University of Iceland
Activity: Examination
Melanoma represents the deadliest form of skin cancer and originates from the pigment-producing melanocyte. The Microphthalmia-associated transcription factor (MITF) is indispensable for the establishment of fully differentiated melanocytes. Melanoma cells have been suggested to exploit the transcriptional activity of MITF to foster cancer progression. However, the role of MITF in regulating the formation of melanoma cells and to mediate metastasis is complex. The scope of this thesis was to study the impact of long term depletion of MITF in melanoma cell lines. Our findings revealed that MITF knock out (KO) melanoma cells exhibit reduced proliferation, migration and invasion potential compared to the control cell line. Transcriptomic analysis revealed a gain of extracellular matrix and neural crest related genes in MITF-KO cells, and, conversely melanocyte specific pigmentation genes were lost. Interestingly, the major components of focal adhesions, including Paxillin and Focal adhesion kinase were induced upon MITF depletion, thus explaining the phenotypic effects. Furthermore, the expression of the histone modifiers PRDM7and SETDB2was reduced in the MITF-KO cells. Consistently, the respective change in histone modification was observed in the promoters of genes that showed differential expression in MITF-KO cells. Together, we showed that the loss of MITF reprograms melanoma cells such that they lose proliferative capacity and gain expression of extracellular matrix and focal adhesion genes. This reprogramming is both due to direct effects of MITF itself but is also regulated by epigenetic modifiers that are under MITF regulation.
The pigment producing melanocytes are derived from the neural crest cells and form precursor cells called melanoblasts. These cells proliferate and migrate to their destination in skin, hair and other organs where they differentiate into melanocytes which produce the pigment melanin. The Microphthalmia-associated transcription factor (MITF) is indispensable for the establishment of fully differentiated melanocytes. MITF controls the expression of genes required for melanocyte survival, proliferation and differentiation. Melanoma cells thus exploit the transcriptional activity of MITF to foster cancer progression. However, the role of MITF in regulating the metastatic potential of melanoma cells is complex and often leads to inconsistent findings. The scope of this thesis was to further elucidate the functional role of MITF in melanoma development. Our findings revealed that MITF knock out (KO) melanoma cells exhibit a reduced proliferation rate compared to the control cell line. Consistent with that, the expression of cell cycle regulators that are known targets of MITF was affected in the KO cells. Surprisingly, both the migration and invasion potential of MITF-KO cells were significantly reduced compared to empty vector cell lines. In contrast, transient depletion of MITF did not affect the invasion potential of melanoma cells. RNA-sequencing followed by differential gene expression analysis revealed a gain of extracellular matrix and neuronal related genes in MITF-KO cells, whereas pigmentation genes that are specific to melanocyte differentiation were lost. Accordingly, the gene expression profile of MITF-KO cells was negatively correlated with the gene signature of melanocytes, whereas a positive correlation was observed with neural crest and Schwann cell signature. Interestingly, the major components of focal adhesions, including paxillin (PAX) and focal adhesion kinase (FAK) are direct targets of MITF and their expression was induced upon MITF depletion. Well in line, the number of FAK and PAX positive focal points were increased in MITF-KO and transient knockdown cell lines. Additionally, expression of N-cadherin was increased in both models whereas E-cadherin was decreased, reflecting marks of mesenchymal transition. Published ChIPseq data show that both E- and N-cadherins have MITF binding sites in their introns and promoter regions, suggesting that they are direct MITF targets. The level of histone modifications reflected major changes in gene expression observed in the MITF-KO cells. The H3K4me3 active mark and the H3K9me3 repressive mark were both altered in the promoter of genes that showed differential expression in MITF-KO cells. Consistent with this, the expression of the histone modifiers PRDM7 and SETDB2 was reduced in the MITF-KO cells. Taken together, we conclude that the loss of MITF drives cells into a de-differentiated state that is governed by a neural crest-like transcriptional program. Furthermore, the maintenance of neural crest gene repertoire is facilitated by epigenetic modifiers that are under MITF regulation. The second part of this thesis investigated the genome wide overlapping targets of the transcription factors MITF, IRF4 and TFEB. All three factors have been shown to collaborate in regulating gene expression in melanoma and melanocytes. Chromatin immunoprecipitation sequencing was performed using eGFP tagged TFEB and IRF4 proteins and genome wide binding sites were identified for each factor. Analysis of the overlapping target genes revealed that MITF and TFEB share binding to lysosomal genes whereas MITF and IRF4 share binding to pigmentation and immue-related genes. Statistically differentially bound sites were identified for each factor, in which MITF displayed unique binding to genes related to neuronal development. TFEB showed exclusive binding to genes related to DNA metabolic processes, whereas IRF4 revealed exlusive binding to genes involved in the hematopoietic system. Thus, the distinct and overlapping binding regions of each factor were identified, which enabled us to dissect the co-operative and independent transcriptional activity of each factor.