Deazaneplanocin A Is a Promising Drug to Kill Multiple Myeloma Cells in Their Niche

Tumoral plasma cells has retained stemness features and in particular, a polycomb-silenced gene expression signature. Therefore, epigenetic therapy could be a mean to fight for multiple myeloma (MM), still an incurable pathology. Deazaneplanocin A (DZNep), a S-adenosyl-L-homocysteine hydrolase inhibitor, targets enhancer of zest homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2) and is capable to induce the death of cancer cells. We show here that, in some MM cell lines, DZNep induced both caspase-dependent and -independent apoptosis. However, the induction of cell death was not mediated through its effect on EZH2 and the trimethylation on lysine 27 of histone H3 (H3K27me3). DZNep likely acted through non-epigenetic mechanisms in myeloma cells. In vivo, in xenograft models, and in vitro DZNep showed potent antimyeloma activity alone or in combination with bortezomib. These preclinical data let us to envisage new therapeutic strategies for myeloma.     

3-Deazaneplanocin A (DZNep), an Inhibitor of the Histone Methyltransferase EZH2, Induces Apoptosis and Reduces Cell Migration in Chondrosarcoma Cells

Objective

Growing evidences indicate that the histone methyltransferase EZH2 (enhancer of zeste homolog 2) may be an appropriate therapeutic target in some tumors. Indeed, a high expression of EZH2 is correlated with poor prognosis and metastasis in many cancers. In addition, 3-Deazaneplanocin A (DZNep), an S-adenosyl-L homocysteine hydrolase inhibitor which induces EZH2 protein depletion, leads to cell death in several cancers and tumors. The aim of this study was to determine whether an epigenetic therapy targeting EZH2 with DZNep may be also efficient to treat chondrosarcomas.

Methods

EZH2 expression was determined by immunohistochemistry and western-blot. Chondrosarcoma cell line CH2879 was cultured in the presence of DZNep, and its growth and survival were evaluated by counting adherent cells periodically. Apoptosis was assayed by cell cycle analysis, Apo2.7 expression using flow cytometry, and by PARP cleavage using western-blot. Cell migration was assessed by wound healing assay.

Results

Chondrosarcomas (at least with high grade) highly express EZH2, at contrary to enchondromas or chondrocytes. In vitro, DZNep inhibits EZH2 protein expression, and subsequently reduces the trimethylation of lysine 27 on histone H3 (H3K27me3). Interestingly, DZNep induces cell death of chondrosarcoma cell lines by apoptosis, while it slightly reduces growth of normal chondrocytes. In addition, DZNep reduces cell migration.

Conclusion

These results indicate that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treat chondrosarcomas.     

Induction of erythroid differentiation of human K562 cells by 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives

In this paper we report the synthesis of twelve 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives and the results obtained on their effects in inducing erythroid differentiation of human leukemic K562 cells. The data obtained demonstrate that two of the newly synthetized compounds are able to induce erythroid differentiation of K562 cells. In addition, these same compounds potentiate K562 erythroid differentiation induced by cytosine arabinoside, retinoic acid and mithramycin. Inducers of erythroid differentiation stimulating fetal gamma-globin synthesis could be considered for possible use in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes.     

Emodin can induce K562 cells to erythroid differentiation and improve the expression of globin genes

In China, the traditional Chinese medicine “YiSui ShenXu Granule” has been used for treating β-thalassemia over 20 years and known to be effective in clinic. Several purified components from “YiSui ShenXu Granule” are tested in K562 cells to reveal its effect on globin expression and erythroid differentiation, and one of the purified components, emodin, was demonstrated to increase the expression of α-, ε-, γ-globin, CD235a, and CD71 in K562 cells. Moreover, the increase of their expression is emodin concentration-dependent. The mRNA and microRNA (miRNA) expression profiles are further analyzed and 417 mRNAs and 35 miRNAs with differential expression between untreated and emodin-treated K562 cells were identified. Among them, two mRNAs that encode known positive regulators of erythropoiesis, ALAS2, and c-KIT respectively, increased during emodin-induced K562 erythroid differentiation, meanwhile, two negative regulators, miR-221 and miR-222, decreased during this process. These results indicate that emodin can improve the expression of globin genes in K562 cells and also induce K562 cells to erythroid differentiation possibly through up-regulating ALAS2 and c-KIT and down-regulating miR-221 and miR-222.     

PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation In Situ

Background

Lymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression.

Methodology/Principal Findings

We performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors.

Conclusions/Significance

This study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.     

利用K562红细胞分化模型和cDNA芯片技术筛选参与红细胞分化的新基因(英文)

以氯高铁血红素 (hemin)诱导K5 6 2分化作为体外红细胞分化模型 ,结合cDNA大规模测序、生物信息学分析、基因芯片杂交和NorthernBlot分析等技术 ,筛选红细胞分化相关的新基因 .首先利用大规模测序技术从人胚肾cDNA文库中随机挑选克隆测得 192个EST(expressedsequencetags)片段 ,经在线生物信息学分析 ,得到 79个代表新基因的未知EST片段 ,并在NCBI(NationalCenterofBiotechnologyInformation)dbEST库中登录 .利用 79个ESTcDNA片段制备了基因芯片 .提取分化前后的K5 6 2细胞的mRNA作为荧光标记反转录的模板 ,反转录后的探针用于DNA芯片杂交 .分析杂交后的结果 ,得到了 2个差异表达较明显的基因 ,GenBank登录号分别为AF147772 (187bp)和AF4 776 2(6 30bp) ,并分别命名为EDRG1和EDRG2 (erythroiddifferentiationrelatedgene 1and 2 ) ,相似性检索表明它们属全新基因 ,基因组草图测序数据库检索表明了两个基因的染色体定位 .随后的Northern印迹用于验证了在分化前后的K5 6 2细胞中差异表达 .提示这两个基因参与了红细胞分化过程 .RT PCR检测了EDRG1和EDRG2在人胚胎多组织中的表达 .结果提示 ,EDRG1可能与多种胚组织的正常发育相关 ,尤其在胚脑中高丰度表达 ,而EDRG2则可能参与了胚心和胚肾的组织生成 .生物     

EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers

Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers. In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by shRNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation between EZH2 and DLC1 expression was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors.     

Phosphorylation of ELAVL1 (Ser219/Ser316) mediated by PKC is required for erythropoiesis

Elavl1 (also known as HuR), an RNA binding protein highly conserved between zebrafish and human, regulates gene expression by stabilizing target mRNA. Our previous studies have uncovered that the predominant isoform elavl1a is required for zebrafish embryonic erythropoiesis. However, the exact mechanism of how elav11 spatiotemporally stabilizes target mRNAs to regulate specific erythropoiesis is not yet understood. Here we show that phosphorylation of elavl1a at Ser219 and Ser316 by PKC is necessarily required for cytosolic shuttling from the nucleus to stabilize gata1 mRNA and thus promotes erythropoiesis. Knockdown of elavl1a resulted in the hindrance of erythropoiesis and Hemin-induced erythroid differentiation of human myeloid leukemia K562 cells. Interestingly, inhibition of PKC reproduced the phenotype seen during zebrafish embryogenesis and erythroid differentiation of myeloid leukemia. Mechanistically, Hemin induced elavl1a export from nuclear to cytoplasmic space in K562 cells in a manner dependent on phosphorylation on Ser219 and Ser316, as overexpression of elavl1a with mutations on Ser219 and Ser316 resulted in erythropoiesis failure. Additionally, co-administration of low doses of elavl1a morpholino (MO) and three PKC inhibitors showed a combined effect in zebrafish embryonic erythropoiesis dysplasia. In conclusion, our study reveals that PKC-mediated phosphorylation of elavl1a at Ser219 and Ser316 sites controls its nucleo-cytoplasmic translocation in zebrafish, thereby regulating embryonic erythropoiesis.     

Regulation of erythroid differentiation by miR-376a and its targets

Lineage differentiation is a continuous process during which fated progenitor cells execute specific programs to produce mature counterparts. This lineage-restricted pathway can be controlled by particular regulators, which are usually exclusively expressed in certain cell types or at specific differentiation stages. Here we report that miR-376a participates in the regulation of the early stages of human erythropoiesis by targeting cyclin-dependent kinase 2 (CDK2) and Argonaute 2 (Ago2). Among various human leukemia cell lines, miR-376a was only detected in K562 cells which originated from a progenitor common to the erythroid and megakaryotic lineages. Enforced expression of miR-376a or silencing of CDK2 and Ago2 by RNAi inhibits erythroid differentiation of K562 cells. Hematopoietic progenitor cells transduced with miR-376a showed a significant reduction of their erythroid clonogenic capacity. MiR-376a is relatively abundant in erythroid progenitor cells, where it reduces expression of CDK2 and maintains a low level of differentiation due to cell cycle arrest and decreased cell growth. Following erythroid induction, miR-376a is significantly down-regulated and CDK2 is released from miR-376a inhibition, thereby facilitating the escape of progenitor cells from the quiescent state into erythroid differentiation. Moreover, our results establish a functional link between miR-376a and Ago2, a key factor in miRNA biogenesis and silencing pathways with novel roles in human hematopoiesis.     

Annexin1 regulates the erythroid differentiation through ERK signaling pathway

K562 cells can be used as a model of erythroid differentiation on being induced by hemin. We found that the level of annexin1 gene expression was notably increased during this indicated process. To test the hypothesis that annexin1 can regulate erythropoiesis, K562 cell clones in which annexin1 was stably increased and was knocked down by RNAi were established, respectively. With analysis by hemoglobin quantification, benzidine staining, and marker gene expression profile determination, we confirmed that hemin-induced erythroid differentiation of K562 cells was modestly stimulated by overexpression of annexin1 while it was significantly blocked by knock down of annexin1. Further studies revealed that the mechanisms of annexin1 regulation of the erythroid differentiation was partially related to the increased ERK phosphorylation and expression of p21(cip/waf), since specific inhibitor of MEK blocked the function of annexin1 in erythroid differentiation. We concluded that annexin1 exerted its erythropoiesis regulating effect by ERK pathway.     

Vitamin C promotes anti-leukemia of DZNep in acute myeloid leukemia

The epigenetic treatment by 3-Deazaneplanocin A (DZNep), a histone methyltransferase inhibitor, shows great potential against acute myeloid leukemia (AML). However, the variant sensitivity and incomplete response to DZNep are commonly observed. Here, we reveal that vitamin C (Vc) dramatically promotes DZNep response against leukemic cells in different cell lines and primary AML samples. Vc enhances apoptosis and differentiation induced by DZNep in different AML cell lines in vitro and reduces leukemia progression in vivo. At the molecular level, Vc downregulates an enzyme of serine synthesis named D-3-phosphoglycerate dehydrogenase (PHGDH), as well as BCL2, an anti-apoptotic gene. Over-expression of PHGDH reverses the Vc-enhanced anti-leukemic effect of DZNep in AML cells. Therefore, our findings provide an effective approach to reduce the resistance against epigenetic treatment by Vc, which shows a potential improvement of their combination in AML patients.