A cytochrome c ELISA Kit (Invitrogen, USA) was used to measure the release of cytochrome c from mitochondria, according to the manufacturers protocol

A cytochrome c ELISA Kit (Invitrogen, USA) was used to measure the release of cytochrome c from mitochondria, according to the manufacturers protocol. To prepare the cytosolic fraction, cells were suspended in subcellular fractionation buffer (250?mM sucrose, 20?mM HEPES (pH 7.5), 10?mM NaCl, 1.5?mM MgCl2, 1?mM EDTA, 1?mM EGTA) and cell suspensions were homogenised by PF-4800567 passage through a syringe with a 25-gauge needle. This shift toward apoptosis, and away from cell-cycle arrest, in the presence of an ALK inhibitor and a p53 activator, is mediated by inhibition of the ALKCAKTCFOXO3a axis leading to a specific upregulation of SOX4. SOX4 cooperates with p53 to upregulate the pro-apoptotic protein PUMA. These data therefore suggest a novel combination therapy strategy for treating ALK-driven neuroblastomas. Neuroblastoma (NB) is a common paediatric solid tumour, and is the cause of 9.1% of cancer-related deaths in children1,2. NB is classified into three groups according to The International Neuroblastoma Risk Group (INRG) classification system3. The high-risk group is characterised by amplification of the MYCN oncogene, and has a poor prognosis, with a 5-year survival rate of 40C50%. Although multimodal chemotherapeutic and immunotherapeutic strategies have improved survival in patients with high-risk disease, newer and better therapeutic strategies are still required4. Anaplastic lymphoma kinase (ALK) belongs to the family of receptor tyrosine kinases (RTKs). The gene encoding ALK is the most frequently mutated gene in NB, and is a cause of familial NB5C8. Therefore, ALK inhibitors have the potential to be effective therapeutic agents for NBs harbouring ALK mutations. Additionally, they may be effective in NBs harbouring amplification of the gene, which currently represent ~2% of total NB cases9,10. Most of the NBs with an amplification of ALK also harbour an amplification of MYCN, and have very poor prognosis10. Crizotinib, a first generation ALK inhibitor, has been studied in clinical trials for the treatment of paediatric cancer (“type”:”clinical-trial”,”attrs”:”text”:”NCT01606878″,”term_id”:”NCT01606878″NCT01606878)11. However, it had limited efficacy, due to primary resistance to crizotinib in NBs10,12. Alectinib is a second-generation ALK inhibitor, which overcomes the acquired resistance ascribed FGF5 to gatekeeper mutations13. However, the efficacy of this second-generation ALK inhibitor in NBs harbouring ALK amplifications remains unclear. Resistance to ALK inhibitors has also become a critical issue in the treatment of non-small cell lung carcinoma (NSCLC) bearing the ALK-fusion gene14,15. Unfortunately, the mechanism by which NBs harbouring amplification of the gene become resistant to ALK inhibitors is poorly understood. The function of the tumour suppressor protein p53 is often found to be inactivated in various tumours, either through mutations or by perturbation of its regulatory pathways16C18. p53 is a transcription factor which, following activation in response to various cellular stresses, induces the expression of various p53-target genes (e.g., p21, PUMA, BAX, and NOXA) that act to prevent tumour development. Although p53 is frequently found to be mutated in various PF-4800567 tumours, some types of tumours, including NB, retain wild-type p53; p53 status has been found to be associated with response to chemotherapy in some cases19,20. Therefore, activation of p53 is a promising therapeutic strategy for the treatment of various types of tumours21. The p53 protein is ubiquitinated by a variety of E3 ubiquitin ligases, including MDM2, and subsequently degraded through the ubiquitinCproteasomal system18. Nutlin-3a, and its derivative RG-7112, are p53 activators that can suppress the p53CMDM2 interaction, and lead to the stabilisation of p53 by preventing its proteasomal degradation22,23. These compounds activate the p53 pathway in cancer cells harbouring wild-type p53, but they have been shown to have limited efficacy in patients21. Although NBs almost always retain wild-type p53, the effect of p53 activators on resistance to ALK inhibitors in ALK-driven NBs remains unclear. In this study, we found that the p53 activators suppress the re-growth of ALK-driven NBs, which are resistant to ALK inhibitors, and reveal the mechanisms by which p53 activators enhance the efficacy of the ALK inhibitors. Results The ALK PF-4800567 inhibitor induces cell-cycle arrest but not cell death in ALK-driven NB cells We first assessed the effect of two ALK inhibitors, crizotinib and alectinib, on cell viability in ALK-driven NBs including the ALK-amplified NB39-nu cell line, the ALK-amplified NB1 cell line, and the ALK-mutated SHSY5Y cell line. Both ALK inhibitors abrogated cell proliferation in the two ALK-driven NB.