Phd in MOLECULAR MEDICINE

Thesis title: Combined treatment with ErbB receptors and Hh signalling pathways inhibitors is superior than single treatment in inhibiting the growth of malignant mesothelioma both in vitro and in vivo

Malignant mesothelioma (MM) is an aggressive neoplasia with low survival rates that originates from the mesothelium cells lining of the serous vaginal cavities, such as pleura, pericardium and peritoneum. MM mainly affects men (70-80%) between the fifth and seventh decade of life, with a mortality ratio between male and female of 4:1. The development of MM is associated with a multifactorial etiology, but the main cause identified in the pathogenesis of MM is professional exposure to asbestos. Due to the poor prognosis and severity of the disease, the average survival of patients without treatment is around 9-12 months, but even following therapy it does not exceed 22 months. Despite the current multimodal therapies, 5-year survival is achieved in only 5% of patients and the therapeutic strategies for the treatment of MM are referred to as ‘life-extending treatments’. In agreement with new findings and with awareness of resistance of MM to conventional therapies and the poor patient survival following traditional chemotherapy, novel molecular targeted therapies for MM treatment have been identified. Novel approaches include the use of inhibitors of mTOR, folate, receptor tyrosine kinase, ciclooxygenase and angiogenesis, synthetic lethal treatment, miRNA replacement, oncoviral therapies, and immunotherapy alone or in combination with chemotherapy. Among the other signal transduction pathways, ErbB receptors and Hh signaling are deregulated in MM. Thus molecules involved in these signaling pathways could be used to develop novel therapeutic target therapy approaches. To this aim the in vitro and in vivo anticancer effects of combined treatment with Afatinib (inhibitor of the tyrosine kinase activity of EGF receptors) and GANT61 (inhibitor of the Hedgehog pathway) have been analysed in MM cells for the first time. In vitro effects of Afatinib and GANT61, used as single or combined treatment, on cell growth, cell death, cell cycle regulation and autophagy was evaluated in human and murine cell lines. Three human malignant mesothelioma cell lines, having different histotype (MM-B1, biphasic phenotype; MM-F1, fibromatous phenotype; H-Meso-1, epithelioid phenotype), and a murine epithelioid MM cell line (#40a) have been used. The effects of Afatinib and GANT61, used as single or combined treatment on the modulation of the activation and expression of molecules involved in signal transduction pathways deregulated in MM, such as ErbB receptors, MAPKs (Mitogen-activated protein (MAP) kinases) and Akt, were also evaluated. Finally, the in vivo antitumor activity of Afatinib and GANT61 used as single or combined treatment was analysed in C57BL/6 mice intraperitoneally transplanted with mouse MM syngeneic cells #40a and treated simultaneously with the two molecules. Our results showed that treatment with AFA and GANT, used alone and in combination, significantly inhibits the growth of MM cells in a dose- and time-dependent manner. The growth inhibition induced by the combined treatment AFA+GANT was superior to the effect obtained upon single treatment with GANT or AFA alone. As regards the effect of the two single treatments on cell proliferation, it must be considered that AFA is certainly a more powerful compound than GANT in its anticancer effects on MM. The inhibition of cell growth was mainly due to the activation of extrinsic pathway of apoptosis. The combined treatment AFA+GANT significantly increased the activation of apoptotic process compared to single treatments. Apoptosis was inhibited by the use of anuniversal caspase inhibitor, Z-VAD-FMK. Z-VAD-FMK significantly reduced the number of subG1-phase cells compared to single treatment in all MM cell lines. AFA increased the Bax/Bcl-2 ratio in H-Meso-1, MM-B1, #40a cell lines but not in MM-F1 cell lines. This effect was amplified by the combined treatment. It is worth of knowing that AFA was able to increase apoptosis in MM-F1 cell line and this effect was potentiated by GANT. The combination of the two compounds showed a significant synergistic effect when used at the highest concentrations for 48 and 72 hours. To corroborate the in vitro findings, C57BL/6 mice were intraperitoneally inoculated with syngeneic mouse MM cells (# 40a) and intraperitoneally treated with AFA, GANT, or AFA+GANT. Our results showed that AFA, GANT and AFA+GANT treatments were able to significantly interfere with the in vivo tumor growth of mouse MM cells transplanted into the peritoneum compared to control mice. Moreover, AFA, GANT and AFA+GANT were able to induce a significant increase in the mean survival and reduction of the tumor volume compared to control mice. To note that AFA+GANT were more effective in inducing a significant increase in the mean survival of mice and reduction of the abdominal circumference compared to the GANT- and AFA-treated mice. All treatments, were able to significantly increase the expression of LC3-II in all cell lines compared to DMSO-treated cells demonstrating the formation of autophagosomes and induction of autophagy. In addition, the levels of SQSTM/p62 increased in GANT- and AFA-treated H-Meso-1, MM-F1, MM-B1 and #40a cells suggesting a block of the autophagic flux. The combined treatment AFA+GANT was not able to decrease the levels of p62 suggesting that the increased antitumor effect by the combined treatment might not be due to the increase of the autophagic flux. AFA downregulated the expression of EGFR and ErbB2 in H-Meso-1 cell line while diminished that of ErbB2 in all the cell lines. However, AFA inhibited Erk1, Erk2 and p54 JNK activation in all human and mouse cell lines. Of note GANT potentiated the inhibitory effect of AFA on ERk1 and ERk2 phosporylation in H-Meso-1 cells. AFA had an opposite effect on p38 activation since it increased p38 phosphorylation in H-Meso-1 cell line, but decreased it in MM-B1 and #40a cell lines. On the other hand, GANT further inhibited p38 phosphorylation in the mouse #40a cell line when added simultaneously to AFA. In addition, AFA inhibited AKT expression in H-Meso-1, MM-B1 and #40a cell lines but not in MM-F1. The combined treatment AFA+GANT significantly reduced the expression of AKT in MM-F1 cell line where AFA did not affect AKT expression. In addition, AKT expression was significantly decreased with the combined treatment as compared to single AFA treatment in H-Meso-1 and in the #40a cell line. Accordingly, the additional effect of GANT to AFA in inhibiting signalling pathways might be different depending on the different histotypes of the MM cell lines. GANT inhibited the Hh pathway activity in H-Meso-1 and MM-F1 cells. However, we could not detect inhibition of it in MM-B1 cells. This cell lines showed a very low basal Hh pathway activity. On the other hand, AFA increased the Hh pathway activity in all the cell lines analysed, thus suggesting that the inhibition of the EGFR pathway can induce the activation of alternative pathways in MM cells such as the Hh pathway, that could be activated most likely through a non-canonical pathway. The combined treatment was able to inhibit the stimulatory effect of AFA on Hh pathway in the Hh-dependent cells H-Meso-1 and MM-F1. It has been demonstrated that Hh showed an important role in mediating resistance to EGFR-inhibitors through the induction of mesenchymal properties. We demonstrated for the first time that the combined treatment with ErbB receptors and Hh signalling pathways inhibitors is superior than single treatment in inhibiting the growth of MM both in vitro and in vivo. Our findings support the use of the Hh/GLI pathway inhibitor in combination with the ErbB receptors pathway inhibitor for the treatment of MM. Our study demonstrated that combined treatment with two inhibitors blocking two different pathways involved in neoplastic transformation and progression is superior than the single treatment blocking one pathway in inhibiting tumor growth in vitro and in vivo in MM. This study may have new clinical implications for the development of new target therapy approaches for malignant mesothelioma.