Characterization of RMS after conditional expression of oncKRas in Ptch

To analyze the effects of the expression of oncKRas on the growth of Hh-associated RMS 65 Ptch^del/+KRasG12D^fl/-Myf5CreERT^tg/- mice were randomized in two groups.

5.3.3.1. oncKras decreases the expression of Gli transcription factors, increases pErk and pAkt levels and enhances the expression of early muscle differentiation markers in RMS of Ptch mutant mice

As already seen in the study of Ptch^del/+NRasG12D^fl/-Myf5creERT^tg/-mice oncRAS can influence HH signaling activity. In order to evaluate whether this was also true for RMS of Ptch^del/+KRasG12D^fl/-Myf5creERT^tg/-mice the expression of Gli1-3 was measured in the reference tissue skeletal muscle and RMS from tamoxifen-induced (n=5) and uninduced (n=2) mice (Figure 38). Preliminary expression analysis shows that the expression of all three Glis is suppressed in tumors of induced Ptch^del/+KRasG12D^fl/-Myf5creERT^tg/-mice. However the suppression was not significant.

Figure 38: oncKRas decreases the expression of Glis in RMS of Ptch mutant mice. Quantification of the Gli1, Gli2 and Gli3 expression levels measured by qRT-PCR analyses on cDNA from RMS and skeletal muscle of uninduced and tamoxifen-induced Ptch^del/+KRasG12D^fl/-Myf5creERT^tg/- mice. For normalization of the data see Figure 32. All data are displayed as mean ± SEM. Statistical significance was tested by using Mann-Whitney test.

Furthermore a western blot analysis from uninduced and tamoxifen-induced Ptch^del/+KRasG12D^fl/-Myf5creERT^tg/-mice was performed (see Chapter 4.16.3). To see whether active KRas signaling can influence Raf/Mek/Erk and PI3K/Akt signaling the phosphorylation status of Erk and Akt was examined. Figure 39 shows that the expression of oncKRas in Ptch mutant RMS results in increased phosphorylation of Erk and Akt, which

Gli1 Gli2 Gli3

0.0 0.5 1.0 1.5

rel. expression wtRas vs. oncRas

indicates that active KRas signaling in oncRas Ptch KRasG12D Myf5creERT mice results in the activation of the Raf/Mek/Erk and PI3K/Akt pathways.

wtRas oncKRas

RMS RMS

Akt [60kDa]

pAkt [60kDa]

Erk [44/42kDa]

pErk [44/42kDa]

Hsc70 [70kDa]

Figure 39: oncKRas increases pErk and pAkt levels in RMS of Ptch mutant mice. Western blot analyses of Akt, pAkt, Erk, pErk in protein lysates from RMS isolated from wtRas and oncKRas

Ptch^del/+KRasG12D^fl/-Myf5creERT^tg/-mice. Hsc70 expression levels served as loading control.

Next the expression of muscle differentiation markers were measured by qRT PCR in skeletal muscle and RMS of wtRas (n=2) and oncKRas (n=5) Ptch^del/+KRasG12D^fl/-Myf5creERT tg/-mice. The preliminary data illustrated in Figure 40 show an increased expression of the early differentiation markes MyoD and Myf5 and a suppressed expression of the late differentiation marker Myogenin in RMS expressing oncogenic KRas compared to the control. Although the modulations are not significant (probably due to the small sample size) the data indicate that oncogenic KRas may cause a more undifferentiated RMS phenotype i.e. it causes expression of myogenesis proliferation and determination markers and concomitantly block late muscle differentiation.

Figure 40: Slightly increased expression of the myogenesis proliferation and determination markers and decreased muscle differentiation marker in oncRas RMS of Ptch mutant mice. Quantification of the MyoD, Myf5 and Myogenin expression levels measured by qRT-PCR analyses on cDNA from RMS and skeletal muscle of uninduced and tamoxifen-induced Ptch^del/+KRasG12D^fl/-Myf5creERT^tg/- mice.

Normalization was done as described in Figure legend 32. All data are displayed as mean ± SEM.

Statistical significance was tested by using Mann-Whitney test.

5.3.3.2. oncKRas increases incidence and shortens latency time of RMS in Ptch mutant mice

In order to investigate if the above mentioned modulation of oncKRas has an influence on the tumor incidence, latency time RMS multiplicity, the mice were monitored weekly (see chapter 4.17.4) for up to 200 days. As illustrated in Table 20 and Figure 41, the analysis of

Ptch^del/+KRasG12D^fl/-Myf5CreERT^tg/- mice shows significant differences in tumor

development. Thus, in mice expressing oncogenic KRas the tumor incidence is significantly increased when compared to the control (82.2 % vs 49.3 %, respectively) (P = 0.0013 for RMS by log-rank test). In addition the tumor-latency time is significantly decreased (79.7 days vs 102.8 days, respectively) (P = 0.0018 for RMS by Gehan-Breslow-Wilcoxon test).

However the oncKRas does not influence the tumor multiplicity.

MyoD Myf5 Myogenin 0.0

0.5 1.0 1.5 2.0

rel. expression wtRas vs. oncRas

Table 20: Influence of oncKRas on RMS development of Ptch KRasG12D

fl/-Myf5CreERT^tg/- uninduced 28 13

(49.3 %) 6 (46.2 %) 1.615 ± 0.241 103

Figure 41: oncKRas significantly increases tumor incidence and shortens latency time. (A) Kaplan Mayer Curve with the RMS free survival of wtRas (black) and oncKRas (grey) Ptch^del/+KRasG12D fl/-Myf5CreERT^tg/- mice. Every event represents the detection of the first RMS in a mouse. (B) Graph shows tumor multiplicity as RMS/animal in wtRas and oncKRas Ptch^del/+KRasG12D^fl/-Myf5CreERT^tg/- mice.

Statistical significance of the RMS-free survival was tested by log-rank test (P = 0.0013), of the latency time by Gehan-Breslow-Wilcoxon test (P = 0.0018) and of the multiplicity by Chi-squared test (P =

calculated (Figure 36). The results show that oncKRas does not significantly increase the number of Ki67 positive nuclei compared to RMS from wtRas animal (4.21 % ± 2.252 % vs 3.782 % ± 1.188 %). This indicates that oncogenic KRas signaling does not or only moderately increase the proliferative capacity of Ptch deficient RMS.

A B C

Figure 42: oncKRas does not significantly increase the proliferation rate of RMS of Ptch mutant mice. Ki-67 stainings of tumors derived from Ptch^del/+KRasG12D^fl/-Myf5CreERT^tg/- mice expressing (A) wtRas (20x magnification) or (B) oncKRas (20x magnification). (C) The plot show the percentage of Ki67 positive nuclei per paraffin section of wtRas and oncRas mice. All data are displayed as mean ± SEM. The cells were counted using image processing software FIJI. 3 (6 pictures per section per RMS were analyzed.

More than 1000 cells were counted). Statistical significance was analyzed using Mann-Whitney test.

As revealed by H&E staining (see chapter 4.16.5) oncKRas expression also does not obviously change the histology of RMS of Ptch mutant mice (Figure 43).

wtRas

(n=7) oncRas (n=9) 0

5 10 15 20

Ki-67 positive cells [%]

A B

C D

Figure 43: No differences in the tissue structure of RMS with and without active KRas signaling.

H&E stainings of tumors derived from Ptch^del/+KRasG12D^fl/-Myf5CreERT^tg/- mice expressing wtRas (A) 10x magnification (scale bar = 100 µm), (B) 20x magnification (scale bar = 50 µm) or oncKRas (C) 10x magnification (scale bar = 100 µm) and (D) 20x magnification (scale bar = 50 µm).

5.3.4. Preliminary characterization of RMS after conditional expression of