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The role of Hedgehog signaling and its interaction with EGFR-pathway in cutaneous squamous cell carcinoma

Natalia Khizanishvili
Doctoral thesis
Date of Examination: 2020-08-06
Date of issue: 2020-07-22
Advisor: Hahn, Heidi Prof. Dr.
Referee: Schön, Margarete Prof. Dr.
Referee: Meyer, Thomas Prof. Dr.

Persistent Address: http://hdl.handle.net/21.11130/00-1735-0000-0005-1427-9

 

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Abstract

English

Cutaneous squamous cell carcinoma (cSCC) is the second most common non-melanoma skin cancer and its incidence is increasing worldwide. This highlights the importance of a better understanding of the molecular mechanisms of cSCC to improve the current treatment strategies. Both, epidermal growth factor receptor (EGFR)-signaling and Hedgehog (HH) signaling, are involved in cSCC. In this thesis, we tried to investigate the role of HH signaling and its interaction with the EGFR-pathway in cSCC. The activity of HH signaling was assessed based on the mRNA expression of its main target GLI1. Since previous studies from our research group revealed that EGF treatment downregulates GLI expression via MEK, we used the specific ERK1/2 inhibitor SCH772984 to investigate if the downregulation is mediated downstream of MEK via ERK. The data revealed that EGF-induced GLI1 downregulation is restored to basal level by ERK inhibition in MET-1 and MET-4 cell lines, but not in SCL-1 cells. These results suggest that the observed EGF-induced GLI1 downregulation is mediated via ERK in MET-1 and MET-4 cells, but via MEK in SCL-1 cell line. We also observed that ERK inhibition does not alter the proliferation or metabolic activity of the investigated cell lines. In the next part of this thesis, we focused on the role of HH signaling in migration and epithelial-to-mesenchymal transition (EMT) of cSCC. For this purpose, we analyzed EGF-treated cells or cells in which GLI1 was knocked down or was overexpressed. Migration was measured by insert migration assay. Because SCL-1 cells did not show any migratory capacity, we focused on MET-1 and MET-4 cell lines. The data revealed that EGF-treatment, as well as a GLI1-knockdown, increased the migratory capacity of MET-4 cells. However, these experiments should be repeated since they were performed only twice. Unfortunately, we were unable to transfect MET-1 with GLI1 targeting siRNA. We also tried to investigate the impact of GLI1 on EMT by analyzing the expression of the mesenchymal markers ZEB1, ZEB2, TWIST, VIM, SNAIL and the epithelial marker E-cadherin (CDH). The expression of EMT-markers was measured in EGF-treated cells and in cells in which GLI1 was knocked-down or as overexpressed. However, different results were obtained when normalizing the gene expression data to different housekeeper genes. Therefore, the current data are difficult to interpret, and additional investigations are needed to specify the results.
Keywords: Hedgehog signaling; squamous cell carcinoma

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