MPM is an aggressive tumor with an ominous prognosis, due to the unavailability of effective therapeutic regimens. Albeit being considered a rare form of cancer, the incidence of MPM is on the rise, due to worldwide exposure to asbestos over the past decades. A better understanding of the molecular mechanisms involved in MPM carcinogenesis is urgently needed to design successful therapies that could offer MPM patients a real clinical benefit. The activation of mTOR pathway is a prognostic factor for MPM, in particular P-mTOR expression during induction chemotherapy was associated with shorter overall survival [
8]. Moreover, mTOR inhibition has shown evidences of efficacy in MPM preclinical models [
22,
23]. In the present work, we confirmed that mTOR pathway is activated in MPM. With respect to the histologic subtype, in contrast with a previous report showing a prevalent mTOR expression in the epithelioid forms [
8], we observed that the presence of an activated mTOR protein is unrelated to MPM histotypes. We further report that the phosphorylation of mTOR is not a consequence of the occurrence of somatic mutations in upstream mediators, e.g., PIK3CA, KRAS, BRAF and EGFR. These results suggest that, in MPM, the activation of mTOR signalling is triggered mainly by environmental and/or metabolic factors, among which a direct exposure to biopersistent fibers, such as asbestos [
24]. We here demonstrated, for the first time to our knowledge, that pleural mesothelioma expresses activated ERM, and that the expression of its principal component ezrin is crucial for the motility and local aggressiveness of MPM cells. ERM proteins are cytoplasmic linkers between transmembrane proteins and the actin cytoskeleton with an active role in signal trasduction [
11,
12]. In particular, ezrin has been reported as responsible for cell survival signals transduced through the PI3K/AKT/mTOR pathway [
25]. In a previous study we obtained ERM dephosphorylation by means of treatment with multikinase inhibitor sorafenib in preclinical models of osteosarcoma [
17]. Based on these assumptions, we investigated whether a combined inhibition of mTOR pathway and sorafenib treatment could provide an effective strategy for MPM management. Despite a generally low efficacy of everolimus as a single agent, we reported a strong synergism of the two drugs in inhibiting cell proliferation
in vitro. The combined treatment was capable to strongly induce apoptosis, by the induction of a ROS burst as we previously observed in sarcoma cell lines [
26]. Consistently, we observed that sorafenib activates the energy sensor AMPK [
27], further inducing mTOR pathway blockage. In the present work with mesothelioma cells, we further characterized apoptotic signalling induced by sorafenib and everolimus treatment. We demonstrated that sorafenib as single agent and even more in combination with everolimus induced mitochondrial ROS production, being this event related to apoptosis induction. Accordingly, treatment with a ROS scavenger protected MPM cells from apoptosis. Moreover, we observed p38 MAPK and c-Jun activation upon sorafenib and everolimus treatment inferring their specific role as downstream mediators of sorafenib and everolimus-induced apoptosis. In fact, we demonstrated that the inhibition with specific p38 chemical inhibitor SB202190 protected MPM cells from sorafenib- everolimus induced apoptosis. Again, according to previous results obtained in sarcoma models [
17], we demonstrated that sorafenib was capable to inhibit also in MPM cells the phosphorylation of ERM, both as a single agent and in combination suggesting ERM as potential novel direct or indirect therapeutic targets of this drug. On the contrary, everolimus induced a weak activation of ERM when administered as a single agent. We hypothesized that this effect might be triggered by the activation of AKT [
19,
28] that, in turn, phosphorylates the tumor suppressor protein merlin [
29]. In fact, this phosphorylation induced the dissociation of merlin from the complex with ezrin, moesin and CD44 allowing its switch toward an activated growth-promoting form [
30]. Being merlin an interesting biomarker in MPM [
31], reciprocal ezrin and merlin coordination in MPM onset and their influence on response to drugs deserve further investigations. The mTOR pathway is involved in ezrin- induced malignant phenotype [
15], moreover the mTOR inhibition has shown antitumor effect in mesothelioma preclinical models [
5,
6,
22,
23]. To further investigate the effect of combining sorafenib and everolimus treatment in
in vivo models of MPM, we set up MSTO-H211 xenografts into NOD/SCID mice. We observed that low doses of sorafenib and everolimus (physiologically achievable in human plasma and capable of inducing significant pharmacodynamic effect), as single agents and in combination, significantly reduced tumor growth without impairing the general health conditions of the treated mice. At the end of the experiments, no sign of adverse events was seen after necroscopy. Moreover, it will be important to investigate this treatment against tumors grown orthotopically to obtain insights on the effect on specific MPM microenvironment.