Epithelial-Mesenchymal Transcription Factors in Melanoma

Loss of E-cadherin is thought to be an important, initial step in early phases of melanomagenesis. Re-expression of E-cadherin consolidates invasive behaviour of melanoma cells and reconstitutes the influence of cells from the tumor microenvironment, in particular keratinocytes, on melanoma cells. A plethora of signaling factors influences the expression of cadherins, the activity thereof at the transcriptional level is regulated by epithelial- mesenchymal transcriptional regulators (EMTRs), like Snail, Slug or Twist. One of the best characterized signaling factors to the pathogenesis of melanoma is hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF is constitutively expressed by melanoma cells, but may also be derived by cells from the microenvironment. Thus stimulation occurs in autocrine as well as paracrine loops. The importance of HGF/SF is demonstrated by the development of sporadic melanomas with metastases in transgenic mice, overexpressing HGF/SF. Previous work from our lab has shown that HGF/SF downregulates E-cadherin and new data suggest that this effect may be achieved through a stage- dependent up- or down-regulation of EMTRs. The intent of this project is to continue investigations on these transcriptional regulators with respect to stage-specific expression, to study mechanisms of HGF/SF induced changes in those transcription factors and to identify co-repressors or-activators of Slug, Snail and Twist. This study aims in understanding the fundamental process of a sequential, stage-specific activation or downregulation of all three EMTRs through HGF/SF, which results in the dynamic regulation of cadherins in melanomagensis. It may also have implications for other tumor entities and determines the influence of EMTRs in the course of tumor progression. The following hypothesis - Hierarchy of epithelial-mesenchymal transcriptional regulators in malignant melanoma - will be examined in two specific aims. Specific Aim 1: Binding specificities and dynamics of Snail, Slug and Twist to `E-boxes` of E- and N-cadherin in melanocytic cells and changes thereof after exposure to HGF/SF. Functional implications of over- or down- regulation of Slug and Twist for early melanoma development and progression. Specific Aim 2: Mechanisms of distinct regulations of Snail, Slug and Twist through HGF/SF signaling.

Epithelial-mesenchymal transition is a key factor in tumor development. Upon activation of several transcription factors, so called epithelial-mesenchymal transition regulators (EMTRs), the conversion from an epithelial to a mesenchymal phenotype is initiated. How these EMTRs are affecting adhesion, migration and invasion as single factors is well known, however, whether these factors act in a hierarchical fashion in order to drive progression has not been investigated so far. To this end we show that a growth factor important for driving melanoma development leads to different expression levels of EMTRs in a stage dependent manner. The modulation of expression levels has functional consequences, which implies a more aggressive phenotype of melanoma cells. Further we observed for the first time that one EMTR is indeed regulating another EMTR at the transcriptional level. This leads to an additive effect in driving migration and changes in adhesion. We propose a model of a site directed activation of EMTRs, which enables melanoma cells to gain momentum in acquiring a more mesenchymal phenotype through enhanced regulation of a cascade of EMTRs driving conversion.