Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide profiling of EZH2-interacting RNAs reveals lncRNA MALAT1 inhibiting PCDH10 by recruiting PRC2 in metastatic gastric cancer cells

ABSTRACT: Polycomb protein complexes are critical for stem cell renewal and tumorigenesis. Some components of Polycomb repressive complex 2 (PRC2) have been frequently found to be overexpressed in a variety of human cancers and contribute to tumor initiation and development partially by transcriptional silencing of tumor suppressor genes. However, the exact underlying mechanisms still remain elusive. To further characterize PRC2-directed transcriptome and explore its implication in tumorigenesis, here we performed a genome-wide profiling of EZH2-associated, a key component of PRC2, transcriptome in human gastric cancer cell lines by RNA immunoprecipitation sequencing (RIP-seq). We observed that the EZH2-interacting transcripts are distributed in protein-coding, intergenic, intronic and promoter regions and transcribed from both strands. Many transcripts are originated from cancer-related loci and transcribed from active promoters. Particularly, we demonstrated that EZH2-associated lncRNA MALAT1 tethers EZH2 to PCDH10 promoter for its transcriptional repression and contributes to gastric cellular invasion and migration.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Homo sapiens

SUBMITTER: Jun Wu

PROVIDER: E-MTAB-3767 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We studied transcriptional changes by Affymetrix human microarrays in DLBCL cell lines as a result of treatment with GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 In eukaryotes, epigenetic post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is responsible for repressing target gene expression through methylation of histone H3 on lysine 27 (H3K27). Over-expression of EZH2 is implicated in tumorigenesis and correlates with poor prognosis in multiple tumor types. Recent reports have identified somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The Y641 residue is the most frequently mutated residue, with 22% of GCB (Germinal Cell B-cell) DLBCL and FL harboring mutations at this site. These lymphomas exhibit increased H3K27 tri-methylation (H3K27me3) due to altered substrate preferences of the mutant enzymes. However, it is unknown whether direct inhibition of EZH2 methyltransferase activity alone will be effective in treating lymphomas carrying activating EZH2 mutations. Herein, we demonstrate that GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and dramatically inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma. 10 DLBCL cell lines (7 mutant and 3 wild type EZH2), that were differentially sensitive to GSK126 in proliferation assays, were treated for 72 hours, in duplicate (n=2), with either DMSO (vehicle) or 500nM of GSK126, a potent selective EZH2 inhibitor. EZH2 mutant cell lines are Pfeiffer, KARPAS-422, WSU-DLCL2, SU-DHL-10, SU-DHL-6, DB and SU-DHL-4. EZH2 wildtype cell lines are HT, OCI-LY-19 and Toledo.

Project description:Background & Aims: E-cadherin expression disruption is commonly observed in epithelial cancers and metastasis. Such event is also recognized as a crucial step in gastric cancer (GC) initiation and progression. As aberrant expression of microRNAs often perturb the normal expression and function of pivotal cancer-related genes, we characterized and dissected a pathway initiated by loss of microRNA-101 that causes E-cadherin dysfunction through upregulation of EZH2 expression in GC. Methods: Microarrays were used to profile the expression of microRNAs in human GC. Array-CGH revealed DNA copy number changes that were validated by genomic quantitative PCR and Snapshot. Expression levels of microRNAs, mRNA and protein were determined by quantitative-real time PCR and western-blot/co-immunofluorescence. CDH1 inactivating mechanisms were analyzed. Gain and loss of function experiments were done in KatoIII cells. E-cadherin functionality was assessed by immunofluorescence and flow cytometry. Results: MiR-101 expression was significantly decreased in tumors in comparison with normal gastric mucosas (P<.0001). In 65% of the analyzed GC cases, miR-101 downregulation was caused by deletions and/or microdeletions at miR-101-2 locus. EZH2 overexpression and consequent loss/aberrant E-cadherin expression was found in concomitance with miR-101 downregulation in 41% of the analyzed GC cases. This occurred preferentially in cases retaining allele(s) untargeted by classical CDH1 inactivating mechanisms. MiR-101 gain of function experiments or direct inhibition of EZH2, led to a strong depletion of endogenous EZH2 protein and consequent rescue of functional E-cadherin at the cell membrane, mimicking results obtained with clinical GC samples. Conclusions: Deletions and/or microdeletions at miR-101-2 locus underlying mature miR-101 downregulation and consequent EZH2 overexpression represented a novel cascade of genetic events leading to E-cadherin disruption, preferentially affecting the intestinal-type of GC. In the present study, 37 primary GCs and a pool of 10 normal gastric mucosas were used to acquire the expression of 703 known human mature miRNAs deposited in the Sanger miRBase Sequence Database, Release 10.0 (http://microrna.sanger.ac.uk), as well as 393 novel human miRNAs discovered through deep sequencing and validated by qRT-PCR and array profiling (NCodeM-bM-^DM-" Human miRNA microarray probe set V3 from Invitrogen). The 10 normal gastric mucosa RNA samples were pooled to obtain the normal gastric reference sample, which was labelled and hybridised four times to check arrays' reproducibility.

Project description:The goal of the RNA-sequencing experiment was to identify differentially expressed genes, ontologies, and pathways based on cell cycle traverse rate (CCTR) in pre-treatment malignant samples from patients with primary operable invasive triple-negative breast cancer (TNBC). TNBC patients belonged to the well-characterized Nottingham Primary Breast Carcinoma series, the database of which was prospectively maintained for another study, although its design accommodates the present retrospective analysis. Most TNBCs exhibited high Nottingham grade and about half of patients exhibited positive lymph nodes. All patients received chemotherapy with cyclophosphamide-methotrexate-5-fluoruracil after surgery (radical mastectomy or breast-conserving surgery). Stained slides used for quantitation of Ki67 index (KI) and mitotic index (MI) were independently scored by two pathologists blinded to clinical annotation, and the scores were averaged. Cell cycle traverse rate (CCTR) was defined as the ratio of the percentage of mitotic cells to the percentage of Ki67-positive cells in full-face formalin-fixed paraffin-embedded sections from primary tumors. To estimate percentages of mitotic cells for each sample, the average mitotic counts for each mitotic score category were first determined, followed by a conversion into percentage after accounting for sample cellularity. Separate slides from formalin-fixed paraffin-embedded malignant samples were used for RNA-sequencing based on the availability of sufficient tissue for analysis. Only tissue from pre-marked tumor areas in these slides were used for RNA-sequencing. RNA was extracted from deparaffinized tissue and first- and second-strand synthesis were performed using commercial kits. Pair-read sequencing was performed using the Illumina HiSeq2500 instrument. The Human Genome Assembly GRCh38.P10 was used as the reference genome for mapping sequence reads. Transcript expression was quantified using Salmon. Differentially expressed genes, ontologies, and pathways were determined based on mean CCTR as well as the optimal CCTR to predict breast cancer-specific survival.

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Genome-wide profiling of EZH2-interacting RNAs reveals lncRNA MALAT1 inhibiting PCDH10 by recruiting PRC2 in metastatic gastric cancer cells

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