Untersuchungen zur Rolle von Wnt5a beim Basalzellkarzinom

Abstract

Basal cell carcinoma (BCC) is the most common tumour in humans. In almost all BCC analyzed so far the Hedgehog/Patched (Hh/Ptch) signalling pathway is pathologically activated. In the vast majority of cases this is due to inactivating mutations in the tumour suppressor gene Ptch. In addition to activated Hh/Ptch signalling, overexpression of Wnt5a has been described in BCC. Therefore this gene also seems to play an important role in this tumour. The aim of this work was to analyze the regulation of Wnt5a expression by Hh/Ptch-signalling. Furthermore, the effects of Wnt5a on keratinocyte and BCC-cell lines and on the activity of the three Wnt-signalling-cascades were investigated. For this purpose murine BCC derived from the Ptchfloxflox; ERT2+/--mouse-model, human BCC, the keratinocyte cell lines HaCaT (human) and C5N (murine), and the BCC cell line ASZ001 (murine) were employed and analyzed by qRT-PCR, Western Blot and immunohistochemistry. Quantitative RT-PCR analyses revealed that Wnt5a is overexpressed in native, non-microdissected human and murine BCC. As shown by other members of our group, Wnt5a is expressed tumour-extrinsically by cells of the tumour stroma. The tumour cells themselves do not express Wnt5a. These results are consistent with my observation that Wnt5a expression in keratinocytes and in the BCC-cell-line ASZ001 is not stimulated by activation of Hh-signalling. Furthermore I was able to show that the tumours and the analyzed cell lines overexpress the Wnt5a-receptors Fzd2, Fzd4 and Ror2. As Wnt5a is a secreted molecule which may influence the behaviour of BCC cells when being expressed by stromal cells, I incubated ASZ001 with recombinant Wnt5a. This led to the expression of the differentiation marker K10 in tumour cells. Changes in the proliferation marker Pcna were not detected. Finally, Western-Blot-Analysis of murine BCC showed that Wnt/Calcium (Ca2+)-signalling and possibly Wnt/JNK/AP1-signalling are active in the tumours, whereas the Wnt/β-Catenin-pathway seems to be inactive. Due to these data and by taking additional results of our group into account, the current hypothesis with respect to the role of Wnt5a in BCC is the following: Wnt5a is induced in the BCC stroma by unknown mechanisms. Wnt5a binds to its receptors on BCC cells and activates Wnt/Ca2+-dependent signalling and possibly the Wnt/JNK/AP1-pathway. The activation of the Wnt/Ca2+-pathway and the accompanying increase of the intracellular Ca2+ concentration lead to the induction of tumour differentiation, which is reflected in increased expression of the differentiation marker K10. Since our group showed that differentiation is accompanied by BCC regression, it is possible that Wnt5a plays a tumour suppressive role in this tumour.