| dc.description.abstracteng | Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma with poor prognosis. RMS, and especially the embryonal subtype (ERMS), show activation of Hedgehog (HH) signaling. In patients with Gorlin syndrome, who have an increased susceptibility to ERMS, activation of the HH pathway is due to mutations in the HH receptor PATCHED (PTCH). This is different in sporadic RMS, in which mutations in components of the HH pathway are very rare.
Because the HH pathway seems to be a good target for RMS therapy, we tested the effects of several SMOOTHENED (SMO) inhibitors on cell lines derived from sporadic human RMS. The response of the cells was very heterogeneous and some drugs paradoxically induced proliferation at certain concentrations or showed anti-proliferative effects without inhibition of HH signaling activity. Therefore we hypothesized that the antitumoral effects of SMO inhibitors may rely on off-target effects. In addition, our group found evidence for an interaction between HH and PI3K/AKT/mTOR signaling in RMS.
Since the majority of RMS show phosphorylation of AKT or S6 and since an interaction between the PI3K/AKT/mTOR and HH signaling pathways is well described in the literature, this thesis elucidated the importance of PI3K/AKT/mTOR signaling in RMS and in regulation of HH signaling activity. Furthermore the effectiveness of PI3K/AKT/mTOR inhibitors alone and in combination with SMO inhibitors was analyzed. For this purpose cell lines from sporadic human ERMS and the Ptch+/- mouse model for ERMS were employed. In addition, we tried to unravel if the anti-cancer effects of SMO inhibitors that were not associated with a downregulation of HH signaling activity were off-target effects.
The data show that cell lines from sporadic human ERMS are rather unresponsive to stimulation by the SHH ligand or SMO agonist. They are also only moderately responsive to SMO inhibitors, which apparently can exert off-target effects. However, they are highly responsive to PI3K/AKT/mTOR inhibitors, especially to the PI3K inhibitor pictilisib. Furthermore, PI3K/AKT/mTOR inhibitors can inhibit HH signaling activity in ERMS cells and when combined with SMO inhibitors they can induce cooperative anti-cancer effects.
Because the investigated human ERMS cell lines in most likelihood do not harbor mutations that cause activation of HH signaling upstream of SMO and because the cells were only moderately responsive to SMO inhibitors, we hypothesized that SMO inhibitors alone are only useful in those ERMS that are caused by mutations upstream of SMO. To approach this hypothesis, we made use of ERMS-like tumors of Ptch mutant mice. Indeed, cultured Ptch+/-
Abstract IV
ERMS cells respond to SMO inhibitors, which goes along with efficient reduction of Hh signaling activity. In addition, and as in human ERMS cells, efficient reduction of Hh signaling activity and antitumor effects are also seen with the PI3K inhibitor pictilisib. Furthermore, the combination of SMO inhibitors plus pictilisib synergistically induces anti-cancer effects. When applied orally to tumor bearing Ptch+/- mice, SMO inhibitors stop tumor growth and downregulate Gli1 expression, which correlates with reduction of tumor size. Tumor growth is also stopped by pictilisib. However, in contrast to cultured Ptch+/- ERMS cells and human ERMS cell lines and probably due to microenvironmental factors, pictilisib does not affect Gli1 expression.
In summary, the experiments of this thesis demonstrate that SMO inhibitors and concomitant reduction of HH signaling activity exhibit strong antitumoral effects in ERMS that harbor mutations upstream of SMO, whereas their antitumoral activity in cell lines derived from sporadic human ERMS (which most likely do not harbor mutations upstream of SMO) is very variable and not always associated with HH signaling inhibition. In contrast, PI3K/AKT/mTOR inhibitors e.g. pictilisib induce a strong antitumoral response irrespective of the mutational status of the HH pathway and in some cases also may allow for lowering the dose of SMO inhibitors. Together, these data suggest that PI3K/AKT/mTOR inhibitors are in general a better and probably a more secure therapeutic option in patients with sporadic ERMS and that a treatment with SMO inhibitors requires either pretesting e.g. in patient-derived short-term RMS cultures or a screen for PTCH mutations. | de |