ZEB1 is a central mediator of the Epithelial-Mesenchymal Transition

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.Carcinomas are solid tumors arising from epithelial tissue, and account for the majority of cancer deaths in the United States. In most occurrences of carcinoma, it is the metastases that kill, not the primary tumor. The Epithelial-Mesenchymal Transition (EMT) provides a model by which tightly associated epithelial cancer cells can disseminate to distant sites. Many factors are known to trigger the EMT, but the extent to which the observed phenotypes represent a common process is unknown. There is also little appreciation of the extent to which EMT-inducing factors interact with one another or act on common or redundant pathways. In this study, I sought a common gene expression signature of the EMT by comparing five mesenchymal cell lines independently derived from the same parental epithelial line using different EMT-inducing factors. The resultant EMT core signature strongly suggested a common pathway is involved. Bioinformatics analysis revealed the transcription factor ZEBI to be a possible mediator of this common pathway. ZEB1 was found to be both sufficient to induce EMT and necessary for maintaining the mesenchymal phenotype in the same cells. ZEBI and miR-200 were known to reciprocally regulate each other, but their relative importance to the EMT phenotype had never been directly tested. I found that ZEB1 induced EMT regardless of miR-200c levels, thereby excluding the model in which miR-200c downregulation is a necessary step for the EMT. I also show evidence that EMT induced by the transcription factor Snail works at least in part through ZEB1.by Kong Jie Kah.Ph.D

Role of miRNAs in Cancer

MicroRNAs are the best representatives of the non-coding part of the genome and their functions are mostly linked to their target genes. During the process of carcinogenesis, both dysregulation of microRNAs and their target genes can explain the development of the disease. However, most of the target genes of microRNAs have not yet been elucidated. In this book, we add new information related to the functions of microRNAs in various tumors and their associated targetome

pig-1 MELK and ced-3 Caspase cooperate to control cell polarity in the C. elegans NSM neuroblast

Snail-like genes encode zinc-finger transcription factors that play essential roles in development, and one of their well-known functions is the epithelial-mesenchymal transition (EMT) induction. Many studies performed in organisms ranging from Drosophila melanogaster to mammals have reported that Snail transcription factors regulate various aspects of stem cell development, such as cell polarity and cell cycle progression. However, the mechanisms through which Snail-like genes regulate these developmental processes are not completely understood. To uncover these mechanisms, I studied the neurosecretory motor neuron neuroblast (NSMnb) lineage during C. elegans embryogenesis. In the NSMnb lineage, we have previously found that CES-1 Snail controls cell cycle progression by regulating expression of the gene cdc-25.2 CDC25. However, the mechanism by which ces-1 controls the asymmetric division of the NSMnb is unknown. By analyzing CES-1 ChIP-seq data acquired from the modENCODE Project, we identified more than 3,000 potential targets of CES-1 Snail. From the potential candidates that are involved in regulating asymmetric cell division, pig-1 was found to play an essential role in asymmetric NSMnb division. pig-1 encodes the sole C. elegans ortholog of Maternal Embryonic Leucine-zipper kinase (MELK) kinase. Through genetic studies, I confirmed that pig-1 acts downstream of ces-1 to control the asymmetric positioning of the NSMnb cleavage plane. Furthermore, by using a single-copy transcriptional reporter of pig-1, I observed that loss of ces-1 increases the transcriptional level of pig-1, while gain of ces-1 activity decreases the level of pig-1. Therefore, I conclude that CES-1 Snail regulates asymmetric positioning of the NSMnb cleavage plane by repressing expression of the gene pig-1. In the NSMnb, CES-1 Snail coordinates the cell cycle through cdc-25.2 and asymmetric positioning of the cleavage plane through pig-1 to ensure asymmetric cell division and the generation of two daughter cells of different sizes and fates: the larger NSM, which survives, and the smaller NSM sister cell (NSMsc), which dies. Apart from influencing the positioning of the cleavage plane, ces-1 and pig-1 also play roles in controlling the orientation of the NSMnb cleavage plane and in specifying the fate of the daughter cell, NSMsc. On the other hand, I show that ced-3, which encodes a Caspase and which usually executes cell death in C. elegans, also plays a role in regulating the asymmetric positioning of the NSMnb cleavage plane. Loss of ced-3 alone did not affect the asymmetric positioning of the NSMnb cleavage plane at lateral-dorsal side, but loss of both ced-3 and pig-1 reversed the cleavage plane to the medial-ventral side and generated a small NSM and a large NSMsc. This indicates that in the NSMnb lineage, ced-3 may have other functions in addition to executing cell death in the smaller daughter (NSMsc). Furthermore, I confirmed that this function is dependent on the Caspase activity of CED-3 protein. Taken together, ces-1 Snail and pig-1 MELK are two key factors that coordinate cell polarity and cell fate in the NSMnb lineage during C. elegans embryogenesis. In addition, ced-3 Caspase acts in parallel to pig-1 and ces-1 to promote the correct positioning of the cleavage plane in the NSMnb

Snails as intermediate hosts for parasitic infections: host-parasite relationships and intervention strategies

A fundamental prerequisite in the fight against medically and veterinary important parasites transmitted by intermediate host snails is a good knowledge of their life cycles, host specificity and geographical distribution. With scientists around the world collecting material from the wild and generating vast amounts of sequencing data, there is a huge opportunity to expand our knowledge of host-parasite relationships from the comfort of an office chair. With these motivations in mind, a bioinformatics tool was developed that has proven to be time efficient and accurate for the rapid identification of hidden parasites in publicly available datasets. Several dozen hidden parasite infections were discovered from the 2150 gastropod datasets tested, and some of these relationships have not yet been described. With our better understanding and the rapid progress in development of molecular and genetic methods, new avenues are opening for the control and eradication of diseases caused by vector-borne parasites. To study crucial parasite-snail interactions and eventually try to interfere with the infection, it is desirable to edit the host genome. Thus, in the framework of this work, preliminary experiments for the development of the CRISPR/Cas9 protocol in Biomphalaria glabrata, the intermediate host of the dangerous blood fluke Schistosoma mansoni, were also performed. The most significant findings in this case are the proof-of-concept of cultivation of B. glabrata embryos in glass capillaries using natural egg fluid and the demonstration that dilution of this fluid or complete replacement by other culture media are not suitable for successful cultivation. I also show that the Diaphanous gene, which has been used in the past to optimize CRISPR/Cas9 in another snail model, is not suitable for our model. The ultimate goal of the development of this molecular-genetic toolbox is the eradication of schistosomiasis by replacing susceptible populations in nature with resistant populations using gene drive technology. Although disrupted by COVID-19 pandemic, this work’s contribution to progress in the fight against helminthic parasitic infections is considerable

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis

<p>Abstract</p> <p>Background</p> <p>Sessile bivalves of the genus <it>Mytilus </it>are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of <it>M. galloprovincialis</it>, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes.</p> <p>Results</p> <p>We have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in <it>M. galloprovincialis</it>. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with <it>Vibrio splendidus </it>at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the <it>Vibrio</it>-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways.</p> <p>Conclusions</p> <p>The Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on <it>Vibrio</it>-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the <it>Mytilus </it>species to an evolving microbial world.</p

Characterisation of the bacterial flora associated with the grey field slug Deroceras reticulatum and assessment of its suitability as a target for biological control

The field slug Deroceras reticulatum is a major pest in UK agriculture and amidst growing concern and regulatory pressures surrounding chemical molluscicides, innovation is required to advance the current repertoire of slug controls. This study set out to investigate the bacteria associated with D. reticulatum to assess their importance to the slug and potential as a target for biological control. Slug gut bacterial isolates identified using the phenotypical API system (BioMérieux) and 16S rRNA gene sequencing, were mainly soil-dwelling organisms of the phyla Proteobacteria and Bacteriodetes some of which may be important in human or plant disease. A ribosomal intergenic spacer analysis (RISA) was developed to study microbial communities in the slug gut. Slugs had an average species richness of 12 and comparing the bacterial communities in slugs from different locations yielded a mean similarity of 0.159 (Jaccard index) which was significantly lower than similarity indices of slugs collected within a single location (Mean Jaccard index 0.205, p<0.001, ANOVA). Cloning and sequencing of RISA bands common to slugs and slug eggs, but absent from the surrounding soil and plants identified bacteria for future investigation as potential beneficial symbionts. Bacteria extracted from the slug gut were tested for sensitivity to 16 antibiotics and greatest inhibition of growth was observed for chloramphenicol, gentamicin and tetracycline. These antibiotics administered to slugs by feeding and injection caused a reduction in gut-associated bacteria in plate counts, and in bacterial 16S rDNA quantities estimated by real-time quantitative PCR. Field collected D. reticulatum has a large transient gut bacterial population which is reduced upon starvation to a low background level. No significant detrimental effect of antibiotic treatment on the fitness and survival of the slugs was seen, in some instances control slugs suffered greater mortality than slugs that had been injected with antibiotic. Slugs that died during bioassays had a significantly greater amount of bacterial 16S rDNA in their gut than slugs that were sacrificed as healthy individuals suggesting the presence of a bacterial pathogen. This study has found little evidence that a bacterial symbiont may exist and be important for optimal fitness and survival of D. reticulatum, but insight into slug associated bacteria will be valuable in the direction of future studies in this field

OvMark: a user-friendly system for the identification of prognostic biomarkers in publically available ovarian cancer gene expression datasets

Background: Ovarian cancer has the lowest survival rate of all gynaecologic cancers and is characterised by a lack of early symptoms and frequent late stage diagnosis. There is a paucity of robust molecular markers that are independent of and complementary to clinical parameters such as disease stage and tumour grade. METHODS: We have developed a user-friendly, web-based system to evaluate the association of genes/miRNAs with outcome in ovarian cancer. The OvMark algorithm combines data from multiple microarray platforms (including probesets targeting miRNAs) and correlates them with clinical parameters (e.g. tumour grade, stage) and outcomes (disease free survival (DFS), overall survival). In total, OvMark combines 14 datasets from 7 different array platforms measuring the expression of ~17,000 genes and 341 miRNAs across 2,129 ovarian cancer samples. RESULTS: To demonstrate the utility of the system we confirmed the prognostic ability of 14 genes and 2 miRNAs known to play a role in ovarian cancer. Of these genes, CXCL12 was the most significant predictor of DFS (HR = 1.42, p-value = 2.42x10-6). Surprisingly, those genes found to have the greatest correlation with outcome have not been heavily studied in ovarian cancer, or in some cases in any cancer. For instance, the three genes with the greatest association with survival are SNAI3, VWA3A and DNAH12. CONCLUSIONS/IMPACT: OvMark is a powerful tool for examining putative gene/miRNA prognostic biomarkers in ovarian cancer (available at http://glados.ucd.ie/OvMark/index.html). The impact of this tool will be in the preliminary assessment of putative biomarkers in ovarian cancer, particularly for research groups with limited bioinformatics facilities

Hallmarks of Cancer Applied to Oral and Oropharyngeal Carcinogenesis: A Scoping Review of the Evidence Gaps Found in Published Systematic Reviews

In 2000 and 2011, Hanahan and Weinberg published two papers in which they defined the characteristics that cells must fulfil in order to be considered neoplastic cells in all types of tumours that affect humans, which the authors called “hallmarks of cancer”. These papers have represented a milestone in our understanding of the biology of many types of cancers and have made it possible to reach high levels of scientific evidence in relation to the prognostic impact that these hallmarks have on different tumour types. However, to date, there is no study that globally analyses evidencebased knowledge on the importance of these hallmarks in oral and oropharyngeal squamous cell carcinomas. For this reason, we set out to conduct this scoping review of systematic reviews with the aim of detecting evidence gaps in relation to the relevance of the cancer hallmarks proposed by Hanahan andWeinberg in oral and oropharyngeal cancer, and oral potentially malignant disorders, and to point out future lines of research in this field

Role of WT1 in Ischaemic Angiogenesis

Ischaemia causes irreversible tissue damage in cardiovascular disease. Since regenerative angiogenesis fails to consistently induce sufficient reperfusion to facilitate repair, targeted manipulation of angiogenesis is clinically desirable. The Wilms’ tumour suppressor (Wt1) is a transcription factor which regulates numerous genes and cellular processes, including many intrinsic to angiogenesis. We hypothesise that WT1 in the endothelium influences the angiogenic function of endothelial cells. WT1 was identified in endothelial and non-endothelial cells comprising vessel outgrowths generated by cultured aortic rings from WT1-GFP reporter mice. Inducible deletion of WT1 from the endothelium (VE-Wt1 KO) significantly delayed angiogenesis in this assay (p<0.05 relative to controls). In vivo, WT1 expression was evident in vascular endothelial and perivascular cells of the hindlimb as early as 3 days following femoral artery ligation to induce ischaemia, often in cells expressing epithelial and mesenchymal markers simultaneously. However, VE-Wt1 KO had no effect on hindlimb reperfusion (laser Doppler; days 0-28) or on vessel density (day 28). Similarly, VE-Wt1 KO had no effect on vessel density or expression of angiogenic factors (qRT-PCR) in sponges inserted subcutaneously in mice (20 days). To further understand the role of WT1 in angiogenesis, transcriptomic RNA expression analysis was performed in WT1+ and WT1- cells isolated (FACs) from sponges after implantation in WT1-GFP mice. WT1+ cells exhibited higher expression of genes involved in a number of processes relevant to tissue repair, including angiogenesis (p=3.11x10-8), wound healing (p=3.45x10-7) and epithelial-to-mesenchymal transition (EMT) (p=5.86x10-4). These results shed new light on the role of WT1 in ischaemic angiogenesis. In concurrence with previously published work, we show that deletion of endothelial WT1 can delay angiogenesis however, WT1 is not just instrumental in endothelial cells in this context. WT1 has a broader role in tissue repair in ischaemia, in part through regulation of cell transition (EMT). This work has improved our understanding of the regulatory role of WT1 in angiogenesis and repair, while revealing a number of novel insights into the function of WT1. This highlights WT1 as a potentially beneficial therapeutic target to facilitate regeneration in cardiovascular disease