Association of distinct type 1 bone morphogenetic protein receptors with different molecular pathways and survival outcomes in neuroblastoma

Neuroblastoma (NB) is a paediatric cancer that arises in the sympathetic nervous system. Patients with stage 4 tumours have poor outcomes and 20% of high-risk cases have MYCN amplification. The bone morphogenetic proteins (BMPs) play roles in sympathetic neuritogenesis, by signalling through bone morphogenetic protein receptor (BMPR)2 and either BMPR1A or BMPR1B. Alterations in BMPR2 expression have been reported in NB; it is unknown if the expression of BMPR1A or BMPR1B is altered. We report lower BMPR2 and BMPR1B, and higher BMPR1A, expression in stage 4 and in MYCN-amplified NB. Kaplan–Meier plots showed that high BMPR2 or BMPR1B expression was linked to better survival, while high BMPR1A was linked to worse survival. Gene ontology enrichment and pathway analyses revealed that BMPR2 and BMPR1B co-expressed genes were enriched in those associated with NB differentiation. BMPR1A co-expressed genes were enriched in those associated with cell proliferation. Moreover, the correlation between BMPR2 and BMPR1A was strengthened, while the correlation between BMPR2 and BMPR1B was lost, in MYCN-amplified NB. This suggested that differentiation should decrease BMPR1A and increaseBMPR1Bexpression.Inagreement,nervegrowthfactortreatmentofculturedsympatheticneuronsdecreasedBmpr1aexpressionandincreasedBmpr1bexpression.Overexpression of dominant negative BMPR1B, treatment with a BMPR1B inhibitor and treatment with GDF5, which signals via BMPR1B, showed that BMPR1B signalling is required for optimal neuritogenesis in NB cells, suggesting that loss of BMPR1B may alter neuritogenesis. The present study shows that expression of distinct BMPRs is associated with different survival outcomes in NB

From neurons to neuroblastoma: dietary flavonoids as regulators of life, death & differentiation

Neuroblastoma (NB) is an embryonal malignancy that arises from cells of sympathoadrenal lineage during the development of the nervous system. Patients classified as stage 4 with aggressive tumors are considered to belong to a high-risk group. MYCN-amplification is detected as a hallmark of NB in 20% of high-risk cases. Based on the International Neuroblastoma Risk Group (INRG), the relapse survival rate of high-risk patients with MYCN-amplification is only 4%. Despite extensive studies, metastasis and tumor relapse lead to debilitating morbidity and frequently death, in high-risk NB patients. Moreover, NB chemotherapy can result in very debilitating long-term sequelae. Errors in the molecular mechanisms that control the development of cell of the sympathetic lineage have been linked to NB development. One group of signals, known as the bone morphogenetic proteins (BMPs), plays an important role in sympathetic neuritogenesis, by signaling through bone morphogenetic protein receptor (BMPR)2 and either BMPR1A or BMPR1B. Alterations in BMPR2 expression have been reported in NB, but it is unknown if the expression of BMPR1A or BMPR1B is altered. The use of neurotrophic factors such as nerve growth factor (NGF) and growth differentiation factor (GDF)5 to promote cell differentiation has been reported in postnatal day (P)1 mouse NC cultures and MYCN-amplified NB SK-N-BE(2), respectively. GDF5 induces cell differentiation in NB cells through activation of BMP-Smad signaling. However, recombinant BMPs are expensive to produce and their clinical use is limited, due to several factors. Therefore, there is an urgent need to identify novel therapeutic agents that could induce cell differentiation in post-chemotherapy tissue with the minimum side effects. Recently, several screening studies have identified small molecules that modulate the BMP pathway. These include natural dietary compounds, including flavonoids and in particular the chalcone subfamily. Interestingly, flavonoids have potential anti-tumor properties with selective cytotoxicity to cancer cells. Additionally, chalcones, which are the most potent flavonoid subclass, have also demonstrated anti-tumor activity in some contexts. This thesis first investigated, in chapter 2, the association of distinct type1 BMPRs with different molecular pathways and survival outcomes in NB. Lower expression of BMPR2 and BMPR1B, and higher BMPR1A expression, were observed in stage 4 and in MYCN-amplified NB, and they were associated with poor survival. In contrast, higher expression of BMPR2 and BMPR1B were associated with better survival. Regarding MYCN status, the correlation between BMPR2 and BMPR1A was strengthened, while the correlation between BMPR2 and BMPR1B was lost, in MYCN-amplified NB. This study therefore showed that expression of distinct BMPRs is associated with different survival outcomes in NB. In chapter 3, the effects of low doses of the chalcones isoliquiritigenin (ISLQ) and 4-hydroxychalcone (4HC), small molecule activator of BMP-Smad signaling, on the morphological differentiation of NB cells, as well as the molecular mechanism of their action, was investigated. ISLQ and 4HC promoted neurite growth in NB cells by activation of BMP-Smad signaling. Moreover, treatment with these chalcones lead to a significant reduction of cell aggregates (tumor-like aggregations) in SK-N-BE(2) cells. After these findings, in chapter 4, the cytotoxic effect of ISLQ in MYCN-amplified NB cells was investigated in detail. ISLQ induced cell death in MYCN-amplified NB cells SK-N-BE(2) and IMR-32 through an elevation of oxidative stress and necroptosis. Combined treatment of ISLQ with the chemotherapy agent cisplatin led to a greater cell toxicity in MYCN-amplified NB cells. In chapter 5, the cytotoxic effect of 4HC on NB cells, and its mode of action, was examined. 4HC demonstrated selective cytotoxicity on MYCN-amplified NB cells, which were more sensitive to 4HC than non-amplified SH-SY5Y cells. Cell death induced by 4HC was shown to result from glutathione (GSH) depletion, elevation of oxidative stress and mitochondrial impairment. This thesis, therefore, demonstrates the role of various types of BMPRs in different molecular pathways and survival outcomes in NB, It also highlights the potential of dietary chalcones for either the induction of NB cell differentiation at lower concentrations, through the activation of BMP-Smad signaling, or induction of selective NB cell death at higher concentrations, by elevation of oxidative stress

The dietary flavonoid isoliquiritigenin is a potent cytotoxin for human neuroblastoma cells

Neuroblastoma (NB) is the most common extracranial solid tumor of early childhood; it accounts for approximately 8–10% of all childhood cancers and is the most common cancer in children in the first year of life. Patients in the high-risk group have a poor prognosis, with relapses being common and often refractory to drug treatment in those that survive. Moreover, the drug treatment itself can lead to a range of long-term sequelae. Therefore, there is a critical need to identify new therapeutics for NB. Isoliquiritigenin (ISLQ) is a naturally-occurring, dietary chalcone-type flavonoid with a range of biological effects that depend on the cell type and context. ISLQ has potential as an anticancer agent. Here we show that ISLQ has potent cytotoxic effects on SK-N-BE(2) and IMR-32 human NB cells, which carry amplification of the MYCN gene, the main prognostic marker of poor survival in NB. ISLQ was found to increase cellular reactive oxygen species (ROS). The cytotoxic effect of ISLQ was blocked by small molecule inhibitors of oxidative stress-induced cell death, and by the antioxidant N-acetyl-l-cysteine (NAC). Combined treatment of either SK-N-B-E(2) or IMR-32 cells with ISLQ and the anticancer agent cisplatin resulted in loss of cell viability that was greater than that induced by cisplatin alone. This study provides proof-of-principle that ISLQ is a potent cytotoxin for MYCN-amplified human NB cells. This is an important first step in rationalizing the further study of ISLQ as a potential adjunct therapy for high-risk NB

Biallelic variants in HECT E3 paralogs, HECTD4 and UBE3C, encoding ubiquitin ligases cause neurodevelopmental disorders that overlap with Angelman syndrome

Pathogenic variants in genes encoding ubiquitin E3 ligases are known to cause neurodevelopmental syndromes. Additional neurodevelopmental disorders associated with the other genes encoding E3 ligases are yet to be identified