| dc.description.abstracteng | Rhabdomyosarcoma (RMS) are the most common pediatric soft tissue sarcomas. RMS of the embryonal subtype (ERMS) are characterized by high expression of markers of an activated Hedgehog (HH) signaling cascade, i.e. they overexpress GLI1, GLI2 and PTCH. However, they very infrequently show pathway-activating mutations e.g. in PTCH, although inherited PTCH/Ptch mutations act as ERMS-drivers both in humans and mice. This is different for oncogenic mutations in the 3 RAS genes (H-, K-and NRAS; oncRAS), which are a very common in ERMS. Since the literature provides evidence for crosstalks between HH and RAS signaling in other tumor entities, this thesis aimed at a better understanding of a potential interaction of HH and RAS signaling in ERMS and of their roles in ERMS pathogenesis, growth and aggressiveness. This is particulary important for improvement of treatment strategies, which currently show only moderate efficiencies and sometimes cause severe side effects.
First, the role of oncRAS isoforms in regulation of HH targets and associated cellular responses were studied in human cell lines derived from sporadic (full-blown) ERMS. The in vitro data from TE617.T, RUCH-2 and RD highlight that oncRAS isoforms decrease the expression of the major HH-target GLI1 in dependency of ERK. GLI1 downregulation was also seen on protein level in both the nucleus and cytoplasm. This indicates that oncRAS/ERK probably act on GLI1 transcription. In addition, oncRAS/ERK also regulate the expression of other HH pathway members e.g. of SHH, PTCH and GLI2, which however depends on the oncRAS isoform and the individual cell line. Although oncRAS mutations can modify the expression of SHH, the cell lines are in all likeliness not able to secrete HH ligands and are not HH-responsive. Together, this supports an oncRAS-mediated non-canonical suppression of HH signaling, or at least of GLI1/GLI1 expression, in sporadic ERMS.
Interestingly, oncRAS isoforms increase proliferation and tumorigenicity of ERMS cell lines despite downregulation of GLI1/GLI1. This indicates that proliferation of the cells is independent of HH signaling or at least of GLI1/GLI1. Since oncRAS-mediated GLI1-downregulation is only moderate in xenografts, it is likely that the tumor microenvironment also influences HH signaling.
OncKRas and oncHRas also aggravate formation of Hh/Ptch-driven ERMS from heterozygous Ptchdel/+ mice. This is in contrast to oncNRas, which induces expression of myogenic markers without otherwise affecting the tumor. This suggests that the 3 oncRas isoforms have divergent functions in ERMS, at least in Hh/Ptch-driven tumors. Interestingly, these oncRas-mediated changes are only seen when the oncRas genes are expressed at a very early ERMS stage, i.e. in ERMS precursor lesions. However, they do not occur when the mutations are induced at the full-blown tumor stage. Since, according to the literature, oncRas mutations themselves do not drive ERMS formation and since none of the oncRas genes significantly influence Hh signaling activity in Hh/Ptch-driven ERMS, these findings open the possibility that the mutational status of PTCH/Ptch may influence the importance of oncRAS mutations in ERMS. On the other hand it is possible that oncRAS mutations are rather passenger mutations, which only affect already initiated ERMS precursor cells and/or cancer stem cells (CSCs), but not the ERMS bulk populations. Indeed, a preliminary analysis shows that cell lines from sporadic (full-blown) ERMS have a subpopulation, which is positive for the CSC marker CD133. Of whether full-blown ERMS of heterozygous Ptchdel/+ mice lack this population is not clear at the moment and further experiments need to be done to shed light on these hypothesis. Together, these interesting results help to better understand the pathology of ERMS and hopefully will also be valuable to improve current therapies for this childhood sarcoma. | de |