High Enhancer Of Zeste Homolog 2 Expression Research Articles

The potential of EZH2 expression to facilitate treatment choice in stage II colorectal adenocarcinoma.

The current selection criteria of patients with stage II colorectal carcinoma (CRC) suitable for adjuvant therapy are not satisfactory. Enhancer of zeste homolog 2 (EZH2) has been demonstrated to be over-expressed in CRC. However, data regarding the role of EZH2 in CRC survival remains controversial, and little is known about it in stage II CRC. Thus, we conducted this study to investigate the clinical significance of EZH2 expression in stage II CRC. Cases with stage II CRC resected between 2015 and 2018 were retrospectively reviewed. EZH2 expression was analyzed by immunohistochemistry using tissue microarrays. The relationship between EZH2 expression and clinicopathological variables was analyzed. Survival curves were estimated by the Kaplan-Meier approach. We found high EZH2 expression in 134 of 221 analyzable stage II tumors (60.63%). No significant associations were observed between EZH2 expression and common clinicopathological factors. Survival analyses showed that cases receiving surgery alone had inferior overall survival (OS) than those receiving surgery and chemotherapy (P=0.0075) in stage II CRC with high EZH2 expression, however, metastasis-free survival (MFS) was similar between these two subgroups. Treatment choice had no impact on the survival of stage II CRC with low EZH2 expression. The OS of stage II CRC with high EZH2 expression improved more strikingly with surgery and adjuvant chemotherapy than with surgery alone, which suggests the potential of EZH2 expression as a biomarker to help identify a subgroup of early-stage CRC benefiting from surgery and adjuvant chemotherapy. More large-scale studies are warranted to corroborate this finding and to further evaluate the predictive nature of EZH2.

MiR-4448/Girdin/Akt/AMPK axis inhibits EZH2-mediated EMT and tumorigenesis in small-cell lung cancer.

Small-cell lung cancer (SCLC) shows high enhancer of zeste homolog 2 (EZH2) expressions. EZH2-mediated epigenetics promote epithelial-mesenchymal transition (EMT), enhancing invasive and metastatic potential in malignancies. MicroRNAs (miRNAs), small noncoding RNAs, modulate EMT, determining tumor phenotypes. However, the association between miRNAs and EZH2 in SCLC remains to be clarified-we aimed to identify a novel tumorigenic mechanism through miRNAs, EZH2, and EMT in SCLC, leading to future therapeutic applications. We analyzed EZH2 and E-cadherin expressions in lung cancer cell lines and tumor tissues from 34 SCLC patients and confirmed EZH2 siRNA-mediated EMT inhibition. miRNA expression profiles were compared between EZH2 knockdown SCLC cells and negative control SCLC cells using miRNA array. We identified a target miRNA of EZH2 showing expressional differences in EZH2-knockdown cells and analyzed the impact of the miRNA on EZH2-mediated EMT and tumorigenesis. All SCLC cells showed increased EZH2 and decreased E-cadherin expressions. SCLC tissues had higher EZH2 and lower E-cadherin expressions than other lung cancer tissues. miRNA array revealed that miR-4448 expression increased in EZH2-knockdown SCLC cells. miR-4448 overexpression reduced tumor cell growth and prevented EMT. miR-4448 bound to the 3'UTR of the girdin gene and suppressed its expression, thereby decreasing Akt phosphorylation at Ser473. Attenuated Akt phosphorylation resulted in AMP-activated protein kinase (AMPK) phosphorylation at Thr172 and 183, enhancing EZH2 phosphorylation at Thr311. SCLC characterized high EZH2 expression and promoted EMT, compared with non-small cell lung cancer. miR-4448 inhibited Girdin expression, reducing Akt phosphorylation, and enhancing AMPK and EZH2 phosphorylation. Eventually, miR-4448 prevented EZH2-mediated EMT and tumorigenesis by modulating the Girdin/Akt/AMPK axis in SCLC. miR-4448 might be a potential SCLC inhibitor.

MicroRNA let-7c-5p Alleviates in Hepatocellular Carcinoma by Targeting Enhancer of Zeste Homolog 2: A Study Intersecting Bioinformatic Analysis and Validated Experiments.

Enhancer of zeste homolog 2 (EZH2)gene has a prognostic role in hepatocellular carcinoma (HCC). This study aimed to identify the role of microRNAs (miRNAs) let-7c-5p by targeting EZH2 in HCC. We downloaded gene and miRNA RNA-seq data from The Cancer Genome Atlas (TCGA) database. Differences in EZH2 expression between different groups were analyzed and the association of EZH2 expression with HCC prognosis was detected using Cox regression analysis. The miRNA-EZH2-pathway network was constructed. Dual-luciferase reporter assay was performed to detect the hsa-let-7c-5p-EZH2. Cell proliferation, migration, invasion, and apoptosis were detected by CCK-8, Wound healing, Transwell, and Flow cytometry, respectively. RT-qPCR and Western blot were used to detect the expression of let-7c-5p and EZH2. EZH2 was upregulated in HCC tumors (P < 0.0001). Cox regression analysis showed that TCGA HCC patients with high EZH2 expression levels showed a short survival time [hazard ratio (HR) = 1.677, 95% confidence interval (CI) 1.316-2.137; P < 0.0001]. Seven miRNAs were negatively correlated with EZH2 expression and were significantly downregulated in HCC tumor samples (P < 0.0001), in which hsa-let-7c-5p was associated with prognosis in HCC (HR = 0.849 95% CI 0.739-0.975; P = 0.021). We identified 14 immune cells that showed significant differences in EZH2 high- and low-expression groups. Additionally, let-7c-5p inhibited HCC cell proliferation, migration, and invasion and reversed the promoted effects of EZH2 on HCC cell malignant characteristics. hsa-let-7c-5p-EZH2 significantly suppressed HCC malignant characteristics, which can be used for HCC prognosis.

Comparative analysis of EZH2, p16 and p53 expression in uterine carcinosarcomas.

Introduction: The role of p16 and p53 immunohistochemistry in the diagnosis of rare and aggressive uterine carcinosarcoma (UCS) has been well established. However, enhancer of zeste homolog 2 (EZH2), a histone methyltransferase and a member of the polycomb group family is a relatively new biomarker, with limited published data on its significance in this tumor type. The goal of this study was to examine EZH2 expression in UCS and its components, in correlation with morphological features, and p16 and p53 staining patterns. Methods: Twenty-eight UCSs were included in the study. EZH2, p16 and p53 immunoreactivity were assessed independently by two pathologists in both tumor components (epithelial and mesenchymal). EZH2 and p16 immunostains were scored semiquantitatively: based on the percentage and intensity of tumor cell staining a binary staining index ("high- or low-expressing") was calculated. The p53 staining pattern was evaluated as wild-type or aberrant (diffuse nuclear, null, or cytoplasmic expression). Statistical tests were used to evaluate the correlation between staining patterns for all three markers and the different tumor components and histotypes. Results: High EZH2 and p16 expression and aberrant p53 patterns were present in 89.3% 78.6% and 85.7% of the epithelial component and in 78.6%, 62.5% and 82.1% of the mesenchymal component, respectively. Differences among these expression rates were not found to be significant (p > 0.05). Regarding the epithelial component, aberrant p53 pattern was found to be significantly (p = 0.0474) more frequent in the serous (100%) than in endometrioid (66.6%) histotypes. Within the mesenchymal component, p53 null expression pattern occurred significantly (p = 0.0257) more frequently in heterologous sarcoma components (71.4%) compared to the homologous histotype (18.8%). Conclusion: In conclusion, EZH2, p16 and p53 seem to play a universal role in the pathogenesis of UCS; however, a distinctive pattern of p53 expression appears to exist between the serous and endometrioid carcinoma components and also between the homologous and heterologous sarcoma components.

EZH2: An Accomplice of Gastric Cancer.

Gastric cancer is the fifth most common cancer and the third leading cause of cancer deaths worldwide. Understanding the factors influencing the therapeutic effects in gastric cancer patients and the molecular mechanism behind gastric cancer is still facing challenges. In addition to genetic alterations and environmental factors, it has been demonstrated that epigenetic mechanisms can also induce the occurrence and progression of gastric cancer. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressor complex 2 (PRC2), which trimethylates histone 3 at Lys-27 and regulates the expression of downstream target genes through epigenetic mechanisms. It has been found that EZH2 is overexpressed in the stomach, which promotes the progression of gastric cancer through multiple pathways. In addition, targeted inhibition of EZH2 expression can effectively delay the progression of gastric cancer and improve its resistance to chemotherapeutic agents. Given the many effects of EZH2 in gastric cancer, there are no studies to comprehensively describe this mechanism. Therefore, in this review, we first introduce EZH2 and clarify the mechanisms of abnormal expression of EZH2 in cancer. Secondly, we summarize the role of EZH2 in gastric cancer, which includes the association of the EZH2 gene with genetic susceptibility to GC, the correlation of the EZH2 gene with gastric carcinogenesis and invasive metastasis, the resistance to chemotherapeutic drugs of gastric cancer mediated by EZH2 and the high expression of EZH2 leading to poor prognosis of gastric cancer patients. Finally, we also clarify some of the current statuses of drug development regarding targeted inhibition of EZH2/PRC2 activity.

EZH2-regulated immune risk score prognostic model predicts outcome of clear cell renal cell carcinoma.

The enhancer of zeste homolog 2 (EZH2) plays an important role in the tumor microenvironment (TME), and EZH2 in shaping the epigenetic landscape of CD8+ T cell fate and function, with a particular emphasis on cancer. Here, high EZH2 expression always leads to less CD8+ T cell infiltration. However, clear cell renal cell carcinoma (ccRCC) is reportedly a "hot" tumor, with contradictory high EZH2 expression. Our goal was to construct a EZH2-regulated immune risk score prognostic model to predict ccRCC outcomes, and provide a prospect of clinical EZH2 inhibitors in fine-tuning T cell responses with immune therapy. We downloaded and analyzed The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), TISIDB database, and WebGestalt for ccRCC patients, EZH2-related tumor-infiltrating lymphocytes and immunomodulators. R packages "limma", "BiocManager", and "preprocessCore", etc. were downloaded to prepare CIBERSORT files, immune cells heatmap, multivariable Cox model and survival analysis. The EZH2-regulated immune risk model's prognostic ability was calculated by receiver operating characteristic (ROC) and area under the curve (AUC) analyses in R studio. EZH2 was highly expressed and related to poor outcome in ccRCC. However, high-expression EZH2 was not related to a "cool" tumor. Of the 49 immunomodulators significantly regulated by EZH2, forest plot showed 26 immunomodulators signatures independently associated with overall survival. The EZH2-regulated immune-risk score prognostic model was an independent prognostic factor (AUC =0.816), especially combined with clinicopathologic parameters in ccRCC overall survival prediction. The EZH2-regulated immune-risk score prognostic model was an independent prognostic factor, with good accuracy and predictability, and could provide experimental data to the clinical area.

Expression of EZH2 and H3K27me3 predicts tumor biology of urothelial carcinoma.

Enhancer of zeste homolog 2 (EZH2) is one of the major epigenetic modifiers involved in the transcriptional repression of target genes through trimethylation of H3K27 (lysine 27 residue of histone H3). Deregulated expression of both EZH2 and H3K27me3 has been implicated in the biological behavior and prognostic outcome of various malignancies. To assess the role of EZH2 and H3K27me3 in the carcinogenesis of urothelial carcinoma of urinary bladder. One hundred fifty consecutive urothelial carcinoma cases of urinary bladder (54.7% high-grade) were included in this study. Immunohistochemical analysis for EZH2 and H3K27me3 was performed on whole tissue sections. A multiplication score obtained by multiplying staining intensity and proportion of positively stained neoplastic cells was used for assessment. EZH2 showed a significant correlation with the tumor grade and lamina propria invasion (p < 0.001). The cases with high EZH2 expression showed a significantly high proliferative index (Mean- 32.7%; p < 0.001). In contrast, negative and low expression of H3K27me3 was significantly more common in high-grade cases (p = 0.006). The expression of H3K27me3 was significantly associated with lamina propria (p = 0.01) and deep muscle invasion (p = 0.007). EZH2 showed a significantly higher expression in the high-grade invasive areas as compared to the high-grade non-invasive areas of the same tumor (p = 0.03). This study establishes an important role of the key epigenetic regulators EZH2 and H3K27me3 in the pathobiology of urothelial carcinomas. Strong expression of EZH2 and weak expression of H3K27me3 are associated with higher grade, proliferative index and invasive behavior.

Targeting EZH2 Promotes Chemosensitivity of BCL-2 Inhibitor through Suppressing PI3K and c-KIT Signaling in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is one of the most common hematological malignancies with high heterogeneity, characterized by a differentiating block at the early progenitor stage. The selective BCL-2 inhibitor, Venetoclax (Ven), has shown exciting clinical results in a certain group of AML patients. However, Ven alone is insufficient to reach an enduringly complete response, which leads to the concern of Ven resistance. Alternative combined therapies with Ven are demanded in AML. Here, we reported the synergistic effect and molecular mechanism of the enhancer of zeste homolog 2 (EZH2) inhibitor DZNeP with Ven in AML cells. Results showed that the combination of DZNeP with Ven significantly induces cell proliferation arrest compared to single-drug control in AML cells and primary samples, and CalcuSyn analysis showed their significant synergy. The combination also significantly promotes apoptosis and increases the expression of pro-apoptotic proteins. The whole transcriptome analysis showed that phosphoinositide-3-kinase-interacting protein1 (PIK3IP1), the PI3K/AKT/mTOR signaling suppressor, is upregulated upon DZNeP treatment. Moreover, EZH2 is upregulated but PIK3IP1 is downregulated in 88 newly diagnosed AML cohorts compared to 70 healthy controls, and a higher expression of EZH2 is associated with poor outcomes in AML patients. Particularly, the combination of DZNeP with Ven dramatically eliminated CD117 (c-KIT) (+) AML blasts, suggesting the effect of the combination on tumor stem cells. In summary, our data indicated that DZNeP increases the sensitivity of Ven in AML by affecting PI3K and c-KIT signaling in AML. Our results also suggested that the therapeutic targeting of both EZH2 and BCL-2 provides a novel potential combined strategy against AML.

Mutually exclusive expression of EZH2 and H3K27me3 in non-small cell lung carcinoma

Enhancer of zeste homolog 2 (EZH2) epigenetically represses gene expression via trimethylation of lysine 27 on histone 3 (H3K27me3). Non-small cell carcinoma (NSCLC) has been reported to show high EZH2 and low H3K27me3 expression compared to normal lung tissues, but there are no studies examining the expression of EZH2 and H3K27me3 simultaneously with immunohistochemistry. In the present study, the expression of EZH2 and H3K27me3 was examined in surgically resected NSCLC. We enrolled 27 cases of squamous cell carcinoma (SCC), 73 cases of Lepidic, 77 of Papillary/Acinar, 51 of Solid, 31 of Micropapillary, and 12 of Mucinous subtypes of adenocarcinoma. First, we examined the expression of EZH2 and H3K27me3 in normal and metaplastic bronchial epithelium adjacent to NSCLC. Normal bronchial epithelium showed EZH2 expression in a limited number of basal cells and H3K27me3 expression in surface differentiated cells with cilia or mucus. In metaplastic bronchial epithelium, the number of EZH2-positive cells increased in multilayered basal cells, and H3K27me3-positive cells were observed in the superficial layer. Then, EZH2 and H3K27me3 expression was analyzed in NSCLC. Abundant EZH2 and rare H3K27me3 expression was detected in SCC, Papillary/Acinar, Solid and Micropapillary subtypes. In Mucinous subtype, EZH2 expression was hardly detected, and H3K27me3 expression was detected in almost all tumor cells. EZH2-expressing and H3K27me3-expressing tumor cells were similarly observed in Lepidic subtype, but double immunofluorescence revealed that EZH2 and H3K27me3 expression pattern was mutually exclusive. No co-expression of EZH2 and H3K27me3 was detected in all examined subtypes. To our knowledge, there have been no reports describing mutually exclusive expression pattern of EZH2 and H3K27me3.

Abstract 5381: Combinatory effect of EZH2 inhibitor and dopamine D1 receptor agonist enhance inhibition of triple negative breast cancer cell growth

Abstract Treatment for Triple Negative Breast Cancer remains a challenge due to its aggressive behavior; poor clinical outcome and disposition to therapeutic resistance. The diverse clinical and molecular characteristics drives high level of heterogeneity in breast cancer. Overall change in the epigenome triggers oncogenic activity of enhancer of zeste homolog 2 (EZH2) causing changes in gene expression that leads to cancer progression. EZH2 is the catalytic member of the Polycomb group protein complex that provides the active site for covalent methylation reactions. High expression of EZH2 is strongly associated with aggressive TNBC phenotypes, compared to other non-TNBCs. Development of resistance to EZH2 inhibitors limit their therapeutic use. Studies have shown that dopamine receptor (DR) agonists and antagonists alter TNBC cell growth in both in vitro and in vivo models. Our objective was to investigate if a combination of a D1R agonist with an EZH2 inhibitor was effective to attenuate TNBC cell proliferation. Docking studies were performed using Schrodinger software and visualization software to determine the affinity of GSK126 and SKF38393 towards EZH2. TNBC cells were treated individually and combined with EZH2 inhibitor, GSK126, and D1 agonist SKF38393 for cell viability and immunoprecipitation. Insilco analysis using docking studies revealed that SKF38393 actively interacts in the same region where GSK126 binds to EZH2 i.e. the catalytic site. Combination treatment of TNBC cells with GSK126 and SKF38393 caused synergistic inhibition of cell viability, suppressed the H3K27 trimethylation activity, and inhibited invasion of TNBC cells. Immunoprecipitation of EZH2 in the presence of GSK126 and SKF38393 dissociated the association between EZH2, EED, and SUZ12 in MDA-MB-231 cells. We report that combination of GSK126 and SKF38393 may have an enhanced efficacy in inhibiting the catalytic function of EZH2. The combined treatment of TNBC cells with GSK126 and SKF38393 inhibit TNBC proliferation by disrupting EZH2 functions. (This work is supported by DOD: W81XWH2010065, for Eswar Shankar) Citation Format: Prem Prakash Kushwaha, Shiv Verma, Gautham Sarathy, Anmol Kumar, Sanjay Gupta, Bhuvaneswari Ramaswamy, Sarmila Majumder, Eswar Shankar. Combinatory effect of EZH2 inhibitor and dopamine D1 receptor agonist enhance inhibition of triple negative breast cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5381.

Enhancer of Zeste Homolog 2 (EZH2) Is a Marker of High-Grade Neuroendocrine Neoplasia in Gastroenteropancreatic and Pulmonary Tract and Predicts Poor Prognosis.

Simple SummaryNeuroendocrine neoplasms most frequently arise in the gastroenteropancreatic and pulmonary tract and show an increasing incidence and prevalence. The prognosis and treatment depend on tumor proliferation and clinical behavior. Highly proliferating grade 3 neoplasms especially, show a wildly divergent therapy response and prognosis. In particular, it is crucial to securely separate the more indolent G3 tumors from the more aggressive carcinomas. Currently, this distinction is based on a combination of clinical, morphologic, immunohistochemical, and molecular biomarkers. However, none of these markers allow for a reliable distinction, and additional markers are needed. EZH2 has attracted increasing interest in different tumor entities. We aimed to analyze the expression of EZH2 in different neuroendocrine neoplasms and to correlate the expression with clinical parameters and survival. We demonstrate that EZH2 is nearly exclusively expressed in highly proliferative neoplasms and is a robust biomarker for identifying aggressive G3 tumors with poor prognosis.Tumor grading is a robust prognostic predictor in patients with neuroendocrine neoplasms (NEN) and guides therapy, especially in tumors with high proliferation. NEN can be separated into well-differentiated and poorly differentiated types. The more aggressive NEN have been further separated into neuroendocrine tumors (NET G3) with a better prognosis and neuroendocrine carcinomas (NEC) with a worse prognosis. Despite this distinction’s tremendous clinical and therapeutic relevance, optimal diagnostic biomarkers are still lacking. In this study, we analyzed the protein expression and prognostic impact of Enhancer of Zeste Homolog 2 (EZH2) by immunohistochemistry in 219 tissue samples of gastroenteropancreatic (GEP-NEN) and pulmonary NEN (P-NEN). EZH2 was almost exclusively expressed in NEN with a proliferation rate above 20% (G3), while all low-grade tumors were nearly negative. Among high-grade NEN, 65% showed high and 35% low expression of EZH2. In this group, the high expression of EZH2 was significantly associated with poor overall survival and NEC histology. Interestingly, EZH2 seems to act independently of Polycomb Repressive Complex 2 (PRC2) in NEN. In conclusion, we propose EZH2 as a robust biomarker for distinguishing between NET G3 and NEC among gastroenteropancreatic and pulmonary NEN.

Genome-wide gain-of-function screening identifies EZH2 mediating resistance to PI3Kα inhibitors in oesophageal squamous cell carcinoma.

Phosphoinositide‐3 kinase alpha (PI3Kα) has been confirmed to be a potential therapeutic target for esophageal squamous cell carcinoma (ESCC), while the potency of PI3Kα inhibitors is often attenuated by concurrent oncogenic signalling pathways. We performed genome‐wide gain‐of‐function screening with a CRISPR‐SAM library and identified enhancer of zeste homolog 2 (EZH2) rendering ESCC cells resistant to the PI3Kα inhibitor CYH33. Enhanced expression of EZH2 frequently occurs in ESCC and is related to poor prognosis. Overexpression of full‐length EZH2 but not methyltransferase‐deficient EZH2 conferred resistance to CYH33, while downregulating EZH2 expression restored sensitivity. EZH2 expression was negatively related to the activity of CYH33 against the proliferation of ESCC cell lines and patient‐derived cells. Transcriptomic analysis revealed that EZH2 abrogated CYH33‐mediated cell cycle regulation. EZH2 epigenetically suppressed the transcription of CDKN1A, promoting RB phosphorylation and cell cycle progression. Concurrently targeting EZH2 significantly potentiated CYH33 to inhibit the growth of ESCC cells and patient‐derived xenografts accompanied by enhanced cell cycle arrest. Taken together, our study demonstrated that an EZH2‐p21‐RB axis remodeled cell cycle regulation and rendered resistance to PI3Kα inhibitors in ESCC. Simultaneously targeting PI3Kα and EZH2 may provide an effective strategy for ESCC therapy with high expression of EZH2.

Combined Detection of RUNX3 and EZH2 in Evaluating Efficacy of Neoadjuvant Therapy and Prognostic Value of Middle and Low Locally Advanced Rectal Cancer.

ObjectiveThis article investigated whether Runt-Related Transcription Factor 3 (RUNX3) and enhancer of zeste homolog 2 (EZH2) can be used to evaluate the clinical efficacy of neoadjuvant therapy and prognosis of locally advanced rectal cancer (LARC). MethodsEighty LARC patients admitted to the Tianjin Medical University Cancer Institute/Hospital and First Affiliated Hospital of Hebei North University from Jan 2015 to Jan 2016 were enrolled. The patients were followed up for 60 months through hospital visits. All patients received neoadjuvant chemoradiotherapy (long range radiotherapy + oral capecitabine) + total mesorecta excision (TME) surgery. The clinical efficacy of the treatments was evaluated through endoscopic, radiography, and tumor regression grade (TRG). In addition, expression level of RUNX3 and EZH2 was quantified via immunohistochemistry. The association of RUNX3 and EZH2 with clinicopathological characteristics of advanced tumors and efficacy of neoadjuvant therapy was explored. Logistic regression analysis was performed to identify predictors of efficacy of neoadjuvant chemoradiotherapy. Survival curve was used to evaluate the impact of RUNX3 and EZH2 on the prognosis of LARC patients.ResultsA total of 80 patients diagnosed with LARC were enrolled in the study. Expression of RUNX3 was elevated in 25 (31.25%) patients, whereas expression of EZH2 was upregulated in 44 (55.00%) patients. Analysis of tumor regression identified 10 cases with TRG grade 0 (pathologic complete response, PCR), 24 cases with TRG grade 1, 35 cases with TRG grade 2, and 11 cases with TRG grade 3. Furthermore, 38 cases had significant down-staging, and 42 cases showed no significant down-staging as revealed by endoscopy and imaging. Patients with high expression of RUNX3 showed better tumor regression response and down-staging compared with those with low expression of RUNX3 (P < 0.001, P < 0.001). Moreover, patients with low EZH2 expression achieved TRG grade 0 and 1 response and down-staging effect compared with those with high expression of EZH2 (P < 0.001, P < 0.001). Logistic regression analysis showed that high expression of RUNX3, low expression of EZH2, and clinical N (cN) stage were good predictors of tumor regression response and down-staging. The 5-year disease free survival (DFS) and overall survival (OS) were 48.75 (39/80) and 58.75% (47/80), respectively. The 5-year DFS and OS of patients with high RUNX3 expression were significantly higher than low RUNX3 expression, whereas the 5-year DFS and OS of patients with high EZH2 expression were significantly lower than low EZH2 expression (P < 0.001). Univariate survival analysis showed that RUNX3 expression, EZH2 expression, cN, clinical T (cT), pathological T (pT) and pathological N (pN) were significantly correlated with the 5-year DFS and 5-year OS. Multivariate survival analysis showed that EZH2 expression and PN were good predictors of 5-year DFS and 5-year OS, whereas RUNX3 was a good predictor of 5-year DFS but not 5-year OS.ConclusionsExpression level of RUNX3 and EZH2 accurately predicts clinical efficacy of neoadjuvant chemoradiotherapy and the prognosis of LARC patients, suggesting that RUNX3 and EZH2 can be used as pivotal clinical predictors for LARC.

Relationship between Expression of Runt-related Transcription Factor 3 and Enhancer of zeste Homolog 2 Proteins and Sensitivity to Neoadjuvant Chemotherapy in Locally Advanced Rectal Cancer

Objective To investigate the expression and correlation of Runt-related transcription factor 3(RUNX3)and enhancer of zeste homolog 2(EZH2)in rectal cancer,and to reveal the relationship between the expression of RUNX3 and EZH2 and the sensitivity of XELOX regimen to neoadjuvant chemotherapy in locally advanced rectal cancer patients. Methods The carcinoma and paracancerous tissues of 31 patients with rectal adenocarcinoma and no preoperative antitumor therapy were selected as cancer group and paracancer group,respectively.The relative mRNA levels of RUNX3 and EZH2 in the two groups were measured by real-time quantitative reverse transcription-polymerase chain reaction,and the protein levels were determined by immunohistochemical assay.The expression of RUNX3 and EZH2 was compared between cancer tissue and paracancerous tissue.The pre-treatment wax blocks of 26 patients with locally advanced rectal cancer who received 3 cycles of XELOX regimen as neoadjuvant chemotherapy before surgery were selected as the pre-neoadjuvant therapy group,and the postoperative pathological wax blocks were selected as the post-neoadjuvant treatment group.Tumor regression grade(TRG)was determined to evaluate the efficacy of neoadjuvant therapy.Immunohistochemical assay was used to detect the protein levels of RUNX3 and EZH2 in the two groups,and then the relationship between the expression patterns of the two proteins and the efficacy of neoadjuvant chemotherapy was analyzed. Results Compared with paracancerous tissue,the cancer tissue showed down-regulated mRNA level and reduced positive protein expression rate of RUNX3,while up-regulated mRNA level(P=0.001)and increased positive protein expression rate of EZH2(P=0.022).The mRNA levels of RUNX3 and EZH2 in the cancer group were negatively correlated(r=-0.599,P=0.000).Twelve patients who received neoadjuvant chemotherapy reached TRG0-TRG2,and the overall effective rate of neoadjuvant chemotherapy was 46.15%(12/26).Compared with pre-neoadjuvant therapy group,the post-neoadjuvant therapy group had increased positive expression rate of RUNX3 protein(P=0.163)and decreased positive expression rate of EZH2 protein(P=0.095).In the pre-neoadjuvant therapy group,the effective rate of neoadjuvant chemotherapy was 75.00%(9/12)in patients with positive RUNX3 expression,77.78%(7/9)in patients with negative EZH2 expression,and 100%(7/7)in patients with positive expression of RUNX3 and negative expression of EZH2.Multivariate Logistic regression showed that the expression of RUNX3 protein was the factor influencing the efficacy of neoadjuvant chemotherapy. Conclusions The positive expression rate of RUNX3 in cancer tissue was lower than that in paracancerous tissue,while that of EZH2 showed the opposite trend.Neoadjuvant chemotherapy may affect the expression of RUNX3 and EZH2 in rectal cancer.The patients with high RUNX3 expression and low EZH2 expression in locally advanced rectal cancer were more sensitive to neoadjuvant chemotherapy with XELOX regimen.

EZH2 presents a therapeutic target for neuroendocrine tumors of the small intestine

Small intestinal neuroendocrine tumors (SI-NETs) are slow-growing tumors that seem genetically quite stable without highly recurrent mutations, but are epigenetically dysregulated. In contrast to the undetectable expression of the enhancer of zeste homolog 2 (EZH2) histone methyltransferase in the enterochromaffin cells of the small intestine, we found high and differential expression of EZH2 in primary SI-NETs and corresponding metastases. Silencing EZH2 in the SI-NET cell line CNDT2.5 reduced cell proliferation and induced apoptosis. Furthermore, EZH2 knockout inhibited tumor progression in a CNDT2.5 SI-NET xenograft mouse model, and treatment of SI-NET cell lines CNDT2.5 and GOT1 with the EZH2-specific inhibitor CPI-1205 decreased cell viability and promoted apoptosis. Moreover, CPI-1205 treatment reduced migration capacity of CNDT2.5 cells. The EZH2 inhibitor GSK126 also repressed proliferation of CNDT2.5 cells. Recently, metformin has received wide attention as a therapeutic option in diverse cancers. In CNDT2.5 and GOT1 cells, metformin suppressed EZH2 expression, and inhibited cell proliferation. Exposure of GOT1 three-dimensional cell spheroids to CPI-1205 or metformin arrested cell proliferation and decreased spheroid size. These novel findings support a possible role of EZH2 as a candidate oncogene in SI-NETs, and suggest that CPI-1205 and metformin should be further evaluated as therapeutic options for patients with SI-NETs.

Novel Natural Inhibitors Targeting Enhancer of Zeste Homolog 2: A Comprehensive Structural Biology Research.

The enhancer of zeste homolog 2 (EZH2) is a methylated modification enzyme of Histone H3-Lys 27. The high expression of EZH2 in cells is closely related to the progression, invasion, and metastasis of neoplasm. Therefore, this target is gradually becoming one of the research hot spots of tumor pathogenesis, and the inhibitors of the EZH2 enzyme are expected to become new antitumor drugs. This study used a series of virtual screening technologies to calculate the affinity between the compounds obtained from the ZINC15 database and the target protein EZH2, the stability of the ligand–receptor complex. This experiment also predicted the toxicity and absorption, distribution, metabolism, and excretion (ADME) properties of the candidate drugs in order to obtain compounds with excellent pharmacological properties. Finally, the ligand–receptor complex under in vivo situation was estimated by molecular dynamics simulation to observe whether the complex could exist steadily in the body. The experimental results showed that the two natural compounds ZINC000004217536 and ZINC000003938642 could bind tightly to EZH2, and the ligand–receptor complex could exist stably in vivo. Moreover, these two compounds were calculated to be nontoxic. They also had a high degree of intestinal absorption and high bioavailability. In vitro experiments confirmed that drug ZINC000003938642 could inhibit the proliferation and migration of osteosarcoma, which could serve as potential lead compounds. Therefore, the discovery of these two natural products had broad prospects in the development of EZH2 inhibitors, providing new clues for the treatment or adjuvant treatment of tumors.

Prognostic significance of EZH2 and ARID1A expression in urothelial carcinoma: an immunohistochemical study

ABSTRACT Enhancer of zeste homolog 2 (EZH2) and AT-rich interactive domain 1A (ARID1A) expression in urothelial carcinoma (UC) has not been well studied. ARID1A is a novel tumor suppressor gene coding for a chromatin remodeling protein that is mutated in urinary bladder cancer. The enhancer of zeste homolog 2 (EZH2) is a transcriptional repressor involved in gene silencing. Amplification of EZH2 has been reported in several malignancies. This study analyzed the immunohistochemical expression of EZH2 and ARID1A in 56 cases of UC that included (n = 21) cases of radical cystectomy and (n = 35) cases of transurethral resections of bladder tumor (TURBT) with muscle fibers and immunotherapy with adjuvant intravesical bacillus Calmette-Guerin (BCG). The predicting role of both markers for tumor recurrence and recurrence-free survival (RFS) was also analyzed. High EZH2 marker expression was observed in 75% of cases while 78.6% of cases had low ARID1A marker expression. There was a significant negative correlation between the two markers where high EZH2 and low ARID1A expression was significantly associated with higher tumor grade, stage, presence of muscle invasion, lymph node metastasis, presence of concomitant carcinoma in situ (CIS) and higher incidence of recurrence with shorter RFS rate. It was concluded that EZH2 and ARID1A play a role in tumor carcinogenesis and differentiation and could be considered as independent prognostic factors in UC and for use as a potential therapeutic target.

The significance of enhancer of zeste homolog 2 (EZH2) expression in spindle cell lesions of the breast.

Spindle cell lesions of the breast are rare entities and pose a diagnostic challenge for pathologists due to overlapping morphologic and immunohistochemical features. We evaluated EZH2 expression in various benign (fibromatosis (n = 8), myofibroblastoma (n = 7), neurofibroma (n = 1), nodular fasciitis (n = 5), benign phyllodes tumor (n = 18)) and malignant (malignant phyllodes tumor (n = 8), metaplastic breast carcinoma (n = 16) and angiosarcoma (n = 8)) spindle cell lesions as a potential diagnostic and therapeutic marker. The EZH2 expression was evaluated semi-quantitatively to categorize the cases as 'low' and 'high' expression. All benign lesions showed low EZH2 expression, whereas high EZH2 expression was observed in the majority (28/32; 88%) of malignant lesions. The study results suggest that EZH2 may be used both as an additional diagnostic tool to reach an accurate diagnosis of the spindle cell lesions of the breast and as a therapeutic target for the malignant lesions.

Evaluation of EZH2 and ERRα in colorectal carcinoma: an immunohistochemical study.

The combined immunohistochemical evaluation of EZH2 (enhancer of zeste homolog 2) and ERRα (estrogen-related receptor α), in relation to clinicopathological prognostic factors and patients' outcome, has not been performed yet in colorectal carcinoma (CRC). In order to achieve this aim, 120 samples were extracted; 60 cases of CRC; and 60 samples from normal colonic tissue. Our study showed that 63.3% and 38.3% of CRC cases reveal high EZH2 and high ERRα nuclear expression, respectively. 6.6% and 8.3% of normal colonic mucosa samples express low EZH2 and low ERRα nuclear expression, respectively. High EZH2 and high ERRα expression correlate with late tumor stages (p = 0.001 each), high grade (p = 0.001, p = 0.009 respectively), positive lymph node involvement (p = 0.001, p = 0.002 respectively) and larger tumor size (p = 0.001 each). There is a moderate highly statistically significant agreement (κ = 0.467, p = 0.001) between EZH2 and ERRα immunohistochemical expression. By Kaplan Meier analysis, high EZH2 and high ERRα show statistically significant shorter overall survival, and progression free survival than cases with low EZH2 and low ERRα immunohistochemical expression, respectively. Thus, EZH2 and ERRα might serve as potential promising prognostic markers in CRC.

Overexpression of miR-101 suppresses collagen synthesis by targeting EZH2 in hypertrophic scar fibroblasts.

BackgroundMicroRNA-101 (miR-101) is a tumor suppressor microRNA (miRNA) and its loss is associated with the occurrence and progression of various diseases. However, the biological function and target of miR-101 in the pathogenesis of hypertrophic scars (HS) remains unknown.MethodsWe harvested HS and paired normal skin (NS) tissue samples from patients and cultured their fibroblasts (HSF and NSF, respectively). We used quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), fluorescence in situ hybridization (FISH), enzyme-linked immunosorbent assays (ELISA) and Western blot analyses to measure mRNA levels and protein expression of miR-101, enhancer of zeste homolog 2 (EZH2), collagen 1 and 3 (Col1 and Col3) and α-smooth muscle actin (α-SMA) in different in vitro conditions. We also used RNA sequencing to evaluate the relevant signaling pathways and bioinformatics analysis and dual-luciferase reporter assays to predict miR-101 targets. We utilized a bleomycin-induced fibrosis mouse model in which we injected miR-101 mimics to evaluate collagen deposition in vivo.ResultsWe found low expression of miR-101 in HS and HSF compared to NS and NSF. Overexpressing miR-101 decreased Col1, Col3 and α-SMA expression in HSF. We detected high expression of EZH2 in HS and HSF. Knockdown of EZH2 decreased Col1, Col3 and α-SMA in HSF. Mechanistically, miR-101 targeted the 3′-untranslated region (3′UTR) of EZH2, as indicated by the decreased expression of EZH2. Overexpressing EZH2 rescued miR-101-induced collagen repression. MiR-101 mimics effectively suppressed collagen deposition in the bleomycin-induced fibrosis mouse model.ConclusionsOur data reveal that miR-101 targets EZH2 in HS collagen production, providing new insight into the pathological mechanisms underlying HS formation.