RBM15-Mediated N6-Methyl Adenosine (m6A) Modification of EZH2 Drives the Epithelial-Mesenchymal Transition of Cervical Cancer.

RBM15 functions as an oncogene in multi-type cancers. However, the reports on the roles of RBM15 in cervical cancer are limited. The purpose of this study was to investigate the potentials of RBM15 in cervical cancer. RT-qPCR was conducted to determine mRNA levels. Western was carried out to detect protein expression. CCK-8, colony formation and EdU assays were conducted to determine cell proliferation. Scratch and transwell assays were conducted to determine cell migration and invasion. MeRIP assay was conducted to determine N6-methyl adenosine (m6A) levels. Luciferase assay was conducted to verify the m6A sites of EZH2 and binding sites between EZH2 and promoter of FN1. ChIP assay was conducted to verify the interaction between EZH2 and FN1. The results showed that RBM15 was upregulated in cervical cancer patients and cells. Moreover, high levels of RBM15 predicted poor clinical outcomes. RBM15 knockdown inhibited the proliferation and epithelial-mesenchymal transition (EMT) of cervical cancer cells. RBM15 promoted the m6A modification of EZH2 as well as its protein translation. Additionally, EZH2 bound to the promoter of fibronectin 1 (FN1) and EZH2-FN1 axis is the cascade downstream of RBM15. Overexpressed EZH2 antagonized the effects of RBM15 knockdown and promoted the aggressiveness of cervical cancer cells. In summary, RBM15/EZH2/FN1 signaling cascade induces the proliferation and EMT of cervical cancer. Therefore, RBM15/EZH2/FN1 signaling may be a promising strategy for cervical cancer.

The Cell Cycle: a Key to Unlock EZH2-targeted Therapy Resistance.

SUMMARY: In this issue, a study by Kazansky and colleagues explored resistance mechanisms after EZH2 inhibition in malignant rhabdoid tumors (MRT) and epithelioid sarcomas (ES). The study identified genetic alterations in EZH2 itself, along with alterations that converge on RB1-E2F-mediated cell-cycle control, and demonstrated that inhibition of cell-cycle kinases, such as Aurora Kinase B (AURKB) could bypass EZH2 inhibitor resistance to enhance treatment efficacy. See related article by Kazansky et al., p. 965 (6).

Targeting dependency on a paralog pair of CBP/p300 against de-repression of KREMEN2 in SMARCB1-deficient cancers.

SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, is the causative gene of rhabdoid tumors and epithelioid sarcomas. Here, we identify a paralog pair of CBP and p300 as a synthetic lethal target in SMARCB1-deficient cancers by using a dual siRNA screening method based on the "simultaneous inhibition of a paralog pair" concept. Treatment with CBP/p300 dual inhibitors suppresses growth of cell lines and tumor xenografts derived from SMARCB1-deficient cells but not from SMARCB1-proficient cells. SMARCB1-containing SWI/SNF complexes localize with H3K27me3 and its methyltransferase EZH2 at the promotor region of the KREMEN2 locus, resulting in transcriptional downregulation of KREMEN2. By contrast, SMARCB1 deficiency leads to localization of H3K27ac, and recruitment of its acetyltransferases CBP and p300, at the KREMEN2 locus, resulting in transcriptional upregulation of KREMEN2, which cooperates with the SMARCA1 chromatin remodeling complex. Simultaneous inhibition of CBP/p300 leads to transcriptional downregulation of KREMEN2, followed by apoptosis induction via monomerization of KREMEN1 due to a failure to interact with KREMEN2, which suppresses anti-apoptotic signaling pathways. Taken together, our findings indicate that simultaneous inhibitors of CBP/p300 could be promising therapeutic agents for SMARCB1-deficient cancers.

Thyroid papillary carcinoma combined with primary follicular lymphoma: a case report.

BACKGROUND: Papillary thyroid carcinoma (PTC) stands out as the most prevalent epithelial malignant thyroid tumor. Thyroid primary follicular lymphoma (PFL) represents a rare malignant tumor originating from mesenchymal tissues. The concurrent occurrence of PTC and PFL is exceptionally rare, particularly in the context of Hashimoto's thyroiditis, presenting significant challenges in clinical diagnosis and treatment. CASE DEMONSTRATION: A 44-year-old female patient presented with a neck mass persisting for over 1 month. The patient underwent surgery, and the incised tissues were subjected to pathology examinations, along with immunohistochemistry and next-generation sequencing tests suggestive of an EZH2 gene mutation in the tumor cells. The final pathological diagnosis confirmed the presence of PTC combined with PFL. Following a 27-month follow-up, the patient displayed no signs of recurrence or metastasis. CONCLUSIONS: The concurrent occurrence of PTC and PFL poses notable challenges in clinical practice, requiring careful consideration in diagnosis and treatment. Herein, we present a rare case of PTC combined with PFL featuring an EZH2 gene mutation, which can be easily overlooked in the context of Hashimoto's thyroiditis. The patient's favorable response to surgical and radiotherapeutic interventions underscores the importance of accurate diagnosis and tailored treatment strategies in similar cases.

EZH2 Expression in Head-and-Neck Squamous Cell Cancer in Young Patients.

EZH2 (Enhancer of zeste homolog 2) promotes tumor growth and survival through numerous mechanisms and is a promising target for novel therapeutic approaches. We aimed to characterize the expression of EZH2 in the tumors of young head-and-neck squamous cell cancer (HNSCC) patients in comparison with the general HNSCC patient population. We used formalin-fixed, paraffin-embedded tissue blocks from 68 random young HNSCC patients (≤39 years, median age: 36 years; diagnosed between 2000 and 2018), which were compared with the samples of 58 age- and gender-matched general HNSCC subjects (median age: 62 years; all diagnosed in the year 2014). EZH2 and p53 expression of the tumors was detected using immunohistochemical staining. Lower EZH2 expression was found to be characteristic of the tumors of young HNSCC patients as opposed to the general population (median EZH2 staining intensity: 1 vs. 1.5 respectively, p < 0.001; median fraction of EZH2 positive tumor cells: 40% vs. 60%, respectively, p = 0.003, Mann-Whitney). Cox analysis identified a more advanced T status (T3-4 vs. T1-2), a positive nodal status, and alcohol consumption, but neither intratumoral EZH2 nor p53 were identified as predictors of mortality in the young patient group. The lower EZH2 expression of young HNSCC patients' tumors discourages speculations of a more malignant phenotype of early-onset tumors and suggests the dominant role of patient characteristics. Furthermore, our results might indicate the possibility of an altered efficacy of the novel anti-EZH2 therapies in this patient subgroup.

Tumor-Intrinsic Enhancer of Zeste Homolog 2 Controls Immune Cell Infiltration, Tumor Growth, and Lung Metastasis in a Triple-Negative Breast Cancer Model.

Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic type of tumor. TNBC is often enriched in tumor-infiltrating neutrophils (TINs), which support cancer growth in part by counteracting tumor-infiltrating lymphocytes (TILs). Prior studies identified the enhancer of zeste homolog 2 (EZH2) as a pro-tumor methyltransferase in primary and metastatic TNBCs. We hypothesized that EZH2 inhibition in TNBC cells per se would exert antitumor activity by altering the tumor immune microenvironment. To test this hypothesis, we used CRISPR to generate EZH2 gene knockout (KO) and overexpressing (OE) lines from parent (wild-type-WT) 4T1 cells, an established murine TNBC model, resulting in EZH2 protein KO and OE, respectively. In vitro, EZH2 KO and OE cells showed early, transient changes in replicative capacity and invasiveness, and marked changes in surface marker profile and cytokine/chemokine secretion compared to WT cells. In vivo, EZH2 KO cells showed significantly reduced primary tumor growth and a 10-fold decrease in lung metastasis compared to WT cells, while EZH2 OE cells were unchanged. Compared to WT tumors, TIN:TIL ratios were greatly reduced in EZH2 KO tumors but unchanged in EZH2 OE tumors. Thus, EZH2 is key to 4T1 aggressiveness as its tumor-intrinsic knockout alters their in vitro secretome and in vivo primary tumor growth, TIN/TIL poise, and metastasis.

Long non-coding RNA HOTAIR: from pan-cancer analysis to colorectal cancer-related uridine metabolism.

Long non-coding RNAs (lncRNAs) are involved significantly in the development of human cancers. lncRNA HOTAIR has been reported to play an oncogenic role in many human cancers. Its specific regulatory role is still elusive. And it might have enormous potential to interpret the malignant progression of tumors in a broader perspective, that is, in pan-cancer. We comprehensively investigated the effect of HOTAIR expression on tumor prognosis across human malignancies by analyzing multiple cancer-related databases like The Cancer Genome Atlas (TCGA) and Tumor Immune Estimation Resource (TIMER). Bioinformatics data indicated that HOTAIR was overexpressed in most of these human malignancies and was significantly associated with the prognosis of patients with cancer, especially in colorectal cancer (CRC). Subsequently, this study further clarified the utility of HOTAIR that downregulation of its expression could result in reduced proliferation and invasion of CRC cells. Mechanistically, HOTAIR upregulated the metabolic enzymes UPP1 by recruiting histone methyltransferase EZH2, thereby increasing the tumor progression. Our results highlight the essential role of HOTAIR in pan-cancer and uridine bypass, suggesting that the HOTAIR/EZH2/UPP1 axis might be a novel target for overcoming CRC. We anticipate that the role of HOTAIR in metabolism could be important in the context of CRC and even exploited for therapeutic purposes.

Repressive H3K27me3 drives hyperglycemia-induced oxidative and inflammatory transcriptional programs in human endothelium.

BACKGROUND: Histone modifications play a critical role in chromatin remodelling and regulate gene expression in health and disease. Histone methyltransferases EZH1, EZH2, and demethylases UTX, JMJD3, and UTY catalyse trimethylation of lysine 27 on histone H3 (H3K27me3). This study was designed to investigate whether H3K27me3 triggers hyperglycemia-induced oxidative and inflammatory transcriptional programs in the endothelium. METHODS: We studied human aortic endothelial cells exposed to high glucose (HAEC) or isolated from individuals with diabetes (D-HAEC). RT-qPCR, immunoblotting, chromatin immunoprecipitation (ChIP-qPCR), and confocal microscopy were performed to investigate the role of H3K27me3. We determined superoxide anion (O2-) production by ESR spectroscopy, NF-κB binding activity, and monocyte adhesion. Silencing/overexpression and pharmacological inhibition of chromatin modifying enzymes were used to modulate H3K27me3 levels. Furthermore, isometric tension studies and immunohistochemistry were performed in aorta from wild-type and db/db mice. RESULTS: Incubation of HAEC to high glucose showed that upregulation of EZH2 coupled to reduced demethylase UTX and JMJD3 was responsible for the increased H3K27me3. ChIP-qPCR revealed that repressive H3K27me3 binding to superoxide dismutase and transcription factor JunD promoters is involved in glucose-induced O2- generation. Indeed, loss of JunD transcriptional inhibition favours NOX4 expression. Furthermore, H3K27me3-driven oxidative stress increased NF-κB p65 activity and downstream inflammatory genes. Interestingly, EZH2 inhibitor GSK126 rescued these endothelial derangements by reducing H3K27me3. We also found that H3K27me3 epigenetic signature alters transcriptional programs in D-HAEC and aortas from db/db mice. CONCLUSIONS: EZH2-mediated H3K27me3 represents a key epigenetic driver of hyperglycemia-induced endothelial dysfunction. Targeting EZH2 may attenuate oxidative stress and inflammation and, hence, prevent vascular disease in diabetes.

EZH2 as a major histone methyltransferase in PDGF-BB-activated orbital fibroblast in the pathogenesis of Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is an extra-thyroidal complication of Graves' disease which can lead to vision loss in severe cases. Currently, treatments of GO are not sufficiently effective, so novel therapeutic strategies are needed. As platelet-derived growth factor (PDGF)-BB induces several effector mechanisms in GO orbital fibroblasts including cytokine production and myofibroblast activation, this study aims to investigate the roles of histone lysine methyltransferases (HKMTs) in PDGF-BB-activated GO orbital fibroblasts by screening with HKMTs inhibitors library. From the total of twelve selective HKMT inhibitors in the library, EZH2, G9a and DOT1L inhibitors, DZNeP, BIX01294 and Pinometostat, respectively, prevented PDGF-BB-induced proliferation and hyaluronan production by GO orbital fibroblasts. However, only EZH2 inhibitor, DZNeP, significantly blocked pro-inflammatory cytokine production. For the HKMTs expression in GO orbital fibroblasts, PDGF-BB significantly and time-dependently induced EZH2, G9a and DOT1L mRNA expression. To confirm the role of EZH2 in PDGF-BB-induced orbital fibroblast activation, EZH2 silencing experiments revealed suppression of PDGF-BB-induced collagen type I and α-SMA expression along with decreasing histone H3 lysine 27 trimethylation (H3K27me3) level. In a more clinically relevant model than orbital fibroblast culture experiments, DZNeP treated GO orbital tissues significantly reduced pro-inflammatory cytokine production while slightly reduced ACTA2 mRNA expression. Our data is the first to demonstrate that among all HKMTs EZH2 dominantly involved in the expression of myofibroblast markers in PDGF-BB-activated orbital fibroblast from GO presumably via H3K27me3. Thus, EZH2 may represent a novel therapeutics target for GO.

Exosome miR-101-3p derived from bone marrow mesenchymal stem cells promotes radiotherapy sensitivity in non-small cell lung cancer by regulating DNA damage repair and autophagy levels through EZH2.

BACKGROUND AND OBJECTIVE: The morbidity rate of non-small cell lung cancer (NSCLC) increases with age, highlighting that NSCLC is a serious threat to human health. The aim of this study was mainly to describe the role of exosomal miR-101-3p derived from bone marrow mesenchymal stem cells (BMSCs) in NSCLC. METHODS: A549 or NCI-H1703 cells (1×105/mouse) were injected into nude mice to establish an NSCLC animal model. RTqPCR, Western blotting and comet assays were used to assess the changes in gene expression, proteins and DNA damage repair. RESULTS: miR-101-3p and RAI2 were found to be expressed at low levels in NSCLC, while EZH2 was highly expressed. In terms of function, miR-101-3p downregulated EZH2. In addition, exosomal miR-101-3p derived from BMSCs promoted the expression of RAI2, inhibited DNA damage repair, and inhibited the activation of the PI3K/AKT/mTOR signaling pathway by inhibiting EZH2, thereby promoting autophagy and decreasing cell viability and finally enhancing the sensitivity of NSCLC to radiotherapy and inhibiting the malignant biological behavior of NSCLC. CONCLUSION: Exosomal miR-101-3p derived from BMSCs can inhibit DNA damage repair, promote autophagy, enhance the radiosensitivity of NSCLC, and inhibit the progression of NSCLC by inhibiting EZH2.

EZH2 Promotes Multiple Myeloma Progression via STAT3 Pathway Activation.

BACKGROUND: Multiple myeloma (MM) is a malignant disorder of plasma cells in the bone marrow. MM causes the clonal proliferation of terminally differentiated plasma cells and the accumulation of monoclonal plasma cells. The enhancer of zeste homolog 2 (EZH2) has been proven to play a significant role in disease development and could act on the signal transducers and activators of the transcription 3 (STAT3) signaling pathway. This pathway contributes to the pathogenesis and maintenance of malignancies. This study aimed to explore the effect of EZH2 on MM progression and the role of the STAT3 pathway in this process. The goal was to increase knowledge and provide further insights about the pathogenesis of MM and identify novel targets for potential therapies. METHODS: The abnormal expression of EZH2 in MM cell lines was tested through real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and western blot analysis. Based on the MM cell line H929, transfection was used to modify EZH2 expression, followed by the subsequent evaluation of induced alteration in STAT3 activation. The STAT3 phosphorylation activator colivelin and inhibitor stattic were used for promoting and inhibiting the STAT3 activation, respectively. Colony-forming assay, transwell migration assay, and flow cytometry were used to explore cell proliferation, cell migration, and cell apoptosis, respectively. RESULTS: Both the EZH2 mRNA and protein were over-expressed in multiple MM cell lines including H929 (p < 0.001), U266 (p < 0.01), RPMI-8226 (p < 0.01) and MM.1S (p < 0.001). Increased EZH2 promoted cell proliferation (p < 0.001) and migration (p < 0.001) and simultaneously inhibited cell apoptosis (p < 0.001), which could be reversed by inhibited STAT3 activation (p < 0.001). In contrast, promoted STAT3 activation increased cell proliferation (p < 0.001) and migration (p < 0.001), while simultaneously inhibiting cell apoptosis (p < 0.001), despite decreased EZH2 expression. CONCLUSIONS: The effect of EZH2 and STAT3 pathways on MM regulation was revealed and verified. EZH2 promoted the progression of MM cells by activating the STAT3 pathway. The EZH2 and STAT3 pathways could be potential targets for effective MM treatment.

Unlocking adult T-cell leukemia/lymphoma's epigenetic secrets: delving into the mechanism and impact of EZH1/2 inhibition.

Epigenetic modifications, particularly through methylation of DNA packaging histones, play a pivotal role in controlling gene expression. Aberrant patterns of histone methylation have been associated with the development and progression of hematological malignancies. Unraveling the impact of aberrant histone marks on gene expression and leukemogenesis has spurred a concerted effort to develop clinically effective epigenetic therapies. In malignancies associated with the accumulation of histone H3 lysine trimethylation (H3K27me3), one such intervention involves preventing the deposition of this repressive histone mark by inhibiting the histone-modifying enzymes EZH1 and EZH2. While inhibition of EZH1/2 has demonstrated efficacy in both preclinical studies and clinical trials in various cancers, studies delineating the dynamic effect of EZH1/2 inhibition on H3K27me3 and disease relapse in clinical samples are lacking. In a recent publication, Yamagishi et al. explore how responses of a patient with adult T-cell leukemia/lymphoma to valemetostat, an EZH1/2 inhibitor, are associated with changes in H3K27me3, chromatin accessibility and gene expression, and how these changes can be circumvented in relapsed disease.

Stat5b/Ezh2 axis governs high PD-L1 expressing tolerogenic dendritic cell subset in autoimmune diabetes.

Dendritic cells (DCs) are specialized antigen-presenting cells that play an important role in inducing and maintaining immune tolerance. The altered distribution and/or function of DCs contributes to defective tolerance in autoimmune diseases such as type 1 diabetes (T1D). In human T1D and in NOD mouse models, DCs share some defects and are often described as less tolerogenic and excessively immunogenic. In the NOD mouse model, the autoimmune response is associated with a defect in the Stat5b signaling pathway. We have reported that expressing a constitutively active form of Stat5b in DCs of transgenic NOD mice (NOD.Stat5b-CA), re-established their tolerogenic function, restored autoimmune tolerance and conferred protection from diabetes. However, the role and molecular mechanisms of Stat5b signaling in regulating splenic conventional DCs tolerogenic signature remained unclear. In this study, we reported that, compared to immunogenic splenic DCs of NOD, splenic DCs of NOD.Stat5b-CA mice exhibited a tolerogenic profile marked by elevated PD-L1 and PD-L2 expression, reduced pro-inflammatory cytokine production, increased frequency of the cDC2 subset and decreased frequency of the cDC1 subset. This tolerogenic profile was associated with increased Ezh2 and IRF4 but decreased IRF8 expression. We also found an upregulation of PD-L1 in the cDC1 subset and high PD-L1 and PD-L2 expression in cDC2 of NOD.Stat5b-CA mice. Mechanistically, we demonstrated that Ezh2 plays an important role in the maintenance of high PD-L1 expression in cDC1 and cDC2 subsets and that Ezh2 inhibition resulted in PD-L1 but not PD-L2 downregulation which was more drastic in the cDC2 subset. Additionally, Ezh2 inhibition severely reduced the cDC2 subset and increased the cDC1 subset and Stat5b-CA.DC pro-inflammatory cytokine production. Together our data suggest that the Stat5b-Ezh2 axis is critical for the maintenance of tolerogenic high PD-L1-expressing cDC2 and autoimmune tolerance in NOD.Stat5b-CA mice.

Identification and functional analysis of the hub Ferroptosis-Related gene EZH2 in diabetic kidney disease.

BACKGROUND: Diabetic kidney disease (DKD) is a common microvascular complication and one of the main causes of death in diabetes. Ferroptosis, an iron-dependent mode of cell death characterized by lipid ROS accumulation, was found to be associated with a number of diseases and has great potential for kidney diseases. It has great value to identify potential ferroptosis-related genes and their biological mechanisms in DKD. METHODS: We obtained the GSE30122 dataset from Gene Expression Omnibus (GEO) database and ferroptosis-related genes from the Ferrdb database. After differential expression analysis, and three machine learning algorithms, the hub ferroptosis-related gene EZH2 was identified. In order to investigate the function of EZH2, Gene Set Enrichment Analysis (GSEA), Gene Set Variation Analysis (GSVA) and single cell analysis were conducted. The expression of EZH2 was validated in DKD patients, HK-2 cell models and DKD mouse models. EZH2 knockdown HK-2 cells and HK-2 cells treated with GSK126 were performed to verify whether EZH2 affected ferroptosis in DKD. CHIP assay was used to detect whether EZH2 regulated ferroptosis by suppressing SLC7A11. Molecular docking was performed to explore EZH2 and four traditional Chinese medicine (Sennoside A, Berberine, Umbelliferone, Platycodin D) related to ferroptosis in DKD treatment. RESULTS: According to the GSE30122 dataset in GEO and ferroptosis-related genes from the Ferrb database, we obtained the hub ferroptosis-related gene EZH2 in DKD via diversified machine learning methods. The increasing of EZH2 expression was shown in single cell analysis, DKD patients, DKD mouse models and high glucose induced DKD cell models. Further study showed that EZH2 knockdown and inhibition can alleviate HG-induced ferroptosis in vitro. CHIP assay showed EZH2-mediated epigenetic silencing regulated the expression of SLC7A11. Molecular docking results showed that EZH2 had strong binding stability with Sennoside A, Berberine, Umbelliferone, and Platycodin D. CONCLUSION: Overall, our data shouwed that histone H3K27 methyltransferase EZH2 could regulate the renal tubular epithelial cell ferroptosis by suppressing SLC7A11 in DKD, which may serve as a credible reliable indicator for diagnosing DKD and a potential target for treatment.

EZH2 mutations in follicular lymphoma distort H3K27me3 profiles and alter transcriptional responses to PRC2 inhibition.

Mutations in chromatin regulators are widespread in cancer. Among them, the histone H3 lysine 27 methyltransferase Polycomb Repressive Complex 2 (PRC2) shows distinct alterations according to tumor type. This specificity is poorly understood. Here, we model several PRC2 alterations in one isogenic system to reveal their comparative effects. Focusing then on lymphoma-associated EZH2 mutations, we show that Ezh2Y641F induces aberrant H3K27 methylation patterns even without wild-type Ezh2, which are alleviated by partial PRC2 inhibition. Remarkably, Ezh2Y641F rewires the response to PRC2 inhibition, leading to induction of antigen presentation genes. Using a unique longitudinal follicular lymphoma cohort, we further link EZH2 status to abnormal H3K27 methylation. We also uncover unexpected variability in the mutational landscape of successive biopsies, pointing to frequent co-existence of different clones and cautioning against stratifying patients based on single sampling. Our results clarify how oncogenic PRC2 mutations disrupt chromatin and transcription, and the therapeutic vulnerabilities this creates.

siRNA treatment targeting integrin α11 overexpressed via EZH2-driven axis inhibits drug-resistant breast cancer progression.

BACKGROUND: Breast cancer, the most prevalent cancer in women worldwide, faces treatment challenges due to drug resistance, posing a serious threat to patient survival. The present study aimed to identify the key molecules that drive drug resistance and aggressiveness in breast cancer cells and validate them as therapeutic targets. METHODS: Transcriptome microarray and analysis using PANTHER pathway and StemChecker were performed to identify the most significantly expressed genes in tamoxifen-resistant and adriamycin-resistant MCF-7 breast cancer cells. Clinical relevance of the key genes was determined using Kaplan-Meier survival analyses on The Cancer Genome Atlas dataset of breast cancer patients. Gene overexpression/knockdown, spheroid formation, flow cytometric analysis, chromatin immunoprecipitation, immunocytochemistry, wound healing/transwell migration assays, and cancer stem cell transcription factor activation profiling array were used to elucidate the regulatory mechanism of integrin α11 expression. Tumour-bearing xenograft models were used to demonstrate integrin α11 is a potential therapeutic target. RESULTS: Integrin α11 was consistently upregulated in drug-resistant breast cancer cells, and its silencing inhibited cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) while restoring sensitivity to anticancer drugs. HIF1α, GLI-1, and EZH2 contributed the most to the regulation of integrin α11 and EZH2 expression, with EZH2 being more necessary for EZH2 autoinduction than HIF1α and GLI-1. Additionally, unlike HIF1α or EZH2, GLI-1 was the sole transcription factor activated by integrin-linked focal adhesion kinase, indicating GLI-1 as a key driver of the EZH2-integrin α11 axis operating for cancer stem cell survival and EMT. Kaplan-Meier survival analysis using The Cancer Genome Atlas (TCGA) dataset also revealed both EZH2 and integrin α11 could be strong prognostic factors of relapse-free and overall survival in breast cancer patients. However, the superior efficacy of integrin α11 siRNA therapy over EZH2 siRNA treatment was demonstrated by enhanced inhibition of tumour growth and prolonged survival in murine models bearing tumours. CONCLUSION: Our findings elucidate that integrin α11 is upregulated by EZH2, forming a positive feedback circuit involving FAK-GLI-1 and contributing to drug resistance, cancer stem cell survival and EMT. Taken together, the results suggest integrin α11 as a promising prognostic marker and a powerful therapeutic target for drug-resistant breast cancer.

EZH2-dependent epigenetic reprogramming in the central nucleus of amygdala regulates adult anxiety in both sexes after adolescent alcohol exposure.

Alcohol use and anxiety disorders occur in both males and females, but despite sharing similar presentation and classical symptoms, the prevalence of alcohol use disorder (AUD) is lower in females. While anxiety is a symptom and comorbidity shared by both sexes, the common underlying mechanism that leads to AUD and the subsequent development of anxiety is still understudied. Using a rodent model of adolescent intermittent ethanol (AIE) exposure in both sexes, we investigated the epigenetic mechanism mediated by enhancer of zeste 2 (EZH2), a histone methyltransferase, in regulating both the expression of activity-regulated cytoskeleton-associated protein (Arc) and an anxiety-like phenotype in adulthood. Here, we report that EZH2 protein levels were significantly higher in PKC-δ positive GABAergic neurons in the central nucleus of amygdala (CeA) of adult male and female rats after AIE. Reducing protein and mRNA levels of EZH2 using siRNA infusion in the CeA prevented AIE-induced anxiety-like behavior, increased H3K27me3, decreased H3K27ac at the Arc synaptic activity response element (SARE) site, and restored deficits in Arc mRNA and protein expression in both male and female adult rats. Our data indicate that an EZH2-mediated epigenetic mechanism in the CeA plays an important role in regulating anxiety-like behavior and Arc expression after AIE in both male and female rats in adulthood. This study suggests that EZH2 may serve as a tractable drug target for the treatment of adult psychopathology after adolescent alcohol exposure.

LncRNA DLG5-AS1 facilitates breast cancer cell proliferation and invasion by promoting EZH2-mediated transcriptional silencing of SFRP1.

Rapid proliferation and metastasis of breast cancer contributed to poor clinical prognosis. Accumulating evidence revealed that the dysregulation of long noncoding RNAs (lncRNAs) was associated with breast cancer progression. However, the role of lncRNA DLG5-AS1 in breast cancer has not been established. Here, we investigated the mechanisms of DLG5-AS1 in the development of breast cancer. We found that the expression of DLG5-AS1 was significantly upregulated in breast cancer tissues and cell lines. DLG5-AS1 interference markedly restrained AU565 cell proliferation, invasion, the expression of apoptosis related (caspase3 and caspase8) and Wnt/ß-catenin pathway related proteins (wnt5a, ß-Catenin and c-Myc), as well as promoted cell apoptosis, whereas DLG5-AS1 overexpression showed an opposite effects. In addition, DLG5-AS1 could directly bind with miR-519 b-3p. We also found that enhancer of zeste homolog 2 (EZH2) is a direct target of miR-519 b-3p, and DLG5-AS1 upregulated EZH2 expression by inhibiting the expression of miR-519 b-3p. EZH2 restrained secreted frizzled related protein 1 (SFRP1) expression through inducing H3 histone methylation in its promoter. MiR-519 b-3p overexpression or SFRP1 knockdown memorably reversed the effects of DLG5-AS1 overexpression on cell functions and Wnt/ß-Catenin pathway related protein expression. Finally, in vivo experiments demonstrated that silencing of DLG5-AS1 inhibited xenograft tumor development in mice. Taken together, these findings demonstrated that DLG5-AS1 facilitated cell proliferation and invasion by promoting EZH2-mediated transcriptional silencing of SFRP1 in breast cancer.

NEAT1 regulates VSMC differentiation and calcification in as long noncoding RNA NEAT1 enhances phenotypic and osteogenic switching of vascular smooth muscle cells in atherosclerosis via scaffolding EZH2.

Atherosclerosis (AS) is a significant contributor to cardio-cerebrovascular ischemia diseases, resulting in high mortality rates worldwide. During AS, vascular smooth muscle cells (VSMCs) play a crucial role in plaque formation by undergoing phenotypic and osteogenic switching. Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has previously been identified as a nuclear regulator that promotes tumorigenesis and metastasis, but its role in regulating VSMCs in AS remains unclear. Our study aimed to investigate the biological functions and specific mechanisms of NEAT1 in regulating VSMCs in AS. We found that NEAT1 was upregulated in the aortas of AS mouse models and dedifferentiated primary VSMCs. Silencing NEAT1 in vitro attenuated the proliferation, migration, and osteogenic differentiation of VSMCs, while NEAT1 overexpression had the opposite effect. Furthermore, NEAT1 promoted VSMC osteogenic differentiation and vascular calcification in both in vivo and in vitro vascular calcification models. We also discovered that NEAT1 directly activates enhancer of zeste homolog 2 (EZH2), an epigenetic enzyme that suppresses the expression of senescence- and antimigration-related genes, by translocating it into the nucleus. CUT&Tag assay revealed that NEAT1 guides EZH2 to the promoters of senescence-related genes (P16, P21, and TIMP3), methylating local histones to reduce their transcription. Our findings suggest that NEAT1 functions in AS by modulating the epigenetic function of EZH2, which enhances the proliferation, migration, and osteogenic differentiation of VSMCs. This study provides new insights into the molecular mechanisms underlying the pathogenesis of AS and highlights the potential of NEAT1 as a therapeutic target of AS.NEW & NOTEWORTHY Our study demonstrates that the upregulation of long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) promotes proliferation and migration during phenotypic switching of vascular smooth muscle cells in atherosclerosis. We also provide in vivo and in vitro evidence that NEAT1 accelerates vascular calcification. Our findings identified the direct interaction between enhancer of zeste homolog 2 (EZH2) and NEAT1 during atherosclerosis. NEAT1 is necessary for EZH2 to translocate from the cytoplasm to the nucleus, where EZH2 epigenetically inhibits the expression of genes related to senescence and antimigration.

Telomere dysfunction alters intestinal stem cell dynamics to promote cancer.

Telomere dynamics are linked to aging hallmarks, and age-associated telomere loss fuels the development of epithelial cancers. In Apc-mutant mice, the onset of DNA damage associated with telomere dysfunction has been shown to accelerate adenoma initiation via unknown mechanisms. Here, we observed that Apc-mutant mice engineered to experience telomere dysfunction show accelerated adenoma formation resulting from augmented cell competition and clonal expansion. Mechanistically, telomere dysfunction induces the repression of EZH2, resulting in the derepression of Wnt antagonists, which causes the differentiation of adjacent stem cells and a relative growth advantage to Apc-deficient telomere dysfunctional cells. Correspondingly, in this mouse model, GSK3ß inhibition countered the actions of Wnt antagonists on intestinal stem cells, resulting in impaired adenoma formation of telomere dysfunctional Apc-mutant cells. Thus, telomere dysfunction contributes to cancer initiation through altered stem cell dynamics, identifying an interception strategy for human APC-mutant cancers with shortened telomeres.