| dc.description.abstracteng | Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and occurs as two major subtypes; embryonal (ERMS) and alveolar RMS (ARMS). Therapies of RMS typically combine surgical excision or local irradiation with chemotherapy. However, the survival rate for metastatic disease is approximately 40 % for ERMS and 10-30 % for ARMS. This highlights the need to evaluate novel therapeutic strategies that can improve the prognosis of these patients.
Like many other cancers, RMS show aberrant hedgehog (HH) signaling activity. This implicates that RMS might be sensitive towards small molecule inhibitors directed against components of the HH pathway. Components of the canonical HH signaling cascade are the HH ligand, its receptor Patched (PTCH), the signaling partner of PTCH Smoothened (SMO) and the family of GLI transcription factors. An approved readout for active HH signaling is the transcriptional level of GLI1.
Various drugs targeting the HH pathway have been developed and most of them inhibit the pathway at the level of SMO. In the first part of this thesis potential antitumoral effects of the SMO antagonists GDC-0449, LDE225, HhA and cyclopamine were compared using two ERMS (RD, RUCH-2) and two ARMS (RMS-13, Rh41) cell lines.
The data strongly suggest that GLI1 transcription and thus HH pathway activity in the used RMS cell lines is regulated independently of SMO by a noncanonical mechanism. Nevertheless, some SMO antagonists (i.e. HhA and LDE225) exerted strong antitumoral properties that were not associated with GLI1 expression. Interestingly, the mode of action of these compounds differed remarkably from each other. In addition, the observed effects were concentration dependent and the responsiveness of the RMS cell lines was heterogeneous. Thus, LDE225 and HhA induced strong proapoptotic and antiproliferative effects in most of the RMS cell lines. Moreover, the analysis revealed that LDE225 can activate caspase 3, HhA can block AKT activity and both drugs can stimulate autophagy (measured by LC3-II levels). GDC-0449 was less effective compared to LDE225 and HhA. It also paradoxically induced proliferation of some cell lines. This was similar for cyclopamine that induced cellular proliferation, never repressed HH signaling activity, showed no proapoptotic effects and activated AKT in most of the cell lines. In conclusion the data suggest that LDE225 and HhA are probably the most potent SMO inhibitors since they exerted antitumoral effects in almost all analyzed RMS cell lines.
Besides HH signaling activity, RMS often show activation of the PI3K/AKT/mTOR pathway. Moreover, both pathways can interact with each other in several types of cancer. Hence, in the second part of this thesis SMO antagonists were combined with PI3K/AKT/mTOR inhibitors to investigate potential cooperative antitumoral effects. In order to block PI3K/AKT/mTOR signaling the dual PI3K/mTOR inhibitor PI103, the specific PI3K inhibitor GDC-0941, the pure AKT inhibitor MK-2206 and the mTOR inhibitors everolimus and rapamycin were applied.
The findings indicate that HH signaling in RMS cell lines is rather regulated in a noncanonical manner by the PI3K/AKT/mTOR pathway. Moreover, HhA was the most efficient SMO antagonist and showed several cooperative antitumoral effects upon combination with PI3K/AKT/mTOR inhibitors. The most promising combination was HhA plus GDC-0941 since this combination showed cooperative antiproliferative and proapoptotic effects in almost all cell lines. In addition, this combination was superior to single drug treatments in RMS bearing Ptch+/– mice.
Taken together, the findings indicate that inhibition of HH signaling by a SMO antagonist plus an inhibitor of the PI3K/AKT/mTOR pathway may be an efficient strategy in order to improve therapy of RMS. | de |