Towards a quantitative understanding of the MITF-PIAS3-STAT3 connection

<p>Abstract</p> <p>Background</p> <p>Expression of the two transcription factors microphthalmia-associated transcription factor (MITF) and signal transducer and activator of transcription 3 (STAT3) are tightly connected to cell proliferation and survival, and are important for melanocyte development. The co-regulation of MITF and STAT3 via their binding to a common inhibitor Protein Inhibitor of Activated STAT3 (PIAS3) is intriguing. A better quantitative understanding of this regulation is likely to be important for elucidation of the melanocyte biology.</p> <p>Results</p> <p>We present a mathematical model describing the MITF-PIAS3-STAT3 signalling network. A default parameter set was developed, partly informed by the literature and partly by constraining the model to mimic reported behavioural features of the system. In addition, a set of experiment-specific parameters was derived for each of 28 experiments reported in the literature. The model seems capable of accounting for most of these experiments in terms of observed temporal development of protein amounts and phosphorylation states. Further, the results also suggest that this system possesses some regulatory features yet to be elucidated.</p> <p>Conclusions</p> <p>We find that the experimentally observed crosstalk between MITF and STAT3 via PIAS3 in melanocytes is faithfully reproduced in our model, offering mechanistic explanations for this behaviour, as well as providing a scaffold for further studies of MITF signalling in melanoma.</p

Regulation of protein kinase CbetaII (PKCbetaII) gene expression in chronic lymphocytic leukaemia (CLL) cells

Chronic lymphocytic leukaemia (CLL) cells are derived from mature B lymphocytes and are distinctive with respect to overexpression of the classical protein kinase C isoform protein kinase CβII (PKCβII), which is encoded by PRKCB. Expression of PKCβII in CLL plays a vital role in the pathogenesis of the malignant cells in this disease, and within the microenvironment cells where it provides signals for the production of factors which support the survival of CLL cells. In CLL cells PRKCB transcription is stimulated by vascular endothelial growth factor (VEGF) through a mechanism involving activated PKCβII. However, at the beginning of this thesis the molecular regulatory mechanism(s) governing expression of the PKCβ gene were poorly described. Thus, to characterise the factors regulating PRKCB transcription in CLL cells I used different approaches including mithramycin treatment, a drug which intercalates into GC-rich areas of DNA to inhibit binding of specificity protein 1 (Sp1), specific Sp1 siRNA, promoter function assays and site directed mutagenesis and chromatin immunoprecipitation (ChIP). Experiments using these techniques showed that Sp1 has a direct role in driving expression of the gene coding for PKCβII in CLL cells. My results also show that Sp1 is highly associated with the PRKCB promoter in CLL cells compared to that in normal B cells, and suggest that this is likely because of the presence of histone marks permissive of gene activation. Examination of other transcription factors such as Sp3, MITF, RUNX1 and E2F1 that potentially bind the PRKCB promoter showed that they have static or indirect effects in regulating transcription of this gene. The exception to this is STAT3 which my data suggests plays a role in suppressing PKCβ gene expression in CLL cells. Exploration of the mechanism through which VEGF induces PRKCB transcription revealed that this growth factor stimulates increased association of Sp1 and decreased association of STAT3 with the PRKCB promoter. Thus, VEGF-stimulated activation of PKCβII may play a role in this process. Taken together, Sp1 is the major driver for overexpression of PKCβII in CLL cells, and because this transcription factor is also overexpressed in these cells, the mechanisms I describe controlling PRKCB transcription potentially provide a foundation for further study of other genes contributing to the phenotype of CLL cells that are regulated by this pleiotropic transcription factor

Dysregulation of Non-Coding RNAs: Roles of miRNAs and lncRNAs in the Pathogenesis of Multiple Myeloma

The dysregulation of non-coding RNAs (ncRNAs), specifically microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), leads to the development and advancement of multiple myeloma (MM). miRNAs, in particular, are paramount in post-transcriptional gene regulation, promoting mRNA degradation and translational inhibition. As a result, miRNAs can serve as oncogenes or tumor suppressors depending on the target genes. In MM, miRNA disruption could result in abnormal gene expression responsible for cell growth, apoptosis, and other biological processes pertinent to cancer development. The dysregulated miRNAs inhibit the activity of tumor suppressor genes, contributing to disease progression. Nonetheless, several miRNAs are downregulated in MM and have been identified as gene regulators implicated in extracellular matrix remodeling and cell adhesion. miRNA depletion potentially facilitates the tumor advancement and resistance of therapeutic drugs. Additionally, lncRNAs are key regulators of numerous cellular processes, such as gene expression, chromatin remodeling, protein trafficking, and recently linked MM development. The lncRNAs are uniquely expressed and influence gene expression that supports MM growth, in addition to facilitating cellular proliferation and viability via multiple molecular pathways. miRNA and lncRNA alterations potentially result in anomalous gene expression and interfere with the regular functioning of MM. Thus, this review aims to highlight the dysregulation of these ncRNAs, which engender novel therapeutic modalities for the treatment of MM.</p

A systems model of phosphorylation for inflammatory signaling events

published_or_final_versio

A Novel Link Between Abl Family Kinases and NM23-H1 During Metastatic Progression

Cancer patient mortality is caused by the ability of tumor cells to invade the extracellular matrix and metastasize. Our lab was the first to identify the role of Abl family of non-receptor tyrosine kinases (c-Abl and Arg) in the progression of solid tumor cancers. In our previous studies, we showed that high c-Abl/Arg activity promotes proliferation, invasion, and metastasis in melanoma and breast cancer cells lines. Here, we demonstrate that our previous findings are clinically relevant by showing increased c-Abl/Arg kinase activity in primary melanoma tumor tissue in comparison to low activity as compared to benign nevi. Additionally, in breast cancer tissue, we found aggressive tumor subtypes (triple-negative and high-grade breast cancer) had increased c-Abl/Arg activity as compared to less aggressive subtypes. To define the mechanism by which c-Abl and Arg promote melanoma and breast cancer metastasis, we searched for novel pathways by which c-Abl and Arg promote invasion, a key step in metastasis. Significantly, we found that c-Abl and Arg decrease the expression of non-metastatic protein, NM23-H1, a metastasis suppressor that is lost during metastatic progression. We demonstrate that NM23-H1 is localized and degraded within the lysosome via proteases, cathepsins L and B. Moreover, we show that c-Abl and Arg upregulate cathepsin mRNA levels and activate the cathepsins, which in-turn degrade NM23-H1. We demonstrate that this pathway is functionally significant as c-Abl and Arg require the downregulation of NM23-H1 to promote invasion in melanoma and breast cancer cell lines. We show that the pathway is clinically significant as c-Abl/Arg activity is inversely correlated with NM23-H1 expression in mouse lung metastases, as well as in human primary melanoma and primary breast cancer tissue. In summary, we are the first to demonstrate novel crosstalk between oncogenic and metastasis suppressor signaling pathways, and provide evidence that pharmacological inhibition of Abl family kinases in melanoma and breast cancer patients may prevent metastatic progression by stabilizing a metastasis suppressor

Sphingolipids

Although sphingolipids are ubiquitous components of cellular membranes, their abundance in cells is generally lower than glycerolipids or cholesterol, representing less than 20% of total lipid mass. Following their discovery in the brain—which contains the largest amounts of sphingolipids in the body—and first description in 1884 by J.L.W. Thudichum, sphingolipids have been overlooked for almost a century, perhaps due to their complexity and enigmatic nature. When sphingolipidoses were discovered, a series of inherited diseases caused by enzyme mutations involved in sphingolipid degradation returned to the limelight. The essential breakthrough came decades later, in the 1990s, with the discovery that sphingolipids were not just structural elements of cellular membranes but intra- and extracellular signaling molecules. It turned out that their lipid backbones, including ceramide and sphingosine-1-phosphate, had selective physiological functions. Thus, sphingolipids emerged as essential players in several pathologies including cancer, diabetes, neurodegenerative disorders, and autoimmune diseases. The present volume reflects upon the unexpectedly eclectic functions of sphingolipids in health, disease, and therapy. This fascinating lipid class will continue to be the subject of up-and-coming future discoveries, especially with regard to new therapeutic strategies

Understanding of retinal degeneration through the lens of high throughput gene expression

The retina is comprised of an intricate network of neurons, including the light-sensing photoreceptors, and supporting glia. Retinal glia maintain tissue homeostasis, while the underlying retinal-pigmented epithelium (RPE) and vascular choroid synergise to ensures a constant nutrient supply and waste removal from the highly metabolically active photoreceptors. Photoreceptor degeneration is central to almost all retinal degenerative diseases, including the increasingly prevalent and currently untreatable Age-Related Macular Degeneration (AMD). Oxidative stress and retinal inflammation are established as drivers of degeneration and are instigated by retinal glia and immune cells from circulation and choroid. Although reductionist approaches and histological observations have unravelled some of the molecular players driving photoreceptor degeneration, the application of high-throughput methods is required to penetrate the bewildering molecular complexity of retinal degeneration. To this end, the work presented in this thesis leverages powerful RNA profiling technologies to uncover novel gene expression patterns in mRNA and regulatory microRNA (miRNA) underpinning retinal degeneration. This thesis integrates data from 7 bulk miRNA/mRNA datasets and 3 single-cell RNA sequencing datasets (scRNAseq) to probe the transcriptomes of (1) the whole degenerating retina, as well as (2) effector cells driving degeneration including Muller glia and choroidal melanocytes and (3) extracellular vesicles (EV) as follow: Published results [1] presented in Chapter 3 describe changes in the mRNA and miRNA in the mouse retina following retinal degeneration induced by photo-oxidative damage. miRNA are short, non-coding RNAs working within a protein complex (RNA-induced silencing complex - RISC) to repress the translation of their mRNA targets. This chapter demonstrates that retinal degeneration is underpinned by a shift in miRNA and mRNA expression towards a proinflammatory state. Simultaneously, retinal degeneration alters miRNA binding sites within pro-inflammatory glial mRNAs allowing targeting by RISC-bound, neuronal miRNA. Published results [2] in Chapter 4 explore the role of EVs in retinal degeneration. EVs are membrane-bound vesicles secreted by nearly all cells as a form of intercellular communication between anatomically separated cells. This chapter developed the first published method for EV isolation from mouse retinas. Using this method, retinal EVs were found to be enriched with neuronal miRNA, and retinal degeneration results in a depletion of EV bioavailability. Lastly, this chapter demonstrates that inhibition of EV biogenesis accelerates retinal degeneration and results in impaired miRNA trafficking. Chapter 5 the explores miRNA-mRNA interactions in Muller glia using data integration from miRNA expression and scRNAseq. These results demonstrate that Muller glia activate a transient gene expression programme driving proliferation and pluripotency early in degeneration, then a shift towards a sustained pro-inflammatory programme follows. Published work [3] in Chapter 6 focuses on the response of choroidal melanocytes to inflammation. The choroid is a reservoir of and entry portal for immune cells in retinal degeneration, yet the relevance of the melanocytes (non-immune, highly abundant choroidal cells) in retinal degeneration is unknown. This chapter demonstrates that choroidal melanocytes have extensive immunomodulatory properties that are activated by both inflammatory challenge and retinal degeneration. In summary, this thesis uncovers novel tissue-level and cell-level gene expression patterns and regulatory networks altered in retinal degeneration, potentially providing the impetus for future therapeutics tacking complex retinal degenerations such as AMD

Targeting STAT3 and STAT5 in Cancer

Every minute, 34 new patients are diagnosed with cancer globally. Although over the past 50 years treatments have improved and survival rates have increased dramatically for several types of cancers, many remain incurable. Several aggressive types of blood and solid cancers form when mutations occur in a critical cellular signaling pathway, the JAK-STAT pathway; (Janus Kinase-Signal Transducer and Activator of Transcription). Currently, there are no clinically available drugs that target the oncogenic STAT3/5 proteins in particular or their Gain of Function hyperactive mutant products. Here, we summarize targeting approaches on STAT3/5, as the field moves towards clinical applications as well as we illuminate on upstream or downstream JAK-STAT pathway interference with kinase inhibitors, heat shock protein blockers or changing nuclear import/export processes. We cover the design paradigms and medicinal chemistry approaches to illuminate progress and challenges in understanding the pleiotropic role of STAT3 and STAT5 in oncogenesis, the microenvironment, the immune system in particular, all culminating in a complex interplay towards cancer progression

The identification of microRNAs to predict glioma prognosis

Until now, personalised medicine for patients in oncology has been focused on the use of DNA-based techniques such as mutation detection and fluorescence in situ hybridisation, fluorescence-activated cell sorting and immuno-staining for classifying tumours. MicroRNAs are short non-coding RNAs that are involved in post-translational regulation of gene expression. Their expression levels are often altered in cancer. Due to their functional importance and stability in biological samples, they represent another tool that could be used to aid patient management. Glioblastoma is a disease that has had little improvement in survival over the past decade in comparison to other cancers. A number of new drugs have been explored but even successful trials have shown limited success. This thesis is focused on identification of microRNAs as signatures for prognosis prediction in glioblastoma. It is separated into four parts; the identification of a microRNA signature that can be used to predict prognosis in glioblastoma; the alignment of glioblastoma microRNA expression with the microRNA expression of oligodendrocyte precursors and its involvement in patient outcome; the use of the expression pattern of the most abundant and robust prognostic microRNA in glioma (miR-9) to delineate glioblastoma subtype and finally the identification of a microRNA signature to predict prognosis in patients treated with the anti-angiogenic drug bevacizumab. The research aims to create signatures suitable for clinical practice, with a small number of predictors, and where possible the function of the microRNAs has been predicted and reviewed to provide confirmation of their role in glioma biology. The key findings of this research are the formation of robust signatures using microRNAs in a disease where few markers are available and proof of a technique that can be used in future drug studies to improve performance at clinical trials

SUMOYLATION OF NEMO AND NF-KB ACTIVATION BY AN RNA HELICASE, DP103 DEFINES THE METASTATIC POTENTIAL OF HUMAN BREAST CANCERS

Ph.DDOCTOR OF PHILOSOPH