Cathepsin K deficiency prevented stress-related thrombosis in a mouse FeCl3 model.

BACKGROUND: Exposure to chronic psychological stress (CPS) is a risk factor for thrombotic cardiocerebrovascular diseases (CCVDs). The expression and activity of the cysteine cathepsin K (CTSK) are upregulated in stressed cardiovascular tissues, and we investigated whether CTSK is involved in chronic stress-related thrombosis, focusing on stress serum-induced endothelial apoptosis. METHODS AND RESULTS: Eight-week-old wild-type male mice (CTSK+/+) randomly divided to non-stress and 3-week restraint stress groups received a left carotid artery iron chloride3 (FeCl3)-induced thrombosis injury for biological and morphological evaluations at specific timepoints. On day 21 post-stress/injury, the stress had enhanced the arterial thrombi weights and lengths, in addition to harmful alterations of plasma ADAMTS13, von Willebrand factor, and plasminogen activation inhibitor-1, plus injured-artery endothelial loss and CTSK protein/mRNA expression. The stressed CTSK+/+ mice had increased levels of injured arterial cleaved Notch1, Hes1, cleaved caspase8, matrix metalloproteinase-9/-2, angiotensin type 1 receptor, galactin3, p16IN4A, p22phox, gp91phox, intracellular adhesion molecule-1, TNF-α, MCP-1, and TLR-4 proteins and/or genes. Pharmacological and genetic inhibitions of CTSK ameliorated the stress-induced thrombus formation and the observed molecular and morphological changes. In cultured HUVECs, CTSK overexpression and silencing respectively increased and mitigated stressed-serum- and H2O2-induced apoptosis associated with apoptosis-related protein changes. Recombinant human CTSK degraded γ-secretase substrate in a dose-dependent manor and activated Notch1 and Hes1 expression upregulation. CONCLUSIONS: CTSK appeared to contribute to stress-related thrombosis in mice subjected to FeCl3 stress, possibly via the modulation of vascular inflammation, oxidative production and apoptosis, suggesting that CTSK could be an effective therapeutic target for CPS-related thrombotic events in patients with CCVDs.

LINC MIR503HG Controls SC-ß Cell Differentiation and Insulin Production by Targeting CDH1 and HES1.

Stem cell-derived pancreatic progenitors (SC-PPs), as an unlimited source of SC-derived ß (SC-ß) cells, offers a robust tool for diabetes treatment in stem cell-based transplantation, disease modeling, and drug screening. Whereas, PDX1+/NKX6.1+ PPs enhances the subsequent endocrine lineage specification and gives rise to glucose-responsive SC-ß cells in vivo and in vitro. To identify the regulators that promote induction efficiency and cellular function maturation, single-cell RNA-sequencing is performed to decipher the transcriptional landscape during PPs differentiation. The comprehensive evaluation of functionality demonstrated that manipulating LINC MIR503HG using CRISPR in PP cell fate decision can improve insulin synthesis and secretion in mature SC-ß cells, without effects on liver lineage specification. Importantly, transplantation of MIR503HG-/- SC-ß cells in recipients significantly restored blood glucose homeostasis, accompanied by serum C-peptide release and an increase in body weight. Mechanistically, by releasing CtBP1 occupying the CDH1 and HES1 promoters, the decrease in MIR503HG expression levels provided an excellent extracellular niche and appropriate Notch signaling activation for PPs following differentiation. Furthermore, this exhibited higher crucial transcription factors and mature epithelial markers in CDH1High expressed clusters. Altogether, these findings highlighted MIR503HG as an essential and exclusive PP cell fate specification regulator with promising therapeutic potential for patients with diabetes.

bHLH transcription factors Hes1, Ascl1 and Oligo2 exhibit different expression patterns in the process of physiological electric fields-induced neuronal differentiation.

BACKGROUND: Recent studies have shown that the expression of bHLH transcription factors Hes1, Ascl1, and Oligo2 has an oscillating balance in neural stem cells (NSCs) to maintain their self-proliferation and multi-directional differentiation potential. This balance can be disrupted by exogenous stimulation. Our previous work has identified that electrical stimulation could induce neuronal differentiation of mouse NSCs. METHODS: To further evaluate if physiological electric fields (EFs)-induced neuronal differentiation is related to the expression patterns of bHLH transcription factors Hes1, Ascl1, and Oligo2, mouse embryonic brain NSCs were used to investigate the expression changes of Ascl1, Hes1 and Oligo2 in mRNA and protein levels during EF-induced neuronal differentiation. RESULTS: Our results showed that NSCs expressed high level of Hes1, while expression of Ascl1 and Oligo2 stayed at very low levels. When NSCs exited proliferation, the expression of Hes1 in differentiated cells began to decrease and oscillated at the low expression level. Oligo2 showed irregular changes in low expression level. EF-stimulation significantly increased the expression of Ascl1 at mRNA and protein levels accompanied by an increased percentage of neuronal differentiation. What's more, over-expression of Hes1 inhibited the neuronal differentiation induced by EFs. CONCLUSION: EF-stimulation directed neuronal differentiation of NSCs by promoting the continuous accumulation of Ascl1 expression and decreasing the expression of Hes1.

Silencing Lnc-HES1-10 Inhibits Osteosarcoma Cells Proliferation, Invasive Ability, and Metastasis.

BACKGROUND: Long noncoding RNA (LncRNA) play a vital role in the development and pathophysiology of osteosarcoma (OS). However, the LncRNA activated by HES1-10 in OS has not been furthered investigated. This present study aims to show the possible function of Lnc-HES1-10 in OS. METHODS: Cell proliferation in vitro were assessed by the MTT assay, whereas the migration and invasion abilities of OS cell lines were measured by wound-healing migration assay and transwell invasion assay, respectively. Quantitative reverse transcriptase polymerase chain reaction and western blot analysis was used to detected the expression level of HES1-10. RESULTS: The present study demonstrated that the Lnc-HES1-10 is overexpressed in OS and associated with poor prognosis of patients. In addition, the results revealed that Lnc-HES1-10 is overexpressed in MG63 and 143B OS cell lines and promote proliferation on both cell lines in vitro. Furthermore, migration and invasion abilities of MG63 and 143B cells are suppressed after silencing Lnc-HES1-10. CONCLUSION: Our finding demonstrates that HES1-10 plays a crucial role in regulating OS growth and metastasis.

Loss of HES1 expression is associated with extracellular matrix remodeling and tumor immune suppression in KRAS mutant colon adenocarcinomas.

The loss of HES1, a canonical Notch signaling target, may cooperate with KRAS mutations to remodel the extracellular matrix and to suppress the anti-tumor immune response. While HES1 expression is normal in benign hyperplastic polyps and normal colon tissue, HES1 expression is often lost in sessile serrated adenomas/polyps (SSAs/SSPs) and colorectal cancers (CRCs) such as those right-sided CRCs that commonly harbor BRAF or KRAS mutations. To develop a deeper understanding of interaction between KRAS and HES1 in colorectal carcinogenesis, we selected microsatellite stable (MSS) and KRAS mutant or KRAS wild type CRCs that show aberrant expression of HES1 by immunohistochemistry. By comparing the transcriptional landscapes of microsatellite stable (MSS) CRCs with or without nuclear HES1 expression, we investigated differentially expressed genes and activated pathways. We identified pathways and markers in the extracellular matrix and immune microenvironment that are associated with mutations in KRAS. We found that loss of HES1 expression positively correlated with matrix remodeling and epithelial-mesenchymal transition but negatively correlated with tumor cell proliferation. Furthermore, loss of HES1 expression in KRAS mutant CRCs correlates with a higher M2 macrophage polarization and activation of IL6 and IL10 immunosuppressive signature. Identifying these HES1-related markers may be useful for prognosis stratification and developing treatment for KRAS-mutant CRCs.

The Notch1/Hes1 signaling pathway affects autophagy by adjusting DNA methyltransferases expression in a valproic acid-induced autism spectrum disorder model.

As a pervasive neurodevelopmental disease, autism spectrum disorder (ASD) is caused by both hereditary and environmental elements. Research has demonstrated the functions of the Notch pathway and DNA methylation in the etiology of ASD. DNA methyltransferases DNMT3 and DNMT1 are responsible for methylation establishment and maintenance, respectively. In this study, we aimed to explore the association of DNA methyltransferases with the Notch pathway in ASD. Our results showed Notch1 and Hes1 were upregulated, while DNMT3A and DNMT3B were downregulated at the protein level in the prefrontal cortex (PFC), hippocampus (HC) and cerebellum (CB) of VPA-induced ASD rats compared with Control (Con) group. However, the protein levels of DNMT3A and DNMT3B were augmented after treatment with 3,5-difluorophenacetyl-L-alanyl-S-phenylglycine-2-butyl ester (DAPT), suggesting that abnormal Notch pathway activation may affect the expression of DNMT3A and DNMT3B. Besides, our previous findings revealed that the Notch pathway may participate in development of ASD by influencing autophagy. Therefore, we hypothesized the Notch pathway adjusts autophagy and contributes to ASD by affecting DNA methyltransferases. Our current results showed that after receiving the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-2'dc), the VPA + DAPT+5-Aza-2'dc (V + D + Aza) group exhibited reduced social interaction ability and increased stereotyped behaviors, and decreased expression of DNMT3A, DNMT3B and autophagy-related proteins, but did not show changes in Notch1 and Hes1 protein levels. Our results indicated that the Notch1/Hes1 pathway may adjust DNMT3A and DNMT3B expression and subsequently affect autophagy in the occurrence of ASD, providing new insight into the pathogenesis of ASD.

Early induction of Hes1 by bone morphogenetic protein 9 plays a regulatory role in osteoblastic differentiation of a mesenchymal stem cell line.

Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9.

Downregulation of a potential therapeutic target NPAS2, regulated by p53, alleviates pulmonary fibrosis by inhibiting epithelial-mesenchymal transition via suppressing HES1.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease and a severe form of pulmonary fibrosis. Epithelial-mesenchymal transition (EMT) of alveolar epithelial cells is induced in response to epithelial injury, which leads to the accumulation of extracellular matrix in the lung parenchyma and contributes to pulmonary fibrosis. NPAS2 (neuronal PAS domain protein 2) is significantly increased in the lung tissues of IPF patients according to microarray dataset GSE10667 and NPAS2 is downregulated in differentiated human pulmonary type 2 epithelial cells in vitro based on microarray dataset GSE3306 from Gene Expression Omnibus (GEO). In this study, we demonstrated that NPAS2 was increased in bleomycin (BLM)- induced fibrotic lungs in mice. Knockdown of NPAS2 inhibited EMT in primary mouse lung alveolar type 2 epithelial (pmATII) cells and human lung alveolar type 2 epithelial cell line A549 cells under BLM challenge in vitro. Moreover, the silence of NPAS2 alleviated the BLM-induced pulmonary fibrosis in a murine model. Mechanistically, NPAS2 promotes EMT through positively regulating hairy and enhancer of split 1 (HES1) expression. In this study, we present novel findings that have not been previously reported, emphasizing that p53 transcriptionally activates NPAS2 in ATII cells and overexpression of NPAS2 weakens the effects of TP53 knockdown on EMT of pmATII and A549 cells. Our results suggest NPAS2 is a novel target gene of p53 in regulating BLM-mediated EMT in ATII cells and pulmonary fibrosis.

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.

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.

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

BACKGROUND: Class 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. METHODS: The 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. RESULTS: Our 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. CONCLUSION: Our 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.

HES1-mediated down-regulation of miR-138 sustains NOTCH1 activation and promotes proliferation and invasion in renal cell carcinoma.

BACKGROUND: Although the aberrant activation of NOTCH1 pathway causes a malignant progression of renal cell carcinoma (RCC), the precise molecular mechanisms behind the potential action of pro-oncogenic NOTCH1/HES1 axis remain elusive. Here, we examined the role of tumor suppressive miR-138-2 in the regulation of NOTCH1-HES1-mediated promotion of RCC. METHODS: This study employed bioinformatics, xenotransplant mouse models, ChIP assay, luciferase reporter assay, functional experiments, real-time PCR and Western blot analysis to explore the mechanisms of miR-138-2 in the regulation of NOTCH1-HES1-mediated promotion of RCC, and further explored miR-138-2-containing combination treatment strategies. RESULTS: There existed a positive correlation between down-regulation of miR-138 and the aberrant augmentation of NOTCH1/HES1 regulatory axis. Mechanistically, HES1 directly bound to miR-138-2 promoter region and thereby attenuated the transcription of miR-138-5p as well as miR-138-2-3p. Further analysis revealed that miR-138-5p as well as miR-138-2-3p synergistically impairs pro-oncogenic NOTCH1 pathway through the direct targeting of APH1A, MAML1 and NOTCH1. CONCLUSIONS: Collectively, our current study strongly suggests that miR-138-2 acts as a novel epigenetic regulator of pro-oncogenic NOTCH1 pathway, and that the potential feedback regulatory loop composed of HES1, miR-138-2 and NOTCH1 contributes to the malignant development of RCC. From the clinical point of view, this feedback regulatory loop might be a promising therapeutic target to treat the patients with RCC.

HES1 deficiency impairs development of human intestinal mesenchyme by suppressing WNT5A expression.

Serious intestinal side-effects that target the NOTCH-HES1 pathway in human cancer differentiation therapy make it necessary to understand the pathway at the human organ level. Herein, we endogenously introduced HES1-/- mutations into human embryonic stem cells (hESCs) and differentiated them into human intestinal organoids (HIO). The HES1-/- hESCs retained ES cell properties and showed gene expression patterns similar to those of wild-type hESCs when they differentiated into definitive endoderm and hindgut. During the formation of the HES1-/- lumen we noted an impaired development of mesenchymal cells in addition to the increased differentiation of secretory epithelium. RNA-Seq revealed that inhibited development of the mesenchymal cells may have been due to a downregulation of WNT5A signaling. Overexpression of HES1 and silencing of WNT5A in the intestinal fibroblast cell line CCD-18Co indicated that HES1 was involved in the activation of WNT5A-induced fibroblast growth and migration, suggesting the likelihood of the Notch pathway in epithelial-mesenchymal crosstalk. Our results facilitated the identification of more precise underlying molecular mechanisms displaying distinct roles in HES1 signaling in stromal and epithelial development in human intestinal mucosa.

EGFL7 Secreted By Human Bone Mesenchymal Stem Cells Promotes Osteoblast Differentiation Partly Via Downregulation Of Notch1-Hes1 Signaling Pathway.

BACKGROUND: Epidermal growth factor-like domain protein 7 (EGFL7) is a secreted protein that is differentially expressed in the bone microenvironment; however, the effect of EGFL7 on the osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) is largely unknown. METHODS: EGFL7 expression in the fracture microenvironment was analyzed based on the Gene Expression Omnibus (GEO) database. Knockdown of EGFL7 by small interfering RNA (siRNA) and in vitro stimulation with recombinant human EGFL7 (rhEGFL7) protein were used to assess alterations in downstream signaling and changes in the osteogenic differentiation and proliferation of hBMSCs. A γ-secretase inhibitor was used to further explore whether inhibition of Notch signaling rescued the osteogenic-inhibitory effect of EGFL7 knockdown in hBMSCs. A femoral defect model was established to verify the effect of recombinant mouse EGFL7 on bone healing in vivo. RESULTS: EGFL7 expression increased during hBMSC osteogenesis. Knockdown of EGFL7 impaired hBMSC osteogenesis and activated Notch1/NICD/Hes1 signaling. rhEGFL7 promoted hBMSC osteogenesis and downregulated Notch1 signaling. The osteoblast-inhibitory effect of EGFL7 knockdown was rescued by Notch1 signaling inhibition. Recombinant EGFL7 led to enhanced bone healing in mice with femoral defects. CONCLUSIONS: EGFL7 promotes osteogenesis of hBMSCs partly via downregulation of Notch1 signaling.

HES1 promoter activation dynamics reveal the plasticity, stemness and heterogeneity in neuroblastoma cancer stem cells.

Notch signaling and its downstream gene target HES1 play a critical role in regulating and maintaining cancer stem cells (CSCs), similar to as they do during embryonic development. Here, we report a unique subclass of Notch-independent Hes-1 (NIHes-1)-expressing CSCs in neuroblastoma. These CSCs maintain sustained HES1 expression by activation of HES1 promoter region upstream of classical CBF-1 binding sites, thereby completely bypassing Notch receptor-mediated activation. These stem cells have self-renewal ability and potential to generate tumors. Interestingly, we observed that NIHes-1 CSCs could transition to Notch-dependent Hes-1-expressing (NDHes-1) CSCs where HES1 is expressed by Notch receptor-mediated promoter activation. We observed that NDHes-1-expressing CSCs also had the potential to transition to NIHes-1 CSCs and during this coordinated bidirectional transition, both CSCs gave rise to the majority of the bulk cancer cells, which had an inactive HES1 promoter (PIHes-1). A few of these PIHes-1 cells were capable of reverting into a CSC state. These findings explain the existence of a heterogenic mode of HES1 promoter activation within the IMR-32 neuroblastoma cell line and the potential to switch between them. This article has an associated First Person interview with the first authors of the paper.

Loss of Hes1 in embryonic stem cells caused developmental disorders in retinal pigment epithelium morphogenesis and specification.

Hairy and enhancer of split homolog-1 (Hes1) is a member of an extensive family of basic helix-loop-helix (bHLH) proteins and plays a crucial role in neurogenesis, myogenesis, hematopoiesis, and sex determination. It has been reported that Hes1 is essential for precursors maintenance, optic cup-stalk boundary maintenance, and morphogenesis of the retina. However, it still reminds questions about the role and mechanism of Hes1 in the development of retinal pigment epithelial cells. In our study, We generated Hes1-/- human embrsyonic stem cells, and attempted to induce them into retinal pigment epithelial cells by our previous protocol, found that the cells induced by Hes1-/- hESCs hardly expressed RPE-related genes, and rarely appeared RPE cell morphology. Additionally, Hes1 may affect the development of RPE cells via Wnt4 pathway by analyzing the RNA-seq data of differently expressed genes between normal RPE cells development and Hes1-/- RPE cells development. Overall, depletion of Hes1 may result in the failure of Wnt4 signal activation, and contributed to the developmental disorder in retinal pigment epithelium morphogenesis and specification.

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

BACKGROUND & AIMS: 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. METHODS: We used genetically engineered dual-recombinase mouse models for inducing Hes1 deletion under various conditions. RESULTS: 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. CONCLUSIONS: Our findings highlight the context-dependent roles of HES1 in the adult pancreas and pancreatic cancer.

Dysregulation of SIRT3 SUMOylation Confers AML Chemoresistance via Controlling HES1-Dependent Fatty Acid Oxidation.

Sirtuin 3 (SIRT3) deacetylase is a key regulator for chemoresistance in acute myeloid leukemia (AML) cells due to its capability of modulating mitochondrial metabolism and reactive oxygen species (ROS). SIRT3 is de-SUMOylated by SUMO-specific peptidase 1 (SENP1), which enhances its deacetylase activity. Therefore, dysregulation of SIRT3 SUMOylation may lead to fortified chemoresistance in AML. Indeed, SIRT3 de-SUMOylation was induced by chemotherapeutic agents, which in turn, exacerbated resistance against chemotherapies in AML by activating SIRT3 via preventing its proteasome degradation. Furthermore, RNA-seq revealed that expression of a collection of genes was altered by SIRT3 de-SUMOylation including inhibition of transcription factor Hes Family BHLH Transcription Factor 1 (HES1), a downstream substrate of Notch1 signaling pathway, leading to increased fatty acids oxidation (FAO). Moreover, the SENP1 inhibitor momordin-Ic or HES1 overexpression synergized with cytarabine to eradicate AML cells in vitro and in xenograft mouse models. In summary, the current study revealed a novel role of SIRT3 SUMOylation in the regulation of chemoresistance in AML via HES1-dependent FAO and provided a rationale for SIRT3 SUMOylation and FAO targeted interventions to improve chemotherapies in AML.

Identification and targeting of a HES1-YAP1-CDKN1C functional interaction in fusion-negative rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), a cancer characterized by features of skeletal muscle, is the most common soft-tissue sarcoma of childhood. With 5-year survival rates among high-risk groups at < 30%, new therapeutics are desperately needed. Previously, using a myoblast-based model of fusion-negative RMS (FN-RMS), we found that expression of the Hippo pathway effector transcriptional coactivator YAP1 (YAP1) permitted senescence bypass and subsequent transformation to malignant cells, mimicking FN-RMS. We also found that YAP1 engages in a positive feedback loop with Notch signaling to promote FN-RMS tumorigenesis. However, we could not identify an immediate downstream impact of this Hippo-Notch relationship. Here, we identify a HES1-YAP1-CDKN1C functional interaction, and show that knockdown of the Notch effector HES1 (Hes family BHLH transcription factor 1) impairs growth of multiple FN-RMS cell lines, with knockdown resulting in decreased YAP1 and increased CDKN1C expression. In silico mining of published proteomic and transcriptomic profiles of human RMS patient-derived xenografts revealed the same pattern of HES1-YAP1-CDKN1C expression. Treatment of FN-RMS cells in vitro with the recently described HES1 small-molecule inhibitor, JI130, limited FN-RMS cell growth. Inhibition of HES1 in vivo via conditional expression of a HES1-directed shRNA or JI130 dosing impaired FN-RMS tumor xenograft growth. Lastly, targeted transcriptomic profiling of FN-RMS xenografts in the context of HES1 suppression identified associations between HES1 and RAS-MAPK signaling. In summary, these in vitro and in vivo preclinical studies support the further investigation of HES1 as a therapeutic target in FN-RMS.

Ischemic preconditioning/ischemic postconditioning alleviates anoxia/reoxygenation injury via the Notch1/Hes1/VDAC1 axis.

Ischemic preconditioning (IPC), and ischemic postconditioning (IPost) have a significant protective effect on myocardial ischemia/reperfusion (MI/R) injury by alleviating oxidative stress and mitochondrial disturbances, although the underlying molecular mechanisms are unclear. The study was to demonstrate that cardioprotection against anoxia/reoxygenation (A/R) injury is transduced via the Notch1/Hes1/VDAC1 signaling pathway. Using mass spectrometry and tandem affinity purification (TAP), to screen for differentially expressed proteins associated with Hes1, followed by standard bioinformatics analysis. The co-immunoprecipitation (Co-IP) assay confirmed an interaction between Hes1 and VDAC1 proteins. H9c2 cells were transfected with Hes1 adenoviral N-terminal TAP vector (AD-NTAP/Hes1) and Hes1-short hairpin RNA adenoviral vector (AD-Hes1-shRNA) to establish A/R injury, IPC, and IPost models, respectively. The expression of Hes1 and VDAC1 proteins were measured by western blot analysis, while the levels of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and apoptosis were evaluated by flow cytometry. AD-NTAP/Hes1 can activate the exogenous protein expression of Hes1, thus decreasing creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) activity and promoting cell viability. The study found that VDAC1 was a potential target protein for Hes1 and the overexpression of Hes1 protein expression downregulated protein expression levels of VDAC1, reduced ROS production, stabilized ΔΨm, and inhibited apoptosis in H9c2 cells. Additionally, downregulation of Hes1 protein expression also upregulated VDAC1 protein expression, increased ROS production, imbalanced ΔΨm, promoted cell apoptosis, and attenuated the cardioprotection afforded by IPC and IPost. The Notch1/Hes1 signaling pathway activated by IPC/IPost can directly downregulate the protein expression of VDAC1 and consequently relieve A/R injury.