## 4 Discussion  
The development of new combination therapies for RMS is of great interest for circumventing resistance mechanisms and improving clinical efficacy [30]. In the present study, we show that inhibition and hyperactivation of the Akt/Src/Cav1 signaling pathway respectively increase and decrease the oxidative stress on RMS cells, suggesting that modulation of this pathway may play a crucial role in protection and sensitization to IR therapy. Figure 6: The combination of statins and actinomycin D promotes chemosensitization in RMS lines. (**A**,**B**) Cells (1.5 \(\times\) 10\({}^{3}\) for myrAktl and RDCav1, or 2 \(\times\) 10\({}^{3}\) for RD and RH30) were seeded in triplicates in 96-well plates 24 h prior to treatment with the indicated compounds. Cell survival was evaluated by neutral red assay after 48 h (\(n\) = 2). Data are mean \(\pm\) SEM, *\(p\)-value \(<\) 0.05; **\(p\)-value \(<\) 0.001; ***\(p\)-value \(<\) 0.0001; one-way ANOVA test vs. vehicle-treated cells. #\(p\)-value \(<\) 0.05; ***\(p\)-value \(<\) 0.001; ***\(p\)-value \(<\) 0.0001; one-way ANOVA test vs. single treatment. (**C**,**D**) Cells (4 \(\times\) 10\({}^{4}\) for myrAktl and RDCav1 or 6 \(\times\) 10\({}^{4}\) for RD and RH30) were plated in duplicates into 6-well plates. FACS analysis was performed on all RMS lines after treatment with lovastatin or simvastatin and actinomycin D for 72 h (\(n\) = 2). When intracellular ROS levels increase markedly, as occurs in response to radiotherapy, activation of this pathway appears to play a crucial role in facilitating ROS detoxification to promote cell survival. As illustrated in Figure 7, we propose a model in which the survival of irradiated cells is favored in a Darwinian manner through the activation of Akt1, which, by orchestrating the increased expression of Src and Cav1, fuels a positive regulatory mechanism that facilitates its own phosphorylation. This improves resistance to IR-induced oxidative stress and repair of DNA damage, as documented by increased catalase expression, lower levels of ROS, and reduction of DSBs. It should be noted that increased phosphorylation of Akt1, Src, and Cav1 was detected in the irradiated RH30 FPRMS line, suggesting that the antioxidant role of this signaling might be conserved in most radioresistant RMS, although this aspect requires further investigation. ROS generation is one of the major mechanisms responsible for the therapeutic effect of IR and almost 50% of chemotherapeutic drugs. Like many other cancers, RMS is considered a tumor with high oxidative stress [31]. Activation of Akt and Src signaling after radiotherapy has been frequently detected in cancer [32], and Akt1 is considered to be a key determinant of intrinsic resistance in several cancer types [33]. Here, we show for the first time that Src and Cav1 are both downstream targets of Akt1 in RD cells. Based on this, we assume that Akt1 increases the phosphorylated form of Cav1 through a mechanism that is known to be Src-kinase-dependent [17; 34]. It has been shown that Src-kinase activates and phosphorylates Cav1 under conditions of oxidative stress, suggesting that Src/Cav1 may act as sensors of oxidative stress [35]. In its phosphorylated form, Cav1 can move from the plasma membrane, where it binds a plethora of proteins, to other cell compartments,  
Figure 7: The Akt1/Src/Cav1 network increases ROS detoxification and DNA repair in RMS cells. Representative scheme of the protein network that facilitates ROS neutralization through increased catalase expression and DNA repair. Statin treatment induces high levels of ROS and radiosensitization by reducing Akt1 and Cav1 activation. thereby producing altered internalization and trafficking of numerous molecules, including a variety of RTKs and G-protein-coupled receptors [35; 36].