Chips and ASAP1. Functional genomic of the tumour metastasis

The differential screening method of Suppression Subtractive Hybridisation (SSH) has been used to identify genes associated with tumour progression and metastasis. Together 268 genes have been found to be up-regulated in the highly metastatic cell lines ASML and MTLy in comparison to its non-metastatic counterparts. In my thesis I have screened this group of genes to identify those that are differentially expressed in further tumour progression systems and show a correlation to the metastatic phenotype. This was achieved with the help of the DNA microarray technology. All the 268 genes were bound on glas chips and hybridised with fluorescent labeled cDNA isolated out of the non-metastatic cell line G and the highly metastatic cell line MatLylu. As a result of the hybridisation assays 40 genes have been identified showing association to the metastatic phenotype in varying tumour progression systems. Numerous of these genes have already been described in the literature as being associated to tumour progression or metastasis. Some of the identified genes have not been reported being related to tumour progression or metastasis and some of these genes are completely unknown (novels). One of these genes which has not been reported being related to tumour progression or metastasis is ASAP1. This gene was chosen for further studies. ASAP1 is a multiple domain protein that can interact with numerous proteins. Known interaction partners of ASAP1 are the tyrosine kinases Src, Crk FAK and Pyk2. Interactions with ARF1 and ARF5, two members of the Ras-superfamily have been described in the literature already. Further known interaction partners of ASAP1 are the phospholipid PIP2 and the scaffold protein CIN85 a regulator of the EGF receptors trafficking. By interacting with the proteins ASAP1 can regulate important cell functions like membrane remodeling, cytoskeleton organization, phospholipid metabolism and cell growth. An association to the neoplastic phenotype of the ASAP1 expression was revealed by in situ hybridisations with human cDNA and by immunohistochemistry assays using generated anti huASAP1 antibodies. To get an insight into the function of ASAP1, the phenotype of 1AS-Bsp73 cells transiently and stably overexpressing ASAP1 was studied. For these assays a newly identified constitutive active alternative spliced variant of ASAP1 (rASAP1c) was used. Further studies concerning the phenotype of highly metastatic ASML-Bsp73 cells transiently and stably overexpressing ASAP1 were performed. (orig.)