Embryonic sympathoblasts transiently express TrkB in vivo and proliferate in response to brain-derived neurotrophic factor in vitro

BACKGROUND: Nerve growth factor and neurotrophin-3 are involved in the development of sympathetic neurons; however, whether brain derived neurotrophic factor also plays a role is not known. The purpose of this study was to determine whether BDNF and its receptor, TrkB, are expressed during the development of paravertebral sympathetic ganglia in vivo and to determine the effect of BDNF in vitro. RESULTS: As neural crest cells coalesce to form sympathetic ganglia, TrkB-positive cells are seen in both chicken and mouse embryos. In chicken embryos, TrkB-expressing cells first appear at Hamburger-Hamilton Stage (St) 27 and they co-express HNK-1, confirming that they are migrating neural crest cells. The TrkB-positive cells lack neural markers at this stage; however, they migrate with other neurally differentiating cells that are TrkA and TrkC-positive. By St. 29/30, TrkB-positive cells begin to express the neural specific markers Hu C/D and Islet-1; eventually, all TrkB positive cells commence neural differentiation. By St. 34, TrkB and TrkC staining are lost. BDNF transcript expression parallels that of TrkB. In the mouse, TrkB-positive cells surround newly formed sympathetic ganglia and a small number of TrkB positive cells that co-express tyrosine hydroxylase are seen within ganglia between E13.5-15. In cell culture, many cells from St. 29–30 chicken lumbar sympathetic ganglia express neural markers and are dividing, indicating that they are sympathoblasts. Sympathoblasts and neurons require both nerve growth factor and neurotrophin-3 for survival. BDNF increases the number of cells expressing neural markers in culture by increasing number of cells that incorporate bromodeoxyuridine. In contrast, most TrkB-positive sympathetic cells in vivo are not actively proliferating between E6–E8. CONCLUSION: Developing paravertebral sympathetic ganglia in avian and murine embryos contain a subpopulation of sympathoblasts that transiently express TrkB and ultimately commence neuronal differentiation. These TrkB expressing sympathoblasts are not actively dividing in vivo; yet, when placed in vitro, will divide in response to BDNF. This suggests that the availability of BDNF in vivo fails to reach a threshold necessary to induce proliferation. We suggest that excess TrkB stimulation of sympathoblasts in vivo may lead to the genesis of neuroblastoma

The MDM2 inhibitor CGM097 combined with the BET inhibitor OTX015 induces cell death and inhibits tumor growth in models of neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, with amplification of the oncogene MYCN being a hallmark of high-risk disease and poor prognosis. Although less frequent, overexpression of MYC is similarly an indicator of poor prognosis. Most NB tumors initially respond to chemotherapy, however, a large number of these cases relapse, resulting in chemoresistant disease. After relapse there is growing evidence of p53 inactivation, which suggests a role for p53 in NB’s chemo-sensitivity. Highly mutated in other cancer types, p53 mutations in NB are rare. With p53 mutations being rare, recent work has suggested that dysregulation of the negative regulator of p53, MDM2, may be a mechanism of p53 suppression in NB. MYC/MYCN and MDM2 have been shown to interact and contribute to NB growth and disease progression. In vitro treatment of NB cells with MDM2 inhibitors has shown promise in increasing the expression of p53, leading to a decrease in proliferation, and increasing apoptosis. BET (Bromodomain and Extra-Terminal domain) inhibitors have also been shown to be effective in treating NB cells in vitro, decreasing MYC/MYCN expression, and resulting in increased apoptosis and differentiation. Our study focuses on the combined treatment of a MDM2 inhibitor (CGM097) with a BET inhibitor (OTX015) resulting in greater p53 activation, lower expression of MYC family proteins and a subsequent synergistic increase in NB cell death

BKM120 displays strong antitumor activity in tumor-bearing mice and prolongs survival.

<p>(A) BKM120 inhibits tumor growth in DAOY MB xenograft mouse study. Group 1: Vehicle control; Group 2: BKM120 at 30 mg/kg; Group 3: BKM120 at 60 mg/kg. BKM120 treatment was administered by oral gavage daily for 60 days (n = 9 per group). (B) Growth curves of individual mouse in each group, with <i>p</i> value of 0.00086 on day 60; and with <i>p</i> value of 0.01 at tumor size of 2500 mm³. (C) Kaplan–Meier plot of the xenograft study. BKM120 significantly prolonged mouse survival in DAOY MB xenograft mouse study (<i>p</i> = 0.0003). Arrow indicates termination of treatment for all the groups. Experiments were repeated two times with 9 mice in each group.</p

BKM120 induces apoptotic markers in MB cells.

<p>MB cells were treated with three concentrations of BKM120 for 24 and 48 hrs. Cell lysates were collected and western blot analysis was performed to measure Cleaved Caspase-3 and cleaved PARP. Level of total Caspase-3 and PARP were also examined. Actin was used as overall loading control. Optic density of bands were quantified using Image Lab Software (Bio-Rad). The relative band intensities of Cleaved Caspase-3, Cleaved PARP, total Caspase-3 and total PARP were normalized to actin and vehicle control (value = 1).</p

Genomic expression levels of PI3K in MB cells.

<p>Heat Map of RNA expression (MAS5 scores) of PI3K class I catalytic subunits and mTOR in MB cell lines.</p

Evaluation of BKM120-mediated cytotoxicity in twelve MB cell lines.

<p><b>(A)</b> MB cells were treated with BKM120 ranging from 31.25 nM to 4 μM over a course of 48 hours. Cell viability was determined by CellTiter-Glo assay. Percent cell viability was plotted and IC₅₀ values were calculated by fitting the data to a four-parameter, variable slope dose-response model in GraphPad. Experiments were averages of three replicates. (B) PIK3CA gene expression MAS5 scores and BKM120 IC₅₀ values in all MB cell lines.</p

BKM120 decreases cellular glycolytic metabolic activity in MB cell lines.

<p>MB cells were treated with BKM120 ranging from 31.25 nM to 4 μM over a course of 48 hours. ATP/cell assays showed a decrease in ATP level per cell after 48 hours of BKM120 treatment, which indicates a decrease in cellular glycolytic metabolic activity in MB cell lines in a dose-dependent manner. Data are presented as percent of vehicle control. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001.</p