Unbiased transcription factor CRISPR screen identifies ZNF800 as master repressor of enteroendocrine differentiation.

Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels, satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens for genes that program cell fate.

MicroRNA-30c-2-3p negatively regulates NF-κB signaling and cell cycle progression through downregulation of TRADD and CCNE1 in breast cancer.

Nuclear Factor kappa B (NF-κB) signaling is frequently deregulated in a variety of cancers and is constitutively active in estrogen receptor negative (ER-) breast cancer subtypes. These molecular subtypes of breast cancer are associated with poor overall survival. We focused on mechanisms of NF-κB regulation by microRNAs (miRNAs), which regulate eukaryotic gene expression at the post-transcriptional level. In a previous genome-wide miRNA screen, we had identified miR-30c-2-3p as one of the strongest negative regulators of NF-κB signaling. Here we have uncovered the underlying molecular mechanisms and its consequences in breast cancer. In vitro results show that miR-30c-2-3p directly targets both TNFRSF1A-associated via death domain (TRADD), an adaptor protein of the TNFR/NF-κB signaling pathway, and the cell cycle protein Cyclin E1 (CCNE1). Ectopic expression of miR-30c-2-3p downregulated essential cytokines IL8, IL6, CXCL1, and reduced cell proliferation as well as invasion in MDA-MB-231 breast cancer cells. RNA interference (RNAi) induced silencing of TRADD phenocopied the effects on invasion and cytokine expression caused by miR-30c-2-3p, while inhibition of CCNE1 phenocopied the effects on cell proliferation. We further confirmed the tumor suppressive role of this miRNA using a dataset of 781 breast tumors, where higher expression was associated with better survival in breast cancer patients. In summary we have elucidated the mechanism by which miR-30c-2-3p negatively regulates NF-κB signaling and cell cycle progression in breast cancer.

Combined DNA methylation and gene expression profiling in gastrointestinal stromal tumors reveals hypomethylation of SPP1 as an independent prognostic factor.

Gastrointestinal stromal tumors (GISTs) have distinct gene expression patterns according to localization, genotype and aggressiveness. DNA methylation at CpG dinucleotides is an important mechanism for regulation of gene expression. We performed targeted DNA methylation analysis of 1.505 CpG loci in 807 cancer-related genes in a cohort of 76 GISTs, combined with genome-wide mRNA expression analysis in 22 GISTs, to identify signatures associated with clinicopathological parameters and prognosis. Principal component analysis revealed distinct DNA methylation patterns associated with anatomical localization, genotype, mitotic counts and clinical follow-up. Methylation of a single CpG dinucleotide in the non-CpG island promoter of SPP1 was significantly correlated with shorter disease-free survival. Hypomethylation of this CpG was an independent prognostic parameter in a multivariate analysis compared to anatomical localization, genotype, tumor size and mitotic counts in a cohort of 141 GISTs with clinical follow-up. The epigenetic regulation of SPP1 was confirmed in vitro, and the functional impact of SPP1 protein on tumorigenesis-related signaling pathways was demonstrated. In summary, SPP1 promoter methylation is a novel and independent prognostic parameter in GISTs, and might be helpful in estimating the aggressiveness of GISTs from the intermediate-risk category.

MicroRNA-519a is a novel oncomir conferring tamoxifen resistance by targeting a network of tumour-suppressor genes in ER+ breast cancer.

Tamoxifen is an endocrine therapy which is administered to up to 70% of all breast cancer patients with oestrogen receptor alpha (ERα) expression. Despite the initial response, most patients eventually acquire resistance to the drug. MicroRNAs (miRNAs) are a class of small non-coding RNAs which have the ability to post-transcriptionally regulate genes. Although the role of a few miRNAs has been described in tamoxifen resistance at the single gene/target level, little is known about how concerted actions of miRNAs targeting biological networks contribute to resistance. Here we identified the miRNA cluster, C19MC, which harbours around 50 mature miRNAs, to be up-regulated in resistant cells, with miRNA-519a being the most highly up-regulated. We could demonstrate that miRNA-519a regulates tamoxifen resistance using gain- and loss-of-function testing. By combining functional enrichment analysis and prediction algorithms, we identified three central tumour-suppressor genes (TSGs) in PI3K signalling and the cell cycle network as direct target genes of miR-519a. Combined expression of these target genes correlated with disease-specific survival in a cohort of tamoxifen-treated patients. We identified miRNA-519a as a novel oncomir in ER+ breast cancer cells as it increased cell viability and cell cycle progression as well as resistance to tamoxifen-induced apoptosis. Finally, we could show that elevated miRNA-519a levels were inversely correlated with the target genes' expression and that higher expression of this miRNA correlated with poorer survival in ER+ breast cancer patients. Hence we have identified miRNA-519a as a novel oncomir, co-regulating a network of TSGs in breast cancer and conferring resistance to tamoxifen. Using inhibitors of such miRNAs may serve as a novel therapeutic approach to combat resistance to therapy as well as proliferation and evasion of apoptosis in breast cancer.

EGF activates TTP expression by activation of ELK-1 and EGR-1 transcription factors.

BACKGROUND: Tristetraprolin (TTP) is a key mediator of processes such as inflammation resolution, the inhibition of autoimmunity and in cancer. It carries out this role by the binding and degradation of mRNA transcripts, thereby decreasing their half-life. Transcripts modulated by TTP encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. TTP can also modulate neoplastic phenotypes in many cancers. TTP is induced and functionally regulated by a spectrum of both pro- and anti-inflammatory cytokines, mitogens and drugs in a MAPK-dependent manner. So far the contribution of p38 MAPK to the regulation of TTP expression and function has been best described. RESULTS: Our results demonstrate the induction of the gene coding TTP (ZFP36) by EGF through the ERK1/2-dependent pathway and implicates the transcription factor ELK-1 in this process. We show that ELK-1 regulates ZFP36 expression by two mechanisms: by binding the ZFP36 promoter directly through ETS-binding site (+ 883 to +905 bp) and by inducing expression of EGR-1, which in turn increases ZFP36 expression through sequences located between -111 and -103 bp. CONCLUSIONS: EGF activates TTP expression via ELK-1 and EGR-1 transcription factors.

MicroRNA-200c represses migration and invasion of breast cancer cells by targeting actin-regulatory proteins FHOD1 and PPM1F.

MicroRNA-200c (miR-200c) has been shown to suppress epithelial-mesenchymal transition (EMT), which is attributed mainly to targeting of ZEB1/ZEB2, repressors of the cell-cell contact protein E-cadherin. Here we demonstrated that modulation of miR-200c in breast cancer cells regulates cell migration, cell elongation, and transforming growth factor ß (TGF-ß)-induced stress fiber formation by impacting the reorganization of cytoskeleton that is independent of the ZEB/E-cadherin axis. We identified FHOD1 and PPM1F, direct regulators of the actin cytoskeleton, as novel targets of miR-200c. Remarkably, expression levels of FHOD1 and PPM1F were inversely correlated with the level of miR-200c in breast cancer cell lines, breast cancer patient samples, and 58 cancer cell lines of various origins. Furthermore, individual knockdown/overexpression of these target genes phenocopied the effects of miR-200c overexpression/inhibition on cell elongation, stress fiber formation, migration, and invasion. Mechanistically, targeting of FHOD1 by miR-200c resulted in decreased expression and transcriptional activity of serum response factor (SRF), mediated by interference with the translocation of the SRF coactivator mycocardin-related transcription factor A (MRTF-A). This finally led to downregulation of the expression and phosphorylation of the SRF target myosin light chain 2 (MLC2) gene, required for stress fiber formation and contractility. Thus, miR-200c impacts on metastasis by regulating several EMT-related processes, including a novel mechanism involving the direct targeting of actin-regulatory proteins.

[Degradation of eukaryotic transcripts. Role and regulation of tristetraprolin activity]. / Degradacja transkryptów eukariotycznych. Funkcja i regulacja tristetraproliny.

The kinetics of transcripts turnover is a very important aspect of the regulation of the amount of newly synthesized proteins. Transcripts can serve as a template for protein synthesis as long as they remain in the cytoplasm, bound to the ribosomes. Degradation of mRNAs effectively influences quantity of transcripts in this pool. The process of mRNA degradation, similarly to transcription, is precisely regulated, mainly by proteins which interact with mRNA. These proteins are responsible both for transcripts stabilization and degradation. In this article we have summarized known pathways of mRNA degradation. We have also reviewed the present state of knowledge on tristetraprolin, one of the best characterized proteins that takes part in the transcripts turnover.