Prokineticin signaling is required for the maintenance of a de novo population of c-KIT⁺ cells to sustain neuroblastoma progression.

High cellular heterogeneity within neuroblastomas (NBs) may account for the non-uniform response to treatment. c-KIT(+) cells are frequently detected in NB, but how they influence NB behavior still remains elusive. Here, we used NB tumor-initiating cells to reconstitute NB development and demonstrated that c-KIT(+) cells are de novo generated and dynamically maintained within the tumors to sustain tumor progression. c-KIT(+) NB cells express higher levels of neural crest and stem cell markers (SLUG, SOX2 and NANOG) and are endowed with high clonogenic capacity, differentiation plasticity and are refractory to drugs. With serial transplantation assays, we found that c-KIT expression is not required for tumor formation, but c-KIT(+) cells are more aggressive and can induce tumors ninefold more efficiently than c-KIT(-/low) cells. Intriguingly, c-KIT(+) cells exhibited a long-term in vivo self-renewal capacity to sustain the formation of secondary and tertiary tumors in mice. In addition, we showed that Prokineticin signaling and mitogen-activated protein kinase pathways are crucial for the maintenance of c-KIT(+) cells in tumor to promote NB progression. Our results highlight the importance of this de novo population of NB cells in sustainable growth of NB and reveal specific signaling pathways that may provide targets leading to more effective NB therapies.

RNAi-mediated stathmin suppression reduces lung metastasis in an orthotopic neuroblastoma mouse model.

Metastatic neuroblastoma is an aggressive childhood cancer of neural crest origin. Stathmin, a microtubule destabilizing protein, is highly expressed in neuroblastoma although its functional role in this malignancy has not been addressed. Herein, we investigate stathmin's contribution to neuroblastoma tumor growth and metastasis. Small interfering RNA (siRNA)-mediated stathmin suppression in two independent neuroblastoma cell lines, BE(2)-C and SH-SY5Y, did not markedly influence cell proliferation, viability or anchorage-independent growth. In contrast, stathmin suppression significantly reduced cell migration and invasion in both the neuroblastoma cell lines. Stathmin suppression altered neuroblastoma cell morphology and this was associated with changes in the cytoskeleton, including increased tubulin polymer levels. Stathmin suppression also modulated phosphorylation of the actin-regulatory proteins, cofilin and myosin light chain (MLC). Treatment of stathmin-suppressed neuroblastoma cells with the ROCKI and ROCKII inhibitor, Y-27632, ablated MLC phosphorylation and returned the level of cofilin phosphorylation and cell invasion back to that of untreated control cells. ROCKII inhibition (H-1152) and siRNA suppression also reduced cofilin phosphorylation in stathmin-suppressed cells, indicating that ROCKII mediates stathmin's regulation of cofilin phosphorylation. This data demonstrates a link between stathmin and the regulation of cofilin and MLC phosphorylation via ROCK. To examine stathmin's role in neuroblastoma metastasis, stathmin short hairpin RNA (shRNA)\luciferase-expressing neuroblastoma cells were injected orthotopically into severe combined immunodeficiency-Beige mice, and tumor growth monitored by bioluminescent imaging. Stathmin suppression did not influence neuroblastoma cell engraftment or tumor growth. In contrast, stathmin suppression significantly reduced neuroblastoma lung metastases by 71% (P<0.008) compared with control. This is the first study to confirm a role for stathmin in hematogenous spread using a clinically relevant orthotopic cancer model, and has identified stathmin as an important contributor of cell invasion and metastasis in neuroblastoma.

Cyclooxygenase inhibitors differentially modulate p73 isoforms in neuroblastoma.

p73 encodes multiple functionally distinct isoforms. Proapoptotic TAp73 isoforms contain a transactivation (TA) domain, and like p53, have tumor suppressor properties and are activated by chemotherapies to induce cell death. In contrast, antiapoptotic DeltaNp73 isoforms lack the TA domain and are dominant-negative inhibitors of p53 and TAp73. DeltaNp73 proteins are overexpressed in a variety of tumors including neuroblastoma. Thus, identification of drugs that upregulate TAp73 and/or downregulate DeltaNp73 represents a potential therapeutic strategy. Here, we report that cyclooxygenase (COX) inhibitors induce apoptosis independent of p53, and differentially modulate endogenous p73 isoforms in neuroblastoma and other tumors. COX inhibitor-mediated apoptosis is associated with the induction of TAp73beta and its target genes. COX inhibitors also downregulate the alternative-spliced DeltaNp73(AS) isoforms, Deltaexon2 and Deltaexon2/3. Furthermore, forced expression of DeltaNp73(AS) results in diminished apoptosis in response to the selective COX-2 inhibitor celecoxib. Celecoxib-mediated downregulation of DeltaNp73(AS) is associated with decreased E2F1 levels and diminished E2F1 activation of the p73 promoter. These results provide the first evidence that COX inhibitors differentially modulate p73 isoforms leading to enhanced apoptosis, and support the potential use of COX inhibitors as novel regulators of p73 to enhance chemosensitivity in tumors with deregulated E2F1 and in those with wild-type (wt) or mutant p53.

Cyclooxygenase inhibitors modulate the p53/HDM2 pathway and enhance chemotherapy-induced apoptosis in neuroblastoma.

Cyclooxygenase-2 (COX-2) is upregulated in many tumors including neuroblastoma, and its overexpression has been implicated in resistance to p53-dependent apoptosis. Although p53 is rarely mutated in neuroblastoma, the p53 protein is rendered inactive via several mechanisms including sequestration in the cytoplasm. Here, we show that COX inhibitors inhibit the growth of neuroblastoma and when combined with low doses of chemotherapy, exert synergistic effects on neuroblastoma cells. Following COX inhibitor treatment, HDM2, which targets p53 for ubiquitin-mediated degradation, is downregulated, resulting in an attenuation of p53 ubiquitination and an increase in p53 half-life. The level of HDM2 phosphorylation at ser166, which influences both HDM2 and p53 subcellular distribution, is markedly diminished in response to COX inhibitors and is associated with increased p53 nuclear localization. Combining COX inhibitors with low-dose chemotherapy potentiates apoptosis and p53 stability, nuclear localization, and activity. p53 knockdown by siRNA resulted in the rescue of COX-inhibitor-treated cells, indicating that COX inhibitor-induced apoptosis is, at least in part, p53-dependent. Taken together, these results provide the first evidence that COX inhibitors enhance chemosensitivity in neuroblastoma via downregulating HDM2 and augmenting p53 stability and nuclear accumulation.

Cloning and characterization of the human neural cell adhesion molecule, CNTN4 (alias BIG-2).

We report the isolation and characterization of human contactin 4 (CNTN4), a brain-derived, immunoglobulin superfamily molecule-2 (alias BIG-2) as a candidate gene responsible for the differentiation potential of human neuroblastoma cells. Northern blot analysis showed highest CNTN4 expression in testes, thyroid, small intestine, uterus and brain. Induction of CNTN4 mRNA expression in human neuroblastoma tumor cells treated with retinoic acid correlated with a block in retinoid-induced neuritogenesis. Our findings suggest a role for human contactin 4 protein in the response of neuroblastoma cells to differentiating agents.

Expression of multiple endocrine neoplasia 2B RET in neuroblastoma cells alters cell adhesion in vitro, enhances metastatic behavior in vivo, and activates Jun kinase.

Point mutations, deletions, and recombinations of the RET proto-oncogene are associated with several inherited human diseases of neural crest-derived cells: Hirschsprung's disease, familial medullary thyroid carcinoma, and the multiple endocrine neoplasia (MEN) syndromes, types 2A and 2B. RET expression is restricted to normal and malignant cells of neural crest origin, such as human neuroblastoma cells. To better understand the role of the activated RET oncogene in neural crest cells, we transfected two adherent human neuroblastoma tumor cell lines with oncogenic MEN2 mutant RET cDNAs. Transfectant clones from both cell lines overexpressing MEN2B RET demonstrated a marked increase in the cell fraction growing in suspension. Both control and MEN2B cells formed tumors at the site of injection in all cases. However, mice injected with MEN2B cells developed lung metastases at a much higher frequency than control mice. Only RET protein derived from MEN2A transfectant cells had increased autokinase activity, whereas MEN2B transfectant cells demonstrated selective activation of the mitogen-activated protein kinase, Jun kinase-1 (Jnk1). These results indicate a biochemical signaling pathway that may link oncogenic RET with the metastatic process.