Untersuchung spezifischer Inhibitoren der PI3K-Signalkaskade zur Therapie des Lymphangioms
Investigation of specific inhibitors of the PI3K signal cascade for the therapy of lymphangioma
von Hannah Leonore Blesinger
Datum der mündl. Prüfung:2020-07-20
Erschienen:2020-07-06
Betreuer:Prof. Dr. Jörg Wilting
Gutachter:Prof. Dr. Jörg Wilting
Gutachter:Prof. Dr. Philipp Ströbel
Gutachter:Prof. Dr. Rainer Mausberg
Dateien
Name:20200622_Dissertation_ediss_Blesinger.pdf
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Format:PDF
Zusammenfassung
Englisch
Lymphatic malformations (LM; lymphangiomas) are benign congenital malformation disorders of the lymphatic system and are characterized by cystic lymphatic enlargements. A distinction is made between the macrocystic, microcystic and mixed forms of LM. The LM occur mainly in the head, mouth and neck area, but can also be found on the trunk, extremities and in the mediastinum. The cysts are lined with lymph endothelial cells (LEC), which express typical endothelium (CD31, VE Cadherin) and lymph endothelial cell markers (podoplanin, PROX1, LYVE 1). The main therapeutic options include surgical resection and sclerotherapy of the affected areas. So far, there is no causal standard therapy. However, recent studies have shown that there are mutations activating the catalytic α subunit of the PIK3CA gene (phosphatidylinositol 4.5 bisphosphate 3 kinase catalytic subunit alpha) in LM samples. At Seattle Children's Hospital, LM tissue from 31 patients was examined for activating PIK3CA mutations. 74% of those affected showed specific mutations, typically in exon 8, 10 or 21. The exact cell type in which the mutations occurred was not determined. The Institute of Anatomy and Cell Biology at the UMG, LM tissue was taken from five patients in culture. LECs and fibroblasts were isolated from the LM tissue and cultured separately. Dr. S. Kaulfuß from the Institute of Human Genetics (UMG) examined the cells for PIK3CA mutations in exons 8, 10 and 21. Typical activating, monoallelic PIK3CA were found in four of five LEC samples mutations; no mutations were detectable in the fibroblasts. In one cell line (Ly-LEC2) there was a deletion in exon 2 (Glu109del), but not in the patient's fibroblasts. In addition to the LM tissue, lymph endothelial cells from juvenile donors (PromoCell, Heidelberg) were also examined. There were no mutations in exons 8, 10 and 21 of the PIK3CA. In the protein detection experiments carried out (Western blot), a significantly increased expression of pAKT in the cell lines Ly-LEC1, Ly-LEC2 and Ly-LEC12 could be demonstrated in contrast to the healthy cell lines HD-LEC C3 and HD-LEC C4. This shows that the PI3K AKT mTOR signaling pathway is highly activated in the mutated LECs. In order to determine the proliferation of the healthy HD-LECs and the mutated Ly-LECs and to test the influence of inhibitors, proliferation assays were carried out on six LEC-lines (three healthy vs. three sick) over 72 h. Specific inhibitors of PIK3CA and the downstream kinases mTOR and AKT, as well as other tyrosine kinases, were used to specifically inhibit the proliferation of the Ly-LEC. The aim is to find a therapeutic window for the treatment of LM patients with inhibitors that inhibit the growth and angiogenesis of Ly - LEC in vitro. The proliferation experiments show partly different and partly similar effects of the eight different inhibitors on the healthy LECs and the LM-LECs. The following inhibitors were tested in the present work: PI3K inhibitors - Wortmannin, BKM120, LY294002, CAL-101; mTOR inhibitor - rapamycin; AKT inhibitor – MK-2206 and the kinase inhibitors - dasatinib and sorafenib. Rapamycin inhibits cell growth as the only inhibitor in both concentrations used in the nanomolar range in a highly significant way compared to the solvent control (DMSO). In the case of the other inhibitors, the higher concentration used (in the micromolar range) inhibits the cell lines significantly in almost all cases in comparison to the DMSO control (exception LY294002 with Ly-LEC2; CAL-101 with Ly-LEC2 and HD-LEC C2, sorafenib at HD-LEC C3). At the lower concentration there is no significant inhibition (exception BKM120 for Ly-LEC12 and HD-LEC C2; MK-2206 for Ly-LEC12, HD-LEC C2 and HD-LEC C4; sorafenib for Ly-LEC1 and HD-LEC C2). In general, there was no clearly specific inhibition of the LM-LECs (Ly-LECs) without influencing the healthy HD-LECs. Only with MK-2206 was there a reduction in the mutated LECs below the initial value, which was not the case with the healthy LECs. Inhibition of the proliferation of the lymphatic endothelial cells by PI3K-/ mTOR-/ AKT- or tyrosine kinase inhibitors is possible in a certain concentration. However, the inhibitors affect the proliferation of both LM LECs and healthy LECs. It is discussed whether the findings of the investigations can be transferred directly to the in vivo situation. Treatment of LM patients, as is already taking place with rapamycin, should be strictly and closely monitored.
Keywords: PI3K; Lymphatic malformation; PIK3CA; Lymphangioma