Expression of GLI1, SHH, pS6, pAKT, pERK and EGF in human cSCC samples . 66

besides HH and PTCH1, cSCC were reported to express GLI1, the major target of active HH signaling (Celebi et al., 2016). Our lab also has tested the antibodies described by Celebi and colleagues. However, in our hands the respective antibodies gave very unspecific signals. We here have used an antibody against SHH from abcam that detects the N-terminal end of SHH and stained 10 human cSCC specimens. Due to the lack of an adequate GLI1 antibody the staining for GLI1 was done by GLI1 ISH. Using adjacent slides, the tumors were also stained with antibodies against pERK (readout for MEK/ERK pathway activation), pAKT (readout for PI3K/AKT activity), pS6 (hallmark of active mTOR signaling) and EGF.

As shown in Fig. 5 A the anti-SHH antibody showed positive staining in ductal epithelium of pancreatic adenocarcinoma that was used as a positive control (Fig. 5 A). Similar expression pattern of SHH in pancreatic cancer has been described in the literature (Thayer et al., 2003).

However, the antibody stained neither cSCC tumor cells nor cSCC stroma (Fig. 5 A). This shows that SHH is not expressed by cSCC. In contrast, when we measured HH signaling activity by performing a GLI1 specific ISH on adjacent slides, we found that GLI1 expression was high in the tumor center whereas tumor cells invading the dermis were not or only moderately positive for GLI1 (Fig. 5 A, B). Most interestingly, this staining pattern was inverse to that using an antibody that specifically detects phosphorylation at S240/244 sites of the mTORC1/S6K-downstream target S6 (Fig. 5 B; please note that in the following the term mTORC1 will be substituted by mTOR). Thus, positivity for pS6 was frequently revealed in invading tumor cells, whereas it was low in the tumor center (Fig. 5 B, E). In contrast, the anti-pAKT antibody recognizing pSER473 of AKT did not show any positive staining in tumor cells (Fig. 5 B) and unfortunately positive control tissue (glioblastoma) raised too much background staining (picture not shown). These data question the reliability of this anti-pAKT antibody although Barrette et al. has reported a positive staining of invasive cSCC tissue samples when using it. On the other hand, pERK was mostly expressed in stromal cells surrounding neoplastic tissue with only rare and weak signals detected in single tumor cells (Fig. 5 C). Therefore, the positivity of pERK was adjacent to tumor cells that were GLI1

negative (Fig. 5 D). EGF was expressed all over the tumor with sometimes stronger expression in GLI1-negative areas of tumor cells invading the dermis (Fig. 5 E).

C positive stromal cells positive tumor cells

pERK

pERK pERK

pERK

D

GLI1 pERK

A pancreatic

adenocarcinoma cSCC

GLI1 SHH SHH

cSCC B

*

* *

*

pAKT

*

* *

*

GLI1 pS6

Figure 5: Immunohistochemical analysis of SHH, pS6, pERK, pAKT and EGF as well as in situ hybridization of GLI1 of human cSCC biopsies. A. Representative pictures of adjacent sections from human cSCC (n=10) showing strong GLI1 expression within the tumor mass (as measured by ISH) and no SHH signals (as measured by antibody staining/IHC) throughout the whole tissue. Pancreatic adenocarcinoma served as a positive control for SHH staining. The pictures are presented at 10 x magnification. B. GLI1 ISH and IHC for pS6 and pAKT on adjacent cSCC sections. Inverse pattern of GLI1 mRNA expression and pS6 protein is shown.

No specific signal for pAKT was detected. Pictures are shown at 10 x magnification. Black asterisks indicate the areas strongly positive for pS6 but negative for GLI1. White asterisks show the regions weakly positive for pS6 but strongly expressing GLI1. C. Representative pictures showing pERK positive stromal (left) and tumor (right) cells. Upper panel 10 x and lower panel 20 x magnification. Arrows point to pERK positive cells. D.

Representative pictures showing GLI1 positive tumors surrounded by pERK positive stromal cells, 10 x magnification. E. Representative pictures of adjacent cSCC sections showing overlapping or inverse staining for GLI1, EGF and pS6. White asterisks indicate areas positive for GLI1, EGF and pS6, black asterisks indicate the regions that are EGF positive but GLI1 and pS6 negative. Invasion front of the tumor, which is positive for EGF and pS6 but negative for GLI1, is indicated with an arrow. The pictures are shown at 10 x magnification. All staining reactions were performed using either AEC (red) or DAB (brown) in case of IHC and using BM-purple (blue) in case of ISH. The expression pattern and staining intensity were assessed by a pathologist.

Due to the lack of SHH staining in tumor tissue but GLI1 positivity in the tumor center (Fig. 5 A), we hypothesized that HH/GLI signaling in cSCC might be activated in a noncanonical way. The opposing expression pattern of GLI1 and pS6 (Fig. 5 B) also may indicate that HH/GLI and mTOR pathways may regulate each other in a negative manner. Moreover, both HH/GLI and mTOR activation may be driven by EGF signaling as EGF staining sometimes overlapped both the GLI1 and pS6 positive areas (Fig. 5 E). On the other hand, EGF positivity overlapped GLI1 negative areas (black asterisks and black arrows in Fig. 5 E), which may indicate a negative regulation of GLI1 by EGF. In addition, there might also be a negative interaction between GLI1 expressing tumor cells and pERK positive stromal cells. Thus, as shown very impressively in Fig. 5 D, pERK positive stromal cells surround the tumor areas, which are GLI1 negative (Fig. 5 D). As already stated we were not sure about the reliability of the pAKT staining.

E

pS6

*

*

* GLI1

*

EGF

*

*

6.2. Basal expression of components of the HH/GLI, PI3K/AKT/mTOR and MEK/ERK pathways in cSCC cell lines

To investigate the role of HH/GLI, PI3K/AKT/mTOR (please note, mTOR is frequently activated by AKT and in the following will be sometimes combined with PI3K/AKT signaling) and MEK/ERK signaling and a putative interaction between them in cSCC, we performed in vitro experiments involving initially 6 different cSCC cell lines, namely, SCL-I, SCL-II, SCC-13, SCC-12, MET-1 and MET-4 as well as HaCaT ras II-4 transformed cells also resembling the cSCC phenotype (Boukamp et al., 1990).

6.2.1. Expression of canonical HH/GLI pathway components on mRNA level

We first checked to what extent the used cell lines express the major HH/GLI pathway components GLI1, GLI2, GLI3, SMO, PTCH and SHH. For this purpose, we performed qPCR. As shown in Fig. 6 almost all pathway components were expressed at a variable level in all 7 cell lines. The exception was SHH, which was markedly expressed only by MET-1 and MET-4 cells. In addition, GLI3 was not expressed in SCC-13 that also expressed SMO at extremely low levels. SMO was also not expressed in SCL-II cells. We also compared the expression levels of cSCC cell lines to primary keratinocytes as well as immortalized HaCaT cells commonly used in studies on human keratinocytes, to see whether the HH/GLI pathway is upregulated in cSCC. The data show that primary keratinocytes expressed low levels of GLI1 and GLI2 but none of the other pathway components, whereas HaCaT cells expressed GLI1, low levels of GLI2, PTCH and SMO. These data indicate that both primary keratinocytes and immortalized keratinocyte cell line express only moderate amounts of GLI2 and completely lack GLI3, which are important factors for activation of both noncanonical and canonical HH signaling. Out of all cell lines, MET-1 and MET-4 expressed the highest levels of GLI2, GLI3, SMO and SHH. Moreover, we also noted relatively high level of GLI1 expression in these two cell lines (Fig. 6). Therefore, we chose them as representative cell lines for further analysis. Furthermore, we chose the cell line SCL-I that – as the human tumor samples – does not express SHH, but otherwise all components necessary for stimulation of the canonical (and noncanonical) HH pathway.

Figure 6: Basal expression of canonical HH/GLI pathway components in cSCC/cSCC-like cell lines and keratinocytes. qPCR showing relative expression levels of GLI1, GLI2, GLI3, PTCH, SMO and SHH in the cSCC cells lines SCL-I, MET-1, MET-4, SCL-II, SCC-13, SCC-12, in the cSCC-like cell line HaCaT ras II-4 cell (black bars), as well as in the keratinocyte cell line HaCaT and primary human keratinocytes (keratinocytes) (grey bars). The target gene expression was normalized to the 18S rRNA housekeeping gene. The data are presented as mean +/- SEM of one experiment measured in triplicates.

6.2.2. Expression of pERK, pAKT and pS6 on protein level

We next checked the basal levels of pAKT, pERK and pS6 as readouts for active PI3K/AKT, MEK/ERK and mTOR signaling pathways, respectively. All examined cell lines showed high levels of phosphorylated S6 protein when compared to the total amount of S6 or HSC70

loading control. Also, all cell lines expressed pAKT and pERK, however to a variable extent.

In detail, SCL-I cells highly expressed pAKT and pERK, SCL-II cells showed strong expression of only pERK while HaCaT ras II-4 and MET-1 cells displayed a moderate expression of pAKT and quite low pERK levels. In the remaining cell lines pERK and pAKT levels were rather low (Fig. 7). Interestingly, in the cell lines showing the highest level of pERK i.e. SCL-I and SCL-II (Fig. 7) we had measured the lowest level of GLI1 by qPCR (see Fig. 6). Taken together, all cSCC cell lines seem to differ between each other, but most of them express all major components of the HH pathway and show activation of PI3K/AKT/mTOR and MEK/ERK signaling pathways, which are downstream effector pathways of RTKs.

Figure 7: Basal activity of PI3K/AKT, MEK/ERK and mTOR signaling pathways in cSCC/cSCC-like cell lines. Representative Western Blot showing activity of AKT/pAKT, ERK/pERK and S6/pS6 in the cSCC-like HaCaT ras II-4 cell line and in the cSCC cell lines SCC-12, MET-1, MET-4, SCL-I, SCL-II and SCC-13.

HSC70 served as a loading control. The size of the proteins (in kDa) is indicated on the left side of the blot.