The apoptosis resistance of a keratinocytic cell line and of basal cell carcinoma is mediated by the transcription factor Gli2 via cFlip upregulation

With regard to the fact that many basal cell carcinoma (BCC) bear mutations in a key player of the Hedgehog signal pathway (PTCH), and have thus an imbalance in the Hedgehog mediators, the Gli transcription factors, we studied the relationship between elevated Gli and oncogenicity, focusing especially on apoptosia mechanisms. It has already been shown that Bcl-2, an antagonist of the intrinsic, mitochondrial apoptosis pathway, is a transcriptional target of the pirimary Hedgehog signal mediator, Gli2. Our aim was to locate and define further Gli2 targets that are related to apoptosis. We made use of a human transgenic keratinocytic cell line (HaCat NHis-Gli2) that expresses high levels of Gli2 under the control of a tetracycline (tet)-controlled transactivator. It allowed us to shut on Gli2 expression by culturing the cells in a tetracycline-containing medium. We firstly screened differential gene expression between tet-on and tet-off cells using Affymetric gene chip analysis. It turned out that besides the expected Gli2 targets, also cFlip, a potent Caspase 8 inhibitor, was significantly upregulated upon Gli2 overexpression. We confirmed this result by quantitative RT-PCR on the mRNA level and by Western blot on the protein level, and could observe a time-dependent cFlip upregulation in response to Gli2. As an enzymatically inactive structural homolog of Caspase 8, cFlip blocks the extrinsic, death-ligand induced pathway of apoptosis at the level of the death receptor complex formation.
In a next step, we assessed the apoptosis-inhibitory impact of Gli2 and the role of cFlip. Our HaCat cells NHis-Gli2 cells express the death receptors TR1 and TR2 (Trail receptors 1 and 2) and are thus susceptible to Trail-induced apoptosis (shown by FACS data and apoptosis assays based on DNA fragmentation). Indeed, when we overexpress Gli2, the cells are significantly protected against Trail-induced apoptosis. With other molecules that are related to extrinsic apoptosis being equally expressed (Affymetrix data), we postulated that cFlip must play a considerable role in the Gli2-mediated protective effect. We therefore downregulated cFlip using RNAi technology and found that cells, although expressing high Gli2 levels, lost their protection, pointing to cFlip as a potent player in the Gli2-mediated defence against apoptosis. All apoptosis assays were done by FACS screening of DNA fragmentation (propidium iodide staining), and were confirmed using the APOPercentage TM assay (Biocolor). This assay reports a different step of the apoptotic process, as it stains apoptotic cells in situ using a dye that is taken up only by those cells that flip their membranes inside out. In order to further confirm the apoptosis data, and to prove that cFlip is the key player, we performed a Caspase 8 activity assay and could show that Trail-triggered Caspase 8 activity is significantly reduced in Gli2 overexpressing cells. Caspase 8 activity could be rescued by cFlip downregulation (RNAi) even in the Gli2 overexpressing situation. We thus identified cFlip downregulation (RNAi) even in the Gli2 overexpressing situation. We thus identified cFlip as an important player in the Gli2-mediated apoptosis resistance in our model cell line.
We then in silico analysed the putative cFlip promoter region (so far undefined), and identified several clusters of potential Gli2 binding sites as defined from formerly published transcriptional targets of Gli2 (e.g. Bcl-2). We cloned these clusters into a luciferase expression reporter vector adnw ere able to identify one cluster that reacted on elevated Gli2 levels as a promoter when transfected into our tet-inducible model cell line.
The four potential binding sites in this cluster were analyzed in a gel shift assay, and two of them clearly showed binding to Gli2. We thereby at least partially defined a cFlip promoter region or a cis-element of the cFlip gene.
In a second phase, we addressed the situation in basal cell carcinoma. We were lucky to get a collaboration with Dr. P. Häusermann from the Dermatology Unit of the University Hospital in Basel, who provided us with BCC tissue specimens. We screened protein expression in all BCC specimens in cryosections, and found that in high Gli2 expressing tumors, cFlip was also highly coexpressed. We then used the RNAi technology on cultured pieces of BCC to downregulate Gli2 ex vivo in these tumors, and measured the expression of Gli2 and of its targets Bcl-2 and cFlip. We succeeded to downregulate Gli2 efficiently and found that also the expression of its targets was significantly lowered, confirming that cFlip is atranscriptional target of Gli2.
We then assessed the apoptosis susceptibility of BCC tissue ex vivo under native and Gli2-downregulated conditions. As Trail receptors 1 and 2 were expressed on the BCC tissues tested, we applied soluble Trail on cultured pieces of BCC. We observed a higher cell death in Gli2-downregulated BCCs compared to native tissue, which supports an anti-apoptotic impact of Gli2 via cFlip in BCC.
The results found in NaCat NHis-Gli2 and in BCCs tested point to a tumor defense mechanism, postulating that BCC can escape from the immune system, among other ways by preventing death-ligand induced apoptosis through the upregulation of the anti-apoptotic cFlip.