Chasing non-existent "microRNAs" in cancer.

MicroRNAs (miRNAs) are important regulators of gene expression whose dysregulation is widely linked to tumourigenesis, tumour progression and Epithelial-Mesenchymal Transition (EMT), a developmental process that promotes metastasis when inappropriately activated. However, controversy has emerged regarding how many functional miRNAs are encoded in the genome, and to what extent non-regulatory products of RNA degradation have been mis-identified as miRNAs. Central to miRNA function is their capacity to associate with an Argonaute (AGO) protein and form an RNA-Induced Silencing Complex (RISC), which mediates target mRNA suppression.

Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression.

Background: This study focuses on the role of lysosomal trafficking in prostate cancer, given the essential role of lysosomes in cellular homoeostasis.

Methods: Lysosomal motility was evaluated using confocal laser scanning microscopy of LAMP-1-transfected prostate cells and spot-tracking analysis. Expression of lysosomal trafficking machinery was evaluated in patient cohort databases and through immunohistochemistry on tumour samples.

Nuclear export of circular RNA.

Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells, are formed by back-splicing of precursor mRNAs in the nucleus. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA.

The landscape of alternative polyadenylation during EMT and its regulation by the RNA-binding protein Quaking.

Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3' end anchored RNA sequencing, we mapped the alternative polyadenylation (APA) landscape following Transforming Growth Factor (TGF)-β-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3'UTR lengthening during this cell state transition.

ESRP1 controls biogenesis and function of a large abundant multiexon circRNA.

While the majority of circRNAs are formed from infrequent back-splicing of exons from protein coding genes, some can be produced at quite high level and in a regulated manner. We describe the regulation, biogenesis and function of circDOCK1(2-27), a large, abundant circular RNA that is highly regulated during epithelial-mesenchymal transition (EMT) and whose formation depends on the epithelial splicing regulator ESRP1. CircDOCK1(2-27) synthesis in epithelial cells represses cell motility both by diverting transcripts from DOCK1 mRNA production to circRNA formation and by direct inhibition of migration by the circRNA.

Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia.

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival.

Post-transcriptional Gene Regulation by MicroRNA-194 Promotes Neuroendocrine Transdifferentiation in Prostate Cancer.

Potent therapeutic inhibition of the androgen receptor (AR) in prostate adenocarcinoma can lead to the emergence of neuroendocrine prostate cancer (NEPC), a phenomenon associated with enhanced cell plasticity. Here, we show that microRNA-194 (miR-194) is a regulator of epithelial-neuroendocrine transdifferentiation. In clinical prostate cancer samples, miR-194 expression and activity were elevated in NEPC and inversely correlated with AR signaling.

Insufficiently complex unique-molecular identifiers (UMIs) distort small RNA sequencing.

The attachment of unique molecular identifiers (UMIs) to RNA molecules prior to PCR amplification and sequencing, makes it possible to amplify libraries to a level that is sufficient to identify rare molecules, whilst simultaneously eliminating PCR bias through the identification of duplicated reads. Accurate de-duplication is dependent upon a sufficiently complex pool of UMIs to allow unique labelling. In applications dealing with complex libraries, such as total RNA-seq, only a limited variety of UMIs are required as the variation in molecules to be sequenced is enormous.

Extensive transcriptional responses are co-ordinated by microRNAs as revealed by Exon-Intron Split Analysis (EISA).

Epithelial-mesenchymal transition (EMT) has been a subject of intense scrutiny as it facilitates metastasis and alters drug sensitivity. Although EMT-regulatory roles for numerous miRNAs and transcription factors are known, their functions can be difficult to disentangle, in part due to the difficulty in identifying direct miRNA targets from complex datasets and in deciding how to incorporate 'indirect' miRNA effects that may, or may not, represent biologically relevant information. To better understand how miRNAs exert effects throughout the transcriptome during EMT, we employed Exon-Intron Split Analysis (EISA), a bioinformatic technique that separates transcriptional and post-transcriptional effects through the separate analysis of RNA-Seq reads mapping to exons and introns.

miR-200/375 control epithelial plasticity-associated alternative splicing by repressing the RNA-binding protein Quaking.

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels.

The tyrosine phosphatase PTPN14 (Pez) inhibits metastasis by altering protein trafficking.

Factors secreted by tumor cells shape the local microenvironment to promote invasion and metastasis, as well as condition the premetastatic niche to enable secondary-site colonization and growth. In addition to this secretome, tumor cells have increased abundance of growth-promoting receptors at the cell surface. We found that the tyrosine phosphatase PTPN14 (also called Pez, which is mutated in various cancers) suppressed metastasis by reducing intracellular protein trafficking through the secretory pathway.

Genome-wide identification of miR-200 targets reveals a regulatory network controlling cell invasion.

The microRNAs of the miR-200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago-HITS-CLIP technology for transcriptome-wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR-200a and miR-200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR-200 targets in widespread coordinate control of actin cytoskeleton dynamics.

Specificity protein 1 (Sp1) maintains basal epithelial expression of the miR-200 family: implications for epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is required for the specification of tissues during embryonic development and is recapitulated during the metastatic progression of tumors. The miR-200 family plays a critical role in enforcing the epithelial state with their expression lost in cells undergoing EMT. EMT can be mediated by activation of the ZEB1 and ZEB2 (ZEB) transcription factors, which repress miR-200 expression via a self-reinforcing double negative feedback loop to promote the mesenchymal state.

Epigenetic modulation of the miR-200 family is associated with transition to a breast cancer stem-cell-like state.

The miR-200 family is a key regulator of the epithelial-mesenchymal transition, however, its role in controlling the transition between cancer stem-cell-like and non-stem-cell-like phenotypes is not well understood. We utilized immortalized human mammary epithelial (HMLE) cells to investigate the regulation of the miR-200 family during their conversion to a stem-like phenotype. HMLE cells were found to be capable of spontaneous conversion from a non-stem to a stem-like phenotype and this conversion was accompanied by the loss of miR-200 expression.

Down-regulation of the miRNA-200 family at the invasive front of colorectal cancers with degraded basement membrane indicates EMT is involved in cancer progression.

Cancer progression is a complex series of events thought to incorporate the reversible developmental process of epithelial-to-mesenchymal transition (EMT). In vitro, the microRNA-200 family maintains the epithelial phenotype by posttranscriptionally inhibiting the E-cadherin repressors, ZEB1 and ZEB2. Here, we used in situ hybridization and immunohistochemistry to assess expression of miR-200 and EMT biomarkers in formalin-fixed paraffin-embedded human colorectal adenocarcinomas.

The inducible tissue-specific expression of the human IL-3/GM-CSF locus is controlled by a complex array of developmentally regulated enhancers.

The closely linked human IL-3 and GM-CSF genes are tightly regulated and are expressed in activated T cells and mast cells. In this study, we used transgenic mice to study the developmental regulation of this locus and to identify DNA elements required for its correct activity in vivo. Because these two genes are separated by a CTCF-dependent insulator, and the GM-CSF gene is regulated primarily by its own upstream enhancer, the main objective in this study was to identify regions of the locus required for correct IL-3 gene expression.

An autocrine TGF-beta/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a form of cellular plasticity that is critical for embryonic development and tumor metastasis. A double-negative feedback loop involving the miR-200 family and ZEB (zinc finger E-box-binding homeobox) transcription factors has been postulated to control the balance between epithelial and mesenchymal states. Here we demonstrate using the epithelial Madin Darby canine kidney cell line model that, although manipulation of the ZEB/miR-200 balance is able to repeatedly switch cells between epithelial and mesenchymal states, the induction and maintenance of a stable mesenchymal phenotype requires the establishment of autocrine transforming growth factor-β (TGF-β) signaling to drive sustained ZEB expression.

Transcriptional and epigenetic regulation of the GM-CSF promoter by RUNX1.

The RUNX1 gene, which is essential for normal hematopoiesis, is frequently rearranged by the t(8;21) chromosomal translocation in acute myeloid leukemia. The resulting RUNX1-ETO fusion protein contributes to leukemic progression by directing aberrant association of transcriptional cofactors and epigenetic modifiers to RUNX1 target genes. For example, the GM-CSF gene is activated by RUNX1, but is repressed by RUNX1-ETO.

Transcription-dependent silencing of inducible convergent transgenes in transgenic mice.

Background: Silencing of transgenes in mice is a common phenomenon typically associated with short multi-copy transgenes. We have investigated the regulation of the highly inducible human granulocyte-macrophage colony-stimulating-factor gene (Csf2) in transgenic mice.

Results: In the absence of any previous history of transcriptional activation, this transgene was expressed in T lineage cells at the correct inducible level in all lines of mice tested.

MicroRNAs as regulators of epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) describes the molecular reprogramming and phenotypic changes involved in the conversion of polarised immotile epithelial cells to motile mesenchymal cells. This process allows the remodelling of tissues during embryonic development and is implicated in the promotion of tumor invasion and metastasis. Several recent studies have identified the miR-200 family and miR-205 as key regulators of EMT and enforcers of the epithelial phenotype.

The microRNA-200 family regulates epithelial to mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is implicated in metastasis initiation and has recently been shown to be regulated by the miRNA-200 family and miR-205. Expression of these miRNAs was lost in invasive breast cancer cell lines displaying mesenchymal-like morphology suggesting these microRNAs may play a role in cancer metastasis.

A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition.

Epithelial to mesenchymal transition occurs during embryologic development to allow tissue remodeling and is proposed to be a key step in the metastasis of epithelial-derived tumors. The miR-200 family of microRNAs plays a major role in specifying the epithelial phenotype by preventing expression of the transcription repressors, ZEB1/deltaEF1 and SIP1/ZEB2. We show here that miR-200a, miR-200b, and the related miR-429 are all encoded on a 7.

The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1.

Epithelial to mesenchymal transition (EMT) facilitates tissue remodelling during embryonic development and is viewed as an essential early step in tumour metastasis. We found that all five members of the microRNA-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) and miR-205 were markedly downregulated in cells that had undergone EMT in response to transforming growth factor (TGF)-beta or to ectopic expression of the protein tyrosine phosphatase Pez. Enforced expression of the miR-200 family alone was sufficient to prevent TGF-beta-induced EMT.