| dc.description.abstracteng | Cutaneous squamous cell carcinoma (cSCC) is the second most common non-melanoma skin tumor in humans with increasing incidence. cSCC is a result of malignant transformation of epidermal cells with ultraviolet (UV) radiation being the major cause of the disease. Currently, gold standard therapy involves surgical excision, which in most of the cases is sufficient to remove a primary tumor. However, a subset of patients develops recurrent or metastatic disease, for which there are no treatment options. Therefore, understanding of molecular mechanisms governing cSCC development and progression are pending to propose new targeted therapy.
cSCC is often characterized by overexpression of epidermal growth factor receptor (EGFR), which nowadays is a main target in clinical trials. Besides, cSCCs often display activation of RAS/MEK/ERK, PI3K/AKT and mTOR signaling pathways. Clinical data show that mTOR inhibition markedly reduces the risk of cSCC development in organ transplant recipients (OTRs). Recently, also the Hedgehog (HH) signaling pathway, which is often deregulated in various cancers, has been shown to be involved in cSCC. Thus, mutations in Patched1 (PTCH), the receptor for HH ligands, were described in 15% of the cases and several studies show expression of HH pathway components in cSCC including glioma-associated oncogene homolog 1 (GLI1), the major readout for HH pathway activity. However, the importance of these pathways and their putative crosstalk in cSCC remain elusive.
In this thesis, the role and putative interaction of HH and EGF signaling as well as its downstream effector pathways i.e. MEK/ERK, PI3K/AKT and mTOR were investigated in human cSCC. By in situ hybridization and immunohistochemical analysis we showed that both GLI1 and EGF are strongly expressed in human cSCC tissue sections. However, while nearly the entire tumor was positive for EGF, GLI1 was highly expressed only in the center of the tumor while outer parts and invading cells were GLI1 negative or only moderately positive. As opposed, phosphorylated ribosomal protein S6 (pS6), the main readout for mTOR activity was strongly expressed in outer parts of the tumor and cells invading the dermis while its expression in the tumor center was weak. Moreover, GLI1 positive areas were surrounded by pERK positive stromal cells. Indeed, in cell culture model we showed that co-culture of cSCC cells with fibroblasts resulted in GLI1 inhibition in the tumor cells supporting the role of microenvironment in paracrine regulation of gene expression in cSCC. Together with the lack of Sonic Hedgehog (SHH), the major ligand that activates HH signaling throughout the tumor tissue, the data indicate that GLI1 is regulated rather in a SHH-independent and thus noncanonical way in cSCC.
Indeed, our in vitro data show that canonical HH signaling does not play a role in cSCC. However, the data provide evidence that GLI1 can be regulated via MEK/ERK, PI3K/AKT and mTOR pathways in cSCC cell lines. Thus, pharmacological inhibition of PI3K, AKT or mTOR with specific inhibitors resulted in a significant decrease in GLI1 expression level in SCL-I, MET-1 and MET-4 cells suggesting a positive regulation of GLI1 by these pathways. However, transfection with constitutively active or dominant negative Akt1 variants did not affect GLI1 expression in SCL-I and MET-4 cells thereby questioning the role of AKT in cSCC. On the other hand, MEK1/2 inhibition with UO126 resulted in upregulation of GLI1 expression in SCL-I cells and did not alter GLI1 transcript levels in MET-1 and MET-4 cells. This indicates that MEK/ERK can negatively regulate GLI1 expression in some cSCCs. Interestingly, when we applied EGF ligand to the cells, we noted a strong inhibition of GLI1 transcription in all three cell lines. Moreover, combination of EGF with UO126, led to an upregulation of GLI1 expression level when compared to EGF treatment alone. Together the data suggest that EGF inhibits GLI1 expression via activation of MEK/ERK signaling in cSCC cell lines.
In addition, we analyzed an impact of MEK/ERK, PI3K/AKT, mTOR and HH pathway inhibition on cell viability, proliferation and apoptosis. In general, the data showed that PI3K/AKT inhibitors and the HH pathway inhibitor HhA remarkably reduced cell viability and proliferation, while mTOR and MEK inhibitors as well as the HH pathway inhibitor vismodegib showed only moderate decrease in the percentage of viable or proliferating cells. However, the magnitude of change for each inhibitor was cell line-dependent. Moreover, cytotoxic effects of the inhibitors could not be excluded. Concerning apoptosis, UO126 increased the number of early apoptotic SCL-1 cells. Together with increased GLI1 expression upon treatment with UO126 in this very cell line, it can be speculated that GLI1 can promote apoptosis.
In the last part of this thesis we investigated the role of GLI1 in proliferation of cSCC cells. We showed that neither knockdown nor overexpression of GLI1 affected BrdU incorporation into DNA of replicating cells.
In summary, our data indicate that EGF negatively regulates GLI1 expression via activation of MEK/ERK signaling in cSCC cells. Moreover, we propose that GLI1 is dispensable for cSCC proliferation. | de |