Despite the availability of multiple medication regimens, the survival rate of MPM patients is definitely dramatically poor [ 3 ]. The identification of new therapeutic approaches requires the development of preclinical in-vitro cellular versions able to identify drugs with high probability to be suitable in the clinic. Continuous cancer cell lines are commonly utilized as representative models of a given tumor histotype, but their translational value is often low. In solid and hematologic growths, the presence of TIC/CSC subpopulations is at the basis of cellular heterogeneity. TICs have different phenotype and drug sensitivity compared to bulk of tumor cells, more closely resembling cognate growth profiles than commonly used cell lines, causing inter-tumor plus intra-tumor variability not reproducible in established cell ranges. In this respect, MPM TICs in vitro retain patient-specific qualities and, due to the relatively short time required to obtain their richness in primary cultures, the genotypic and phenotypic adjustments induced by in-vitro growth are minimized [ 39 ]. Hence, TIC cultures, although they pose several technical difficulties, better reproduce the original tumor features than long-term founded cell lines, in particular regarding drug responsiveness.

In this study, we utilized three individual MPM TIC cultures representing a suitable design to improve the value of studies in preclinical research. The number of patient-derived cultures we analyzed is not high due to the difficulties to get postsurgical samples and to establish primary mesothelioma stem cell-enriched cultures (only four of 10 cultures analyzed maintained in-vivo tumorigenicity). Notwithstanding, we believe that this experimental design is highly representative of the MPM phenotype and its biological conduct, and is extremely useful to evaluate the effects of sorafenib on cellular viability and the mechanisms involved.

Sorafenib is reported to exert antitumor effects via both the direct blockade of Raf within the ERK1/2 pathway and the inhibition of multiple RTKs. Nevertheless , the precise pharmacological mechanisms responsible for its effects are still questionable and why sorafenib is not always effective in sufferers remains poorly understood [ 24 ]. Since sorafenib authorization for RCC [ 17 ], HCC [ 18 ], plus DTC [ 19 ], several clinical studies have investigated the particular effectiveness sorafenib in other tumors [ 40 ]. However , probably due to the molecular heterogeneity of these tumors, only few sufferers showed transient benefit from these trials, while disease development occurred in most of the patients; therefore , determinants of medical efficacy of sorafenib need confirmatory challenge. Analogously, following a phase I study which determined safety, pharmacokinetics, plus efficacy of sorafenib in combination with doxorubicin in MPM individuals [ 41 ], two phase II trials reported restricted activity [ 42 , 43 ]. The modest outcome of sorafenib because monotherapy in advanced MPM patients, although similar to that will seen with other VEGFR TKIs [ 42 ], might be because of different factors, including the lack of MPM patient selection as a consequence of the particular absence of predictive markers for drug response. In particular, the particular identification of molecular mechanisms crucial for tumor cellular proliferation affected by sorafenib and, possibly, ensuring drug synergism with cytotoxic drugs should be considered to identify potentially responsive affected person subgroups. Overall, low number of patients entered the reported clinical trials, and the treatment of a molecularly undefined number of MPM patients concurs with disappointing in-vivo effectiveness associated with sorafenib, making their clinical significance uncertain. Moreover, mainly because MPMs are not exclusively driven by gene mutation plus amplification or RTK activation, and because intracellular pathways plus autocrine/paracrine loops coexist and may interact with RTK functions, more molecular investigation and patient selection are warranted.

In this context, patient-derived TIC cultures may represent a novel preclinical design with higher translational predictivity, able to identify a subset of patients whose tumors show molecular characteristics predictive for positive clinical responses.

Previous in-vitro studies provided evidence that will sorafenib targets TICs isolated from cell lines [ 44 ] and primary cultures [ 20 ] in other tumor types. Here we demonstrate that sorafenib also impairs MPM TIC viability, mainly via the inhibited of FGFR1 activity rather than downstream molecules.

Sorafenib effects occur within the nanomolar/low micromolar range in all the TIC cultures analyzed, displaying similar sensitivity profiles and IC 50 values to MPM cell lines [ 12 , 45 ]. These data are in accordance with most studies confirming the cytotoxic effect in the 1– 10  μ Meters range of sorafenib in human cancer cell lines through different tumor types [ 46 ]. Efficacious sorafenib flat screen concentrations achievable in patients are usually reported within the nanomolar range, although in some case concentrations within the micromolar variety were reached [ 47 ], which are similar to the sorafenib levels effective in our cell model. Moreover, the concentrations where sorafenib inhibits FGFR1 autocrine activation in MPM TICs are similar to those reported to affect the RAF/MEK/ERK pathway plus activated RTK in human breast cancer cell lines [ 48 ]. Notwithstanding all these considerations, we have to acknowledge that the dependence on high sorafenib concentration to affect MPM TIC stability in vitro is still an issue when preclinical results need to be translated to the clinical setting, because kinase inhibitors in many cases are associated with nonspecific dose-dependent toxic effects.

In this context, our results displaying a nonhomogeneous sensitivity to the drug among TIC civilizations isolated from different tumors suggest that distinctive molecular functions determine the efficacy of the treatment. In particular, we display that TICs isolated from a human MPM (MM1) are usually highly responsive to sorafenib (IC 50   =  260 nM) due to a constitutive autocrine activation associated with FGFR1, which we identified as the main molecular target during these cells, as discussed in the following. If this observation had been to be confirmed in different patient-derived cultures, we can hypothesize which the identification of autocrine activation of this receptor could signify a starting point to identify subgroup of patients likely to be extremely responsive to sorafenib.

As far as the mechanisms of action in MPM TICs, sorafenib treatment for 24– 48  h induces G1 cell cycle arrest, as also reported in NSCLC [ 49 ] or thyroid cancer cell outlines [ 50 ]. Interestingly, in our experimental model, cell loss of life was mostly observed after 72  h of therapy, indicating that sorafenib inhibition of MPM TIC proliferation precedes the activation of the apoptotic process. Sorafenib-induced apoptosis had been observed in MM3 and MM4 cultures, while apoptotic cellular death, although present, was less relevant in MM1 cells using sorafenib at concentrations corresponding to the IC 50 . Cytotoxic activity of sorafenib in human being cancer cells has been ascribed to downregulation of anti-apoptotic proteins as Mcl-1 [ 20 , 51 ]; accordingly, we noticed that sorafenib downregulates the expression of Mcl-1 out of all three MPM TIC cultures, an event contributing to the proapoptotic activity of this drug.

Ras/Raf/MEK/ERK and PI3K/Akt are the main signal transduction cascades involved in MPM development and progression, and in TIC expansion and survival, while STAT3 contributes to maintain the transformed condition and to promote metastasis [ 52 ]. To delve much deeper into the molecular mechanisms mediating sorafenib activity, we examined its effects on MPM TICs in the presence associated with either EGF or bFGF. EGF is relevant for MPM pathogenesis and proliferation [ 12 , 35 , 52 ]. In MM3 plus MM4 cells, sorafenib slightly attenuates EGF-dependent MEK, ERK1/2, and STAT3 phosphorylation, while Akt activation is transiently inhibited. Even if the extent of inhibition differs among MM1, MM3, and MM4 cells, sorafenib caused an imperfect reduction of EGF effects in all the cultures analyzed.

On their whole, these types of findings suggest that sorafenib-mediated inhibition of EGF-dependent MAPK signaling via Raf inhibition is not fully efficient in MPM TICs, and elevated levels of phosphorylated MEK and ERK1/2 are maintained even when Raf activity is theoretically totally blocked by sorafenib. To analyze the regulation of Raf/MEK/ERK signaling and to exclude that functional alterations and/or variations in the MAPK pathway were responsible for the weak reaction to sorafenib, we used the pan-Raf inhibitor AZ628. AZ628 considerably inhibited EGF-dependent MEK and ERK1/2 phosphorylation in the sorafenib-responsive MM4 TICs. These results suggest that modulation of the MAPK pathway is readily achievable in MPM TICs as well as the low sorafenib efficacy is not due to intrinsic mechanisms associated with Raf activation. Thus, the direct inhibition of Raf seems to play a minor role in the antiproliferative activity of sorafenib in MPM TICs.

Because overexpression of alternative growth factors continues to be proposed as an escape mechanism from targeted therapies [ 53 ], we evaluated the activity of sorafenib in TICs stimulated with bFGF. Interestingly, sorafenib caused an almost full abolishment of bFGF-induced phosphorylation of both MEK plus ERK1/2. Akt and STAT3 activation by bFGF seemed to be inhibited. The differential ability of sorafenib to prevent MAPK cascade activated by bFGF and EGF is extremely suggestive that sorafenib effects in MPM TICs need to be mainly ascribed to a direct inhibition of FGFR tyrosine kinase, rather than downstream effectors such as Raf. This proof is further confirmed by the demonstration that sorafenib straight abrogates ligand-dependent FGFR1 (but not EGFR) phosphorylation.

Thus, we suggest that FGFR1 is a major target of the antiproliferative activity of sorafenib in MPM TICs. The FGFR family, and FGFR1 in particular, is highly expressed in MPM cell lines [ 54 ], and represents an emerging therapeutic target to get cancer treatment [ 55 ]. In several tumors, FGFR signaling drives tumorigenesis being activated by autocrine/paracrine loops [ 56 ]. For example , an aberrant autocrine bFGF circuit is really a key component of downstream ERK1/2 activation and tumor aggressiveness in NSLC [ 57 ], breast cancer [ 58 ], neck and head squamous cell carcinoma [ 59 ], and mesothelioma [ 36 ] cells, and mediates resistance to RTK blockers [ 60 ]. Our data support that FGFR-driven signaling plays a relevant role in the biology of MPM TICs and that sorafenib cytotoxic and proapoptotic effects are primarily ascribed to inhibition of FGFR1 rather than a Raf-dependent system. These data, quite unexpectedly, contrast to what is commonly seen in other tumor types in which sorafenib mainly acts by way of a direct inhibition of Raf kinase. Conversely, in the MPM TICs we analyzed, sorafenib was much more potent upon FGFR1 than on RAF, although the latter kinase is usually active and functional, as shown by the efficient inhibited induced by the selective inhibitor AZ628. Importantly, sorafenib exercise on receptor downstream signaling is commonly considered a key function of this drug to interfere with different RTK activity. All of us show that, at least in MPM TICs, this does not really occur and a direct effect on FGFR1 is mediating the particular antitumor efficacy of sorafenib. Actually, the mechanism associated with action of sorafenib in all three TIC cultures converges on FGFR1, this receptor being instrumental for the reaction to the drug of all the cultures analyzed (MM1, MM3, plus MM4).

Certainly, the observation that sorafenib effects in all TICs are usually mainly mediated by the direct inhibition of FGFR1 exercise can also explain the higher sensitivity of MM1 cells when compared with MM3 and MM4 cells, as regards the antiproliferative process of sorafenib (IC 50 : 0. 26  μ M in MM1 vs 6. 40 and five. 97  μ M in MM3 and MM4). Actually MM1 cells display a constitutive activation of the MAPK pathway mainly dependent on the autocrine activation of FGFR1; in fact , untreated MM1 cells release high levels of bFGF, reaching concentrations about 30-fold higher than those of MM3 plus MM4 cells. Thus, FGFR1 constitutive activity likely symbolizes the main stimulus to MM1 cell proliferation and, as a result, being the key target of sorafenib effects, could figure out the higher sensitivity of these cells. Notably, in MM1 cellular material, the release of large amounts of bFGF also caused a good ultra-rapid turnover of FGFR1 activated form, likely continual by the ligand autocrine loop, resulting in undetectable levels of receptor autophosphorylation even after bFGF exposure by western blot evaluation, as described previously [ 61 ].

The FGFR1 activation/inactivation cycle is certainly thus possibly boosted in cells with a strong bFGF production, although maintaining a sufficient amount of free receptor for holding, as reported [ 62 ], and also observed for chemokine receptors in human glioblastoma TICs [ 63 ].

Taken together, these information support that FGFR-driven signaling plays a relevant role within the proliferation and survival of MPM TICs and symbolizes a common and determining factor mediating sorafenib antiproliferative impacts, in all three cultures analyzed. Moreover, the level of bFGF release and autocrine activation of FGFR1 significantly impacts the particular entity of the antiproliferative response to the drug. Importantly, these types of data also highlight a pivotal Raf-independent mechanism fundamental the cytotoxic and proapoptotic effects of sorafenib is obvious in MPM TICs, in contrast with a direct inhibition associated with Raf proposed in different tumors. In particular, to the best of the knowledge, this is the first study demonstrating the relationship between bFGF production from tumor cells and the susceptibility to sorafenib of a TIC subpopulation. The translation of these results right into a clinical setting could provide indications to select MPM sufferers likely responsive to treatment with sorafenib. The possibility of bFGF-driven expansion and survival of tumor cells via autocrinally controlled pathways may be crucial to screen potentially responsive patients in order to targeted therapies, like sorafenib [ 64 ]. In fact , medical trials testing unselected patients did not report a significant effectiveness of the drug [ 42 , 43 ]. Thus, we propose that the particular tumor dependence on bFGF and the high efficacy of sorafenib in inhibiting the FGFR axis might help with forecasting tumor responsiveness to sorafenib.