Translin facilitates RNA polymerase II dissociation and suppresses genome instability during RNase H2- and Dicer-deficiency.

The conserved nucleic acid binding protein Translin contributes to numerous facets of mammalian biology and genetic diseases. It was first identified as a binder of cancer-associated chromosomal translocation breakpoint junctions leading to the suggestion that it was involved in genetic recombination. With a paralogous partner protein, Trax, Translin has subsequently been found to form a hetero-octomeric RNase complex that drives some of its functions, including passenger strand removal in RNA interference (RNAi). The Translin-Trax complex also degrades the precursors to tumour suppressing microRNAs in cancers deficient for the RNase III Dicer. This oncogenic activity has resulted in the Translin-Trax complex being explored as a therapeutic target. Additionally, Translin and Trax have been implicated in a wider range of biological functions ranging from sleep regulation to telomere transcript control. Here we reveal a Trax- and RNAi-independent function for Translin in dissociating RNA polymerase II from its genomic template, with loss of Translin function resulting in increased transcription-associated recombination and elevated genome instability. This provides genetic insight into the longstanding question of how Translin might influence chromosomal rearrangements in human genetic diseases and provides important functional understanding of an oncological therapeutic target.

Non-coding Natural Antisense Transcripts: Analysis and Application.

Non-coding natural antisense transcripts (ncNATs) are regulatory RNA molecules that are overlapping with as well as complementary to other transcripts. These transcripts are implicated in a broad variety of biological and pathological processes, including tumorigenesis and oncogenic progression. With this complex field still in its infancy, annotations, expression profiling and functional characterisations of ncNATs are far less comprehensive than those for protein-coding genes, pointing out substantial gaps in the analysis and characterisation of these regulatory transcripts. In this review, we discuss ncNATs from an analysis perspective, in particular regarding the use of high-throughput sequencing strategies, such as RNA-sequencing, and summarize the unique challenges of investigating the antisense transcriptome. Finally, we elaborate on their potential as biomarkers and future targets for treatment, focusing on cancer.

Translin-Trax: Considerations for Oncological Therapeutic Targeting.

Dicer-deficient cancers have poor prognoses, which is linked to the degradation of tumour-suppressing miRNA precursors by the Translin-Trax (Tn-Tx) ribonuclease. Inhibition of Tn-Tx potentially offers a new therapeutic intervention point. However, Tn-Tx functions in an array of biological processes, and here we consider how this complexity could influence therapeutic design strategies.

Brachyury: Strategies for Drugging an Intractable Cancer Therapeutic Target.

New approaches to drug discovery are unlocking enormous therapeutic potential residing in cancer-specific molecules. Brachyury is emerging as an exciting new drug target for the rare bone cancer chordoma. Here, recent advances targeting Brachyury in chordoma are discussed and how these might open doors to the targeting of other, more common cancer types.

Meiosis-like Functions in Oncogenesis: A New View of Cancer.

Cancer cells have many abnormal characteristics enabling tumors to grow, spread, and avoid immunologic and therapeutic destruction. Central to this is the innate ability of populations of cancer cells to rapidly evolve. One feature of many cancers is that they activate genes that are normally associated with distinct developmental states, including germ cell-specific genes. This has historically led to the proposal that tumors take on embryonal characteristics, the so called embryonal theory of cancer. However, one group of germline genes, not directly associated with embryonic somatic tissue genesis, is the one that encodes the specific factors to drive the unique reductional chromosome segregation of meiosis I, which also results in chromosomal exchanges. Here, we propose that meiosis I-specific modulators of reductional segregation can contribute to oncogenic chromosome dynamics and that the embryonal theory for cancer cell growth/proliferation is overly simplistic, as meiotic factors are not a feature of most embryonic tissue development. We postulate that some meiotic chromosome-regulatory functions contribute to a soma-to-germline model for cancer, in which activation of germline (including meiosis) functions drive oncogenesis, and we extend this to propose that meiotic factors could be powerful sources of targets for therapeutics and biomonitoring in oncology. Cancer Res; 77(21); 5712-6. ©2017 AACR.

Human germ/stem cell-specific gene TEX19 influences cancer cell proliferation and cancer prognosis.

BACKGROUND: Cancer/testis (CT) genes have expression normally restricted to the testis, but become activated during oncogenesis, so they have excellent potential as cancer-specific biomarkers. Evidence is starting to emerge to indicate that they also provide function(s) in the oncogenic programme. Human TEX19 is a recently identified CT gene, but a functional role for TEX19 in cancer has not yet been defined. METHODS: siRNA was used to deplete TEX19 levels in various cancer cell lines. This was extended using shRNA to deplete TEX19 in vivo. Western blotting, fluorescence activated cell sorting and immunofluorescence were used to study the effect of TEX19 depletion in cancer cells and to localize TEX19 in normal testis and cancer cells/tissues. RT-qPCR and RNA sequencing were employed to determine the changes to the transcriptome of cancer cells depleted for TEX19 and Kaplan-Meier plots were generated to explore the relationship between TEX19 expression and prognosis for a range of cancer types. RESULTS: Depletion of TEX19 levels in a range of cancer cell lines in vitro and in vivo restricts cellular proliferation/self-renewal/reduces tumour volume, indicating TEX19 is required for cancer cell proliferative/self-renewal potential. Analysis of cells depleted for TEX19 indicates they enter a quiescent-like state and have subtle defects in S-phase progression. TEX19 is present in both the nucleus and cytoplasm in both cancerous cells and normal testis. In cancer cells, localization switches in a context-dependent fashion. Transcriptome analysis of TEX19 depleted cells reveals altered transcript levels of a number of cancer-/proliferation-associated genes, suggesting that TEX19 could control oncogenic proliferation via a transcript/transcription regulation pathway. Finally, overall survival analysis of high verses low TEX19 expressing tumours indicates that TEX19 expression is linked to prognostic outcomes in different tumour types. CONCLUSIONS: TEX19 is required to drive cell proliferation in a range of cancer cell types, possibly mediated via an oncogenic transcript regulation mechanism. TEX19 expression is linked to a poor prognosis for some cancers and collectively these findings indicate that not only can TEX19 expression serve as a novel cancer biomarker, but may also offer a cancer-specific therapeutic target with broad spectrum potential.

Translin and Trax differentially regulate telomere-associated transcript homeostasis.

Translin and Trax proteins are highly conserved nucleic acid binding proteins that have been implicated in RNA regulation in a range of biological processes including tRNA processing, RNA interference, microRNA degradation during oncogenesis, spermatogenesis and neuronal regulation. Here, we explore the function of this paralogue pair of proteins in the fission yeast. Using transcript analysis we demonstrate a reciprocal mechanism for control of telomere-associated transcripts. Mutation of tfx1+ (Trax) elevates transcript levels from silenced sub-telomeric regions of the genome, but not other silenced regions, such as the peri-centromeric heterochromatin. In the case of some sub-telomeric transcripts, but not all, this elevation is dependent on the Trax paralogue, Tsn1 (Translin). In a reciprocal fashion, Tsn1 (Translin) serves to repress levels of transcripts (TERRAs) from the telomeric repeats, whereas Tfx1 serves to maintain these elevated levels. This reveals a novel mechanism for the regulation of telomeric transcripts. We extend this to demonstrate that human Translin and Trax also control telomere-associated transcript levels in human cells in a telomere-specific fashion.

Brachyury identifies a class of enteroendocrine cells in normal human intestinal crypts and colorectal cancer.

Normal homeostasis of adult intestinal epithelium and repair following tissue damage is maintained by a balance of stem and differentiated cells, many of which are still only poorly characterised. Enteroendocrine cells of the gut are a small population of differentiated, secretory cells that are critical for integrating nutrient sensing with metabolic responses, dispersed amongst other epithelial cells. Recent evidence suggests that sub-sets of secretory enteroendocrine cells can act as reserve stem cells. Given the link between cells with stem-like properties and cancer, it is important that we identify factors that might provide a bridge between the two. Here, we identify a sub-set of chromogranin A-positive enteroendocrine cells that are positive for the developmental and cancer-associated transcription factor Brachyury in normal human small intestinal and colonic crypts. Whilst chromogranin A-positive enteroendocrine cells are also Brachyury-positive in colorectal tumours, expression of Brachyury becomes more diffuse in these samples, suggesting a more widespread function in cancer. The finding of the developmental transcription factor Brachyury in normal adult human intestinal crypts may extend the functional complexity of enteroendocrine cells and serves as a platform for assessment of the molecular processes of intestinal homeostasis that underpins our understanding of human health, cancer and aging.

Brachyury regulates proliferation of cancer cells via a p27Kip1-dependent pathway.

The T-box transcription factor Brachyury is expressed in a number of tumour types and has been demonstrated to have cancer inducing properties. To date, it has been linked to cancer associated induction of epithelial to mesenchymal transition, tumour metastasis and expression of markers for cancer stem-like cells. Taken together, these findings indicate that Brachyury plays an important role in the progression of cancer, although the mechanism through which it functions is poorly understood. Here we show that Brachyury regulates the potential of Brachyury-positive colorectal cancer cells to proliferate and reduced levels of Brachyury result in inhibition of proliferation, with features consistent with the cells entering a quiescent-like state. This inhibition of proliferation is dependent upon p27Kip1 demonstrating that Brachyury acts to modulate cellular proliferative fate in colorectal cancer cells in a p27Kip1-dependent manner. Analysis of patient derived colorectal tumours reveals a heterogeneous localisation of Brachyury (in the nucleolus, nucleus and cytoplasm) indicating the potential complexity of the regulatory role of Brachyury in solid colorectal tumours.

CancerEST: a web-based tool for automatic meta-analysis of public EST data.

The identification of cancer-restricted biomarkers is fundamental to the development of novel cancer therapies and diagnostic tools. The construction of comprehensive profiles to define tissue- and cancer-specific gene expression has been central to this. To this end, the exploitation of the current wealth of 'omic'-scale databases can be facilitated by automated approaches, allowing researchers to directly address specific biological questions. Here we present CancerEST, a user-friendly and intuitive web-based tool for the automated identification of candidate cancer markers/targets, for examining tissue specificity as well as for integrated expression profiling. CancerEST operates by means of constructing and meta-analyzing expressed sequence tag (EST) profiles of user-supplied gene sets across an EST database supporting 36 tissue types. Using a validation data set from the literature, we show the functionality and utility of CancerEST. DATABASE URL: http://www.cancerest.org.uk.

Identification of a class of human cancer germline genes with transcriptional silencing refractory to the hypomethylating drug 5-aza-2'-deoxycytidine.

Bona fide germline genes have expression restricted to the germ cells of the gonads. Testis-specific germline development-associated genes can become activated in cancer cells and can potentially drive the oncogenic process and serve as therapeutic/biomarker targets; such germline genes are referred to as cancer/testis genes. Many cancer/testis genes are silenced via hypermethylation of CpG islands in their associated transcriptional control regions and become activated upon treatment with DNA hypomethylating agents; such hypomethylation-induced activation of cancer/testis genes provides a potential combination approach to augment immunotherapeutics. Thus, understanding cancer/testis gene regulation is of increasing clinical importance. Previously studied cancer/testis gene activation has focused on X chromosome encoded cancer/testis genes. Here we find that a sub-set of non-X encoded cancer/testis genes are silenced in non-germline cells via a mechanism that is refractory to epigenetic dysregulation, including treatment with the hypomethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor tricostatin A. These findings formally indicate that there is a sub-group of the clinically important cancer/testis genes that are unlikely to be activated in clinical therapeutic approaches using hypomethylating agents and it indicates a unique transcriptional silencing mechanism for germline genes in non-germline cells that might provide a target mechanism for new clinical therapies.

A novel cohort of cancer-testis biomarker genes revealed through meta-analysis of clinical data sets.

The identification of cancer-specific biomolecules is of fundamental importance to the development of diagnostic and/or prognostic markers, which may also serve as therapeutic targets. Some antigenic proteins are only normally present in male gametogenic tissues in the testis and not in normal somatic cells. When these proteins are aberrantly produced in cancer they are referred to as cancer/testis (CT) antigens (CTAs). Some CTA genes have been proven to encode immunogenic proteins that have been used as successful immunotherapy targets for various forms of cancer and have been implicated as drug targets. Here, a targeted in silico analysis of cancer expressed sequence tag (EST) data sets resulted in the identification of a significant number of novel CT genes. The expression profiles of these genes were validated in a range of normal and cancerous cell types. Subsequent meta-analysis of gene expression microarray data sets demonstrates that these genes are clinically relevant as cancer-specific biomarkers, which could pave the way for the discovery of new therapies and/or diagnostic/prognostic monitoring technologies.

Meta-analysis of expression of l(3)mbt tumor-associated germline genes supports the model that a soma-to-germline transition is a hallmark of human cancers.

Evidence is starting to emerge indicating that tumorigenesis in metazoans involves a soma-to-germline transition, which may contribute to the acquisition of neoplastic characteristics. Here, we have meta-analyzed gene expression profiles of the human orthologs of Drosophila melanogaster germline genes that are ectopically expressed in l(3)mbt brain tumors using gene expression datasets derived from a large cohort of human tumors. We find these germline genes, some of which drive oncogenesis in D. melanogaster, are similarly ectopically activated in a wide range of human cancers. Some of these genes normally have expression restricted to the germline, making them of particular clinical interest. Importantly, these analyses provide additional support to the emerging model that proposes a soma-to-germline transition is a general hallmark of a wide range of human tumors. This has implications for our understanding of human oncogenesis and the development of new therapeutic and biomarker targets with clinical potential.

CancerMA: a web-based tool for automatic meta-analysis of public cancer microarray data.

The identification of novel candidate markers is a key challenge in the development of cancer therapies. This can be facilitated by putting accessible and automated approaches analysing the current wealth of 'omic'-scale data in the hands of researchers who are directly addressing biological questions. Data integration techniques and standardized, automated, high-throughput analyses are needed to manage the data available as well as to help narrow down the excessive number of target gene possibilities presented by modern databases and system-level resources. Here we present CancerMA, an online, integrated bioinformatic pipeline for automated identification of novel candidate cancer markers/targets; it operates by means of meta-analysing expression profiles of user-defined sets of biologically significant and related genes across a manually curated database of 80 publicly available cancer microarray datasets covering 13 cancer types. A simple-to-use web interface allows bioinformaticians and non-bioinformaticians alike to initiate new analyses as well as to view and retrieve the meta-analysis results. The functionality of CancerMA is shown by means of two validation datasets.

Meta-analysis of clinical data using human meiotic genes identifies a novel cohort of highly restricted cancer-specific marker genes.

Identifying cancer-specific biomarkers represents an ongoing challenge to the development of novel cancer diagnostic, prognostic and therapeutic strategies. Cancer/testis (CT) genes are an important gene family with expression tightly restricted to the testis in normal individuals but which can also be activated in cancers. Here we develop a pipeline to identify new CT genes. We analysed and validated expression profiles of human meiotic genes in normal and cancerous tissue followed by meta-analyses of clinical data sets from a range of tumour types resulting in the identification of a large cohort of highly specific cancer biomarker genes, including the recombination hot spot activator PRDM9 and the meiotic cohesin genes SMC1beta and RAD21L. These genes not only provide excellent cancer biomarkers for diagnostics and prognostics, but may serve as oncogenes and have excellent drug targeting potential.

The immortal strand hypothesis: still non-randomly segregating opinions.

Abstract Cairns first suggested a mechanism for protecting the genomes of stem cells (SCs) from replicative errors some 40 years ago when he proposed the immortal strand hypothesis, which argued for the inheritance of a so-called immortal strand by an SC following asymmetric SC divisions. To date, the existence of immortal strands remains contentious with published evidence arguing in favour of and against the retention of an immortal strand by asymmetrically dividing SCs. The conflicting evidence is derived from a diverse array of studies on adult SC types and is predominantly based on following the fate of labelled DNA strands during asymmetric cell division events. Here, we review current data, highlighting limitations of such labelling techniques, and suggest how interpretation of such data may be improved in the future.

Biological roles of translin and translin-associated factor-X: RNA metabolism comes to the fore.

Translin, and its binding partner protein TRAX (translin-associated factor-X) are a paralogous pair of conserved proteins, which have been implicated in a broad spectrum of biological activities, including cell growth regulation, mRNA processing, spermatogenesis, neuronal development/function, genome stability regulation and carcinogenesis, although their precise role in some of these processes remains unclear. Furthermore, translin (with or without TRAX) has nucleic-acid-binding activity and it is apparent that controlling nucleic acid metabolism and distribution are central to the biological role(s) of this protein and its partner TRAX. More recently, translin and TRAX have together been identified as enhancer components of an RNAi (RNA interference) pathway in at least one organism and this might provide critical insight into the biological roles of this enigmatic partnership. In the present review we discuss the biological and the biochemical properties of these proteins that indicate that they play a central and important role in eukaryotic cell biology.

A role for recombination in centromere function.

Centromeres are essential for chromosome segregation during both mitosis and meiosis. There are no obvious or conserved DNA sequence motif determinants for centromere function, but the complex centromeres found in the majority of eukaryotes studied to date consist of repetitive DNA sequences. A striking feature of these repeats is that they maintain a high level of inter-repeat sequence identity within the centromere. This observation is suggestive of a recombination mechanism that operates at centromeres. Here we postulate that inter-repeat homologous recombination plays an intrinsic role in centromere function by forming covalently closed DNA loops. Moreover, the model provides an explanation of why both inverted and direct repeats are maintained and how they contribute to centromere function.