The survivin-ran inhibitor LLP-3 decreases oxidative phosphorylation, glycolysis and growth of neuroblastoma cells

Abstract Background Neuroblastoma (NB), the most common extracranial solid malignancy in children, carries a poor prognosis in high-risk disease, thus requiring novel therapeutic approaches. Survivin is overexpressed in NB, has pro-mitotic and anti-apoptotic functions, and impacts on oxidative phosphorylation (OXPHOS) and aerobic glycolysis. The subcellular localization and hence function of survivin is directed by the GTPase Ran. Aim To determine efficacy and modes of action of the survivin-Ran inhibitor LLP-3 as a potential novel therapy of NB. Methods Survivin and Ran mRNA expression in NB tumors was correlated to patient survival. Response to LLP-3 in NB cell lines was determined by assays for viability, proliferation, apoptosis, clonogenicity and anchorage-independent growth. Interaction of survivin and Ran was assessed by proximity-linked ligation assay and their subcellular distribution by confocal immunofluorescence microscopy. Expression of survivin, Ran and proteins important for OXPHOS and glycolysis was determined by Western blot, hexokinase activity by enzymatic assay, interaction of survivin with HIF-1α by co-IP, and OXPHOS and glycolysis by extracellular flux analyzer. Results High mRNA expression of survivin and Ran is correlated with poor patient survival. LLP-3 decreases viability, induces apoptosis, and inhibits clonogenic and anchorage-independent growth in NB cell lines, including those with MYCN amplification, and mutations of p53 and ALK. LLP-3 inhibits interaction of survivin with Ran, decreasing their concentration both in the cytoplasm and the nucleus. LLP-3 impairs flexibility of energy metabolism by inhibiting both OXPHOS and glycolysis. Metabolic inhibition is associated with mitochondrial dysfunction and attenuated hexokinase activity but is independent of HIF-1α. Conclusion LLP-3 attenuates interaction and concentration of survivin and Ran in NB cells. It controls NB cells with diverse genetic alterations, associated with inhibition of OXPHOS, aerobic glycolysis, mitochondrial function and HK activity. Thus, LLP-3 warrants further studies as a novel drug against NB

CHD5 inhibits metastasis of neuroblastoma

CHD5, a tumor suppressor at 1p36, is frequently lost or silenced in poor prognosis neuroblastoma (NB) and many adult cancers. The role of CHD5 in metastasis is unknown. We confirm that low expression of CHD5 is associated with stage 4 NB. Forced expression of CHD5 in NB cell lines with 1p loss inhibited key aspects of the metastatic cascade in vitro: anchorage-independent growth, migration, and invasion. In vivo, formation of bone marrow and liver metastases developing from intravenously injected NB cells was delayed and decreased by forced CHD5 expression. Genome-wide mRNA sequencing revealed reduction of genes and gene sets associated with metastasis when CHD5 was overexpressed. Known metastasis-suppressing genes preferentially upregulated in CHD5-overexpressing NB cells included PLCL1. In patient NB, low expression of PLCL1was associated with metastatic disease and poor survival. Knockdown of PLCL1 and of p53 in IMR5 NB cells overexpressing CHD5 reversed CHD5-induced inhibition of invasion and migration in vitro. In summary, CHD5 is a metastasis suppressor in NB

Additional file 1 of The survivin-ran inhibitor LLP-3 decreases oxidative phosphorylation, glycolysis and growth of neuroblastoma cells

Additional file 1: Suppl. Fig. 1. Uncropped full-length Western blots of Fig. 2A. Suppl. Fig 2. LLP-3 decreases both nuclear and cytoplasmic expression of survivin and Ran in KELLY cells by 24 h. Suppl. Fig. 3. LLP-3 does not consistently alter expression of GLUT1, HK2, PKM2, LDHA, PDK1 and PDHA. Suppl. Fig. 4. Uncropped full-length Western blots of Suppl. Fig. 3. Suppl. Fig. 5. HIF-1α protein does not alter the effect of LLP-3 on NB cell lines. Suppl. Fig. 6. Uncropped full-length Western blots of Suppl. Fig. 5A (A) and Suppl. Fig. 5C (B)

Strong expression of CD57 in U-NB1 neuroblastoma cells promotes clonogenicity and induction of spheres.

<p>(<b>A</b>) <b>Morphology of U-NB1 cells growing in serum-free and serum-replete medium.</b> Appearance of spheres derived from single cells plated in non-adherent plates and grown in serum-free medium containing EGF and bFGF (left panel). Morphology of cells when cultured on collagen-coated plastic in serum-supplemented medium (right panel). Bars correspond to 30 µm. (<b>B</b>) <b>Enhanced clonogenicity of U-NB1 cells strongly expressing CD57.</b> U-NB1 cells were separated by flow cytometry into a CD57^high fraction (cells above the 80^th percentile of CD57 expression) and a CD57^low fraction (cells below the 20^th percentile). CD57 expression of unsorted and sorted cells is shown in histograms (upper panel). Solid lines correspond to CD57, dashed lines to isotype controls. For soft agar clonogenicity assays fractionated U-NB1 cells were seeded at 1000 cells per well into 24-well plates. 28 days after plating colonies were visualised by MTT staining. The numbers of colonies per well are depicted in the graph. The asterisk denotes p<0.05, using Student's t-test. Experiments were repeated three times, with similar results. (<b>C</b>) <b>Enhanced induction of spheres by U-NB1 cells strongly expressing CD57.</b> U-NB1 cells were separated by flow cytometry into CD57^high and CD57^low fractions. Fractionated as well as unsorted cells were plated at very low density in serum-free medium. The percentage of “first generation” spheres growing from single cells was determined. After dissociation of spheres this procedure was repeated twice (“second” and “third generation” spheres, respectively). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042025#s2" target="_blank">Results</a> are shown as box plots (lower panel). Asterisks denote p<0.001, using Student's t-test. Experiments were performed twice, with similar results.</p

gamma-secretase inhibitor I inhibits neuroblastoma cells, with NOTCH and the proteasome among its targets

As high-risk neuroblastoma (NB) has a poor prognosis, new therapeutic modalities are needed. We therefore investigated the susceptibility of NB cells to gamma-secretase inhibitor I (GSI-I). NOTCH signaling activity, the cellular effects of GSI-I and its mechanisms of cytotoxicity were evaluated in NB cells in vitro and in vivo. The results show that NOTCH signaling is relevant for human NB cells. Of the GSIs screened in vitro GSI-I was the most effective inhibitor of NB cells. Both MYCN-amplified and non-amplified NB cells were susceptible to GSI-I. Among the targets of GSI-I in NB cells were NOTCH and the proteasome. GSI-I caused G2/M arrest that was enhanced by acute activation of MYCN and led to mitotic dysfunction. GSI-I also induced proapoptotic NOXA. Survival of mice bearing an MYCN non-amplified orthotopic patient-derived NB xenograft was significantly prolonged by systemic GSI-I, associated with mitotic catastrophe and reduced angiogenesis, and without evidence of intestinal toxicity. In conclusion, the activity of GSI-I on multiple targets in NB cells and the lack of gastrointestinal toxicity in mice are advantageous and merit further investigations of GSI-I in NB

CD57^high cells in Schwannian stroma-poor neuroblastoma of patients.

<p><i>INPC</i>, International Neuroblastoma Pathology Classification <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042025#pone.0042025-Shimada1" target="_blank">[21]</a>; <i>INSS</i>, International Neuroblastoma Staging System <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042025#pone.0042025-Brodeur1" target="_blank">[22]</a>; <i>undifferentiated cells</i>, grade 3 NB cells according to Hughes classification (modified) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042025#pone.0042025-Harms1" target="_blank">[23]</a>; <i>after chemo</i>, after chemotherapy as specified by the NB2004 Trial Protocol; <i>CD57^high</i>, staining of CD57 at least as intense as in natural killer cells of the sample.</p

Expression of CD57 determines clonogenicity and correlates with

<p>induction of spheres <b>of SK-N-BE(2)-C neuroblastoma cells.</b> (<b>A</b>) <b>SK-N-BE(2)-C cells with high expression of CD57 are clonogenic.</b> SK-N-BE(2)-C cells were separated by flow cytometry into CD57^high and CD57^low fractions. For soft agar clonogenicity assays fractionated SK-N-BE(2)-C cells were seeded at 2000 cells per well into 24-well plates. 28 days after plating, colonies were visualised by MTT staining. The numbers of colonies per well are depicted in the graph. The asterisk denotes p<0.05, using Student's t-test. Experiments were repeated three times, with similar results. (<b>B</b>) <b>CD57 expression of SK-N-BE(2)-C cells correlates with induction of spheres.</b> The propensity of single SK-N-BE(2)-C cells to generate spheres when plated at very low density in serum-free medium was compared between unsorted SK-N-BE(2)-C cells (upper panel, bar equals 30 µm), CD57^high cells and CD57^low cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042025#s2" target="_blank">Results</a> are calculated as the percentage of spheres generated from the cells seeded and are shown as box plots (lower panel). Asterisks denote p<0.001, using Student's t-test. Experiments were performed twice, with similar results.</p

Enhanced formation of stroma-poor neuroblastoma by CD57^high U-NB1 cells.

<p>(<b>A</b>) <b>Enhanced tumor formation of CD57^high U-NB1 cells.</b> CD57^high and CD57^low tumor cells were isolated by FACS from one primary orthotopic tumor. 1×10⁴ cells were surgically transplanted into the left adrenal glands of immunodeficient mice (8 animals per group). The development of tumors was monitored by palpation and verified by necropsy when mice had to be killed because of excessively large tumors or when the experiment was terminated at 90 days. The log rank test was used for statistical analysis. The experiment was repeated twice, with similar results. (<b>B</b>) <b>Orthotopic U-NB1 tumors are stroma-poor.</b> Tumors were formalin-fixed, paraffin-embedded and HE-stained. Scale bar equals 200 µm.</p

CD57^high Neuroblastoma Cells Have Aggressive Attributes <em>Ex Situ</em> and an Undifferentiated Phenotype in Patients

<div><h3>Background</h3><p>Neuroblastoma is thought to originate from neural crest-derived cells. CD57 defines migratory neural crest cells in normal development and is expressed in neuroblastoma.</p> <h3>Methodology and Principal Findings</h3><p>We investigated the role of CD57 expression in neuroblastoma cells <em>ex situ</em> and <em>in situ</em>. Compared to CD57^low U-NB1 neuroblastoma cells, CD57^high cells developed tumors with decreased latency after orthotopic transplantation into adrenal glands of mice. In addition, CD57^high U-NB1 and SK-N-BE(2)-C neuroblastoma cells were also more clonogenic, induced more spheres and were less lineage-restricted. CD57^high cells attached better to endothelial cells and showed enhanced invasiveness. While invasion of U-NB1 cells was inhibited by blocking antibodies against CD57, neither invasion of SK-N-BE(2)-C cells nor adhesion of U-NB1 and SK-N-BE(2)-C cells was attenuated. After tail vein injection only CD57^high cells generated liver metastases, while overall metastatic rate was not increased as compared to CD57^low cells. In stroma-poor neuroblastoma of patients CD57^high cells were associated with undifferentiated tumor cells across all stages and tended to be more frequent after chemotherapy.</p> <h3>Conclusion</h3><p>Strong expression of CD57 correlates with aggressive attributes of U-NB1 and SK-N-BE(2)-C neuroblastoma cells and is linked with undifferentiated neuroblastoma cells in patients.</p> </div