Relapse with drug-resistant disease is the main cause of death in MYCN-amplified neuroblastoma patients. MYCN-amplified neuroblastoma cells in vitro are characterized by a failure to arrest at the ...G₁-S checkpoint after irradiation- or drug-induced DNA damage. We show that several MYCN-amplified cell lines harbor additional chromosomal aberrations targeting p53 and/or pRB pathway components, including CDK4/CCND1/MDM2 amplifications, p16INK4A/p14ARF deletions or TP53 mutations. Cells with these additional aberrations undergo significantly lower levels of cell death after doxorubicin treatment compared with MYCN-amplified cells, with no additional mutations in these pathways. In MYCN-amplified cells CDK4 expression is elevated, increasing the competition between CDK4 and CDK2 for binding p21. This results in insufficient p21 to inhibit CDK2, leading to high CDK4 and CDK2 kinase activity upon doxorubicin treatment. CDK4 inhibition by siRNAs, selective small compounds or p19
INK4D
overexpression partly restored G₁-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment. Our results suggest a specific function of p19
INK4D
, but not p16
INK4A
, in sensitizing MYCN-amplified cells with a functional p53 pathway to doxorubicin-induced cell death. In summary, the CDK4/cyclin D-pRB axis is altered in MYCN-amplified cells to evade a G₁-S arrest after doxorubicin-induced DNA damage. Additional chromosomal aberrations affecting the p53-p21 and CDK4-pRB axes compound the effects of MYCN on the G₁ checkpoint and reduce sensitivity to cell death after doxorubicin treatment. CDK4 inhibition partly restores G₁-S arrest and sensitizes cells to doxorubicin-mediated cell death in MYCN-amplified cells with an intact p53 pathway.
Relapse with drug-resistant disease is the main cause of death in MYCN-amplified neuroblastoma patients. MYCN-amplified neuroblastoma cells in vitro are characterized by a failure to arrest at the ...G(1)-S checkpoint after irradiation- or drug-induced DNA damage. We show that several MYCN-amplified cell lines harbor additional chromosomal aberrations targeting p53 and/or pRB pathway components, including CDK4/CCND1/MDM2 amplifications, p16INK4A/p14ARF deletions or TP53 mutations. Cells with these additional aberrations undergo significantly lower levels of cell death after doxorubicin treatment compared with MYCN-amplified cells, with no additional mutations in these pathways. In MYCN-amplified cells CDK4 expression is elevated, increasing the competition between CDK4 and CDK2 for binding p21. This results in insufficient p21 to inhibit CDK2, leading to high CDK4 and CDK2 kinase activity upon doxorubicin treatment. CDK4 inhibition by siRNAs, selective small compounds or p19(INK4D) overexpression partly restored G(1)-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment. Our results suggest a specific function of p19(INK4D), but not p16(INK4A), in sensitizing MYCN-amplified cells with a functional p53 pathway to doxorubicin-induced cell death. In summary, the CDK4/cyclin D-pRB axis is altered in MYCN-amplified cells to evade a G(1)-S arrest after doxorubicin-induced DNA damage. Additional chromosomal aberrations affecting the p53-p21 and CDK4-pRB axes compound the effects of MYCN on the G(1) checkpoint and reduce sensitivity to cell death after doxorubicin treatment. CDK4 inhibition partly restores G(1)-S arrest and sensitizes cells to doxorubicin-mediated cell death in MYCN-amplified cells with an intact p53 pathway.
Wnt signalling is involved in the formation, metastasis and relapse of a wide array of cancers. However, there is ongoing debate as to whether activation or inhibition of the pathway holds the most ...promise as a therapeutic treatment for cancer, with conflicting evidence from a variety of tumour types. We show that Wnt/β-catenin signalling is a bi-directional vulnerability of neuroblastoma, malignant melanoma and colorectal cancer, with hyper-activation or repression of the pathway both representing a promising therapeutic strategy, even within the same cancer type. Hyper-activation directs cancer cells to undergo apoptosis, even in cells oncogenically driven by β-catenin. Wnt inhibition blocks proliferation of cancer cells and promotes neuroblastoma differentiation. Wnt and retinoic acid co-treatments synergise, representing a promising combination treatment for MYCN-amplified neuroblastoma. Additionally, we report novel cross-talks between MYCN and β-catenin signalling, which repress normal β-catenin mediated transcriptional regulation. A β-catenin target gene signature could predict patient outcome, as could the expression level of its DNA binding partners, the TCF/LEFs. This β-catenin signature provides a tool to identify neuroblastoma patients likely to benefit from Wnt-directed therapy. Taken together, we show that Wnt/β-catenin signalling is a bi-directional vulnerability of a number of cancer entities, and potentially a more broadly conserved feature of malignant cells.
Despite intensive study, many mysteries remain about the MYCN oncogene's functions. Here we focus on MYCN's role in neuroblastoma, the most common extracranial childhood cancer. MYCN gene ...amplification occurs in 20% of cases, but other recurrent somatic mutations are rare. This scarcity of tractable targets has hampered efforts to develop new therapeutic options. We employed a multi-level omics approach to examine MYCN functioning and identify novel therapeutic targets for this largely un-druggable oncogene. We used systems medicine based computational network reconstruction and analysis to integrate a range of omic techniques: sequencing-based transcriptomics, genome-wide chromatin immunoprecipitation, siRNA screening and interaction proteomics, revealing that MYCN controls highly connected networks, with MYCN primarily supressing the activity of network components. MYCN's oncogenic functions are likely independent of its classical heterodimerisation partner, MAX. In particular, MYCN controls its own protein interaction network by transcriptionally regulating its binding partners.Our network-based approach identified vulnerable therapeutically targetable nodes that function as critical regulators or effectors of MYCN in neuroblastoma. These were validated by siRNA knockdown screens, functional studies and patient data. We identified β-estradiol and MAPK/ERK as having functional cross-talk with MYCN and being novel targetable vulnerabilities of MYCN-amplified neuroblastoma. These results reveal surprising differences between the functioning of endogenous, overexpressed and amplified MYCN, and rationalise how different MYCN dosages can orchestrate cell fate decisions and cancerous outcomes. Importantly, this work describes a systems-level approach to systematically uncovering network based vulnerabilities and therapeutic targets for multifactorial diseases by integrating disparate omic data types.
Uncontrolled cell cycle entry, resulting from deregulated CDK-RB1-E2F pathway activity, is a crucial determinant of neuroblastoma cell malignancy. Here we identify neuroblastoma-suppressive functions ...of the p19-INK4d CDK inhibitor and uncover mechanisms of its repression in high-risk neuroblastomas. Reduced p19-INK4d expression was associated with poor event-free and overall survival and neuroblastoma risk factors including amplified MYCN in a set of 478 primary neuroblastomas. High MYCN expression repressed p19-INK4d mRNA and protein levels in different neuroblastoma cell models with conditional MYCN expression. MassARRAY and 450K methylation analyses of 105 primary neuroblastomas uncovered a differentially methylated region within p19-INK4d. Hypermethylation of this region was associated with reduced p19-INK4d expression. In accordance, p19-INK4d expression was activated upon treatment with the demethylating agent, 2'-deoxy-5-azacytidine, in neuroblastoma cell lines. Ectopic p19-INK4d expression decreased viability, clonogenicity and the capacity for anchorage-independent growth of neuroblastoma cells, and shifted the cell cycle towards the G1/0 phase. p19-INK4d also induced neurite-like processes and markers of neuronal differentiation. Moreover, neuroblastoma cell differentiation, induced by all-trans retinoic acid or NGF-NTRK1-signaling, activated p19-INK4d expression. Our findings pinpoint p19-INK4d as a neuroblastoma suppressor and provide evidence for MYCN-mediated repression and for epigenetic silencing of p19-INK4d by DNA hypermethylation in high-risk neuroblastomas.
Studies showed that the metabolic unlike the neuroendocrine effects of ghrelin could be abrogated by co-administered unacylated ghrelin. The aim was to investigate the interaction between ghrelin and ...desacyl ghrelin administered intraperitoneally on food intake and neuronal activity (c-Fos) in the arcuate nucleus in non-fasted rats. Ghrelin (13
μg/kg) significantly increased food intake within the first 30
min post-injection. Desacyl ghrelin at 64 and 127
μg/kg injected simultaneously with ghrelin abolished the stimulatory effect of ghrelin on food intake. Desacyl ghrelin alone at both doses did not alter food intake. Both doses of desacyl ghrelin injected separately in the light phase had no effects on food intake when rats were fasted for 12
h. Ghrelin and desacyl ghrelin (64
μg/kg) injected alone increased the number of Fos positive neurons in the arcuate nucleus compared to vehicle. The effect on neuronal activity induced by ghrelin was significantly reduced when injected simultaneously with desacyl ghrelin. Double labeling revealed that nesfatin-1 immunoreactive neurons in the arcuate nucleus are activated by simultaneous injection of ghrelin and desacyl ghrelin. These results suggest that desacyl ghrelin suppresses ghrelin-induced food intake by curbing ghrelin-induced increased neuronal activity in the arcuate nucleus and recruiting nesfatin-1 immunopositive neurons.
Abstract Peripheral ghrelin has been shown to act as a gut–brain peptide exerting a potent orexigenic effect on food intake. The dorsomedial nucleus of the hypothalamus (DMH) is innervated by ...projections from other brain areas being part of the network of nuclei controlling energy homeostasis, among others NPY/AgRP-positive fibers arising from the arcuate nucleus (ARC). The aim of the study was to determine if peripherally administered ghrelin affects neuronal activity in the DMH, as assessed by Fos expression. The number of Fos positive neurons was determined in the DMH, paraventricular nucleus of the hypothalamus (PVN), ARC, ventromedial hypothalamic nucleus (VMH), nucleus of the solitary tract (NTS) and in the area postrema (AP) in non-fasted Sprague–Dawley rats in response to intraperitoneally (ip) injected ghrelin (3 nmol/rat) or vehicle (0.15 M NaCl). Peripheral ghrelin induced a significant increase in the number of Fos-ir positive neurons/section compared with vehicle in the ARC (mean ± SEM: 49 ± 2 vs. 23 ± 2 neurons/section, p = 0.001), PVN (69 ± 5 vs. 34 ± 3, p = 0.001), and DMH (142 ± 5 vs. 83 ± 5, p < 0.001). Fos-ir positive neurons were mainly localized within the ventral part of the DMH. No change in Fos expression was observed in the VMH (53 ± 8 vs. 48 ± 6, p = 0.581), NTS (42 ± 2 vs. 40 ± 3, p = 0.603), and in the AP (7 ± 1 vs. 5 ± 1, p = 0.096). Additional double-labelling with anti-Fos and anti-AgRP revealed that Fos positive neurons in the DMH were encircled by a network of AgRP-ir positive fibers. These data indicate that peripheral ghrelin activates DMH neurons and that NPY-/AgRP-positive fibers may be involved in the response.
Cholecystokinin (CCK) plays a role in the short-term inhibition of food intake. Cocaine- and amphetamine-regulated transcript (CART) peptide has been observed in neurons of the paraventricular ...nucleus (PVN). It has been reported that intracerebroventricular injection of CART peptide inhibits food intake in rodents. The aim of the study was to determine whether intraperitoneally (ip) injected CCK-8S affects neuronal activity of PVN-CART neurons.
Ad libitum fed male Sprague–Dawley rats received 6 or 10
μg/kg CCK-8S or 0.15
M NaCl ip (
n
=
4/group). The number of c-Fos-immunoreactive neurons was determined in the PVN, arcuate nucleus (ARC), and the nucleus of the solitary tract (NTS). CCK-8S dose-dependently increased the number of c-Fos-immunoreactive neurons in the PVN (mean
±
SEM: 102
±
6
vs. 150
±
5 neurons/section,
p
<
0.05) and compared to vehicle treated rats (18
±
7,
p
<
0.05
vs. 6 and 10
μg/kg CCK-8S). CCK-8S at both doses induced an increase in the number of c-Fos-immunoreactive neurons in the NTS (65
±
13,
p
<
0.05, and 182
±
16,
p
<
0.05). No effect on the number of c-Fos neurons was observed in the ARC. Immunostaining for CART and c-Fos revealed a dose-dependent increase of activated CART neurons (19
±
3
vs. 29
±
7;
p
<
0.05), only few activated CART neuron were observed in the vehicle group (1
±
0). The present observation shows that CCK-8S injected ip induces an increase in neuronal activity in PVN-CART neurons and suggests that CART neurons in the PVN may play a role in the mediation of peripheral CCK-8S's anorexigenic effects.
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