6.4.1 Functionality of PI3K/AKT/mTOR signaling inhibitors depends on feedback regulatory loops
At first, all PI3K/AKT/mTOR inhibitors were functional. PI-103, pictilisib and MK-2206 efficiently downregulated pAKT levels, and everolimus and rapamycin efficiently inhibited the phosphorylation of the mTOR target S6 (see Figs.10B and 15).
Furthermore, everolimus and rapamycin enhanced phosphorylation of AKT in RD cells. This phenomenon has already been described237. Thus, upon mTOR inhibition, an IGF-1R-dependent activation of PI3K and subsequent phosphorylation of AKT at Ser473 occurs. Due to this negative feedback loop, the use of mTOR inhibitors for RMS clinical trials (phase II and III clinical trials are ongoing, as described in section 1.1.1) should be carefully reconsidered.
PI-103 and pictilisib efficiently inhibited the phosphorylation of S6 in RD and RUCH-2 cells.
However, MK-2206 lacked this effect as was observed for several other cancer cell lines
238-240. One possible explanation of the missing inhibition of S6 phosphorylation is the possibility of an AKT-independent regulation of mTORC1. However, this would be contrary to what was observed upon pictilisib treatment, which efficiently downregulated pAKT and pS6. Together, the two PI3K inhibitors PI-103 and pictilisib showed higher functionality than AKT or mTOR inhibitors.
6.4.2 Anticancer effects of SMO inhibitors alone and in combination with PI3K/AKT/mTOR inhibitors
Next, the impact of a combined treatment with SMO and PI3K/AKT/mTOR inhibitors on HH signaling activity and on proliferation and apoptosis was investigated in RD and RUCH-2 cells. As already stated, PI3K/AKT/mTOR inhibitors more efficiently reduced HH signaling compared to SMO inhibitors (see Fig. 14). These data indicate that non-canonical HH signaling via the PI3K/AKT/mTOR signaling pathway plays a more prominent role in both ERMS cell lines compared to canonical HH signaling. In addition the data showed that inhibition of canonical HH signaling was not strengthened when SMO inhibitors were combined with PI3K/AKT/mTOR inhibitors. Similar results have been shown in a recent paper when using vismodegib in combination with PI-103249.
We next investigated the impact of the drugs on proliferation (see Figs. 16 and 17) and apoptosis (see Fig. 18). In the settings where cells were treated with single drugs,
vismodegib or sonidegib did not affect proliferation, whereas HhAntag reduced it. In addition, sonidegib and HhAntag – in contrast to vismodegib - induced apoptosis in RD cells. PI-103 and pictilisib efficiently blocked proliferation of RD and RUCH-2 cells and induced apoptosis in RD cells. MK-2206 blocked proliferation of RUCH-2 cells. In contrast, everolimus and rapamycin did not affect proliferation of either cell line. Although these effects are very hard to explain with the markers used in this thesis, it is possible that downregulation of pAKT and pS6 are required for efficient growth inhibition of RD cells. Thus, MK-2206 or everolimus and rapamycin, that did not reduce pS6 or pAKT, respectively, had no anti-proliferative or pro-apoptotic effects in RD cells. On the other hand, MK-2206 blocks proliferation of RUCH-2 cells but not of RD cells, which goes along with a reduction of GLI1 expression in RUCH-2, but not in RD. This may indicate that the lack of pS6 reduction might be compensated by GLI1 reduction in RUCH-2 cells. However, this is pure speculation.
When SMO inhibitors were combined with PI3K/AKT/mTOR inhibitors, cooperative antitumoral effects were observed for several combination treatments. However, these were dependent on the used SMO inhibitor and on the cell line investigated. Thus, in RD cells, combination treatments with vismodegib generally did not evoke cooperative anti-proliferative or pro-apoptotic effects, whereas the combination sonidegib plus pictilisib induced a synergistic reduction of proliferation at concentrations of 50 µM plus 10 µM. Additionally, combinations of 30 µM sonidegib plus PI-103, pictilisib or MK-2206 evoked strong and cooperative pro-apoptotic effects. Unfortunately, these effects were not observed at a concentration of 10 µM sonidegib and GLI1 expression, pAKT and pS6 levels and caspase 3 cleavage were only measured in samples treated with 10 µM sonidegib. Thus, it was not possible to correlate these factors with the cooperative pro-apoptotic effects. Nevertheless, single treatment of RD cells with PI-103, pictilisib or MK-2206 induced cleavage of caspase 3 and apoptosis (see Figs. 15 and 18), suggesting that the cooperative pro-apoptotic effects may be caspase-dependent.
Also the combination of HhAntag plus pictilisib evoked cooperative anti-proliferative effects and the combination of 12.5 µM HhAntag plus 2.5 µM pictilisib acted synergistically (see Fig.
17C). However, the cooperative anti-proliferative effects were not accompanied by any changes in GLI1 expression, pAKT, pS6 or cleavage of caspase 3. Thus, the reason for the cooperative effects has to be based on other factors. HhAntag also evoked strong pro-apoptotic effects when combined with MK-2206. This was indeed accompanied by an enhanced cleavage of caspase 3. However, it remains unclear why the pro-apoptotic effects only occurred upon combination treatment with MK-2206 and not with PI-103 or pictilisib.
Similar to what was seen in RD cells, combination of vismodegib plus PI3K/AKT/mTOR inhibitors did not enhance the anti-proliferative effects in RUCH-2 cells (see Fig. 16). This was also true for sonidegib. Although the combination of sonidegib plus rapamycin cooperatively reduced proliferation, the overall effect of this combination was rather weak compared to e.g. single treatment with pictilisib. In contrast, combination of HhAntag plus PI-103 induced strong and cooperative anti-proliferative effects. However, this effect also could not be traced back to GLI1, pAKT or pS6 levels and remains to be analyzed in the future.
Furthermore the combination of HhAntag plus pictilisib did not enhance the anti-proliferative effects of either single treatment in RUCH-2. However, since pictilisib already reduced proliferation towards the lower detection limit and since the combination of HhAntag plus pictilisib enhanced cleavage of caspase 3 (see Fig. 15C), this combination may indeed exert very potent antitumoral effects, at least in RUCH-2 cells. Furthermore, the combination of HhAntag plus MK-2206 induced strong and cooperative anti-proliferative effects (see Fig.
16C). Because this went along with a more pronounced reduction of pS6 levels and an increase in cleavage of caspase 3 compared to either single treatment (Fig. 15C), the cooperative effects could be due to enhancement of apoptosis. This is similar to a recently published observation showing that MK-2206, which alone does not affect pS6 levels, cooperatively reduces pS6 levels in mouse embryonic fibroblasts when combined with rapamycin240. In addition, the authors of this report also state that, similar to what we have observed, only the parallel reduction of pAKT and pS6 inhibits cell growth. In RUCH-2 cells, cooperative anti-proliferative effects were also seen when HhAntag was combined with rapamycin (see Fig. 16C). This was accompanied by a reduction of pAKT (see Fig. 15C).
Since rapamycin only mildly reduced pAKT levels and HhAntag strengthened this effect, HhAntag may enhance effectiveness of rapamycin via downregulation of AKT activity.
However, the combination HhAntag plus everolimus, which also reduced the level of pAKT, did not show cooperative anti-proliferative effects. Thus, the cooperative anti-proliferative effect of the combination rapamycin plus HhAntag may be based on other mechanisms than pAKT levels.
Together, cell lines derived from sporadic ERMS show PI3K/AKT/mTOR-mediated non-canonical activation of HH signaling. In addition, antitumoral effects in these cells are generally induced by PI3K/AKT/mTOR inhibitors, but not by SMO inhibitors. Although the antitumoral effects of PI3K/AKT/mTOR inhibitors are accompanied by inhibition of non-canonical HH signaling, inhibition of the latter pathway is most probably not much involved in the antitumoral effects. Thus, HhAntag, MK-2006 and the mTOR inhibitors rapamycin or everolimus strongly inhibited GLI1 expression but did not affect proliferation. This is similar to another study showing that application of the GLI1/2 inhibitor GANT-61 is not effective in RD
cells249. Together these data indicate that targeting the HH pathway alone is not very useful in therapy of sporadic ERMS.
In addition, the combination of SMO inhibitors plus PI3K/AKT/mTOR inhibitors is generally not superior to single drug treatment in cell lines derived from sporadic ERMS. The exceptions were the combinations of HhAntag plus PI-103, MK-2206 or rapamycin, which cooperatively inhibited cellular proliferation of at least RUCH-2 cells. This fact also indicates that cooperative antitumoral effects of SMO inhibitors and PI3K/AKT/mTOR inhibitors are highly drug- and cell line-specific.