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Background: Intersecting a genome-wide expression profile of metastatic and nonmetastatic human neuroblastoma xenograft variants with expression profiles of tumours from stage 1 and 4 neuroblastoma patients, we previously characterised hexokinase 2 (HK2) as a gene whose expression was upregulated in both metastatic neuroblastoma variants and tumours from stage 4 neuroblastoma patients. Methods: Local and metastatic neuroblastoma cell variants as well as metastatic neuroblastoma cells genetically manipulated to downregulate the expression of HK2 were utilised for in vitro and in vivo examinations of the involvement of HK2 in neuroblastoma. Results: Hexokinase 2 expression and its activity levels were increased in neuroblastoma metastatic variants as compared with the local variants. The upregulation of HK2 confers upon the metastatic cells high resistance to the antiproliferative effect of the HK2 inhibitor 3-BrPa and to the chemotherapy agent Deferoxamine. The inhibition of HK2 transcript lowered the proliferation and motility of sh-HK2 cells as compared with sh-control cells. Mice that were inoculated with sh-HK2 cells had a lower incidence of local tumours, smaller tumour volumes and a diminished load of lung metastasis compared with mice inoculated with sh-control cells. Conclusions: Hexokinase 2 plays a significant role in shaping the malignant phenotype of neuroblastoma and influences the progression of this disease.

In previous studies, we demonstrated that human neuroblastoma cells are equipped with the machinery to direct their homing to bone marrow. These tumor cells express the CXCR4 receptor for the bone marrow stroma-derived chemokine CXCL12 (SDF-1) and secrete the CXCL12 ligand. The present study was undertaken to explore possible differences in gene-expression patterns between neuroblastoma variants that over-express CXCR4 (designated STH cells) and those which express very little of this receptor (STL cells). The results of the study clearly indicate that these variants show a differential gene-expression profile. They differ in expression of some integrins such as VLA2, VLA3 and VLA6, of neuroendocrine-markers such as CD56 and synaptophysin, in the expression of c-kit and in the secretion of certain cytokines and growth factors such as TNFalpha, SDF-1, VEGF, IL-8, GM-CSF and IP-10. We hypothesize that these differences are due to an autocrine SDF-1alpha-CXCR4 axis.

Lanthionine ketimine (LK) is an endogenous amino acid metabolite thought to form when promiscuous reactions of the transsulfuration pathway create thioether amino acid intermediates that undergo further transamination reactions catalyzed mostly by glutamine transaminase K (kynurenine aminotransferase). Cell penetrating ester derivatives of LKE have potent antioxidant, anti-neuroinflammatory and pro-neurotrophic abilities and reduce correlates of disease in diverse animal models of neurodegenerative pathologies. The precise mechanism of action for LK derivatives has remained elusive though LK-ethyl ester (LKE, also known as XN001) has proven to be a potent enhancer of cell autophagy in cell culture and in vivo. In the course of translational development of XN001 for neurodisease therapy, data was uncovered that this compound potently and selectively inhibits the cyclin dependent kinase-5(Cdk5)/p25 complex with no inhibition of Cdk5/p35. LKE /XN001 also inhibits Cdk5/p25 ability to phosphorylate its target S522 residue in CRMP2 (collapsin response mediator protein-2) in a cell-free system. Furthermore, LKE/XN001 reduces Cdk5-targeted CRMP2 phosphorylation and neurofilament phosphorylation in the Cln3Δex7/8mouse modeling Batten disease (juvenile neuronal ceroid lipofuscinosis) while simultaneously reducing p25 levels in the Cln3Δex7/8mouse cerebellum. Molecular modeling identified a candidate LKE/XN001 binding site on the interface between the Cdk5 activation loop and its p25 binding partner. The putative binding interaction involves a network of hydrogen bonding interactions and a key salt bridge between LKE/XN001 and R156 of Cdk5, which could conceivably encourage a less productive conformation of the holoenzyme. Antagonism of Cdk5/p25 signaling pathways could explain the broad neuro-therapeutic profile of LKE/XN001 and provides a biological rational for translational development of LK analogs for neuropathologies. Disclosure KH is inventor of XN001 and holds equity in XoNovo Ltd.

Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion. In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC) xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs) compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT) processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.

The mechanism by which dormant tumor cells can begin growing after long periods of inactivity and accelerate disease recurrence is poorly understood. The present study characterizes dormant neuroblastoma (NB) cells, as well as metastatic cells, which reside in the same organ microenvironment. A xenograft model of human NB consisting of variants that generate nonmetastatic local tumors in the orthotopic inoculation site and variants that generate lung metastatic NB (MetNB) cells was developed in our laboratory. The present study shows that lungs of mice inoculated with nonmetastatic NB variants contain disseminated neuroblastoma (DisNB) human cells. Both DisNB and MetNB variants expressed a similar tumorigenicty phenotype in vivo , whereas the MetNB variants produced a heavy metastatic load and the DisNB variants produced no or little metastasis. A comparative in vitro characterization of MetNB and DisNB cells revealed similarities and differences. DisNB, but not MetNB cells, expressed the minimal residual disease markers PHOX2B and TH. MetNB cells demonstrated higher migratory capacity, an elevated matrix metalloproteinase (MMP) secretion, and a higher constitutive phosphorylation of extracellular signal-regulated kinase (ERK) than DisNB cells. We suggest that characteristics common to both MetNB and DisNB cells were acquired relatively early in the metastatic process and the characteristics that differ between these variants were acquired later. We hypothesize that the DisNB cells are metastasis precursors, which may progress toward metastasis under certain microenvironmental conditions.

Metastasis is the primary cause of mortality in Neuroblastoma (NB) patients, but the metastatic process in NB is poorly understood. Metastsis is a multistep process that requires the coordinated action of many genes. The identification of genes that promote or suppress tumor metastasis can advance our understanding of this process. In the present study, we utilized a human NB xenograft model comprising local and metastatic NB variants, which was recently developed in our laboratory. We set out to identify molecular correlates of NB metastasis and to determine the clinical relevance of these molecules. We first performed genome-wide expression profiles of metastatic and nonmetastatic NB variants that have an identical genetic background. We found that some of the proteins highly expressed in the metastatic NB variants are localized in the cytoplasm and endoplasmic reticulum. Other proteins are linked to metabolic processes and signaling pathways, thereby supporting the invasive and metastatic state of the cells. Subsequently, we intersected the differentially expressed genes in the human xenografted variants with genes differentially expressed in Stage 1 and Stage 4 primary tumors of NB patients. By using the same gene-expression platform, molecular correlates associated with metastatic progression in primary NB tumors were identified. The resulting smaller gene set was clinically relevant as it discriminated between high- and low-risk NB patients.

Transendothelial migration (TEM) of tumor cells is a crucial step in metastasis formation. The prevailing paradigm is that the mechanism underlying TEM of tumor cells is similar to that of leukocytes involving adhesion molecules and chemokines. Fractalkine (CX3CL1) is a unique membrane-bound chemokine that functions also as an adhesion molecule. CX3CL1 can be cleaved to a soluble fragment, capable of attracting fractalkine receptor (CX3CR1)-expressing cells. In the present study, we asked if CX3CR1 is involved in the TEM of neuroblastoma cells. We demonstrated that biologically functional CX3CR1 is expressed by several neuroblastoma cell lines. Most importantly, CX3CR1-expressing neuroblastoma cells were stimulated by CX3CL1 to transmigrate through human bone-marrow endothelial cells. A dose dependent phosphorylation of ERK1/2 and AKT was induced in CX3CR1-expressing neuroblastoma cells by soluble CX3CL1. In addition to CX3CR1, neuroblastoma cells also express the CX3CL1 ligand. Membrane CX3CL1 expression was downregulated and the shedding of soluble CX3CL1 was upregulated by PKC activation. Taken together, the results of this study indicate that CX3CR1 plays a functional role in transmigration of neuroblastoma cells through bone-marrow endothelium. These results led us to hypothesize that the CX3CR1-CX3CL1 axis takes part in bone-marrow metastasis of neuroblastoma.

Bone marrow stroma cells secrete the chemokine CXCL12 that may support bone marrow metastasis formation by neuroblastoma cells. The present study demonstrates that bone marrow stroma cell lines also secrete CXCL10, a chemokine that was shown in the past to have anti-malignancy functions. A receptor recognized by antibodies against CXCR3 was shown to be expressed by six neuroblastoma cell lines. Further detailed analysis was performed on the NUB6 and SK-NMC neuroblastoma cells, showing that CXCL10 induced potent Erk phosphorylation in a G(alpha)i-dependent manner. The role of a CXCR3-like receptor in Erk phosphorylation was substantiated by the ability of CXCL11, another potent CXCR3 ligand, to induce Erk phosphorylation in the NUB6 and SK-NMC cells. Further characterization of CXCL10 activities indicated that CXCL10 partly inhibited the growth of the NUB6 and SK-NMC cells. Both NUB6 and SK-NMC cells did not migrate to CXCL10, although their migratory machinery was intact, as evidenced by their migration to bone marrow constituents. Altogether, these results suggest that CXCL10 interacts with a CXCR3-like receptor in neuroblastoma cell lines, raising the possibility that following the homing of the tumor cells to the bone marrow (through a CXCL10-independent mechanism), CXCL10 may partly inhibit neuroblastoma cell growth at this site.