The field of cancer genomics is currently being enhanced by the power of Epigenome-wide association studies (EWAS). Over the last couple of years comprehensive sequence data sets have been generated, allowing analysis of genome-wide activity in cohorts of different individuals to be increasingly available. Finding associations between epigenetic variation and phenotype is one of the biggest challenges in biomedical research. Laboratories lacking dedicated resources and programming experience require bioinformatics expertise which can be prohibitively costly and time-consuming. To address this, we have developed a collection of freely available Galaxy tools (Poterlowicz, 2018a), combining analytical methods into a range of convenient analysis pipelines with graphical user-friendly interface.The tool suite includes methods for data preprocessing, quality assessment and differentially methylated region and position discovery. The aim of this project was to make EWAS analysis flexible and accessible to everyone and compatible with routine clinical and biological use. This is exemplified by my work undertaken by integrating DNA methylation profiles of melanoma patients (at baseline and mitogen-activated protein kinase inhibitor MAPKi treatment) to identify novel epigenetic switches responsible for tumour resistance to therapy (Hugo et al., 2015). Configuration files are publicly published on our GitHub repository (Poterlowicz, 2018b) with scripts and dependency settings also available to download and install via Galaxy test toolshed (Poterlowicz, 2018a). Results and experiences using this framework demonstrate the potential for Galaxy to be a bioinformatics solution for multi-omics cancer biomarker discovery tool.

Melanoma skin cancer is the abnormal growth of the melanocytes – the pigmented cells located in the epidermis. The current gold standard diagnostic technique for determining whether a lesion is cancerous involves subjectively examining suspicious lesions and performing an invasive biopsy to confirm melanoma. This method may neglect some lesions or cause scarring from biopsies that turn out to be benign. Thus, impedance-based detection using a multi-electrode device was investigated as a noninvasive technique to diagnose melanoma skin cancer. The multi-electrode device was designed with 8 equally spaced Ag/AgCl electrodes surrounding one central electrode at a 5 mm radius. The electrodes were held in place by a vice-like mechanism using three circular Delrin sections. The electrodes were interfaced to an 8:1 multiplexer and National Instruments Educational Laboratory Virtual Instrumentation Suite (ELVIS) for measurement control and impedance analysis. The ELVIS system, multiplexer, and electrode device were validated for accuracy with various values of resistors and capacitors. Raw and cooked chicken thigh meat and skin were tested to evaluate the capabilities of the electrode device to discern different tissue types and tissue moisture contents by impedance measurements. EpidermTM and Melanoma tissue-engineered skin analogues, provided by MatTek Corporation, were tested to mimic the in situ disease state. The electrode device was found to produce reliable measurements for known electrical components with resistances between 10 ohms and 100 k-ohms and capacitances between 10 nF and 10 uF. The measurements from the chicken tissues and tissue-engineered skin constructs – excluding cooked chicken skin data – fell within the reliable range of the electrode device and were thus considered reliable as well. All analyses concluded that a statistical difference between the impedances of raw meat and raw skin, cooked meat and raw meat, and EpidermTM and Melanoma existed. Therefore, the hypothesis that a multi-electrode device could differentiate between melanoma and healthy skin tissues based on impedance measurements was satisfied.

MEASURING IMPEDANCE OF TISSUES USING A MICROFABRICATED MICROELECTRODE ARRAY By Ashwini Bhat This thesis looks at the possibility of using impedance spectroscopy for differentiating tissue, using a microelectrode array (MEA). The thesis first discusses the background and the motivation for this thesis. It covers the certain basic concepts of the human skin starting from the top epidermis layer all the way to the deep dermis layers of the skin. Then it discusses different types of skin cancer and how they occur, in humans. It also discusses various microfabrication techniques such as oxidation, wet etching, sputtering and photolithography for the creation of a MEA in order to test the tissue. The microfabricated MEA is then used to measure impedance across cooked and raw chicken at different frequencies in order to see if the two types of tissues can be differentiated using their respective impedances. The data shows that the MEA was not able to successfully differentiate the two types of the tissues. It does however list multiple improvements in the fabrication of the MEA and improvements that could be made to the testing procedures which could possible give greater difference in impedance between the two tissues

Genetic splice variants have become of central interest in recent years, as they play an important role in different cancers. Little is known about splice variants in melanoma. Here, we analyzed a genome-wide transcriptomic dataset of benign melanocytic nevi and primary melanomas (n = 80) for the expression of specific splice variants. Using kallisto, a map for differentially expressed splice variants in melanoma vs. benign melanocytic nevi was generated. Among the top genes with differentially expressed splice variants were Ras-related in brain 6B (RAB6B), a member of the RAS family of GTPases, Macrophage Scavenger Receptor 1 (MSR1), Collagen Type XI Alpha 2 Chain (COLL11A2), and LY6/PLAUR Domain Containing 1 (LYPD1). The Gene Ontology terms of differentially expressed splice variants showed no enrichment for functional gene sets of melanoma vs. nevus lesions, but between type 1 (pigmentation type) and type 2 (immune response type) melanocytic lesions. A number of genes such as Checkpoint Kinase 1 (CHEK1) showed an association of mutational patterns and occurrence of splice variants in melanoma. Moreover, mutations in genes of the splicing machinery were common in both benign nevi and melanomas, suggesting a common mechanism starting early in melanoma development. Mutations in some of these genes of the splicing machinery, such as Serine and Arginine Rich Splicing Factor A3 and B3 (SF3A3, SF3B3), were significantly enriched in melanomas as compared to benign nevi. Taken together, a map of splice variants in melanoma is presented that shows a multitude of differentially expressed splice genes between benign nevi and primary melanomas. The underlying mechanisms may involve mutations in genes of the splicing machinery.

<p>Regulators of G protein signaling (RGS) proteins are key negative regulators of Gα signaling, a branch of G-protein-coupled receptor (GPCR)-mediated signal transduction. Approximately 35% of drugs approved by the Food and Drug Administration (FDA) target GPCRs, so it is not surprising that the discovery of RGS proteins has triggered an interest in them as new drug targets. Even though many studies have been shown the involvement of RGS proteins in cancers, there is still a knowledge gap in understanding function and regulation of RGS proteins in these diseases. Consequently, in this thesis, I explored roles of two RGS proteins that have been implicated in cancers.</p> <p>RGS2 is proposed to act as a tumor suppressor in many different cancers, such as breast cancer, bladder, and ovarian cancer. Here, we investigated if RGS2 also plays a tumor suppressor role in UM, whose growth is driven by overactivated Gαq/11 signaling. We found that increased expression levels of RGS2 inhibit cell growth of UM 92.1 and Mel-202 cells. Mechanistically, this cell growth inhibition is dependent on the association between RGS2 and Gαq, but independent of its canonical GTPase-accelerating protein (GAP) activity. Furthermore, RGS2 inhibited the Mitogen-activated protein kinases (MAPK) signaling, downstream of Gαq, while leaving Yes-associated protein 1/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) activation unaffected. These data indicate a tumor suppressor role for RGS2 in UM and proposes RGS2 stabilization as a potential therapeutic targeting strategy. </p> <p>In contrast to RGS2, RGS20 contributes to cancer progression, particularly in breast cancer. However, how RGS20 is regulated is understudied. Palmitoylation, a reversible post-translational modification, regulates functions of other RGS proteins, and RGS20 is predicted to be palmitoylated. We provided direct evidence of RGS20 palmitoylation in cells and validated the palmitoylation site as the conserved cysteine (Cys148) in the RGS domain. Our results showed that palmitoylation on this site does not affect its GAP activity and subcellular localization, but it affects the association between RGS20 and active Gαo, and inhibition of Gαo-mediated signaling. This study serves as a foundation for future studies in furthering understating the role of palmitoylation in RGS20 function and its possible implications in cancer biology. </p>

De-restricted 15/11/2013

Immune checkpoint blockade (ICB) therapies have transformed the treatment landscape for advanced melanoma, extending patient survival and improving quality of life for numerous patients with a disease that was once considered to be universally fatal. However, despite the success of ICB for many patients, over half are either resistant to initial therapy, or develop resistance over time after an initial response. The mechanisms underlying this therapy resistance remain unclear for the majority of patients. We have recently identified loss of the co-stimulatory and adhesion molecule CD58 on melanoma cells as a driver for cancer immune evasion and ICB resistance. In this thesis, we use in vitro co-culture models of patient-derived melanoma cells and tumor infiltrating lymphocytes as well as in vivo patient-derived xenograft models to demonstrate the necessity of CD58 interactions with its ligand CD2 on T cells for T cell activation, tumor infiltration, and effector cytotoxicity. Furthermore, using genome-wide genetic and protein screening approaches, we identify CMTM6 as a positive regulator of CD58, and uncover its role in mediating CD58’s regulation of inhibitory PD-L1 signaling by binding to both proteins and preventing their lysosomal degradation. Thus, CMTM6 co-regulates these co- inhibitory and co-stimulatory signals such that, in the absence of CD58, CMTM6 becomes available to bind and stabilize additional PD-L1, enhancing its inhibitory signals to T cells. Finally, we identify a potential role for CD58 on T cells as a marker of effector memory T cells with enhanced effector and progenitor function. The CD58-CD2 axis therefore serves a multi-faceted, underappreciated role in melanoma cancer immunity, and may serve as a therapeutic target for enhancing anti-tumor T cell responses.

Background: We aim to validate a seven-marker immunohistochemical signature, consisting of Bax, Bcl-X, PTEN, COX-2, (loss of) ß-Catenin, (loss of) MTAP and (presence of) CD20, in an independent patient cohort and test clinical feasibility. Methods: We performed staining of the mentioned antibodies in tissue of 88 primary melanomas and calculated a risk score for each patient. Data were correlated with clinical parameters and outcome (recurrence-free, distant metastasis-free and melanoma-specific survival). Results: The seven-marker signature was able to identify high-risk patients within stages IB-III melanoma patients that have a significantly higher risk of disease recurrence, metastasis, and death. In particular, the high sensitivity of relapse prediction (>94%) in sentinel negative patients (stages IB–IIC) was striking (negative predictive value of 100% for melanoma-specific survival and distant metastasis-free survival, and 97.5% for relapse-free survival). For stage III patients (positive nodal status), the negative predictive value was 100% with the seven-marker signature. Conclusions: The seven-marker signature can help to further select high-risk patients in stages IIB-C but also in earlier stages IB–IIA and be a useful tool for therapy decisions in the adjuvant and future neo-adjuvant settings. Stage III patients with measurable lymph node disease classified as high-risk with the seven-marker signature are potential candidates for neoadjuvant immunotherapy.

Cellular heterogeneity is regarded as a major factor for treatment response and resistance in a variety of malignant tumors, including malignant melanoma. More recent developments of single-cell sequencing technology provided deeper insights into this phenomenon. Single-cell data were used to identify prognostic subtypes of melanoma tumors, with a special emphasis on immune cells and fibroblasts in the tumor microenvironment. Moreover, treatment resistance to checkpoint inhibitor therapy has been shown to be associated with a set of differentially expressed immune cell signatures unraveling new targetable intracellular signaling pathways. Characterization of T cell states under checkpoint inhibitor treatment showed that exhausted CD8+ T cell types in melanoma lesions still have a high proliferative index. Other studies identified treatment resistance mechanisms to targeted treatment against the mutated BRAF serine/threonine protein kinase including repression of the melanoma differentiation gene microphthalmia-associated transcription factor (MITF) and induction of AXL receptor tyrosine kinase. Interestingly, treatment resistance mechanisms not only included selection processes of pre-existing subclones but also transition between different states of gene expression. Taken together, single-cell technology has provided deeper insights into melanoma biology and has put forward our understanding of the role of tumor heterogeneity and transcriptional plasticity, which may impact on innovative clinical trial designs and experimental approaches.

Malignant melanoma is an invasive type of skin cancer with high mortality rates, if not detected promptly. The mortality trends are generally linked to multiple dysplastic nevi, positive family history, genetic susceptibility and phenotypic features including fair skin, freckles, numerous atypical nevi, light coloured hair and eyes, inability to tan and prolonged exposure to ultraviolet radiation B (UVB). To date, the major anti-cancer therapeutics for melanoma include surgery, chemotherapy, radiotherapy, and immunotherapy. Recently, extracellular vesicles, especially exosomes, have been highlighted for their therapeutic benefits in numerous chronic diseases such as cancer. Exosomes display multifunctional properties, including inhibition of cancer cell proliferation and initiation of apoptosis. Hence, this study aimed to evaluate the genotoxicity and cytotoxicity of cord blood stem cell-derived (CBSC) exosomes on 6 samples of peripheral blood lymphocytes taken from healthy individuals and melanoma patients and on 3 samples of melanoma (CHL-1) cells. The limited number of samples was due to the time limitations and restrictions that were in place due to the COVID-19 pandemic. In this in vitro study, the optimal concentration of CBSC-derived exosomes (0, 100, 200, 300, 400 μg/ml protein at 24, 48 and 72h treatments) was confirmed by the CCK-8 assay. CBSC exosomes (300 μg/ml) were used to treat lymphocytes and CHL-1 cells in the Comet assay and evaluated using the real-time polymerase chain reaction (qPCR) and Western blotting (WB). The data of the CCK-8 and Comet assays illustrated that exosomes exerted genotoxic effects on CHL-1 cells (CCK-8 assay, ****p < 0.0001), (Comet assay, *p <0.05, **p < 0.01). However, the data portraying a reduction in the viability of lymphocytes needs further investigation as the number of samples was limited, therefore, further clarification is required. Importantly, no significant adverse effect was observed in healthy lymphocytes when treated with the same exosomes (p = ns). When further challenged with UVA+B radiation, the exosomes did not induce any genoprotective effect on ROS-induced CHL-1 cells, compared to the positive control (p = ns). Our data insinuates that the damage might be caused by inducing apoptosis. The anti-tumourigenic potential of exosomes was observed by activating the p53-mediated apoptotic pathway in CHL-1 cells, up-regulating p53, p21 and caspase 3 and down-regulating BCL-2 at mRNA (**p < 0.01, ***p <0.001, ****p <0.0001) and protein levels (*p < 0.05, **p <0.01). The potency of CBSC exosomes in inhibiting cancer progression in CHL-1 cells whilst causing no harm to the healthy lymphocytes makes it an ideal potential candidate for anti-cancer therapy. More samples are required to evaluate the therapeutic effect of exosomes on lymphocytes from cancer patients to fully understand their mechanism of action.