1, respectively. Taken together, these results suggest that NDRG2 may play a role in controlling the formation of Rad-PCs in the cerebral cortex of adult rats following traumatic injury, and that Notch signaling pathway plays a key role in this process.

Abstract 4869: Furin and Notch signaling pathway as part of the 2OHOA landscape against glioblastoma

Abstract Glioblastoma (GBM) is the most common and aggressive cancer tumor of the central nervous system, with only 12-15 months of patients’ median survival. Melitherapy is a novel therapeutic platform based on the regulation of the membrane’s structure and organization with the consequent modulation of certain cell signals. In this context, 2-hydroxioleic acid (2OHOA, LAM561, INN: idroxioleic acid) has been developed for the treatment of GBM and is currently running a phase IIB/III clinical trial for newly diagnosed GBM patients. 2OHOA modulates the lipid composition and structure of cancer cells, increasing membranes fluidity and altering the activity of membrane-associated proteins, inhibiting the proliferation, inducing ER stress and differentiation, and finally triggering cell death by autophagy. The Notch signaling pathway has been highly related to tumorigenesis and cell survival driving the pathogenesis of GBM. In this work, we studied whether 2OHOA modulates the Notch pathway and its relevance in its mechanism of action (MoA) as an antitumoral drug. For this purpose, the 2OHOA’s effect on different components of the pathway was studied by western-blot, Q-PCR, and confocal microscopy. In addition, Notch receptor processing and NICD formation (final effector) were analyzed by cell fractionation and Notch processing enzymes activity, like furin, was evaluated upon 2OHOA treatment. First, the inhibition of this pathway in GBM cells by 2OHOA was confirmed by (i) lower expression levels of some of its components (Notch1, Notch3, and Jagged) and target genes (Hes1, CD3), (ii) less nuclear presence of NICD and Hes1 after 2OHOA treatment, (iii) accumulation of full-length Notch receptor in the membranes, impairing its processing and the NICD formation. Second, the relevance of Notch pathway in 2OHOA’s MoA was determined by the partial hindering of 2OHOA antiproliferative effect by Hes1 overexpression. Moreover, the reduction of Hes1 expression induced by the drug in different GBM cells correlated positively with their sensitivity to 2OHOA. Finally, 2OHOA downregulated furin-like proteases activity by physical association, an enzyme responsible for the Notch processing first step. All together reveals that the inhibition of Notch pathway by 2OHOA plays a role in its antitumoral effect, and this event is unleashed by the direct furin enzyme inhibition, identifying it as a novel target for this drug in the GBM and other pathologies treatment. Citation Format: Raquel Rodríguez-Lorca, Roberto Beteta-Göbel, Ramón Román, Manuel Torres, Victoria Llado, Pablo V. Escribá, Paula Fernández-García. Furin and Notch signaling pathway as part of the 2OHOA landscape against glioblastoma. [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 4869.