No description available.

No description available.

No description available.

Uveal melanoma is the most common primary intraocular malignant tumour in adults. Despite improvements in the diagnosis and treatment of the primary tumour, patients continue to have the same mortality rate as several decades ago, reflecting our poor understanding of the mechanisms behind the formation of metastases in this disease. The purpose of this study was therefore to characterize an animal model of uveal melanoma and use this model to study the transcriptional changes that cells undergo from culture to intraocular tumour, to circulation and finally to the formation of a metastatic nodule. / Using microarrays we identified 314 changes in transcript abundance between the intraocular tumour and metastatic lesions. Principal Components Analysis was used to cluster these transcripts into four distinct groups. A further 61 gene transcripts showed statistically significant changes between re-cultured cells isolated from the model, with the circulating malignant cells representing an intermediate step between cells isolated from intraocular tumours and metastatic lesions. We have produced a detailed analysis of the molecular changes that take place as human uveal melanoma cells evolve from a primary tumour to metastasis in an animal model, including the decrease in expression of specific melanoma markers. These changes were verified using quantitative real time polymerase chain reaction and three different functional assays. / In addition we sought to describe the genetic changes that are present in these cells. Using comparative genomic hybridization arrays we were able to successfully describe the deletions and amplifications that are present in genomic DNA extracted from paraffin embedded sections of the primary tumour. This represents the first time that archival tissue has successfully been used for this sort of analysis in uveal melanoma. We identified several genomic amplifications and deletions including an area of amplification of Wnt2, which is involved in beta-catenin regulation and C-Met, which plays a role in tumour cell homing to the liver in patients. / To the best of our knowledge, this is the first time that a detailed genetic analysis has been carried out on the progression of uveal melanoma from intraocular tumour, to circulation, to the formation of metastases.

Uveal melanoma is the most common primary malignant intraocular tumour in adults and despite advances in treatment of the primary tumour, the 10-year survival rate remains unchanged. The most frequent cause of death for patients of this disease is liver metastases. Removal of the primary tumour before clinical presentation of metastases, however, has no effect on patient outcome. / In order to understand the interactions between single malignant cells or sub-clinical metastases and affected organs, we have successfully developed a novel animal model of uveal melanoma. We utilized the unique properties of green fluorescent protein, a skin-flap in vivo imaging technique, and nude mice to accomplish this goal. The precision of green fluorescent protein imaging has allowed us to observe single cells interacting with organ tissues and reveal that these malignant cells are only capable of surviving in the liver.

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.

Vemurafenib is a potent serine/threonine kinase inhibitor and is registered as Zelboraf® for the treatment of metastatic melanomas harboring BRAFV600E mutations. There is a large individual variation in drug response and the side effects observed among patients treated with Zelboraf® has proven to be severe.  LC-MS/MS methods were developed to measure vemurafenib and its metabolites in human plasma for prediction of treatment outcome and side effects in order to individualize treatment with Zelboraf®.  A novel, rapid quantification method was developed for vemurafenib using a stable isotope labeled internal standard. The method was validated according to international guidelines with regard to calibration range, accuracy, precision, carry-over, dilution integrity, selectivity, matrix effects, recovery and stability. All parameters met the set acceptance criteria.  The first method suitable for quantifying vemurafenib metabolites in human plasma is presented. Lacking commercially available reference substances, human liver microsomes were used to produce the metabolites. In patient samples at steady-state five previously in vitro identified metabolites were quantified for the first time.

Melanome zählen zu den zehn häufigsten Tumorentitäten weltweit. Bei frühzeitiger Diagnose ist eine Exzision im Gesunden kurativ. Sobald eine Resektion im Gesunden jedoch nicht mehr möglich ist, sinken die Heilungschancen drastisch. Maligne Melanome sprechen wenig auf konventionelle Tumortherapien wie Radiatio und zytostatische Chemotherapie an. Daher werden neue Therapieoptionen in der Melanomtherapie getestet. Neueste Ansätze beziehen sich auf die Modulation von Immunzellen mittels monoklonaler Antikörper sowie die Modifikation der Signaltransduktion über die Mitogen-aktivierte Protein Kinase Kinase (MAPKK = MEK), BRAF und c-KIT. Auch Ionenkanäle stellen eine vielversprechende, zukünftige Option in der Behandlung maligner Melanome dar. Ich konnte zeigen, dass neben der bereits beschriebenen funktionellen Expression des „transient receptor potential“ Kanals TRPM8 in Melanomzelllinien auch TRPA1 in verschiedenen Melanomzelllinien exprimiert und funktionell ist. Die Phytopharmaka Senföl (Allylisothiozyanat; AITC) und Zimtaldehyd zeigen in Melanom-Modellen antitumoröse Effekte. Zudem sind beide Substanzen potente Stimulatoren von TRPA1. In dieser Arbeit wurde untersucht, ob AITC und Zimtaldehyd TRPA1-vermittelt die Proliferation, Apoptose und Migration von Melanomzellen beeinflussen. Das Vorkommen von TRPA1 in verschiedenen Melanomzelllinien wurde auf molekularbiologischer Ebene, mit fluorometrischen Bestimmungen des TRPA1-vermittelten Ca2+-Einstroms sowie in elektrophysiologischen Messungen nachgewiesen. Anschließend wurde die funktionelle Relevanz von TRPA1 bezüglich tumorhemmender Eigenschaften geprüft. Durch die Anwendung von TRPA1-Blockern konnte die AITC- und Zimtaldehyd-induzierte Verminderung der Proliferation nicht aufgehoben werden. Auch bezüglich der Migration und Apoptose konnte keine Korrelation zu einer TRPA1-Modulation festgestellt werden. Daher scheinen die durch AITC und Zimtaldehyd induzierten Effekte höchstwahrscheinlich nicht durch TRPA1 vermittelt zu werden.

Interleukin-2 activates lymphocytes to become highly cytotoxic for a wide range of tumour cell types in vitro (Iymphokine activated killer or LAK cells), and in animal models. However, only limited therapeutic benefit was observed in clinical trials of LAK cell therapy. This project aimed to investigate the molecular and cellular interactions involved in the production and effector functions of LAK cells, to identify factor(s) which might be responsible for the poor clinical responses observed in LAK cell therapy. Tumour cell lines were heterogeneous in their response to killing by cytokines (TNFα, LT, IFNγ and IL-1β), and purified monocytes or lymphocytes, but were consistently highly sensitive to LAK cell cytotoxicity. Autologous monocytes and lymphocytes were not killed by LAK cells, in contrast to human umbilical vein endothelial cells and fibroblasts. Supernatants from LAK cells were considerably less cytotoxic than the effector cells, and physical separation of effector and target cells resulted in inhibition of killing. Lymphocyte and LAK cell cytotoxicity was associated predominantly with the CD8+ (cytotoxic T-cell) lymphocyte sub-population, and was significantly inhibited by anti-TNFα and anti-LT, demonstrating that these cytokines were the primary effector molecules in this system. LAK cells and A375 melanoma cells showed procoagulant activity, predominantly via the tissue factor pathway, and LAK cells also possessed surface factor V. In addition, A375 cells were highly fibrinolytic. Tumour cell killing by LAK cells was inhibited by plasma, and further experiments determined that polymerised fibrin, but not fibrin monomer was responsible. From these results it was suggested that culture of small numbers of cells from tumour biopsies, and the determination of their sensitivity to cytotoxic drugs, cytokines and effector cells may lead to more effective treatment protocols for immunotherapy of individual tumours. In order to enhance the efficacy of immunotherapy, further in vivo research is required to elucidate the interactions between immune effector cells and the coagulation/fibrinolytic systems.

The melanocortin-1 receptor (MC1R) is a G-protein coupled receptor (GPCR) expressed on the surface of the melanocyte. MC1R activation after UV exposure results in the production of the dark eumelanin pigment and the tanning process in humans, providing protection from UV induced DNA damage. MC1R activation has also recently been linked to DNA repair. The MC1R gene is highly polymorphic in Caucasian populations with a number of MC1R variant alleles associated with red hair, fair skin, poor tanning and increased risk of melanoma and non-melanoma skin cancer. These MC1R variant receptors were thought to be loss of function, however the type of defect and the extent of the loss of function for individual variants was relatively unknown before the commencement of this PhD project. Many GPCR mutant proteins are intracellularly retained, resulting in a loss of signalling ability. To determine if this was the case for MC1R variant receptors, the localisation of the wild type and variant MC1R protein was investigated using immunofluorescence and radio-ligand binding on transfected melanocytic cells as well as primary melanocyte strains. For the first time, several MC1R variants including V60L, R151C, I155T, R160W and R163Q, were shown to have reduced cell surface expression compared to wild type MC1R. cAMP assays were used to determine the signalling ability of activated wild type and variant MC1R, importantly, variant receptors with reduced cell surface expression showed corresponding impairment in cAMP signalling. In contrast, the R142H and D294H variants, which have normal cell surface expression but significantly impaired cAMP signalling, are thought to have a defect in G-protein coupling. Some MC1R variants were found to have dominant negative activity on the wild type receptor in co-expression studies, this result may explain the MC1R heterozygote effect on human pigmentation phenotypes. This dominant negative effect resulted in either reduced wild type cell surface expression or reduced G-protein coupling and may be mediated by receptor dimerisation. In order to validate the in vitro studies, comparison of variant receptor characteristics with skin and hair colour data of individuals both homozygous and heterozygous for MC1R variant alleles was performed. This revealed parallels between variant MC1R cell surface expression, functional ability, dominant negative activity and the strength of the effects of variant alleles on human pigmentation. From the in vitro functional studies, it was clear that most variant receptors retained some signaling ability, although the relative abilities varied. An important unanswered question in the literature was whether the phenotype of carriers of the high penetrance MC1R variant alleles was actually representative of complete loss of function for MC1R. Due to the rarity of MC1R null alleles they had only previously been found in the heterozygous state, however we described the phenotype of one individual compound heterozygous for two frameshift mutations resulting in an individual unable to produce any functional MC1R protein. Phenotypic analysis indicated that red hair and fair skin is found in the absence of MC1R. Finally, preliminary studies using low temperature, chemical or pharmacological chaperones indicated that the cell surface expression of some MC1R variants could be rescued in cell transfection experiments. This resulted in a restoration of signaling ability after stimulation with agonist. These studies into the localization and function of MC1R variants have contributed to a greater understanding of the molecular mechanism underlying the association of MC1R with skin cancer risk phenotypes, and may lead to future drug based therapies that are able to rescue the function of MC1R variants that are intracellularly retained.