Regulation of HGF Induced Loss of Adhesion in Melanoma

Hepatocyte growth factor (HGF), a known mitogen, motogen, and morphogen for many epithelial cells, is secreted in a variety of transformed cells and tumors. Expression of HGF in transgenic mice combined with UV radiation led to recapitulation of melanoma development with stages of progression culminating in metastatic disease. HGF is secreted by melanoma cells, but not melanocytes and c-Met, the HGF receptor, is expressed by all melanoyctic cells, enabling autocrine and paracrine activation through HGF. Loss of adhesion is a prerequisite to overcome microenvironmental control. A progressive loss of E-cadherin expression, a major adhesion molecule, occurs during melanoma development and restoration of E-cadherin expression in melanoma cells results in downregulated expression of invasion-relevant adhesion receptors. When autocrine activation was induced continuously with HGF-expressing adenoviruses, adhesion molecules E-cadherin and Desmoglein 1 were decreased in melanocytes and melanoma cells. c-Met was coimmunoprecipitated with E- cadherin, Desmoglein 1 and Plakoglobulin, suggesting that they form a complex that acts to regulate intercellular adhesion. HGF thus decouples melanomas from keratinocytes by downregulating E-cadherin and Desmoglein 1 and allows dissemination of melanocytic cells. This project aims in answering the following questions: - Which pathways are involved in HGF/SF induced downregulation of E-cadherin and Desmoglein 1 in melanocytes and E-cadherin-positive and Desmoglein 1-positive melanoma cells? Especially, which of the already know modulators of the cadherin-catenin complex are activated? - Is the downregulation predominately mediated through cytoplasmic tyrosine kinases, directly through c-Met or through transcriptional repressors? - Are there HGF/SF induced changes in Wnt expression and is there a cross-talk between certain Wnt proteins and HGF? - Are HGF and Wnts concomitantly downregulating E-cadherin and Desmoglein 1 via transcriptional repressors? Results from these studies give new insights into the dynamics of intercellular communication at a very early time- point in melanoma development.

Hepatocyte growth factor (HGF) is important to the pathogenesis of malignant melanoma. Transgenic mice overexpressing HGF develop melanoma at an early stage, especially after exposure to UV irradiation. One of the key effects of HGF is the downregulation of adhesion molecules, like E-cadherin, at the very beginning of melanomagenesis. Loss of E-cadherin is thought to be an initial step in early phases of melanoma development. It enables melanocytes to attain independence from surrounding keratinocytes, through which they are communicating and controlled at normal skin homeostasis. Transcription factors like Snail, Slug or Twist regulate expression levels and functions of adhesion molecules. Thus down-regulation of E-cadherin through HGF may occur by modulating these transcription factors. Whether Snail, Slug or Twist and the adhesion molecule CD44 are modulated by HGF and which signalling pathways are crucial to this regulation has been investigated. This project aimed to characterise (i) the effects of HGF on Snail, Slug and Twist in melanocytic cells, (ii) signalling pathways implicated in HGF mediated changes in Snail, Slug and Twist expression and (iii) whether HGF increases a splice variant of the adhesion molecule CD44, CD44v6, in primary human melanocytes and the functional significance thereof. i) In primary human melanocytes HGF led to up-regulation of Snail and Slug, which then leads to down-regulation of E-cadherin. In melanoma cell lines there is a down-regulation of Slug and an up-regulation of Twist, followed by a concomitant switch in cadherin expression from E- to N-cadherin. ii) In primary human melanocytes signalling through NF-kappaB is essential for Snail and Slug expression. On the contrary in melanoma cell lines, in which signalling pathways are permanently activated, the MAPK and PI3K pathways as well as GSK-3beta are involved in the regulation. iii) Exposure to HGF results in up-regulation of CD44v6 in melanocytes. Increased CD44v6 expression led to activation of the receptor for HGF and to higher migration. Like for Snail and Slug NF-kappa B is crucial for the expression of CD44v6. In summary these results underscore the importance of HGF at the very beginning of melanoma development and identified mechanisms of HGF triggered effects, which implied modulation of transcription factors and an adhesion molecule.