Sox9 Promoter Research Articles

Interaction of SOX5 with SOX9 promotes warfarin-induced aortic valve interstitial cell calcification by repressing transcriptional activation of LRP6

Calcific aortic valve disease (CAVD) is an important health burden due to its increasing prevalence and lack of available approaches. Osteogenic transdifferentiation of aortic valve interstitial cells (AVICs) contributes to valve calcification. SRY-related HMG-box transcription factor 5 (SOX5) is essential for cartilage development. Whether SOX5 is involved in AVIC calcification has not been determined. This study aimed to explore the role of SOX5 in warfarin-induced AVIC calcification. Immunostaining showed decreased SOX5 in human calcific AV and warfarin induced mouse calcific AV tissues compared with human noncalcific AV and control mouse AV tissues. In calcific human AVICs (hAVICs) and porcine AVICS (pAVICs), both knockdown and overexpression of SOX5 inhibited calcium deposition and osteogenic marker gene expression. Protein expression assays and ChIP assays showed that overexpression of SOX5 led to increased recruitment of SOX5 to the SOX9 promoter and resulted in increased mRNA and protein expression of SOX9. Coimmunoprecipitation and immunofluorescence showed that SOX5 binds to SOX9 with its HMG domain in nucleus. Blue Native PAGE showed overexpression of SOX5 led to multimeric complex formation of SOX5 and resulted in decreased binding of SOX5 to SOX9 similar to the results of knockdown of SOX5. Further ChIP and western blotting assays showed that both knockdown and overexpression of SOX5 resulted in SOX9 initiating transcription of anti-calcific gene LRP6 in warfarin-treated pAVICs. Knockdown of LRP6 rescues the anti-calcification effect of SOX5 overexpression. We found that both loss and gain of function of SOX5 lead to the same phenotype: decreased warfarin induced calcification. The stoichiometry of SOX5 is crucial for cooperation with SOX9, SOX9 nuclear localization and subsequent binding of SOX9 to LRP6 promoter. These results suggest that SOX5 is a potential target for the development of anti-calcification therapy.

Abstract P177: Pronethalol Decreases Rbpjk To Reduce Sox2 In Cerebral Arteriovenous Malformation

Cerebral arteriovenous malformations (AVMs) are the common vascular malformations that tend to rupture and cause hemorrhagic strokes. Disruptions in the integrity of vascular endothelium and endothelial cell (EC) differentiation give rise to the formation of cerebral AVMs. In previous studies, we reported that endothelial-mesenchymal transitions (EndMTs) contributed to cerebral AVMs in that ECs lost their identity and gained mesenchymal plasticity to cause lumen disorder. We have shown that unwanted induction of Sry-box 2 (Sox2) signaling was responsible for the EndMTs in cerebral AVMs, and identified the beta-adrenergic antagonist pronethalol as an inhibitor of Sox2 expression that stabilized EC differentiation and lumen formation, thereby limiting the cerebral AVMs. We also showed that the depletion of beta-adrenergic receptors had no effect on the Sox2 expression, which suggested that the beta antagonists exerted their effect through other pathways. However, the nature of these pathways remains unclear. Here, we hypothesize that beta-adrenergic antagonist directly inhibits notch-associated transcription factor recombination signal binding protein for immunoglobulin kappa J (RBPJκ), a key component of Notch signaling, to limit induction of Sox2 so as to improve cerebral AVMs. We treated MGP CRISPR cells, in which matrix Gla protein (Mgp) was depleted by using gene-editing tool of clustered regularly interspaced short palindromic repeats and its associated protein 9, with BMP6. We found that BMP6 induced Sox2, Notch1 and Jagged1 and 2. We further performed gene knockdown to reduce RBPJκ and found that the knockdown of RBPJκ abolished the Sox2 induction. We revealed that pronethalol significantly reduced the expression of RBPJκ but did not affect Notch1, Jagged1 or 2. We showed more enriched DNA-binding around the RBPJκ binding site in the Sox2 promoter of the MGP CRISPR cells than control cells. Excess RBPJκ DNA-binding was abolished by treatment of pronethalol in MGP CRISPR cells. Together, the results suggest that the beta antagonists reduce RBPJκ, thereby preventing excess BMP/Notch signaling from inducing Sox2.

Metabolic nuclear receptors coordinate energy metabolism to regulate Sox9+ hepatocyte fate

SummaryRecent research has indicated the adult liver Sox9+ cells located in the portal triads contribute to the physiological maintenance of liver mass and injury repair. However, the physiology and pathology regulation mechanisms of adult liver Sox9+ cells remain unknown. Here, PPARα and FXR bound to the shared site in Sox9 promoter with opposite transcriptional outputs. PPARα activation enhanced the fatty acid β-oxidation, oxidative phosphorylation (OXPHOS), and adenosine triphosphate (ATP) production, thus promoting proliferation and differentiation of Sox9+ hepatocytes along periportal (PP)-perivenous (PV) axis. However, FXR activation increased glycolysis but decreased OXPHOS and ATP production, therefore preventing proliferation of Sox9+ hepatocytes along PP-PV axis by promoting Sox9+ hepatocyte self-renewal. Our research indicates that metabolic nuclear receptors play critical roles in liver progenitor Sox9+ hepatocyte homeostasis to initiate or terminate liver injury-induced cell proliferation and differentiation, suggesting that PPARα and FXR are potential therapeutic targets for modulating liver regeneration.

H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

Chondrocyte differentiation is a critical process for endochondral ossification, which is responsible for long bone development and fracture repair. Considerable progress has been made in understanding the transcriptional control of chondrocyte differentiation; however, epigenetic regulation of chondrocyte differentiation remains to be further studied. NSD1 is a H3K36 (histone H3 at lysine 36) methyltransferase. Here, we showed that mice with Nsd1 deficiency in Prx1+ mesenchymal progenitors but not in Col2+ chondrocytes showed impaired skeletal growth and fracture healing accompanied by decreased chondrogenic differentiation. Via combined RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we identified sex determining region Y box 9 (Sox9), the key transcription factor of chondrogenic differentiation, as a functional target gene of NSD1. Mechanistically, NSD1 regulates Sox9 expression by modulating H3K36me1 and H3K36me2 levels in the Sox9 promoter region, constituting a novel epigenetic regulatory mechanism of chondrogenesis. Moreover, we found that NSD1 can directly activate the expression of hypoxia-inducible factor 1α (HIF1α), which plays a vital role in chondrogenic differentiation through its regulation of Sox9 expression. Collectively, the results of our study reveal crucial roles of NSD1 in regulating chondrogenic differentiation, skeletal growth, and fracture repair and expand our understanding of the function of epigenetic regulation in chondrogenesis and skeletal biology.

Metformin alleviates intervertebral disc degeneration by upregulating MMP-1 expression via the KDM6A/SOX9/miR-202-3p/MMP-1 signaling pathway

IntroductionMiR-202-3p is involved in the pathogenesis of intervertebral disc degeneration (IDD) via regulating MMP-1 expression in neuronal precursor cells (NPCs). As an activator of miR-202-3p and KDM6A expression, metformin (MET) may regulate the expression of SOX9 via regulating the methylation status of SOX9 promoter. However, the role of MET in the treatment of IDD remains to be explored.Material and methodsQuantitative real-time PCR was performed to analyze the expression of KDM66, SOX9, MMP-1 and miR-202-3p in NPCs and IDD rabbits. Western blot was carried out to evaluate the expression of KDM6A, SOX9 and MMP-1 protein under different conditions. Bisulfite sequencing PCR was used to analyze the DNA methylation of SOX9 promoter. Luciferase assay was carried out to explore the inhibitory effect of miR-202-3p upon MMP-1.ResultsThe expression of KDM6A, SOX9 and MMP-1 was abnormal in IDD cells and rabbits, while the MET treatment restored the normal expression of KDM6A, SOX9 and MMP-1 and miR-202-3p. Mechanistically, MET treatment reduced the level of hypermethylation of SOX9 promoter, thus restoring the expression of SOX9 in IDD cells and rabbits. The elevation in SOX9 expression promoted the expression of miR-202-3p, therefore inhibiting the expression of MMP-1, a downstream target of miR-202-3p.ConclusionsIn this study, we set up cellular and animal models of IDD and treated them with MET to probe the effect of MET on IDD and the signaling pathway of KDM6A/SOX9/miR-202-3p/MMP-1. Our work provided deep insight into the molecular mechanism underlying the therapeutic role of MET in the treatment of IDD.

Decitabine and Cisplatin are Synergistic to Exert Anti-Tumor Effect on Gastric Cancer via Inducing Sox2 DNA Demethylation.

BackgroundCisplatin is a vital chemotherapy regimen for gastric cancer (GC), while partial response is observed (approximately 40%) because of drug resistance. Thus, it is urgent to improve drug sensitivity to improve the therapeutic effect of cisplatin on GC.PurposeThe study was performed to explore the synergistic effect of decitabine and cisplatin in GC.Materials and MethodsCancer and matched adjacent tissues from patients with GC were obtained and quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry were performed to evaluate Sox2 expression level. Methylation-specific PCR (MSP) was performed to assess the effect of 5-aza-2ʹ-deoxycytidine (5-Aza-CdR) on Sox2 promoter. Cell proliferation assay, scratch-wound migration assay and Transwell invasion ability were performed to assess the effect of 5-Aza-CdR on proliferation, migration and invasion ability. Meantime, the effect of 5-Aza-CdR was also investigated in gastric cell lines BGC-823 and nude mouse xenograft tumor model. Finally, the anti-cancer effect of decitabine, cisplatin and their combination treatment were investigated in a BGC-823 and nude mouse xenograft tumor model, Sox2 methylation level, Sox2 expression of BGC-823 and xenograft tumors were analyzed by MSP, qRT-PCR and Western blot.ResultsSox2 expression was significantly associated with different differentiated degrees, depth of invasion (0.0011), lymph node metastasis (0.0013), and TNM stage (0.0002). Next, methylation inhibitor 5-Aza-CdR restored Sox2 expression to promote proliferation, migration and invasion in vitro and in vivo. Finally, cisplatin and decitabine was found to be synergistic to inhibit proliferation of xenograft tumors. Likewise, cisplatin and decitabine were also synergistic to induce Sox2 DNA demethylation to promote Sox2 mRNA and protein expression in BGC-823 and xenograft tumors.ConclusionCisplatin and decitabine could be synergistic to induce Sox2 DNA demethylation to promote expression of the Sox2 gene, which exerted an anti-tumor effect on GC. It may suggest an insight for innovative therapeutics of GC.

Histone demethylase JMJD2D promotes the self-renewal of liver cancer stem-like cells by enhancing EpCAM and Sox9 expression

Cancer stem-like cells (CSCs) contribute to the high rate of tumor heterogeneity, metastasis, therapeutic resistance, and recurrence. Histone lysine demethylase 4D (KDM4D or JMJD2D) is highly expressed in colon and liver tumors, where it promotes cancer progression; however, the role of JMJD2D in CSCs remains unclear. Here, we show that JMJD2D expression was increased in liver cancer stem-like cells (LCSCs); downregulation of JMJD2D inhibited the self-renewal of LCSCs in vitro and in vivo and inhibited the lung metastasis of LCSCs by reducing the survival and the early lung seeding of circulating LCSCs. Mechanistically, JMJD2D promoted LCSC self-renewal by enhancing the expression of CSC markers EpCAM and Sox9; JMJD2D reduced H3K9me3 levels on the promoters of EpCAM and Sox9 to enhance their transcription via interaction with β-catenin/TCF4 and Notch1 intracellular domain, respectively. Restoration of EpCAM and Sox9 expression in JMJD2D-knockdown liver cancer cells rescued the self-renewal of LCSCs. Pharmacological inhibition of JMJD2D using 5-c-8HQ reduced the self-renewal of LCSCs and liver cancer progression. Collectively, our findings suggest that JMJD2D promotes LCSC self-renewal by enhancing EpCAM and Sox9 expression via Wnt/β-catenin and Notch signaling pathways and is a potential therapeutic target for liver cancer.

MBRS-10. QUIESCENT SOX9-POSITIVE CELLS BEHIND MYC DRIVEN MEDULLOBLASTOMA RECURRENCE

Tumor recurrence is the leading cause of death in medulloblastoma, the most frequent malignant pediatric brain tumor. Recurrence occurs when subpopulations of cancer cells evade standard therapy by acquiring features of immune escape, metastatic spread, and treatment resistance. The transcription factor SOX9 correlated with treatment resistance and dissemination in aggressive Group 3 medulloblastoma. By studying paired primary-recurrent medulloblastoma samples and patient-derived xenograft models, we identified rare SOX9-positive slow-cycling, therapy-resistant tumor cells that accumulate in relapses and in metastases. In an inducible transgenic Group 3 tumor model, doxycycline treatment kills all tumor cells by turning MYC off. However, when MYC expression was redirected to the SOX9 promoter, recurrences from rare, dormant SOX9-positive cells developed with 100% penetrance. Expression profiling revealed that recurrences were more inflammatory, metastatic, and showed elevated MGMT methyltransferase levels which depleted recurrent cells when selectively inhibited. Our model explains how recurrences develop from SOX9-induced quiescence in MYC-driven brain cancer.

Sclerostin Antibody Administration Increases the Numbers of Sox9creER+ Skeletal Precursors and Their Progeny.

Blocking the Wnt inhibitor, sclerostin, increases the rate of bone formation in rodents and in humans. On a cellular level, the antibody against sclerostin acts by increasing osteoblast numbers partly by activating the quiescent bone-lining cells in vivo. No evidence currently exists, to determine whether blocking sclerostin affects early cells of the osteoblast lineage. Here we use a lineage-tracing strategy that uses a tamoxifen-dependent cre recombinase, driven by the Sox9 promoter to mark early cells of the osteoblast lineage. We show that, when adult mice are treated with the rat-13C7, an antibody that blocks sclerostin action in rodents, it increases the numbers of osteoblast precursors and their differentiation into mature osteoblasts in vivo. We also show that rat-13C7 administration suppresses adipogenesis by suppressing the differentiation of Sox9creER+ skeletal precursors into bone marrow adipocytes in vivo. Using floxed alleles of the CTNNB1 gene encoding β-catenin, we show that these precursor cells express the canonical Wnt signaling mediator, β-catenin, and that the actions of the rat-13C7 antibody to increase the number of early precursors is dependent on direct stimulation of Wnt signaling. The increase in osteoblast precursors and their progeny after the administration of the antibody leads to a robust suppression of apoptosis without affecting the rate of their proliferation. Thus, neutralizing the Wnt-inhibitor sclerostin increases the numbers of early cells of the osteoblast lineage osteoblasts and suppresses their differentiation into adipocytes in vivo. © 2021 American Society for Bone and Mineral Research (ASBMR).

Histone Lysine Demethylase JMJD2D/KDM4D and Family Members Mediate Effects of Chronic Social Defeat Stress on Mouse Hippocampal Neurogenesis and Mood Disorders.

Depression, anxiety and related mood disorders are major psychiatric illnesses worldwide, and chronic stress appears to be one of the primary underlying causes. Therapeutics to treat these debilitating disorders without a relapse are limited due to the incomplete molecular understanding of their etiopathology. In addition to the well-studied genetic component, research in the past two decades has implicated diverse epigenetic mechanisms in mediating the negative effects of chronic stressful events on neural circuits. This includes the cognitive circuitry, where the dynamic hippocampal dentate gyrus (DG) neurogenesis gets affected in depression and related affective disorders. Most of these epigenetic studies have focused on the impact of acetylation/deacetylation and methylation of several histone lysine residues on neural gene expression. However, there is a dearth of investigation into the role of demethylation of these lysine residues in chronic stress-induced changes in neurogenesis that results in altered behaviour. Here, using the chronic social defeat stress (CSDS) paradigm to induce depression and anxiety in C57BL/6 mice and ex vivo DG neural stem/progenitor cell (NSCs/NPCs) culture we show the role of the members of the JMJD2/KDM4 family of histone lysine demethylases (KDMs) in mediating stress-induced changes in DG neurogenesis and mood disorders. The study suggests a critical role of JMJD2D in DG neurogenesis. Altered enrichment of JMJD2D on the promoters of Id2 (inhibitor of differentiation 2) and Sox2 (SRY-Box Transcription Factor 2) was observed during proliferation and differentiation of NSCs/NPCs obtained from the DG. This would affect the demethylation of repressive epigenetic mark H3K9, thus activating or repressing these and possibly other genes involved in regulating proliferation and differentiation of DG NSCs/NPCs. Treatment of the NSCs/NPCs culture with Dimethyloxallyl Glycine (DMOG), an inhibitor of JMJDs, led to attenuation in their proliferation capacity. Additionally, systemic administration of DMOG in mice for 10 days induced depression-like and anxiety-like phenotype without any stress exposure.

NF‐YA promotes the cell proliferation and tumorigenic properties by transcriptional activation of SOX2 in cervical cancer

NF‐YA is considered as a crucial regulator for the maintenance of cancer stem cell (CSC) and involved in various types of malignant tumours. However, the exact function and molecular mechanisms of NF‐YA in the progression of cervical cancer remains poorly understood. Here, the expression of NF‐YA detected by immunohistochemistry was gradually increased from normal cervical tissues, to the high‐grade squamous intraepithelial lesions, and then to cervical cancer tissues. NF‐YA promoted the cell proliferation and tumorigenic properties of cervical cancer cells as well as tumorsphere formation and chemoresistance in vitro. The luciferase reporter assay combined with mutagenesis analyses and Western blotting showed that NF‐YA trans‐activated the expression of SOX2 in cervical cancer. Furthermore, quantitative chromatin immunoprecipitation (qChIP) and electrophoretic mobility shift assay (EMSA) confirmed that NF‐YA protein directly bound to the CCAAT box region located upstream of the SOX2 promoter. Together, our data demonstrated that NF‐YA was highly expressed in cervical cancer and promoted the cell proliferation, tumorigenicity and CSC characteristic by trans‐activating the expression of SOX2.

Inhibition of LSD1 promotes the differentiation of human induced pluripotent stem cells into insulin-producing cells

BackgroundHuman induced pluripotent stem cells (hiPSCs) represent a potentially unlimited source of pancreatic endocrine lineage cells. Although insulin-producing β cells derived from hiPSCs have been successfully induced, much work remains to be done to achieve mature β cells. Lysine-specific demethylase 1 (LSD1) plays an important role in the regulation of hiPSC self-renewal and differentiation. We propose a new strategy to acquire insulin-producing cells (IPCs) from hiPSCs by knocking down LSD1.MethodsKnockdown of LSD1 in hiPSCs with five shRNA. Assessment of the effects of shRNA on hiPSC proliferation, cell cycle, and apoptosis. Using knockdown hiPSCs with 31.33% LSD1 activity, we achieved a four-step differentiation into IPCs and test its differentiation efficiency, morphology, and marker genes and proteins. We implanted the IPCs into the renal subcapsular of SCID-Beige diabetic mice to evaluate the hypoglycemic effect in vivo. We tested LSD1 and HDAC1 whether they are present in the CoREST complex through IP-WB, and analyzed LSD1, CoREST, HDAC1, H3K4me2/me3, and H3K27me3 protein expression before and after knockdown of LSD1.ResultsDifferentiated hiPSCs were 38.32% ± 3.54% insulin-positive cells and released insulin/C-peptide in response to glucose stimulus in a manner comparable to adult human islets. Most of the IPCs co-expressed mature β cell-specific markers. When transplanted under the left renal capsule of SCID-Beige diabetic mice, these IPCs reversed hyperglycemia, leading to a significant increase in the definitive endoderm cells. IP-WB results showed that LSD1, HDAC1, and CoREST formed a complex in hiPSCs. Chip-PCR results showed that LSD1, HDAC1, and CoREST were enriched in the same district during the SOX17 and FOXA2 promoter region. Inhibition of LSD1 would not affect the level of CoREST but decreased the HDAC1 expressions. The H3K4me2/me3 and H3K9act level of SOX17 and FOXA2 promoter region increased after inhibited of LSD1, and promoted transcriptional activation. The H3K4me2/me3 and H3K9act level of OCT4 and SOX2 promoter region decreased with the transcriptional repressed.ConclusionsLSD1 regulated histone methylation and acetylation in promoter regions of pluripotent or endodermal genes. Our results suggest a highly efficient approach to producing IPCs from hiPSCs.

Pranlukast Antagonizes CD49f and Reduces Stemness in Triple-Negative Breast Cancer Cells.

IntroductionCancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors. CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as CD49f antagonists.Materials and MethodsWe performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clinically tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation.ResultsPranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of CD49f+ MDA-MB-231 cells to laminin, indicating that it antagonizes CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces conformational changes in CD49f that affect its interaction with β1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transactivation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC.ConclusionOur results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients.

Mevalonate Pathway Enzyme HMGCS1 Contributes to Gastric Cancer Progression.

The 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) is a potential regulatory node in the mevalonate pathway that is frequently dysregulated in tumors. This study found that HMGCS1 expression is upregulated in stomach adenocarcinoma samples of patients and tumorspheres of gastric cancer cells. HMGCS1 elevates the expression levels of the pluripotency genes Oct4 and SOX-2 and contributes to tumorsphere formation ability in gastric cancer cells. HMGCS1 also promotes in vitro cell growth and progression and the in vivo tumor growth and lung metastasis of gastric cancer cells. After blocking the mevalonate pathway by statin and dipyridamole, HMGCS1 exerts nonmetabolic functions in enhancing gastric cancer progression. Furthermore, the level and nuclear translocation of HMGCS1 in gastric cancer cells are induced by serum deprivation. HMGCS1 binds to and activates Oct4 and SOX-2 promoters. HMGCS1 also enhances the integrated stress response (ISR) and interacts with the endoplasmic reticulum (ER) stress transducer protein kinase RNA-like endoplasmic reticulum kinase (PERK). Our results reveal that HMGCS1 contributes to gastric cancer progression in both metabolic and nonmetabolic manners.

Lipid availability determines fate of skeletal progenitor cells via SOX9.

The avascular nature of cartilage makes it a unique tissue1–4, but whether and how the absence of nutrient supply regulates chondrogenesis remains unknown. Here, we show that obstruction of vascular invasion during bone healing favours chondrogenic over osteogenic differentiation of skeletal progenitor cells. Unexpectedly, this process is driven by a decreased availability of extracellular lipids. When lipids are scarce, skeletal progenitors activate FoxO transcription factors, which bind to the Sox9 promoter and increase its expression. Besides initiating chondrogenesis, SOX9 acts as a regulator of cellular metabolism by suppressing fatty acid oxidation, and thus adapts the cells to an avascular life. Our results define lipid scarcity as an important determinant of chondrogenic commitment, reveal a role for FoxOs during lipid starvation, and identify SOX9 as a critical metabolic mediator. These data highlight the importance of the nutritional microenvironment in the specification of skeletal cell fate.

The therapeutic effect of bone marrow-derived mesenchymal stem cells on osteoarthritis is improved by the activation of the KDM6A/SOX9 signaling pathway caused by exposure to hypoxia.

Abnormal expression of KDM6A and SOX9 is a key factor in the pathogenesis of osteoarthritis (OA). Cellular treatments of OA with articular cartilage chondrocytes (ACCs) and bone marrow mesenchymal stem cells (BMSCs) are promising, but their underlying mechanisms remain to be explored. The pellet size, weight and sulfated glycosaminoglycan/DNA content of ACCs were measured to evaluate the effect of BMSCs on the chondrogenic differentiation of SCCs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyze the proliferation of ACCs cultured along or cocultured with BMSCs. Quantitative polymerase chain reaction (qPCR) was performed to evaluate the messenger RNA expression of KDM6A, SOX9, type2 collagen, and Aggrecan in ACCs and OA rats. Western blot and immunohistochemistry were performed to analyze the expression of KDM6A and SOX9 proteins. Bisulfite sequencing PCR was performed to assess the DNA methylation level of the SOX9 promoter. Flow cytometry was used to evaluate the apoptotic status of ACCs. The chondrogenic differentiation of ACCs was significantly enhanced by coculturing with BMSCs, especially under a hypoxic condition. The expression of KDM6A, SOX9, type2 collagen, and Aggrecan was remarkably elevated in ACCs cocultured with BMSCs. Also, the DNA methylation of SOX9 promoter was decreased in ACCs cocultured with BMSCs, along with notably reduced apoptosis. Moreover, ACCs cocultured with BMSCs could repair cartilage lesions and prevent the abnormal expression of KDM6A, SOX9, type2 collagen, and Aggrecan in OA rats. In this study, we cocultured ACCs with BMSCs and used them to treat OA rats. Our findings presented a mechanistic basis for explaining the therapeutic effect of BMSCs on OA treatment.

Metastatic Phosphatase PRL-3 Induces Ovarian Cancer Stem Cell Sub-population through Phosphatase-Independent Deacetylation Modulations.

SummaryCancer stem cells (CSCs) are responsible for tumor initiation, chemoresistance, metastasis, and relapse, but the underlying molecular origin of CSCs remains elusive. Here we identified that metastatic phosphatase of regenerating liver 3 (PRL-3) transcriptionally upregulates SOX2 in the expansion of CSC sub-population from normal cancer cells. Mechanistically, SOX2 upregulation is attributed to the binding of the acetylated myocyte enhancer factor 2A (MEF2A) to SOX2 promoter in tumor cells. In parallel, PRL-3 competitively binds to Class IIa histone deacetylase 4 (HDAC4) to facilitate HDAC4 translocation, leading to the disassociation of HDAC4 from MEF2A and histones. The released MEF2A and histones thus remain acetylated and render the subsequent accessibility of the acetylated MEF2A to SOX2 promoter region. Clinical relevance among PRL-3, SOX2, and HDAC4 is validated in ovary cancer samples. Therefore, this PRL-3-HDAC4-MEF2A/histones-SOX2 signaling axis would be a potential therapeutic target in inhibiting ovarian cancer metastasis and relapse.

STEM-17. LOW GRADE ASTROCYTOMA MUTATIONS COOPERATE TO DISRUPT SOX2 GENOMIC ARCHITECTURE AND BLOCK DIFFERENTIATION VIA PREVIOUSLY UNIDENTIFIED ENHANCER ELEMENTS

Abstract Neomorphic mutations in isocitrate dehydrogenase I (IDH1) result in the formation of the oncometabolite 2-hydroxyglutarate (2HG) in a significant subset of gliomas and other tumors including acute myeloid leukemias. Preclinical evidence suggests that gliomas harboring IDH1 mutations undergo widespread, long-lasting modifications to the epigenome that persist following inhibition of 2HG production. However, the exact mechanism underlying gliomagenesis remains unclear. To address difficulties in growing these tumors in culture, our group generated a model of low-grade astrocytoma in human neural stem cells (NSCs). This model, referred to as 3-Hit NSCs, suggested a block in differentiation potential underlies gliomagenesis at the cellular level. This block is rescued by restoration of expression of the transcription factor (sex determining region Y)-box 2 (SOX2), which is transcriptionally downregulated during IDH mutant gliomagenesis. We believe that these changes occur secondary to profound alterations in 3-dimensional chromatin organization around the SOX2 genomic locus. Our preliminary data suggest SOX2 expression in control NSCs depends on 3-dimensional association of its promoter to an uncharacterized, distal enhancer located 600 kb telomeric to the SOX2 gene. We believe this association is disrupted in 3-Hit NSCs due to eviction of chromatin organizer CTCF from its motifs in the SOX2 topologically associating domain (TAD). To test this hypothesis, we used CRISPR-Cas9 technology to excise CTCF motifs immediately upstream of the SOX2 promoter and in the region of the putative enhancer in control NSCs. Excision of such motifs significantly reduced SOX2 mRNA levels and impaired growth of control NSCs. We are currently working on characterizing this novel SOX2-enhancer interaction in native stem cells, as well as tumors that depend on SOX2 expression. This works aims to elucidate the core epigenetic mechanisms underlying IDH mutant gliomagenesis. Our findings will be used to improve therapy in IDH-mutant glioma.

DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor-Positive Breast Cancer Following Endocrine Therapy.

Estrogen receptor (ER)-positive breast cancer recurrence is thought to be driven by tumor-initiating cells (TIC). TICs are enriched by endocrine therapy through NOTCH signaling. Side effects have limited clinical trial testing of NOTCH-targeted therapies. Death-associated factor 6 (DAXX) is a newly identified marker whose RNA expression inversely correlates with NOTCH in human ER+ breast tumor samples. In this study, knockdown and overexpression approaches were used to investigate the role of DAXX on stem/pluripotent gene expression, TIC survival in vitro, and TIC frequency in vivo, and the mechanism by which DAXX suppresses TICs in ER+ breast cancer. 17β-Estradiol (E2)-mediated ER activation stabilized the DAXX protein, which was required for repressing stem/pluripotent genes (NOTCH4, SOX2, OCT4, NANOG, and ALDH1A1), and TICs in vitro and in vivo. Conversely, endocrine therapy promoted rapid protein depletion due to increased proteasome activity. DAXX was enriched at promoters of stem/pluripotent genes, which was lost with endocrine therapy. Ectopic expression of DAXX decreased stem/pluripotent gene transcripts to levels similar to E2 treatment. DAXX-mediated repression of stem/pluripotent genes and suppression of TICs was dependent on DNMT1. DAXX or DNMT1 was necessary to inhibit methylation of CpGs within the SOX2 promoter and moderately within the gene body of NOTCH4, NOTCH activation, and TIC survival. E2-mediated stabilization of DAXX was necessary and sufficient to repress stem/pluripotent genes by recruiting DNMT1 to methylate some promoters and suppress TICs. These findings suggest that a combination of endocrine therapy and DAXX-stabilizing agents may inhibit ER+ tumor recurrence. SIGNIFICANCE: Estradiol-mediated stabilization of DAXX is necessary and sufficient to repress genes associated with stemness, suggesting that the combination of endocrine therapy and DAXX-stabilizing agents may inhibit tumor recurrence in ER+ breast cancer.

Inhibition of EZH2 ameliorates cartilage endplate degeneration and attenuates the progression of intervertebral disc degeneration via demethylation of Sox-9.

BackgroundCartilaginous endplate (CEP) degeneration is considered as one of the major causes of intervertebral disc degeneration (IVDD) which causes low back pain. Recent studies have proved that epigenetic alteration is involved in a variety of diseases. This work explored the role of histone methyltransferase enhancer of zeste homologue 2 (EZH2) in CEP degeneration, as well as its underlying epigenetic mechanisms, and confirmed the effect of EZH2 knockdown on delaying IVDD development.MethodsWestern blotting, immunofluorescence staining, and ChIP assay were applied to demonstrate the molecular mechanism of EZH2 in CEP tissue. The therapeutic potential of EZH2 was investigated using puncture-induced rat models.FindingsThe EZH2 expression was upregulated in human and rat CEP tissue. It was also found that the overexpression of EZH2 suppressed the expression of Collagen II, aggrecan and Sox-9, and promoted the expression of ADTAMTS5 and MMP13 in rat endplate chondrocytes (EPCs), which could be reversed by EZH2 silencing. The correlation between EZH2 and Sox-9 was further explored, while overexpression of Sox-9 could reverse the effect of EZH2 in rat EPCs. Moreover, inhibition of EZH2 upregulated the level of Sox-9 by demethylating H3K27me3 at Sox-9 promoter sites, revealing the regulatory mechanism of EZH2 on Sox-9. Meanwhile, puncture-induced rat models showed that EZH2 knockdown exerted a protective effect on CEP and disc degeneration.InterpretationThis study reveals that EZH2 inhibition is a promising strategy for mitigating the symptoms and progression of IVDD.Funding: This study was funded by the Natural Science Foundation of Zhejiang Province (Y16H060034). Authors declare that the funders had no involvement in the study design, data analysis and interpretation of the results.