Evaluating the Molecular Properties and Function of ANKHD1, and Its Role in Cancer.

Ankyrin repeat and single KH domain-containing protein 1 (ANKHD1) is a large, scaffolding protein composed of two stretches of ankyrin repeat domains that mediate protein-protein interactions and a KH domain that mediates RNA or single-stranded DNA binding. ANKHD1 interacts with proteins in several crucial signalling pathways, including receptor tyrosine kinase, JAK/STAT, mechanosensitive Hippo (YAP/TAZ), and p21. Studies into the role of ANKHD1 in cancer cell lines demonstrate a crucial role in driving uncontrolled cellular proliferation and growth, enhanced tumorigenicity, cell cycle progression through the S phase, and increased epithelial-to-mesenchymal transition. Furthermore, at a clinical level, the increased expression of ANKHD1 has been associated with greater tumour infiltration, increased metastasis, and larger tumours. Elevated ANKHD1 resulted in poorer prognosis, more aggressive growth, and a decrease in patient survival in numerous cancer types. This review aims to gather the current knowledge about ANKHD1 and explore its molecular properties and functions, focusing on the protein's role in cancer at both a cellular and clinical level.

Integration of JAK/STAT receptor-ligand trafficking, signalling and gene expression in Drosophila melanogaster cells.

The JAK/STAT pathway is an essential signalling cascade required for multiple processes during development and for adult homeostasis. A key question in understanding this pathway is how it is regulated in different cell contexts. Here, we have examined how endocytic processing contributes to signalling by the single cytokine receptor in Drosophila melanogaster cells, Domeless. We identify an evolutionarily conserved di-leucine (di-Leu) motif that is required for Domeless internalisation and show that endocytosis is required for activation of a subset of Domeless targets. Our data indicate that endocytosis both qualitatively and quantitatively regulates Domeless signalling. STAT92E, the single STAT transcription factor in Drosophila, appears to be the target of endocytic regulation, and our studies show that phosphorylation of STAT92E on Tyr704, although necessary, is not always sufficient for target transcription. Finally, we identify a conserved residue, Thr702, which is essential for Tyr704 phosphorylation. Taken together, our findings identify previously unknown aspects of JAK/STAT pathway regulation likely to play key roles in the spatial and temporal regulation of signalling in vivo.

A genome-wide RNAi screen identifies MASK as a positive regulator of cytokine receptor stability.

Cytokine receptors often act via the Janus kinase and signal transducer and activator of transcription (JAK/STAT) pathway to form a signalling cascade that is essential for processes such as haematopoiesis, immune responses and tissue homeostasis. In order to transduce ligand activation, cytokine receptors must dimerise. However, mechanisms regulating their dimerisation are poorly understood. In order to better understand the processes regulating cytokine receptor levels, and their activity and dimerisation, we analysed the highly conserved JAK/STAT pathway in Drosophila, which acts via a single receptor, known as Domeless. We performed a genome-wide RNAi screen in Drosophila cells, identifying MASK as a positive regulator of Domeless dimerisation and protein levels. We show that MASK is able to regulate receptor levels and JAK/STAT signalling both in vitro and in vivo We also show that its human homologue, ANKHD1, is also able to regulate JAK/STAT signalling and the levels of a subset of pathway receptors in human cells. Taken together, our results identify MASK as a novel regulator of cytokine receptor levels, and suggest functional conservation, which may have implications for human health.This article has an associated First Person interview with the first author of the paper.

How does methotrexate work?

Developed over 70 years ago as an anti-folate chemotherapy agent, methotrexate (MTX) is a WHO 'essential medicine' that is now widely employed as a first-line treatment in auto-immune, inflammatory diseases such as rheumatoid arthritis (RA), psoriasis and Crone's disease. When used for these diseases patients typically take a once weekly low-dose of MTX - a therapy which provides effective inflammatory control to tens of millions of people worldwide. While undoubtedly effective, our understanding of the anti-inflammatory mechanism-of-action of low-dose MTX is incomplete. In particular, the long-held dogma that this disease-modifying anti-rheumatic drug (DMARD) acts via the folate pathway does not appear to hold up to scrutiny. Recently, MTX has been identified as an inhibitor of JAK/STAT pathway activity, a suggestion supported by many independent threads of evidence. Intriguingly, the JAK/STAT pathway is central to both the inflammatory and immune systems and is a pathway already targeted by other RA treatments. We suggest that the DMARD activity of MTX is likely to be largely mediated by its inhibition of JAK/STAT pathway signalling while many of its side effects are likely associated with the folate pathway. This insight into the mechanism-of-action of MTX opens the possibility for repurposing this low cost, safe and effective drug for the treatment of other JAK/STAT pathway-associated diseases.

Ankyrin repeat and single KH domain 1 (ANKHD1) drives renal cancer cell proliferation via binding to and altering a subset of miRNAs.

Clear cell renal cell carcinoma (ccRCC) represents the most common kidney cancer worldwide. Increased cell proliferation associated with abnormal microRNA (miRNA) regulation are hallmarks of carcinogenesis. Ankyrin repeat and single KH domain 1 (ANKHD1) is a highly conserved protein found to interact with core cancer pathways in Drosophila; however, its involvement in RCC is completely unexplored. Quantitative PCR studies coupled with large-scale genomics data sets demonstrated that ANKHD1 is significantly up-regulated in kidneys of RCC patients when compared with healthy controls. Cell cycle analysis revealed that ANKHD1 is an essential factor for RCC cell division. To understand the molecular mechanism(s) utilized by ANKHD1 to drive proliferation, we performed bioinformatics analyses that revealed that ANKHD1 contains a putative miRNA-binding motif. We screened 48 miRNAs with tumor-enhancing or -suppressing activities and found that ANKHD1 binds to and regulates three tumor-suppressing miRNAs (i.e. miR-29a, miR-205, and miR-196a). RNA-immunoprecipitation assays demonstrated that ANKHD1 physically interacts with its target miRNAs via a single K-homology domain, located in the C terminus of the protein. Functionally, we discovered that ANKHD1 positively drives ccRCC cell mitosis via binding to and suppressing mainly miR-29a and to a lesser degree via miR-196a/205, leading to up-regulation in proliferative genes such as CCDN1. Collectively, these data identify ANKHD1 as a new regulator of ccRCC proliferation via specific miRNA interactions.

Effect of methotrexate on JAK/STAT pathway activation in myeloproliferative neoplasms.

BACKGROUND: The myeloproliferative neoplasms are a group of haematological malignancies characterised by pathological activation of the JAK/STAT (Janus kinase and signal transducer and activator of transcription) intracellular signalling pathway. 50-95% of patients have an acquired mutation (JAK2V617F) causing constitutive activation of JAK2. Our aim was to find new treatments for myeloproliferative neoplasms by identifying compounds that suppress JAK/STAT pathway activation. METHODS: We used a luciferase-based transcriptional assay in the low complexity Drosophila model system to screen a library of 2000 small molecules for modulators of JAK/STAT pathway activation. Screen hits were validated with western blotting in the HDLM-2 Hodgkin's lymphoma cell line. The HEL cell line, in which constitutive JAK/STAT pathway activation is caused by JAK2V617F, was used to determine the relevance of screen hits for treatment of myeloproliferative neoplasms. FINDINGS: Methotrexate and the chemically similar drug aminopterin were independently identified as strong inhibitors of the Drosophila JAK/STAT pathway, an effect conserved to human cells. Methotrexate did not affect protein phosphorylation in other intracellular signalling pathways. Methotrexate caused significant suppression of JAK/STAT activation in HEL cells at a concentration equivalent to that seen in patients taking low-dose oral methotrexate (p≤0·001). INTERPRETATION: Our results suggest that methotrexate is a promising treatment for myeloproliferative neoplasms that could be translated into clinical trials after assessment in primary cells. These results are particularly relevant in myelofibrosis. Inhibitors of JAK1/2 improve symptoms and prolong life in myelofibrosis, but their use is limited by cost. Other existing therapies for myelofibrosis appear no more effective than placebo. Methotrexate might bring the benefits of JAK/STAT pathway inhibition at a lower cost. FUNDING: Cancer Research UK, Yorkshire Cancer Research, UK Medical Research Council, Wellcome Trust, EU Framework Cancer Pathways.

Gα73B is a downstream effector of JAK/STAT signalling and a regulator of Rho1 in Drosophila haematopoiesis.

JAK/STAT signalling regulates many essential developmental processes including cell proliferation and haematopoiesis, whereas its inappropriate activation is associated with the majority of myeloproliferative neoplasias and numerous cancers. Furthermore, high levels of JAK/STAT pathway signalling have also been associated with enhanced metastatic invasion by cancerous cells. Strikingly, gain-of-function mutations in the single Drosophila JAK homologue, Hopscotch, result in haemocyte neoplasia, inappropriate differentiation and the formation of melanised haemocyte-derived 'tumour' masses; phenotypes that are partly orthologous to human gain-of-function JAK2-associated pathologies. Here we show that Gα73B, a novel JAK/STAT pathway target gene, is necessary for JAK/STAT-mediated tumour formation in flies. In addition, although Gα73B does not affect haemocyte differentiation, it does regulate haemocyte morphology and motility under non-pathological conditions. We show that Gα73B is required for constitutive, but not injury-induced, activation of Rho1 and for the localisation of Rho1 into filopodia upon haemocyte activation. Consistent with these results, we also show that Rho1 interacts genetically with JAK/STAT signalling, and that wild-type levels of Rho1 are necessary for tumour formation. Our findings link JAK/STAT transcriptional outputs, Gα73B activity and Rho1-dependent cytoskeletal rearrangements and cell motility, therefore connecting a pathway associated with cancer with a marker indicative of invasiveness. As such, we suggest a mechanism by which JAK/STAT pathway signalling may promote metastasis.

Control of tissue morphology by Fasciclin III-mediated intercellular adhesion.

Morphogenesis is dependent on the orchestration of multiple developmental processes to generate mature functional organs. However, the signalling pathways that coordinate morphogenesis and the mechanisms that translate these signals into tissue shape changes are not well understood. Here, we demonstrate that changes in intercellular adhesion mediated by the transmembrane protein Fasciclin III (FasIII) represent a key mediator of morphogenesis. Using the embryonic Drosophila hindgut as an in vivo model for organogenesis, we show that the tightening of hindgut curvature that normally occurs between embryonic stage 12 and 15 to generate the characteristic shepherd's crook shape is dependent on localised JAK/STAT pathway activation. This localised pathway activity drives the expression of FasIII leading to its subcellular lateralisation at a stage before formation of septate junctions. Additionally, we show that JAK/STAT- and FasIII-dependent morphogenesis also regulates folds within the third instar wing imaginal disc. We show that FasIII forms homophilic intercellular interactions that promote intercellular adhesion in vivo and in cultured cells. To explore these findings, we have developed a mathematical model of the developing hindgut, based on the differential interfacial tension hypothesis (DITH) linking intercellular adhesion and localised surface tension. Our model suggests that increased intercellular adhesion provided by FasIII can be sufficient to drive the tightening of tube curvature observed. Taken together, these results identify a conserved molecular mechanism that directly links JAK/STAT pathway signalling to intercellular adhesion and that sculpts both tubular and planar epithelial shape.

Case report: Effectiveness of low-dose methotrexate monotherapy in post-essential thrombocythemia myelofibrosis.

JAK/STAT pathway signalling is associated with both chronic inflammatory conditions such as psoriasis and haematological malignancies such as the myeloproliferative neoplasms (MPNs). Here we describe a 73yo female patient with a history of chronic plaque psoriasis, post-essential thrombocythemia myelofibrosis (MF) and a quality of life substantially impacted by both conditions. We report that 15 mg oral Methotrexate (MTX) weekly as a monotherapy is well tolerated, provides a substantial clinical improvement for both conditions and significantly improves quality of life. We suggest that the recently identified mechanism of action of MTX as a JAK inhibitor is likely to explain this efficacy and suggest that repurposing MTX for MPNs may represent a clinical- and cost-effective therapeutic option.

Macrophage subpopulation identity in Drosophila is modulated by apoptotic cell clearance and related signalling pathways.

In Drosophila blood, plasmatocytes of the haemocyte lineage represent the functional equivalent of vertebrate macrophages and have become an established in vivo model with which to study macrophage function and behaviour. However, the use of plasmatocytes as a macrophage model has been limited by a historical perspective that plasmatocytes represent a homogenous population of cells, in contrast to the high levels of heterogeneity of vertebrate macrophages. Recently, a number of groups have reported transcriptomic approaches which suggest the existence of plasmatocyte heterogeneity, while we identified enhancer elements that identify subpopulations of plasmatocytes which exhibit potentially pro-inflammatory behaviours, suggesting conservation of plasmatocyte heterogeneity in Drosophila. These plasmatocyte subpopulations exhibit enhanced responses to wounds and decreased rates of efferocytosis when compared to the overall plasmatocyte population. Interestingly, increasing the phagocytic requirement placed upon plasmatocytes is sufficient to decrease the size of these plasmatocyte subpopulations in the embryo. However, the mechanistic basis for this response was unclear. Here, we examine how plasmatocyte subpopulations are modulated by apoptotic cell clearance (efferocytosis) demands and associated signalling pathways. We show that loss of the phosphatidylserine receptor Simu prevents an increased phagocytic burden from modulating specific subpopulation cells, while blocking other apoptotic cell receptors revealed no such rescue. This suggests that Simu-dependent efferocytosis is specifically involved in determining fate of particular subpopulations. Supportive of our original finding, mutations in amo (the Drosophila homolog of PKD2), a calcium-permeable channel which operates downstream of Simu, phenocopy simu mutants. Furthermore, we show that Amo is involved in the acidification of the apoptotic cell-containing phagosomes, suggesting that this reduction in pH may be associated with macrophage reprogramming. Additionally, our results also identify Ecdysone receptor signalling, a pathway related to control of cell death during developmental transitions, as a controller of plasmatocyte subpopulation identity. Overall, these results identify fundamental pathways involved in the specification of plasmatocyte subpopulations and so further validate Drosophila plasmatocytes as a heterogeneous population of macrophage-like cells within this important developmental and immune model.

Advances in genome-wide RNAi cellular screens: a case study using the Drosophila JAK/STAT pathway.

BACKGROUND: Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated. RESULTS: Here we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols. CONCLUSIONS: Our work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens.

Negative regulation of Drosophila JAK-STAT signalling by endocytic trafficking.

Appropriate regulation of signal transduction pathways is essential for normal development and is often disrupted in disease. Therefore, many regulatory mechanisms and feedback loops have evolved to ensure appropriate signalling. One mechanism previously suggested to modulate a range of signal transduction pathways involves the internalisation and destruction of transmembrane receptors by the endocytic trafficking machinery. Strikingly, a recent report has suggested that the endocytic trafficking of the Drosophila JAK-STAT pathway receptor Domeless (Dome) does not act to downregulate pathway activity, but rather is necessary for in vivo signalling. Here, we examine this relationship to address the interaction of Drosophila JAK-STAT pathway signalling and endocytic trafficking. We show that Dome is trafficked through clathrin-mediated endocytosis and a directed RNAi screen identified several components of the endocytic machinery as negative regulators of pathway signalling. We demonstrate that Dome signals both from the plasma membrane and internalised vesicles and show, using knockdown experiments, that endocytic components negatively regulate JAK-STAT signalling in vivo. As such, disruption in endocytic trafficking represents a potent negative regulator of the disease relevant JAK-STAT signalling cascade.

Transcriptional targets of Drosophila JAK/STAT pathway signalling as effectors of haematopoietic tumour formation.

Although many signal transduction pathways have been implicated in the development of human disease, the identification of pathway targets and the biological processes that mediate disease progression remains challenging. One such disease-related pathway is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) cascade whose constitutive misactivation by the JAK2 V617F mutation underlies most human myeloproliferative disorders. Here, we use transcript profiling of Drosophila haemocyte-like cells to identify JAK/STAT target genes, combined with an in vivo model for JAK-induced blood cell overproliferation, to identify the main effectors required for haematopoietic tumour development. The identified human homologues of the Drosophila effectors were tested for potential V617F-mediated transcriptional regulation in human HeLa cells and compared with small interfering RNA-derived data, quantify their role in regulating the proliferation of cancer-derived cell lines. Such an inter-species approach is an effective way to identify factors with conserved functions that might be central to human disease.

Opsoclonus-myoclonus paraneoplastic syndrome in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma can present with epistaxis, cervical lymphadenopathy, audiological symptoms secondary to eustachian tube dysfunction, pain, or neurological symptoms from tumours directly invading the skull base. It is unusual for patients to present with indirect systemic manifestations. Paraneoplastic neurological syndrome can precede clinically overt malignancy by up to 5 years; therefore, a combination of thorough clinical, laboratory and radiological investigations is required to reach a diagnosis. Intravenous immunoglobulin and steroids might improve neurological symptoms initially and prevent irreversible neuronal damage, but treatment of the underlying cancer is important for long-term resolution. Our case adds to a small but growing body of literature related to anti-Ri antibodies, opsoclonus-myoclonus syndrome presentations, and is the first reported association of this combination with nasopharyngeal carcinoma.

Zoledronate Extends Health Span and Survival via the Mevalonate Pathway in a FOXO-dependent Manner.

Over recent decades, increased longevity has not been paralleled by extended health span, resulting in more years spent with multiple diseases in older age. As such, interventions to improve health span are urgently required. Zoledronate (Zol) is a nitrogen-containing bisphosphonate, which inhibits the farnesyl pyrophosphate synthase enzyme, central to the mevalonate pathway. It is already used clinically to prevent fractures in osteoporotic patients, who have been reported to derive unexpected and unexplained survival benefits. Using Drosophila as a model we determined the effects of Zol on life span, parameters of health span (climbing ability and intestinal dysplasia), and the ability to confer resistance to oxidative stress using a combination of genetically manipulated Drosophila strains and Western blotting. Our study shows that Zol extended life span, improved climbing activity, and reduced intestinal epithelial dysplasia and permeability with age. Mechanistic studies showed that Zol conferred resistance to oxidative stress and reduced accumulation of X-ray-induced DNA damage via inhibition of farnesyl pyrophosphate synthase. Moreover, Zol was associated with inhibition of phosphorylated AKT in the mammalian traget of rapamycin pathway downstream of the mevalonate pathway and required dFOXO for its action, both molecules associated with increased longevity. Taken together, our work indicates that Zol, a drug already widely used to prevent osteoporosis and dosed only once a year, modulates important mechanisms of aging. Its repurposing holds great promise as a treatment to improve health span.

Validation of diagnostic criteria for variant Creutzfeldt-Jakob disease.

OBJECTIVE: Variant Creutzfeldt-Jakob disease (vCJD), a novel form of human prion disease, was recognized in 1996. The disease affected a younger cohort than sporadic CJD, and the early clinical course was dominated by psychiatric and sensory symptoms. In an attempt to aid diagnosis and establish standardization between surveillance networks, diagnostic criteria were established. These were devised from the features of a small number of cases and modified in 2000 as the clinical phenotype was established. Since then, only minor changes have been introduced; revalidation of the criteria in the current format is overdue. METHODS: Included in this study are autopsy/cerebral biopsy-proven cases of vCJD referred to the National CJD Surveillance Unit (NCJDSU) between 1995 and 2004 and suspect cases in which an alternative diagnosis was identified following autopsy/cerebral biopsy. RESULTS: Over the 10-year period, 106 definite cases of vCJD and 45 pathologically confirmed "noncases" were identified from the archives of the NCJDSU. The median age at onset of the cases was significantly younger than that of the noncases (27 years [range, 12-74 years] vs 43 years [range, 10-64 years]), and the median duration of illness was significantly shorter (14 months [range, 6-39 months] vs 22 months [range, 2-139 months]). The most commonly identified core clinical feature in cases was dementia; persistent painful sensory symptoms were the least frequent. Eighty-eight of 106 (83%) vCJD cases were retrospectively classified as probable in life, 6 cases were classified as possible. Most cases were classified as probable on the basis of core clinical features and brain magnetic resonance imaging. To date, the diagnostic criteria remain 100% specific, with no autopsy/cerebral biopsy-proven noncases classified as probable in life. INTERPRETATION: This study confirms that the diagnostic criteria for vCJD are sensitive and specific and provide a useful standard framework for case classification in a surveillance setting.

Identification of JAK/STAT signalling components by genome-wide RNA interference.

Signalling pathways mediating the transduction of information between cells are essential for development, cellular differentiation and homeostasis. Their dysregulation is also frequently associated with human malignancies. The Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway represents one such signalling cascade whose evolutionarily conserved roles include cell proliferation and haematopoiesis. Here we describe a systematic genome-wide survey for genes required for JAK/STAT pathway activity. Analysis of 20,026 RNA interference (RNAi)-induced phenotypes in cultured Drosophila melanogaster haemocyte-like cells identified interacting genes encoding 4 known and 86 previously uncharacterized proteins. Subsequently, cell-based epistasis experiments were used to classify these proteins on the basis of their interaction with known components of the signalling cascade. In addition to multiple human disease gene homologues, we have found the tyrosine phosphatase Ptp61F and the Drosophila homologue of BRWD3, a bromo-domain-containing protein disrupted in leukaemia. Moreover, in vivo analysis demonstrates that disrupted dBRWD3 and overexpressed Ptp61F function as suppressors of leukaemia-like blood cell tumours. This screen represents a comprehensive identification of novel loci required for JAK/STAT signalling and provides molecular insights into an important pathway relevant for human cancer. Human homologues of identified pathway modifiers may constitute targets for therapeutic interventions.

Identification of functionally distinct macrophage subpopulations in Drosophila.

Vertebrate macrophages are a highly heterogeneous cell population, but while Drosophila blood is dominated by a macrophage-like lineage (plasmatocytes), until very recently these cells were considered to represent a homogeneous population. Here, we present our identification of enhancer elements labelling plasmatocyte subpopulations, which vary in abundance across development. These subpopulations exhibit functional differences compared to the overall population, including more potent injury responses and differential localisation and dynamics in pupae and adults. Our enhancer analysis identified candidate genes regulating plasmatocyte behaviour: pan-plasmatocyte expression of one such gene (Calnexin14D) improves wound responses, causing the overall population to resemble more closely the subpopulation marked by the Calnexin14D-associated enhancer. Finally, we show that exposure to increased levels of apoptotic cell death modulates subpopulation cell numbers. Taken together this demonstrates macrophage heterogeneity in Drosophila, identifies mechanisms involved in subpopulation specification and function and facilitates the use of Drosophila to study macrophage heterogeneity in vivo.