Cancer cells secrete a variety of factors that induce tumour growth, metastasis, angiogenesis, and alter the tumour stroma to create a more favourable microenviroment. This project will investigate the mechanisms underlying secretion of factors that are important in melanoma progression, and the potential of targeting the secretory pathway as a novel therapeutic strategy.
Melanoma is the most aggressive and deadly form of skin cancer. Although early stage melanoma is curable by surgical removal, metastatic melanoma is notoriously difficult to treat. Targeted therapies (such as the inhibitors of mutant BRAF) have recently had some success treating the advanced disease; however after an initial response resistance occurs and the disease continues to progress. Understanding how melanoma progresses and finding novel therapeutic strategies is thus extremely important. Melanoma is a highly secretory cell type, and the factors released by melanoma cells may act in an autocrine or paracrine fashion on tumour and stroma, where they may modulate the extracellular environment. The cancer "secretome" is increasingly being studied as a source of novel therapeutic targets or diagnostic factors. In the proposed project, we plan to identify new targets through studying the melanoma secretome. In addition, we will investigate the molecular mechanisms underlying the secretion of factors that are already known to be involved in melanoma progression. We will also test drugs that are able to interfere with the secretory pathway (either the secretion of specific factors, or more general inhibitors) in melanoma cells in order to determine whether this may be a viable therapeutic strategy.
Hypothesis and Aims: We hypothesise that the secretory pathway is important in melanoma progression, and that targeting the secretory pathway is a promising strategy for melanoma treatment. Our aims include:
1. Analysing the secretome of human melanoma cells. We will culture melanoma cell lines that represent different stages of progression, as well as highly aggressive drug-resistant cell lines derived from patients that have relapsed after treatment with the BRAF inhibitor. We will perform secretome analysis using mass spectrometry to determine which secreted proteins may be associated with melanoma progression or resistance to the BRAF inhibitor.
2. We will investigate the intracellular trafficking pathways of secreted factors that are important for melanoma progression. Vascular Endothelial cell Growth Factor (VEGF) is known to be secreted at high levels by melanoma cells and induces tumour angiogenesis. Matrix metalloproteinases (MMPs) are also produced by melanoma cells and facilitate cell invasion by degrading the extracellular matrix around the tumour cells. We will create fluorescently tagged versions of these proteins to analyse the intracellular trafficking pathways in melanoma using immunofluorescence and live imaging as well as other molecular biology techniques. We will determine which trafficking proteins regulate the secretion of these proteins.
3. We will test drugs that interfere with the secretory pathway (either alone or in combination with currently used drugs) for inhibition of melanoma growth and metastasis. We will obtain drugs that have already been tested in other cancers and are known to inhibit the secretion of specific factors such as VEGF or inhibit secretion more generally. We will also perform a drug screen of FDA approved compounds in combination with a flow cytometry assay to identify drugs that decrease secretion of factors involved in melanoma progression. We will test these drugs using both in vitro assays for growth and invasion, as well as a mouse melanoma xenograft model.
In order to achieve these aims we already have available a number of well characterised human melanoma cell lines, many of which have been transduced to express the novel fluorescence ubiquitination cell cycle indicator (FUCCI). We have also established an in vitro 3D melanoma spheroid model that better mimics the tumour microenvironment. By using a range of molecular biology and imaging techniques, these studies will provide new insight into a previously overlooked avenue of melanoma biology and may lead to the development of novel therapeutic or diagnostic strategies.
This project will utilise a wide variety of techniques including:
• Widefield de-convolution, confocal and multiphoton microscopy
• Image analysis using specialised software
• Live imaging
• Western blotting
• Cloning fluorescently tagged proteins
• Cell culture (including 3D culture models)
• Real time PCR
• Lentivirus expression vectors
• Plasmid DNA transfection
• Mouse melanoma xenograft models
• Mass spectrometry
• Flow cytometry
• Migration and invasion assays
The opportunity ID for this research opportunity is 1446