Inhibitor of Apoptosis Proteins as Targets for the Treatment of Malignant Rhabdoid Tumours

Abstract

Malignant rhabdoid tumor (MRT) is a rare paediatric cancer which unfortunately is highly refractive to treatment. Retrospective reviews commonly place the survival rate at approximately 30-35 %. Thus, there is an urgent need for the development of new targeted therapies which may improve patient outcomes. Several members of the inhibitor of apoptosis protein (IAP) family have been shown to inhibit apoptosis namely; X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1, cIAP2), livin and survivin. These IAPs have been linked to chemotherapy resistance and their overexpression correlated with a poorer prognosis in several malignancies. Their role in MRT has not yet been established. In the present study, the expression of a number of these IAPs in a panel of MRT cell lines was detected. Also, treatment with the XIAP inhibitor embelin, the SMAC mimetic BV6 and the survivin inhibitor YM155 decreased the viability of these cell lines. Moreover, expression of XIAP, its target caspase 3 and its endogenous inhibitor SMAC was demonstrated in patient samples. In this study, the XIAP inhibitor embelin was demonstrated to synergistically enhance cell death in combination with the front-line chemotherapeutic cisplatin in MRT cells. This was associated with complete depletion of the IAPs, alongside enhanced caspase activation following combination treatment. Likewise, treatment with BV6 was shown to decrease expression levels of a number of IAPs. As per the results seen with embelin, BV6 worked synergistically with cisplatin to enhance cell death in MRT cells. Preliminary results suggest possible ripoptosome involvement in cisplatin and BV6 mediated cell death. Embelin was also found to sensitise MRT cell lines to TRAIL via the extrinsic apoptotic pathway with additional engagement of the intrinsic apoptotic pathway. This was supported by caspase 8/3 and Bid cleavage along with SMAC release into the cytosol. Also, addition of a general caspase inhibitor, or a specific caspase 8 inhibitor prevented cell death. Western blot analysis over a wide range of timepoints suggested that this enhanced cell death likely occurred via a multifactorial mechanism encompassing decreased expression of a number of anti-apoptotic proteins including survivin, FLIPL and in particular Mcl-1, which had reduced levels from as early as 4 hours. The fact that embelin is an inhibitor of XIAP and that siRNA mediated XIAP knockdown also significantly enhanced TRAIL mediated cell death suggests that XIAP inhibition is also partially responsible for the synergistic effects observed. Livin cleavage may also have been a contributing factor. To conclude, this study provides pre-clinical evidence that IAP inhibition may be a therapeutic option in MRT either as a stand-alone or combinatorial treatment. Further studies are warranted and should include examination of IAP targeted therapeutics in an in vivo setting.