Investigation into the Molecular Mechanisms Underlying Chemoresistance in Malignant Rhabdoid Tumours

Abstract

Malignant rhabdoid tumour (MRT) is a rare and aggressive paediatric malignancy associated with poor prognosis and is, unfortunately, highly refractive to treatment. One of the major issues when treating MRT patients is the emergence of chemoresistance. The cellular mechanisms driving chemoresistance in MRT remain elusive. The aim of this study was to determine mechanisms underlying resistance to the commonly utilised chemotherapeutic agent, cisplatin, in MRT cells and whether targeting these mechanisms represents a therapeutic strategy to sensitise cells to cisplatin and improve treatment outcome. This study examined the roles of autophagy, the Nrf2/GSH antioxidant system and the Bcl-2 family of proteins in mediating cisplatin resistance in a panel of MRT cell lines. Autophagy is a catabolic process whereby a cell degrades and recycles its own components. Autophagy has become an area of focus in the study of chemoresistance due to its reported dual role as both a pro-survival and pro-death mechanism. The role of autophagy in mediating chemoresistance in MRT remains poorly understood. Cisplatin was shown to induce autophagy and apoptosis concomitantly in a panel of MRT cell lines. Inhibition of autophagy by late-stage inhibitors, chloroquine and bafilomycin-A1, was shown to significantly enhance cisplatin-induced apoptosis but it should be noted, that off target autophagy-independent effects of these inhibitors cannot be ruled out. In contrast, inhibition of autophagy by targeting early stages of the autophagic process using the pharmacological inhibitor SAR405 or through the genetic knockdown of critical autophagic protein ATG5 by siRNA did not sensitise cells to cisplatin-induced apoptosis. Inhibition of caspase-induced apoptosis with Z-VAD-FMK also inhibited autophagy levels demonstrating a complex interplay between these two pathways. Collectively, these results suggest that autophagy does not appear to elicit a pro-survival effect in the chemotherapeutic response of MRT cells. The balance between oxidative stress mediated by reactive oxygen species (ROS) and the antioxidant system, including the well-known antioxidant glutathione (GSH) and the transcription factor Nuclear erythroid-related factor-2 (Nrf2), has been implicated in chemoresistance. This study evaluated the role of these components in the response of MRT cells to treatment with cisplatin. This study characterised the basal levels of GSH, ROS and Nrf2 in a panel of MRT cell lines and found a correlation between the expression profile of the antioxidant defence system and cisplatin sensitivity. Results showed that treatment with ROS scavenger N-acetylcysteine (NAC) protected cells from cisplatin-induced ROS and apoptosis. Interestingly, depleting GSH levels with the inhibitor buthionine sulphoximine (BSO) enhanced cisplatin-induced ROS and sensitised cells to cisplatin. Inhibition of Nrf2 expression with the small molecule inhibitor ML385 or by siRNA enhanced ROS and sensitised resistant MRT cells to cisplatin. These results suggest that targeting the Nrf2/GSH antioxidant system may present a novel therapeutic strategy to combat chemoresistance in rhabdoid tumours. Finally, this study examined the role of the Bcl-2 family in mediating chemoresistance in MRT. Expression levels of a series of pro- and anti-apoptotic members of the family were characterised to assess whether they may play a role in determination of cisplatin cytotoxicity. Interestingly, cisplatin-sensitive BT12 cells were shown to be deficient in anti-apoptotic Bcl-2, whilst high levels are expressed in the more resistant BT16 and G401 cells. This study then went on to evaluate whether targeting Bcl-2 with the Bcl-2 selective BH3 mimetic ABT-199 could affect the apoptotic response to cisplatin and sensitise MRT cells to cytotoxicity. ABT-199 was found to synergistically enhance cisplatin-induced apoptosis in BT16 cells. This BH3 mimetic was shown to upregulate intracellular and mitochondrial ROS and to downregulate mitochondrial respiration and GSH levels. Moreover, ABT-199 was shown to downregulate anti-apoptotic Mcl-1 in a ROS-dependent manner and inhibition of Mcl-1 degradation and ROS production by the ROS scavenger NAC was shown to reverse the synergistic effect on apoptosis by ABT-199 and cisplatin, proposing that a ROS-dependent mechanism underlies the observed synergy. In conclusion, this study has provided novel insights into the molecular mechanisms underlying chemoresistance in MRT and proposes novel therapeutic avenues in the design of new treatment strategies for MRT.