Expression Levels Of EZH2 Research Articles

CircSYPL1 Promotes the Proliferation and Metastasis of Hepatocellular Carcinoma via the Upregulation of EZH2 Expression by Competing with hsa-miR-506-3p.

Objective Circular RNAs (circRNAs) and microRNAs are crucial for progressing of hepatocellular carcinoma (HCC). Nonetheless, the function or mechanisms of a newly discovered circRNA, circSYPL1, as well as miR-506-3p, in the progression of HCC are mostly unexplained. The purpose of this research was to determine the mechanisms by which circSYPL1 and miR-506-3p regulate the malignant features of HCC. Methods The expression level of circSYPL1 was indeed detected using real-time PCR in HCC cell lines, primary as well as metastatic cancers. To assess the functionality of circSYPL1 upregulation and knockdown, we used proliferation and apoptosis, in addition to migration assays, as well as tumor xenograft and lung metastasis assays. The mechanisms of competing endogenous RNAs with circSYPL1/miR-506-3p/EZH2 were investigated using luciferase as well as RNA pull-down experiments. Lastly, cell proliferation and migration, in addition to tumor xenograft tests, were used to validate the biological significance of the circSYPL1/miR-506-3p/EZH2 signaling axis through overexpression or otherwise silencing. Results circSYPL1 expression was significantly upregulated in HCC cell lines, in addition to primary and metastatic tumors of patients with HCC. Additionally, it may promote HCC initiation, development as well as progression. By knocking down circSYPL1 siRNA, we were able to drastically decrease the aggressiveness of HCC cells. circSYPL1 sponged miR-506-3p to boost EZH2 expression levels, as indicated by luciferase and RNA pull-down assays. Furthermore, circSYPL1 overexpression could upregulate EZH2 expression, while miR-506-3p mimics or EZH2 shRNAs could reverse the circSYPL1-induced malignancy of HCC cells. Conclusion On a mechanistic level, circSYPL1 can interact with miR-506-3p in a competitive manner to upregulate EZH2, hence increasing the aggressiveness of tumors.

EZH2-mediated H3K27me3 promotes autoimmune hepatitis progression by regulating macrophage polarization

Autoimmune hepatitis (AIH) is a chronic progressive liver disease related to abnormal immune stimulation, leading to liver cirrhosis, liver cancer and liver failure. There is an urgent need to find novel biomarkers and potential drug targets for effective treatment of the disease. Although previous studies have shown that EZH2, as a histone methyltransferase, plays critical roles in tumor and autoimmune diseases, its role in autoimmune hepatitis remains largely unknown. In this study, we reported that the EZH2 and H3K27me3 expression level was significantly upregulated in liver tissues during the progression of AIH. High expression of EZH2 enhanced autoimmune hepatitis, immune response and liver fibrosis through H3K27me3. EZH2 inhibition induced the phenotype of hepatic macrophages to switch from M1 to M2 in the development of AIH. These findings indicated that EZH2-mediated H3K27me3 promoted autoimmune hepatitis by regulating the polarization of hepatic macrophages. EZH2 may be a promising therapeutic target for the prevention or treatment of autoimmune hepatitis.

Clinical Value of EZH2 in Hepatocellular Carcinoma and Its Potential for Target Therapy.

Background and objectives: EZH2 is overexpressed in hepatocellular carcinoma (HCC) and is correlated with poor prognosis. However, its clinical significance and molecular mechanism have not been studied in HCC. In this study, clinical and prognostic values of EZH2 was studied using Total Cancer Genome Atlas (TCGA) data and then, these data were confirmed in Huh1 and HepG2 cell lines. Materials and Methods: We used the TCGA database from cBioPortal. In addition, we analyzed EZH2 mRNA levels in HCC cell lines and its correlation with STAT3 and EZH2. Results: According to TCGA, EZH2 had a prognostic value in various cancers, especially in HCC. Furthermore, EZH2 in HCC was correlated with N stage (p = 0.045) and alpha-fetoprotein (AFP) > 20 ng/mL (p < 0.01). However, a negative association between EZH2 and age (p = 0.027) was found. The overall survival result of HCC was significantly poorer in patients with high EZH2 expression. In addition, the recurrence rate was also significantly higher in patients with high expression of EZH2 than those with low expression (χ2 = 16.10, p < 0.001). EZH2 expression was negatively correlated with STAT3 expression among EZH2-associated genes (R = −0.163, p = 0.002). EZH2 expression level was down-regulated to 50% or less compared to the control group treated negative siRNA. MTT assays showed that EZH2-siRNA affected on the viability of HCC cell line significantly. Conclusions: In conclusion, the overexpression of EZH2 was an independent biomarker for poor outcomes of HCC. However, more in vivo studies are required to identify the downstream target genes in HCC to improve our understanding of the biological role of EZH2 in HCC.

Dysregulated Expression of Long Non-Coding RNA MINCR and EZH2 in Colorectal Cancer

Background:As critical regulators, lncRNAs have attracted attention from researchers for diagnostic, prognostic, and therapeutic purposes in human carcinogenesis via interfering with mRNAs such as EZH2. Nevertheless, the potent roles and molecular mechanisms of these RNAs in CRC are not clearly known. Methods:In this study, the tissue expressions of lncRNA MINCR and EZH2 mRNA between colorectal tumors and polyps were compared with the adjacent normal tissues collected from 114 Iranian patients, using real-time PCR method. Furthermore, the correlation of the expression levels of MINCR and EZH2 with other clinical parameters was evaluated. Results:The significant overexpression of MINCR and EZH2 were observed in the CRC tissues compared to control tissues (p < 0.0001). This observation confirmed the association of these expression enhancements with the pathological stage of CRC patients. Conclusion:Our findings revealed that the expression of MINCR significantly alters during CRC development, and it can be identified as a potential biomarker for the detection of CRC.

Prognostic Value of Histone Modifying Enzyme EZH2 in RCHOP-Treated Diffuse Large B-Cell Lymphoma and High Grade B-Cell Lymphoma.

Background: DLBCL represent a heterogeneous group of aggressive diseases. High grade B-cell lymphomas (HGBCL) were recently individualized from DLBCL as a discrete diagnostic entity due to their worse prognosis. Currently, although most patients are successfully treated with RCHOP regimens, 1/3 will either not respond or ultimately relapse. Alterations in histone modifying enzymes have emerged as the most common alterations in DLBCL, but their role as prognostic biomarkers is controversial. We aimed to ascertain the prognostic value of EZH2 immunoexpression in RCHOP-treated DLBCL and HGBCL. Results: We performed a retrospective cohort study including 125 patients with RCHOP-treated DLBCL or HGBCL. EZH2 expression levels did not differ between diagnostic groups or between DLBCL-NOS molecular groups. We found no associations between EZH2 expression levels and outcome, including in the subgroup analysis (GC versus non-GC). Nonetheless, EZH2/BCL2 co-expression was significantly associated with worse outcome (event free survival and overall survival). Conclusion: Although EZH2 mutations are almost exclusively found in GC-DLBCL, we found similar EZH2 expression levels in both DLBCL-NOS molecular groups, suggesting non-mutational mechanisms of EZH2 deregulation. These findings suggest that the use of EZH2 antagonists might be extended to non-GC DLBCL patients with clinical benefit. EZH2/BCL2 co-expression was associated with a worse outcome.

The association between lncRNA H19 and EZH2 expression in patients with EBV-positive laryngeal carcinoma.

SUMMARYObjectiveLaryngeal cancer is the second most common malignancy in the head and neck, with Epstein-Barr virus infection as a risk factor. Our aim is to evaluate correlations between the expression of lncRNA H19 and EBV infection in laryngeal cancer and H19 involvement in neoplastic progression through EZH2 association.Materials and methods30 paired laryngeal tissue specimens (neoplastic and non-neoplastic) were included in the study. Nucleic acid isolation and cDNA synthesis was performed according to the manufacturer’s protocol. EBV DNA and expression of lytic (BZLF1) and latent (LMP1) forms of infection were assessed in PCR assays; expression levels of H19 and EZH2 were quantified in qRT-PCR. Data was analysed using GraphPad Prism 5.0.ResultsHigher H19 relative expression in neoplastic vs paired non-neoplastic samples was found (p < 0.0001) with a significant increase in EBV DNA positive neoplasms (p = 0.0434). An inverse correlation between H19 and EZH2 expression levels was noticed in EBV positive cases. Additionally, increased levels of H19 in LMP1 positive samples compared with those positive for BZLF1 was found (p = 0.0593).ConclusionslncRNA H19 and EZH2 significantly contribute to the development of laryngeal carcinoma, being correlated with EBV infection markers.

Zinc-finger protein 750 mitigates malignant biological behavior of oral CSC-like cells enriched from parental CAL-27 cells.

Oral squamous cell carcinoma (OSCC) is the most commonly occurring oral malignancy. Cancer stem cells (CSCs) are known to be responsible for cancer recurrence and metastasis. Zinc-finger protein 750 (ZNF750) has been reported to inhibit OSCC cell proliferation and invasion. The present study aimed to elucidate the role of ZNF750 in the inhibition of the renewal ability of CSCs derived from the OSCC cell line, CAL-27. The effects of ZNF750 on CSC-like properties were examined using aldehyde dehydrogenase (ALDH), tumor sphere formation and colony formation assays. Reverse transcription-quantitative PCR and western blotting were performed to detect the expression levels of octamer-binding transcription factor 4, sex-determining region Y-box 2, the enhancer of zeste homolog 2 (Ezh2), embryonic ectoderm development (EED) and SUZ12 polycomb repressive complex 2 subunit (SUZ12), and for the identification of genes associated with metastasis. ZNF750 effectively attenuated CSC-like cell self-renewal abilities; ZNF750 decreased the ALDH-positive cell population, tumor sphere and colony formation abilities, cell viability and stemness factors. Furthermore, the expression levels of Ezh2, EED and SUZ12 were decreased by ZNF750. ZNF750 inhibited MMP1, 3, 9 and 13 expression levels, and decreased the cell invasion and migratory abilities. Moreover, the expression of tissue inhibitors of matrix metalloproteinases-1 was increased by ZNF750. However, opposite effects were observed following the knockdown of the ZNF750 gene. Overall, the present study demonstrated that ZNF750 has the potential to inhibit the renewal of CSC-like cells enriched from parental CAL-27 cells.

NEK2 Inhibition Enhances the Efficacy of PD-1/PD-L1 Blockade in Multiple Myeloma

IntroductionThe development of new treatments for high-risk multiple myeloma (HRMM) are needed. The PD-1/PD-L1 axis is one of the chief inhibitory immune checkpoints in antitumor immunity. Despite the success of PD-1 (PDCD1) / PD-L1 (CD274) blockade in some neoplasms, use of it as a monotherapy has failed to improve outcome in RRMM. We have previously demonstrated that the cell-cycle-regulated serine-threonine kinase, NEK2 is elevated in HRMM and that inhibition of NEK2 can overcome drug-resistance and prolong survival of xenografted MM cells. Here, we aimed to investigate the possible role of NEK2 in regulating the immune checkpoint response in MM and development of possible anti-PD1/PDL1 combination therapies.MethodsGene expression profiles and pathway enrichment analyses were conducted on oligonucleotide microarray gene expression profiles from over 1000 primary MM samples to evaluate the correlation of NEK2 and immune checkpoint expression levels. To elucidate the underlying mechanism, we used Nek2 -/- mice crossed with EμMyc mice to generate B cell tumor mouse model with NEK2 deficiency. RNA-sequencing analyses of premalignant B cells was compared between EμMyc/Nek2 WT and EμMyc/Nek2 -/- mice. The hub molecular regulators in the NEK2 correlated pathways were further determined by western blot using NEK2 overexpressing and knockdown cell lines and then verified by co-immunoprecipitation with a NEK2 antibody. Lastly, to establish its clinic significance, the efficacy of INH1 (small compound NEK2 inhibitor), (D)-PPA 1 (peptide-based PD-1/PD-L1 interaction inhibitor) or a PD-L1 (monoclonal antibody) was tested in bone marrow BM mononuclear cells from primary MM patients in-vitro as well as in MM xenografts. Tumor burden and T cell immune responses were monitored by M-spike and mass cytometry.ResultsGene expression profiles demonstrated that CD274 expression was significantly higher in the non-proliferative hyperdiploid (HY) subtype of MM, representing between 25-35% of all MM. NEK2 was negatively correlated with CD274 gene expression across all 7 MM subtypes. Gene set enrichment analysis showed that the IFN-γ signaling pathway, which can induce CD274 expression, was significantly enriched in the HY subtype as well as premalignant B cells from EμMyc/Nek2 -/- mice. Elevated expression of EZH2, a histone methyltransferase gene, is also highly correlated wirth NEK2 levels in primary MM. We found that NEK2 inhibition increases CD274 expression as well as reduced EZH2 expression and H3K27me3 levels in MM cell lines. In contrarst, myeloma cells overexpressing NEK2 showed increased expression and activity of EZH2 and H3K27me3 levels. Thus, NEK2 appears to regulate CD274/PD-L1 expression through EZH2-mediated histone methylation. Next we demonstrated that NEK2 and EZH2 directly interact and that overexpression of NEK2 leads to increased methylation of the CD274/PD-L1 gene. We treated BM mononuclear cells from primary MM with PD-1/PD-L1 inhibitor with and without a NEK2 inhibitor. The combination was most effective at eliminating CD138 + myeloma cells while having no effects on T, B and myeloid cell populations.ConclusionOur study showed that expression of CD274/PD-L1 is suppressed in primary HRMM and that CD274/PD-L1 expression is negatively regulated by NEK2 via EZH2-mediated methylation. Inhibition of NEK2 sensitizes myeloma cells to PD-1/PD-L1 blockade, showing either a synergistic or an additive effect in MM cell cytotoxicity. DisclosuresNo relevant conflicts of interest to declare.

EZH1/2 inhibition augments the anti-tumor effects of sorafenib in hepatocellular carcinoma

Both EZH2 and its homolog EZH1 function as histone H3 Lysine 27 (H3K27) methyltransferases and repress the transcription of target genes. Dysregulation of H3K27 trimethylation (H3K27me3) plays an important role in the development and progression of cancers such as hepatocellular carcinoma (HCC). This study investigated the relationship between the expression of EZH1/2 and the level of H3K27me3 in HCC. Additionally, the role of EZH1/2 in cell growth, tumorigenicity, and resistance to sorafenib were also analyzed. Both the lentiviral knockdown and the pharmacological inhibition of EZH1/2 (UNC1999) diminished the level of H3K27me3 and suppressed cell growth in liver cancer cells, compared with EZH1 or EZH2 single knockdown. Although a significant association was observed between EZH2 expression and H3K27me3 levels in HCC samples, overexpression of EZH1 appeared to contribute to enhanced H3K27me3 levels in some EZH2lowH3K27me3high cases. Akt suppression following sorafenib treatment resulted in an increase of the H3K27me3 levels through a decrease in EZH2 phosphorylation at serine 21. The combined use of sorafenib and UNC1999 exhibited synergistic antitumor effects in vitro and in vivo. Combination treatment canceled the sorafenib-induced enhancement in H3K27me3 levels, indicating that activation of EZH2 function is one of the mechanisms of sorafenib-resistance in HCC. In conclusion, sorafenib plus EZH1/2 inhibitors may comprise a novel therapeutic approach in HCC.

Functional relationship of SNP (Ala490Thr) of an epigenetic gene EZH2 results in the progression and poor survival of ER+/tamoxifen treated breast cancer patients

EZH2 is a classic histone methyltransferase that causes the trimethylation of H3K27 and consequently suppresses the cancer preventive genes. The role of elevated levels of EZH2 expression in breast cancer aggressiveness and poor prognosis is very well established. The present study focusses on the insight of the clinically important SNPs of EZH2 gene in determining the structure-function relationship towards breast cancer susceptibility. For this purpose the EZH2 SNPs (rs41277434A>C and rs201135441C>T (A490T)) were computationally explored and further the prediction outcomes were validated by performing population-based association and pharmacogenetic study in north Indian region of Punjab. The results of the present analysis provided the novel insight of rs201135441C>T (A490T) mutation, that A>T change i.e. nonpolar amino acid to polar amino acid stabilizes the enzyme-substrate (EZH2-Histone) complex which in turn promotes trimethylation over histone 3 (H3) at lysine residue 27 (H3K27me3) and this might be leading to the methylation of the promoter region of various cancer preventive genes, hence may increase the risk of breast cancer susceptibility. Further the association based study of SNP rs201135441C>T (A490T) support the in silico outcomes by revealing that the T mutant allele of rs201135441 has significantly increased the risk of breast cancer susceptibility and also reduce the overall survival and progression free survival of ER+/tamoxifen treated breast cancer patients and triple negative breast cancer (TNBC) patients, respectively. To the best of our knowledge, this is the first study on EZH2 polymorphism with breast cancer. In conclusion, these findings suggest that these variations in the EZH2 gene may have strong clinical significance as they can be targeted for prognosis, prevention and in drug development.

EZH2 knockdown upregulates expression of the genes involved in T-ALL cell differentiation

Background: EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit), as one of the polycyclic group proteins (PcGs), is an epigenetic regulator that plays a crucial role in the pathophysiology of hematologic malignancies through regulating cell differentiation. Also, it is well known that aberrant expression of specific transcription factors can be involved in the pathogenesis of various cancers. Objective: Herein, we aimed to suppress EZH2 expression in MOLT-4 cells, T-ALL (T cell acute lymphoblastic leukemia) cell line, and evaluate the role of EZH2 on the expression of transcription factors that regulate T cell maturation, differentiation, and apoptosis. Methods: EZH2-siRNA was transfected into MOLT-4 cells, and the expression levels of EZH2, NOTCH1, TCF1, IKZF1, and NFATC1 were measured using real-time PCR. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was performed to study the effect of EZH2 knockdown on MOLT-4 cell viability. The apoptosis rate of EZH2-siRNA transfected cells was assessed by flow cytometry. The interaction of mentioned genes was investigated using STRING and GO (gene ontology). Results: Our results have shown that EZH2-siRNA transfection can substantially decrease EZH2 expression in MOLT-4 cells. Besides, EZH2 suppression can upregulate NOTCH1, TCF1, IKZF1, and NFATC1 expression levels. EZH2 knockdown does not affect the viability and apoptosis of MOLT-4 cells. The most remarkable protein-protein interaction of EZH2 has been with NOTCH1. Besides, GO analysis has demonstrated that EZH2, NOTCH1, TCF1, IKZF1, and NFATC1 were located within nucleoplasm and can regulate RNA polymerase II-mediated transcription. Conclusion: Our results have shown that MOLT-4 cells harbor increased expression of EZH2 in comparison with normal human T cells. EZH2 knockdown can upregulate the expression of the transcription factors involved in T cell differentiation. Thus, EZH2 can halt the differentiation of immature lymphoblastic T cells.

METTL3 promotes inflammation and cell apoptosis in a pediatric pneumonia model by regulating EZH2

In recent times, the medical science has developed by leaps and bounds, however, the molecular mechanism of pediatric pneumonia is still unclear. Although prior researches have shown that methyltransferase-like 3 (METTL3) is up-regulated in a variety of inflammatory diseases, its role and mechanism has been rarely studied in pediatric pneumonia, and need to be defined elaborately. In this study, the related molecular mechanism of METTL3 on inflammation and cell apoptosis in a pediatric pneumonia was investigated. Quantitative real-time polymerase chain reaction (qPCR) and western blot assays were employed to examine the mRNA and protein expression level of METTL3 and EZH2 in peripheral blood monocytes from pediatric pneumonia patients or cell model (WI-38). Then, qPCR and ELISA assay were applied to verify the inflammatory response in LPS-treated WI-38 cell lines after knockdown of METTL3. Besides, MTT cell viability assays, flow cytometry, and western blot assays were applied to examine the cell viability and cell apoptosis of LPS-treated WI-38 cell after knockdown of METTL3. Further, the western blot assays were employed to examine the protein expression levels of p-JAK2, JAK2, p-STAT3, STAT3, and EZH2 in LPS-treated WI-38 cell after knockdown of METTL3. Finally, ELISA and western blot were applied to verify the inflammatory response and cell apoptosis of LPS-treated WI-38 cell after knockdown of METTL3 and overexpression of EZH2. In this study, the results showed that METTL3 and EZH2 were highly expressed in pediatric pneumonia patients and cell models (WI-38), respectively. Besides, downregulation of METTL3 inhibited LPS-induced inflammatory response and cell apoptosis. Then, the fact that METTL3 regulates the JAK2/STAT3 signaling pathway through EZH was proved. Furthermore, downregulation of METTL3 inhibits inflammation and apoptosis through EZH2. This study found that METTL3 promotes inflammation and cell apoptosis in a pediatric pneumonia model by regulating EZH2.

MiR-101-3p Serves as a Tumor Suppressor for Renal Cell Carcinoma and Inhibits Its Invasion and Metastasis by Targeting EZH2.

Background The role of miRNAs in renal cell carcinoma (RCC) is not certain. We wanted to study the biological functions and potential mechanisms of miR-101-3p in RCC. Methods miR-101-3p was inhibited in A498 and OSRC-2 (two RCC cell lines). We studied its effect on cell invasion and proliferation. Target EZH2 of miR-101-3p was designated by different methods, including luciferase functional analysis and Western blotting. The expression level of the target gene in treated cells was quantitatively analyzed by quantitative real-time polymerase chain reaction. In addition, induction of miR-101-3p to prevent tumor formation of A498 cells in mice was further studied. Results The overexpression of miR-101-3p significantly inhibited the proliferation, migration, and invasion in two RCC cells. Western blotting and luciferase functional analysis indicated that miR-101-3p regulated the expression of EZH2 in two cell lines. Mice inoculated with A498 and OSRC-2 cells transfected with miR-101-3p mimics showed significantly smaller xenografts and weaker EZH2 expression levels than the control group. Conclusions miR-101-3p inhibited RCC cell proliferation, migration, and invasion by targeting EZH2.

Horizontal transfer of the stemness-related markers EZH2 and GLI1 by neuroblastoma-derived extracellular vesicles in stromal cells

Neuroblastoma (NB) is the most common extracranial pediatric solid cancer originating from undifferentiated neural crest cells. NB cells express EZH2 and GLI1 genes that are known to maintain the undifferentiated phenotype of cancer stem cells (CSC) in NB. Recent studies suggest that tumor-derived extracellular vesicles (EVs) can regulate the transformation of surrounding cells into CSC by transferring tumor-specific molecules they contain. However, the horizontal transfer of EVs molecules in NB remains largely unknown. We report the analysis of NB-derived EVs in bioengineered models of NB that are based on a collagen 1/hyaluronic acid scaffold designed to mimic the native tumor niche. Using these models, we observed an enrichment of GLI1 and EZH2 mRNAs in NB-derived EVs. As a consequence of the uptake of NB-derived EVs, the host cells increased the expression levels of GLI1 and EZH2. These results suggest the alteration of the expression profile of stromal cells through an EV-based mechanism, and point the GLI1 and EZH2 mRNAs in the EV cargo as diagnostic biomarkers in NB.

Abstract 3142: Determination of molecular patterns associated with the expression of the TMPRSS2-ERG fusion and ERG, EZH2, NKX3.1 and SPINK-1 genes to evaluate the clonal origin of multifocal prostate cancer and its association with disease progression

Abstract To represent the molecular heterogeneity of PCa, which could improve medical decision-making, we aim to study it through the expression of ERG, EZH2, NKX3.1 and SPINK-1; as well as the TMPRSS2-ERG fusion status, in order to assess the clonality of the foci in each patient and also the possible association of this alteration with different degrees of differentiation and lymph node metastasis. FFPE samples of different foci with different lesion degrees: High-Grade Prostatic Intraepithelial Neoplasia (n=17), PCa foci with different Gleason scores (n= 53; GL3: (n=23); GL4: (n=23) and GL5: (n=7)), and positive lymph node (LN) (n=13) from radical prostatectomy from twenty patients were used to analyze the presence of TMPRSS2-ERG fusion and expression levels of ERG, EZH2 and NKX3.1 using RT-PCR. The molecular concordance between the different prostatic foci of the same patient was determined, taking into account subclonal events. The LN were compared with prostatic foci to try to establish its clonal origin. Molecular alterations were analyzed by comparing them across all the histopathological groups. The molecular concordance per each alteration was the predominant status (75% to 90%), but the concordance decreased to 50% when all the alterations were taken into account. The ERG(+) subtype was found in 54.7% of the foci and 70% of the patients, and 20% showed overexpression of the EZH2 gene ERG-independent. None of the thirteen analyzed LN showed the same pattern of other prostatic foci from each patient. All the LN samples were characterized by low expression of the NKX3.1 gene; and for some patients, these levels varied in comparison with other prostatic foci samples from the same patient. Only the expression of NKX3.1 was found inversely proportional to the progression of PCa when it was made the analysis by histopathological groups. In this study, the frequency of SPINK-1 cases overexpressed in ERG(-) coincides with that expected (11%), but it was not found to be exclusive with ERG (+), as in other studies that have not taken into account patients from Colombia, a greater relationship was found with ERG (+).The combination of ERG, EZH2 and SPINK-1 overexpression with low expression of NKX3.1 was the predominant pattern in the most advanced lesion (LN), while the opposite pattern was the most frequent in the HGPIN samples. Metastasis have less molecular heterogeneity than pre-neoplastic lesions. We found important molecular heterogeneity in PCa foci that could be related with the prognosis of the disease; for example, low expression of NKX3.1 was associated to metastasis. This highlights the need for searching personalized treatment. Multifocal PCa may have monoclonal or multiclonal origin. Citation Format: Yenifer Yamile Segura Moreno, María Carolina SANABRIA SALAS, Jorge Andrés MESA LÓPEZ DE MESA, Rodolfo VARELA RAMIREZ, Natalia Lizeth ACOSTA VEGA, Martha Lucia SERRANO. Determination of molecular patterns associated with the expression of the TMPRSS2-ERG fusion and ERG, EZH2, NKX3.1 and SPINK-1 genes to evaluate the clonal origin of multifocal prostate cancer and its association with disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3142.

External Stimuli Modulate EZH2 Expression in Chronic Lymphocytic Leukemia. Rationale and Evidence for Synergistic Anti-Tumor Effects of B Cell Signaling and EZH2 Inhibitors

The histone methyltransferase EZH2 induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). We have previously reported that EZH2 is overexpressed in adverse-prognosis CLL and associated with increased cell survival and proliferation. Moreover, we demonstrated that EZH2 pharmacological inhibition induced down-regulation of H3K27me3 levels leading to increased cell apoptosis. Here we sought to investigate if EZH2 expression could be affected by microenvironmental triggering, a key driver of CLL development and evolution.The study group included 10 CLL cases (9 with unmutated and 1 with mutated IGHV genes) that had previously been screened and found responsive to cross-linking of the B cell receptor (BcR) with anti-IgM. CD19+ cells were negatively selected from peripheral blood and stimulated or not through the BcR for 14 hours. Using RQ-PCR we found that stimulation with anti-IgM showed opposing effects on EZH2 mRNA levels, since 5 cases upregulated while the remaining 5 downregulated EZH2. In contrast, stimulation with CpG or CpG/CD40L for 14 hours caused pronounced and consistent upregulation of EZH2 mRNA levels compared to unstimulated cells (FD=8.7, p=0.03 and FD=11.3, p=0.03, respectively; n=6). This effect was also obvious at the protein levels in cells treated with CpG, CD40L and CpG/CD40L for 24 hours, where EZH2 protein was significantly upregulated as revealed by Western blot analysis (FD=3.9, p=0.002; FD=1.7, p=0.005; and FD=2.9, p=0.01, respectively; n=6). In CpG-stimulated cultures, the induction of EZH2 was accompanied by a significant increase in cell viability (FD=2, p=0.002; n=6), supporting the conclusion that EZH2 expression likely contributes to the anti-apoptosis phenotype induced in CLL cells by microenvironmental signals. Prompted by these observations, we then investigated whether B cell signaling inhibition might also modulate EZH2 expression and H3K27me3 levels in vivo by longitudinal profiling of another group of 9 CLL patients that were under ibrutinib treatment. In patients at +1 month under ibrutinib, when, as shown by us and others, CLL cells become anergized through the BcR, we observed a significant decrease in EZH2 protein (FD=3, p=0.0007; n=9) as well a trend for reduced H3K27me3 levels (FD=1.4, p=0.07; n=6) compared to the pre-treatment samples, further highlighting the links between BcR signaling and EZH2 expression and functionality. Next, we assessed ex vivo the effects of ibrutinib on CLL cell viability in cases with differential EZH2 expression [EZH2high (n=7) vs EZH2low (n=6)] and found decreased viability in treated cells compared to control cells (DMSO-exposed) in EZH2high cases (FD=1.9, p=0.0025), with no difference in EZH2low cases.Finally, we examined potential synergistic effects of B cell signaling inhibitors [ibrutinib (IB), idelalisib (IDE)] and EZH2 inhibitors (GSK343, GSK126) in primary CLL cells. To this end, CD19+ cells from 6 CLL cases were first pre-stimulated with CpG for 24 hours and then exposed to either single inhibitors (IB, IDE, GSK343, GSK126) or combinations of B cell signaling and EZH2 inhibitors (IB/GSK343, IDE/GSK343, IB/GSK126). Flow cytometry analysis after 3 days in culture revealed significantly lower CLL cell viability and H3K27me3 levels in CLL cells exposed to combined versus single treatment (FD≥ 1.5, p<0.05).In conclusion, we show that EZH2 expression is affected by signals emanating from CLL microenvironment. Moreover, we provide for the first time evidence that the combination of BcR signaling inhibitors widely used in CLL therapy and EZH2 inhibitors currently tested in phase I/II clinical trials in lymphoid malignancies has synergistic effects, suggesting that targeting both processes might represent a rational and promising therapeutic strategy. DisclosuresStavroyianni:Gilead Sciences: Honoraria; Takeda: Honoraria; Janssen Pharmaceuticals: Honoraria. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria; Janssen Pharmaceuticals: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Papakonstantinou:Gilead: Research Funding; Janssen Pharmaceuticals: Research Funding. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; Novartis SA: Research Funding.

Analysis of the Predictive Value of EZH2 Expression Level on the Clinical Efficacy and Long-term Prognosis of Patients with Primary Gastrointestinal Diffuse Large B-cell Lymphoma

To investigate the predictive value of methyltransferase EZH2 expression level on the clinical efficacy and long-term prognosis of patients with primary gastrointestinal diffuse large B-cell lymphoma (PGI-DLBCL). 161 patients with newly treated PGI-DLBCL in our hospital from August 2013 to July 2019 were selected. The expression level of EZH2 protein was detected by immunohistochemistry, and the short-term efficacy and long-term survival differences of patients with different levels of EZH2 were compared. The predictive values of EZH2 expression level on the short-term efficacy and long-term prognosis of PGI-DLBCL patients were analyzed by Log-rank test and COX risk proportional regression model. Chi-square test and Logistic regression analysis were used to analyze the influencing factors of EZH2 expression level. The complete response (CR) and overal response(OR) rates of those with high EZH2 expression were significantly lower than those with low EZH2 expression (P<0.001). The median OS and PFS of EZH2 high-level and low-level expression group was 37, 31 months and 49, 42 months, respectively. The cumulative OS and PFS rates of the high-level expression group were significantly lower than those of the low-level expression group, and the differences were statistically significant (P<0.05). The high expression levels of H3K27me3, EZH2, BCL-2, BCL-6, c-MYC were closely related to the shortening of OS and PFS, while the high expression level of Ki-67 was closely related to the shortening of OS (P<0.05), of which the high expression levels of H3K27me3, EZH2, BCL-2, and BCL-6 were independent risk factors for shortening of OS and PFS. The expression level of EZH2 was positively correlated with the expression level of H3K27me3, BCL-6, c-MYC and Ki-67 (r=0.741, r=0.837, r=0.809, r=0.772), and the high expression levels of H3K27me3, BCL-6 and Ki-67 were independent factors influencing the high expression of EZH2. In patients with PGI-DLBCL, the high expression of EZH2 significantly reduces the short-term CR and OR rates, which is an independent risk factor for the shortening of long-term OS and PFS rates, and it is independently related to the high expression of H3K27me3 and BCL6.

EZH2 Mediates miR-146a-5p/HIF-1α to Alleviate Inflammation and Glycolysis after Acute Spinal Cord Injury.

Acute spinal cord injury (ASCI) is a severe traumatic disease of the central nervous system, the underlying mechanism of which is unclear. This study was intended to study the role of EZH2 and miR-146a-5p/HIF-1α in inflammation and glycolysis after ASCI, providing reference and basis for the clinical treatment and prognosis of ASCI injury. We used lipopolysaccharide (LPS) to induce inflammation of microglia, and we constructed the ASCI animal model. qRT-PCR detected the relative expression levels of EZH2, HIF-1α, miR-146a-5p, IL-6, TNF-α, IL-17, PKM2, GLUT1, and HK2 in cells and tissues. Western blot was performed to detect the expression levels of EZH2, HIF-1α, H3K27me3, IL-6, TNF-α, IL-17, PKM2, GLUT1, and HK2. ChIP verified the enrichment of H3K27me3 in the miR-146a-5p promoter region. Bioinformatics predicted the binding sites of HIF-1α and miR-146a-5p, and dual-luciferase reporter assay verified the binding of HIF-1α and miR-146a-5p. ELISA detects the levels of inflammatory factors IL-6, TNF-α, and IL-17 in the cerebrospinal fluid of rats. The GC-TOFMS was used to detect the changes of glycolytic metabolites in the cerebrospinal fluid of rats. EZH2 could mediate inflammation and glycolysis of microglia. EZH2 regulates inflammation and glycolysis through HIF-1α. EZH2 indirectly regulated the HIF-1α expression by mediating miR-146a-5p. EZH2 mediates miR-146a-5p/HIF-1α to alleviate inflammation and glycolysis in ASCI rats. In the present study, our results demonstrated that EZH2 could mediate miR-146a-5p/HIF-1α to alleviate the inflammation and glycolysis after ASCI. Therefore, EZH2/miR-146a-5p/HIF-1α might be a novel potential target for treating ASCI.

MicroRNA-124 regulates apoptosis in sevoflurane anesthesia-induced neuroblastoma cells by targeting enhancer of zeste homolog 2

Purpose: To investigate the mechanism of microRNA-124 action on neuroblastoma apoptosis induced by sevoflurane.&#x0D; Methods: MiR-124 expression was assessed in a neuroblastoma cell line (SMS-KAN) using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The role of miR-124 in sevoflurane anesthesia-induced neuroblastoma was studied by cell activity and apoptosis analysis using 3-(4, 5-dimethylthiazolyl-2-yl)-2-5 diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. MiR-124 target protein genes were confirmed via luciferase reporter activity, qRT-PCR, and western blot analysis.&#x0D; Results: miR-124 was upregulated in sevoflurane anesthesia-induced neuroblastoma (p &lt; 0.05). After miR-124 knockdown, apoptosis was significantly reduced and cell viability was enhanced in sevoflurane anesthesia-induced SMS-KAN nerve cells (p &lt; 0.05). Furthermore, a significant reduction of luciferase activity was observed in 293T cells co-transfected with miR-124 mimics and EZH2-wild type (EZH2-WT) (p &lt; 0.05). The mRNA and protein expression levels of EZH2 decreased in SMS-KAN nerve cells transfected with miR-124 mimics (p &lt; 0.05). Overexpression of EZH2 inhibited the apoptosis of SMSKAN cells induced by sevoflurane (p &lt; 0.05). Furthermore, the apoptosis of SMS-KAN cells transfected with miR-124 inhibitor were offset by transfected siEZH2.&#x0D; Conclusion: The results suggest that overexpression of miR-124 suppresses cell proliferation by targeting EZH2 in SMS-KAN cells. Therefore, miR-124 represents a potential target for neuroblastoma therapy

Electroacupuncture promotes the recovery of rats with spinal cord injury by suppressing the Notch signaling pathway via the H19/EZH2 axis.

BackgroundSpinal cord injury (SCI) is a life-changing event with an extremely poor prognosis. In our preliminary studies, electroacupuncture (EA) was found to promote the repair of SCI, which was closely related to the Notch signaling pathway. Therefore, in the present study, we hypothesized that EA protects against SCI by inhibiting the Notch signaling pathway and sought to investigate the underlying molecular mechanisms.MethodsRat and cell models of SCI were established. The expression of long non-coding RNA H19 was measured by real-time quantitative polymerase chain reaction. The expression levels of EZH2, Notch1, Notch3, Notch4, Hes1, and PS1 protein were measured by western blot. Cell apoptosis and viability were analyzed using flow cytometry and Cell Counting Kit-8 assays, respectively. The expressions of glial fibrillary acidic protein (GFAP) and nestin were detected by immunofluorescence staining.ResultsThe expressions of H19, EZH2, and GFAP were significantly increased after SCI but were inhibited by EA; in contrast, nestin expression was significantly decreased by SCI but was restored by EA. Moreover, oxygen-glucose deprivation (OGD) treatment elevated the expression of H19, EZH2, and Notch-related factors as well as apoptosis in PC-12 cells, while suppressing cell viability. Suppressing H19 alleviated the effects of OGD on cell viability and apoptosis, and inhibited the expression of EZH2 and Notch-related factors expression; these effects were reversed by EZH2 overexpression. Finally, EA promoted the recovery of SCI rats and neural stem cell (NSC) proliferation by inhibiting the Notch signaling pathway, which was reversed by H19 overexpression.ConclusionsOur results demonstrated that EA promotes the recovery of SCI rats and increases the proliferation and differentiation of NSCs by suppressing the Notch signaling pathway via modulating the H19/EZH2 axis.