Year: 2015
Type: dissertation
Abstract: Melanoma is one of the most aggressive forms of skin cancer, and is very difficult to treat as resistance to existing treatments almost invariably develops. The disease is genetically heterogenous which further increases the challenges of developing new treatments against it. Exploring how differing levels of different proteins affect the tumor cells is the key in identifying new targets for future treatments. MITF is the master transcription factor of melanocytes, and is shown to be able to control a large portion of the melanocyte genome. While it is only directly mutated in about 20% of melanomas, it is directly or indirectly deregulated in most patients. This, combined with the MITF-m-isoform of the protein having melanocyte-specific expression, makes the gene interesting as potential future drug target against melanoma. Increased knowledge about its targets and its effect on gene expression would provide valuable insights. Here, we demonstrate the effects MITF downregulation have on the tumor suppressor genes AP-2a and AP-2c, the membrane bound growth factor receptors ERBB2 and ERBB3, and the intracellular signaling proteins AKT and ERK. To compare samples, siRNA-mediated gene knockdown in SK-Mel28 melanoma cells was performed by using the transfection methods Photochemical Internalization and Lipofectamine 2000. Photochemical Internalization was shown to significantly affect MITF levels and was abandoned in favor of Lipofectamine 2000 transfection. Knockdown of MITF was shown to reduce levels of AP-2a significantly, while increasing levels of ERBB3. These effects were consistent at both a protein level and RNA level. Furthermore, reduced levels of MITF resulted in increased signaling in the PI3K/AKT-pathway, a pathway that also has been previously shown to be regulated by ERBB3. Lowered levels of AP-2a was found to reduce AKT-signaling. The MAPK/ERK-pathway was also examined, but no change was observed after either MITF or AP-2a siRNA knockdown. MTS assay experiments showed an increase in metabolic activity in cells transfected with MITF siRNA. These results indicate that MITF regulate genes capable of both assisting and preventing melanoma progression.
Author: Eirik Bager Sundmark
Language: English
Topic: Melanoma and MAPK Pathways
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funder: Universitetet i Oslo
OpenAlex ID: W2231769867