Increased susceptibility of cystic fibrosis airway epithelial cells to ferroptosis.

BACKGROUND: Defective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF). Abnormal airway iron homeostasis and the presence of lipid peroxidation products, indicative of oxidative stress, are features of CF lung disease. RESULTS: Here, we report that CF AECs (IB3-1) are susceptible to ferroptosis, a type of cell death associated with iron accumulation and lipid peroxidation. Compared to isogenic CFTR corrected cells (C38), the IB3-1 cells showed increased susceptibility to cell death upon exposure to iron in the form of ferric ammonium citrate (FAC) and the ferroptosis inducer, erastin. This phenotype was accompanied by accumulation of intracellular ferrous iron and lipid peroxides and the extracellular release of malondialdehyde, all indicative of redox stress, and increased levels of lactate dehydrogenase in the culture supernatant, indicating enhanced cell injury. The ferric iron chelator deferoxamine (DFO) and the lipophilic antioxidant ferrostatin-1 inhibited FAC and erastin induced ferroptosis in IB3-1 cells. Glutathione peroxidase 4 (GPX4) expression was decreased in IB3-1 cells treated with FAC and erastin, but was unchanged in C38 AECs. Necroptosis appeared to be involved in the enhanced susceptibility of IB3-1 AECs to ferroptosis, as evidenced by partial cell death rescue with necroptosis inhibitors and enhanced mixed lineage kinase domain-like (MLKL) localisation to the plasma membrane. CONCLUSION: These studies suggest that the increased susceptibility of CF AECs to ferroptosis is linked to abnormal intracellular ferrous iron accumulation and reduced antioxidant defences. In addition, the process of ferroptotic cell death in CF AECs does not appear to be a single entity and for the first time we describe necroptosis as a potential contributory factor. Iron chelation and antioxidant treatments may be promising therapeutic interventions in cystic fibrosis.

Increased susceptibility of cystic fibrosis airway epithelial cells to ferroptosis

BACKGROUND: Defective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF). Abnormal airway iron homeostasis and the presence of lipid peroxidation products, indicative of oxidative stress, are features of CF lung disease. RESULTS: Here, we report that CF AECs (IB3-1) are susceptible to ferroptosis, a type of cell death associated with iron accumulation and lipid peroxidation. Compared to isogenic CFTR corrected cells (C38), the IB3-1 cells showed increased susceptibility to cell death upon exposure to iron in the form of ferric ammonium citrate (FAC) and the ferroptosis inducer, erastin. This phenotype was accompanied by accumulation of intracellular ferrous iron and lipid peroxides and the extracellular release of malondialdehyde, all indicative of redox stress, and increased levels of lactate dehydrogenase in the culture supernatant, indicating enhanced cell injury. The ferric iron chelator defer-oxamine (DFO) and the lipophilic antioxidant ferrostatin-1 inhibited FAC and erastin induced ferroptosis in IB3-1 cells. Glutathione peroxidase 4 (GPX4) expression was decreased in IB3-1 cells treated with FAC and erastin, but was unchanged in C38 AECs. Necroptosis appeared to be involved in the enhanced susceptibility of IB3-1 AECs to ferroptosis, as evidenced by partial cell death rescue with necroptosis inhibitors and enhanced mixed lineage kinase domain-like (MLKL) localisation to the plasma membrane. CONCLUSION: These studies suggest that the increased susceptibility of CF AECs to ferroptosis is linked to abnormal intracellular ferrous iron accumulation and reduced antioxidant defences. In addition, the process of ferroptotic cell death in CF AECs does not appear to be a single entity and for the first time we describe necroptosis as a potential contributory factor. Iron chelation and antioxidant treatments may be promising therapeutic interventions in cystic fibrosis.

Upregulation of LYAR induces neuroblastoma cell proliferation and survival.

The N-Myc oncoprotein induces neuroblastoma by regulating gene transcription and consequently causing cell proliferation. Paradoxically, N-Myc is well known to induce apoptosis by upregulating pro-apoptosis genes, and it is not clear how N-Myc overexpressing neuroblastoma cells escape N-Myc-mediated apoptosis. The nuclear zinc finger protein LYAR has recently been shown to modulate gene expression by forming a protein complex with the protein arginine methyltransferase PRMT5. Here we showed that N-Myc upregulated LYAR gene expression by binding to its gene promoter. Genome-wide differential gene expression studies revealed that knocking down LYAR considerably upregulated the expression of oxidative stress genes including CHAC1, which depletes intracellular glutathione and induces oxidative stress. Although knocking down LYAR expression with siRNAs induced oxidative stress, neuroblastoma cell growth inhibition and apoptosis, co-treatment with the glutathione supplement N-acetyl-l-cysteine or co-transfection with CHAC1 siRNAs blocked the effect of LYAR siRNAs. Importantly, high levels of LYAR gene expression in human neuroblastoma tissues predicted poor event-free and overall survival in neuroblastoma patients, independent of the best current markers for poor prognosis. Taken together, our data suggest that LYAR induces proliferation and promotes survival of neuroblastoma cells by repressing the expression of oxidative stress genes such as CHAC1 and suppressing oxidative stress, and identify LYAR as a novel co-factor in N-Myc oncogenesis.

The Histone Methyltransferase DOT1L Promotes Neuroblastoma by Regulating Gene Transcription.

Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2 DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene-amplified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1, and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc-mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522-33. ©2017 AACR.

Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma.

Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. We used a MYC target gene signature that predicts poor neuroblastoma prognosis to identify the histone chaperone FACT (facilitates chromatin transcription) as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small-molecule curaxin compound CBL0137 markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with standard chemotherapy by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells and suggesting a treatment strategy for MYCN-driven neuroblastoma.

The histone demethylase JMJD1A induces cell migration and invasion by up-regulating the expression of the long noncoding RNA MALAT1.

Patients with neuroblastoma due to N-Myc oncogene amplification have a high frequency of tumor metastasis. However, it is not clear how N-Myc induces cell migration, invasion and metastasis. The histone demethylase JMJD1A activates gene transcription by demethylating the lysine 9 residue of histone H3 (H3K9) at target gene promoters. The long noncoding RNA MALAT1 induces lung cancer cell migration and plays a pivotal role in lung cancer metastasis. Here we demonstrated that N-Myc up-regulated the expression of JMJD1A in N-Myc oncogene-amplified human neuroblastoma cells by directly binding to the JMJD1A gene promoter. Affymetrix microarray studies revealed that the gene second most significantly up-regulated by JMJD1A was MALAT1. Consistent with this finding, RT-PCR and chromatin immunoprecipitation assays showed that JMJD1A bound to the MALAT1 gene promoter and demethylated histone H3K9 at the MALAT1 gene promoter. Moreover, JMJD1A and MALAT1 induced, while the small molecule JMJD1A inhibitor DMOG suppressed, neuroblastoma cell migration and invasion. Taken together, our data identify a novel pathway through which N-Myc causes neuroblastoma cell migration and invasion, and provide important evidence for further development of more potent JMJD1A/MALAT1 inhibitors for the prevention of tumor metastasis.

Enhancing the anticancer effect of the histone deacetylase inhibitor by activating transglutaminase.

Histone deacetylase (HDAC) inhibitors have shown promising anticancer effects in clinical trials. However, a proportion of patients do not respond to HDAC inhibitor therapy. We have previously demonstrated that tissue transglutaminase (TG2) is one of the genes commonly up-regulated by HDAC inhibitors in vitro and in vivo, and that two structurally distinct TG2 protein isoforms, the full-length (TG2-L) and the short form (TG2-S), exert opposing effects on cell differentiation due to difference in transamidation activity. Here we show that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) transcriptionally activates the expression of both TG2-L and TG2-S, and that up-regulation of TG2-L renders neuroblastoma cells less sensitive to SAHA-induced cytotoxicity. Combination therapy with SAHA and the transamidation activator Naringenin, a natural product found in citrus fruits, synergistically enhanced transamidation activity and SAHA-induced cytotoxicity in neuroblastoma cells, but not in normal non-malignant cells. In tumour-bearing N-Myc transgenic mice, SAHA and Naringenin synergistically suppressed tumour progression. Taken together, our data demonstrate that SAHA-induced TG2-L over-expression renders cancer cells less sensitive to SAHA therapy, and suggest the addition of Naringenin to SAHA and probably also other HDAC inhibitors in future clinical trials in cancer patients.

SIRT1 promotes N-Myc oncogenesis through a positive feedback loop involving the effects of MKP3 and ERK on N-Myc protein stability.

The N-Myc oncoprotein is a critical factor in neuroblastoma tumorigenesis which requires additional mechanisms converting a low-level to a high-level N-Myc expression. N-Myc protein is stabilized when phosphorylated at Serine 62 by phosphorylated ERK protein. Here we describe a novel positive feedback loop whereby N-Myc directly induced the transcription of the class III histone deacetylase SIRT1, which in turn increased N-Myc protein stability. SIRT1 binds to Myc Box I domain of N-Myc protein to form a novel transcriptional repressor complex at gene promoter of mitogen-activated protein kinase phosphatase 3 (MKP3), leading to transcriptional repression of MKP3, ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, and N-Myc protein stabilization. Importantly, SIRT1 was up-regulated, MKP3 down-regulated, in pre-cancerous cells, and preventative treatment with the SIRT1 inhibitor Cambinol reduced tumorigenesis in TH-MYCN transgenic mice. Our data demonstrate the important roles of SIRT1 in N-Myc oncogenesis and SIRT1 inhibitors in the prevention and therapy of N-Myc-induced neuroblastoma.