Roberta Vitali

c-Kit is preferentially expressed in MYCN-amplified neuroblastoma and its effect on cell proliferation is inhibited in vitro by STI-571.

Coexpression for c‐Kit receptor and its ligand stem cell factor (SCF) has been described in neuroblastoma (NB) cell lines and tumors, suggesting the existence of an autocrine loop modulating tumor growth. We evaluated c‐Kit and SCF expression by immunohistochemistry in a series of 75 primary newly diagnosed neuroblastic tumors. Immunostaining for c‐Kit was found in 10/75 and for SCF in 17/75, with 5/10 c‐Kit–positive tumors also expressing SCF. For both, c‐Kit and SCF staining were predominantly found in the most aggressive subset of tumors, i.e., those amplified for MYCN: c‐Kit was detected in 8/14 amplified vs. 2/61 single copy (p<0.001), and SCF in 9/14 amplified vs. 8/61 single copy tumors (p<0.001). Furthermore, the association of c‐Kit expression with advanced stage (3 or 4) (p=0.001) and of SCF expression with adrenal primary (p=0.03) was substantiated. The in vitro activity of the tyrosine kinase inhibitor STI‐571 (imatinib mesylate, Gleevec, Glivec) on NB cell lines positive or negative for c‐Kit was also assessed. When cells were grown in 10% fetal calf serum, the 4 c‐Kit‐positive cell lines tested were sensitive to STI‐571 growth inhibition to a different extent (ranging from 30 to 80%); also the c‐Kit‐negative cell line GI‐CA‐N was slightly affected, suggesting that other STI‐571 targets operate in regulating NB proliferation. In addition, c‐Kit‐positive cell lines SK‐N‐BE2(c) and HTLA230, grown in SCF only, remained sensitive (40 and 70% of growth inhibition, respectively), while, in the same conditions, proliferation of the c‐Kit‐negative cell line GI‐CA‐N was not affected. Immunoprecipitation of c‐Kit from cell lysates of SK‐N‐BE2(c) and HTLA230 cells grown in SCF and subsequent western blot analysis of the immunoprecipitates revealed a sharp decrease of c‐Kit phosphorylation after STI‐571 treatment. These data demonstrate that both c‐Kit and SCF are preferentially expressed in vivo in the most aggressive neuroblastic tumors and that their signaling is active in promoting in vitro NB cell proliferation that can be selectively inhibited by treatment with STI‐571.

Fecal HMGB1 is a novel marker of intestinal mucosal inflammation in pediatric inflammatory bowel disease.

OBJECTIVES:High-mobility group box 1 (HMGB1) is a nuclear protein with functions in the regulation of transcription. In inflammatory conditions, HMGB1 is actively secreted from immune cells in the extracellular matrix, where it behaves as a proinflammatory cytokine. The aim of the present study was to investigate the role of HMGB1 in pediatric inflammatory bowel disease (IBD).METHODS:We analyzed the stools of 19 children with Crohns disease (CD), 21 with ulcerative colitis (UC), and 13 controls. The gene/protein expression levels of HMGB1 were assessed in bioptic specimens of all children using real-time PCR and western blot assay. Finally, intracellular localization of the protein was analyzed by western blot, after separation of nuclear and cytoplasmic extracts, and by immunohistochemistry.RESULTS:HMGB1 protein levels were significantly increased (P<0.001) in the stools of patients, but were undetectable in the controls; fecal HMGB1 correlated well with fecal calprotectin levels (r: 0.77 in CD, r: 0.70 in UC; P<0.01); and mRNA and protein expression were unchanged in inflamed bioptic tissues compared with controls. However, by separately analyzing the nuclear and cytoplasmic fraction, we detected the cytoplasmic HMGB1 expression to be significantly enhanced (P<0.01) in the inflamed tissues of the patients. In addition, HMGB1 was significantly detected in 16 patients with inactive disease, whose endoscopic scores showed persisting inflammation, suggesting that it may be a sensitive marker of mucosal inflammation, although the disease is clinically inactive.CONCLUSIONS:It was shown for the first time in our study that HMGB1 is secreted by human inflamed intestinal tissues and abundantly found in the stools of IBD patients. Hence, it can be considered as a novel marker for intestinal inflammation. We can also suggest that the presence of HMGB1 in large amounts in the fecal stream of IBD patients is mainly due to active secretion of the protein stored in the nucleus rather than a “de novo” synthesis.

Down-regulation of insulin-like growth factor I receptor activity by NVP-AEW541 has an antitumor effect on neuroblastoma cells in vitro and in vivo.

Purpose: Signaling through insulin-like growth factor I receptor (IGF-IR) is important for growth and survival of many tumor types. Neuroblastoma is sensitive to IGF. Experimental Design: We assessed the ability of NVP-AEW541, a recently developed small molecule that selectively inhibits IGF-IR activity, for neuroblastoma growth effects in vitro and in vivo. Our data showed that, in a panel of 10 neuroblastoma cell lines positive for IGF-IR expression, NVP-AEW541 inhibited in vitro proliferation in a submicromolar/micromolar (0.4-6.8) range of concentrations. Results: As expected, NVP-AEW541 inhibited IGF-II–mediated stimulation of IGF-IR and Akt. In addition to growth inhibition, the drug also induced apoptosis in vitro. Oral administration of NVP-AEW541 (50 mg/kg twice daily) inhibited tumor growth of neuroblastoma xenografts in nude mice. Analysis of tumors from the drug-treated animals revealed a marked apoptotic pattern and a decrease in microvascularization compared with controls. Interestingly, quantitative real-time PCR detected both in vitro and in vivo a significant down-regulation of mRNA for vascular endothelial growth factor (VEGF) caused by NVP-AEW541. In addition, in Matrigel-coated chambers and in severe combined immunodeficient mice tail vein injected with neuroblastoma cells, tumor invasiveness was significantly reduced by this agent. Analysis of IGF-IR expression in a series of 43 neuroblastoma primary tumors revealed IGF-IR positivity in 86% of cases. Conclusions: Taken together, these data indicate that NVP-AEW541 can be considered as a novel promising candidate for treatment of neuroblastoma patients.

Necroptosis Is Active in Children With Inflammatory Bowel Disease and Contributes to Heighten Intestinal Inflammation

OBJECTIVES:A new caspase-independent mode of programmed cell death, termed necroptosis, has recently been identified. Altered expression of molecules involved in the necroptosis pathway has been shown to trigger intestinal inflammation. The initiation of necroptosis is principally mediated by the release of receptor interacting protein 3 (RIP3) from suppression by caspase-8. Furthermore, it has been suggested that the mixed lineage kinase domain-like (MLKL) factor is an interacting target of RIP3 in active necroptosis. This study aims at investigating the occurrence of necroptosis in children with inflammatory bowel disease (IBD) and its contribution to human intestinal inflammation.METHODS:Biopsy samples were collected from the ileum and colon of 33 children with Crohns disease, 30 with ulcerative colitis, and 20 healthy controls. Ten children with allergic colitis (AC) were used as non-IBD comparators. RIP3, caspase-8, and MLKL protein expression levels were evaluated by western blotting. The adenocarcinoma cell line HT29 was used for in vitro experiments.RESULTS:RIP3 and MLKL increased (P<0.01) in inflamed tissues of IBD and AC patients, whereas caspase-8 was reduced. No variations were observed in uninflamed tissues of patients. The relationship between RIP3 increase, active necroptosis, and intestinal inflammation was confirmed by in vitro analyses.CONCLUSIONS:We show for the first time that necroptosis is strongly associated with intestinal inflammation in children with IBD and contributes to strengthen the inflammatory process. We believe that RIP3 and MLKL could represent attractive targets for the management of human IBD.

Characterization of adherent-invasive Escherichia coli isolated from pediatric patients with inflammatory bowel disease†

Background: Crohns disease (CD) and ulcerative colitis (UC), known as inflammatory bowel diseases (IBD), are characterized by an abnormal immunological response to commensal bacteria colonizing intestinal lumen and mucosa. Among the latter, strains of adherent‐invasive Escherichia coli (AIEC), capable of adhering to and invading epithelium, and to replicate in macrophages, have been described in CD adults. We aimed at identifying and characterizing AIEC strains in pediatric IBD. Methods: In all, 24 CD children, 10 UC, and 23 controls were investigated. Mucosal biopsies, taken during colonoscopy, were analyzed for the presence of AIEC strains by an adhesive‐invasive test. Protein expression of the specific AIEC receptor, the carcinoembryonic antigen‐related cell adhesion molecule 6 (CEACAM6), was evaluated by western blot and immunohistochemistry, while tumor necrosis factor alpha (TNF‐&agr;) and interleukin (IL)‐8 mRNA expression was detected by real‐time polymerase chain reaction (PCR), after bacterial infection. Transmission electron microscopy and trans‐epithelial electric resistance assays were performed on biopsies to assess bacteria‐induced morphological and functional epithelial alterations. Results: Two bacterial strains, EC15 and EC10, were found to adhere and invade the Caco2 cell line, similar to the well‐known AIEC strain LF82 (positive control): they upregulated CEACAM6, TNF‐&agr;, and IL‐8 gene/protein expression, in vitro and in cultured intestinal mucosa; they could also survive inside macrophages and damage the epithelial barrier integrity. Lesions in the inflamed tissues were associated with bacterial infection. Conclusions: This is the first study showing the presence of adhesive‐invasive bacteria strains in the inflamed tissues of children with IBD. Collective features of these strains indicate that they belong to the AIEC spectrum, suggesting their possible role in disease pathogenesis. (Inflamm Bowel Dis 2011)

Slug (SNAI2) down-regulation by RNA interference facilitates apoptosis and inhibits invasive growth in neuroblastoma preclinical models.

Purpose: We assessed the relevance of Slug (SNAI2) for apoptosis resistance and invasion potential of neuroblastoma cells in vitro and in vivo. Experimental Design: We evaluated the effect of imatinib mesylate on invasion and analyzed the genes modulated by imatinib mesylate treatment in neuroblastoma cells. Slug expression, inhibited by imatinib mesylate treatment, was knocked down in neuroblastoma cells by RNA interference, and the effects on invasion and apoptosis were evaluated in vitro. A pseudometastatic model of neuroblastoma in severe combined immunodeficient mice was used to assess the effects of Slug silencing alone or in combination with imatinib mesylate treatment on metastasis development. Results: Microarray analysis revealed that several genes, including Slug, were down-regulated by imatinib mesylate. Slug expression was detectable in 8 of 10 human neuroblastoma cell lines. Two Slug-expressing cell lines were infected with a vector encoding a microRNA to Slug mRNA. Infected cells with reduced levels of Slug were tested for the expression of apoptosis-related genes (p53, Bax, and Bcl-2) identified previously as Slug targets. Bcl-2 was down-regulated in Slug-interfered cells. Slug down-regulation increased sensitivity to apoptosis induced by imatinib mesylate, etoposide, or doxorubicin. Invasion of Slug-silenced cells was reduced in vitro. Animals injected with Slug-silenced cells had fewer tumors than controls and the inhibition of tumor growth was even higher in animals treated with imatinib mesylate. Conclusions: Slug down-regulation facilitates apoptosis induced by proapoptotic drugs in neuroblastoma cells and decreases their invasion capability in vitro and in vivo. Slug inhibition, possibly combined with imatinib mesylate, may represent a novel strategy for treatment of metastatic neuroblastoma.

Silencing of endogenous IGFBP-5 by micro RNA interference affects proliferation, apoptosis and differentiation of neuroblastoma cells.

Signal transduction through the IGF axis is implicated in proliferation, differentiation and survival during development and adult life. The IGF axis includes the IGF binding proteins (IGFBPs) that bind IGFs with high affinity and modulate their activity. In neuroblastoma (NB), a malignant childhood tumor, we found that IGFBP-5 is frequently expressed. Since NB is an IGF2-sensitive tumor, we investigated the relevance and the function of endogenous IGFBP-5 in LAN-5 and in SY5Y(N) cell lines transfected with micro and small interfering RNAs directed to IGFBP-5 mRNA. Cells in which IGFBP-5 expression was suppressed were growth-inhibited and more prone to apoptosis than the parental cell line and controls. Apoptosis was further enhanced by X-ray irradiation. The ability of these cells to undergo neuronal differentiation was impaired after IGFBP-5 inhibition but the effect was reversed by exposure to recombinant IGFBP-5. Together, these data demonstrate the importance of IGFBP-5 for NB cell functions and suggest that IGFBP-5 might serve as a novel therapeutic target in NB.

Expression of insulin-like growth factor-binding protein 5 in neuroblastoma cells is regulated at the transcriptional level by c-Myb and B-Myb via direct and indirect mechanisms.

Neuroblastoma (NB), a malignant childhood tumor deriving from the embryonic neural crest, is sensitive to the growth-stimulating effects of insulin-like growth factors (IGFs). Aggressive cases of this disease often acquire autocrine loops of IGF production, but the mechanisms through which the different components of the IGF axis are regulated in tumor cells remain unclear. Upon conditional expression of c-Myb in a NB cell line, we detected up-regulation of IGF1, IGF1 receptor, and insulin-like growth factor-binding protein 5 (IGFBP-5) expression. Analysis of the IGFBP-5 promoter revealed two potential Myb binding sites at position −59 to −54 (M1) and −429 to −424 (M2) from the transcription start site; both sites were bound by c-Myb and B-Mybin vitro and in vivo. Reporter assays carried out using the proximal region of the human IGFBP-5 promoter demonstrated that c-Myb and B-Myb enhanced transcription. However, site-directed mutagenesis and deletion of the Myb binding sites coupled with reporter assays revealed that M2 but not M1 was important for Myb-dependent transactivation of the IGFBP-5promoter. The double mutant M1/M2 was still transactivated by c-Myb, suggesting the existence of Myb binding-independent mechanisms ofIGFBP-5 promoter regulation. A constitutively active AKT transactivated the IGFBP-5 promoter, whereas the phosphatidylinositol 3-kinase inhibitor LY294002 suppressed it. Moreover, the kinase dead dominant negative K179M AKT mutant was able to inhibit transcription from the M2 and M1/M2IGFBP-5 mutant promoters. Deletion analysis of theIGFBP-5 promoter revealed that the AKT-responsive region lies between nucleotides −334 and −83. Together, these data suggest that the Myb binding-independent transactivation of theIGFBP-5 promoter was due to the activation of the phosphatidylinositol 3-kinase/AKT pathway likely mediated by IGF1 receptor-dependent signals. Finally, IGFBP-5 was able to modulate proliferation of NB cells in a manner dependent on its concentration and on the presence of IGFs.

Clinical Significance of CXC Chemokine Receptor-4 and c-Met in Childhood Rhabdomyosarcoma

Purpose: The CXC chemokine receptor-4 (CXCR4)/stromal-derived factor-1 and c-Met/hepatocyte growth factor axes promote the metastatic potential of rhabdomyosarcoma cell lines in experimental models, but no data are available on their role in rhabdomyosarcoma tumors. The expressions of CXCR4 and c-Met were evaluated in primary tumors and isolated tumor cells in marrow, and were correlated with clinicopathologic variables and survival. Experimental Design: Forty patients with recently diagnosed rhabdomyosarcoma were retrospectively enrolled. CXCR4 and c-Met expression was investigated in primary tumors by immunohistochemistry, in isolated marrow-infiltrating tumor cells using double-label immunocytology. Results were expressed as the mean percentage of immunostained tumor cells. Results: CXCR4 and c-Met were expressed in ≥5% of tumor cells from 40 of 40 tumors, with 14 of 40 cases showing ≥50% of immunostained tumor cells (high expression). High CXCR4 expression correlated with alveolar histology (P = 0.006), unfavorable primary site (P = 0.009), advanced group (P < 0.001), marrow involvement (P = 0.007), and shorter overall survival and event-free survival (P < 0.001); high c-Met expression correlated with alveolar histology (P = 0.005), advanced group (P = 0.04), and marrow involvement (P = 0.02). In patients with a positive diagnosis for isolated tumor cells in marrow (n = 16), a significant enrichment in the percentage of CXCR4-positive (P = 0.001) and c-Met–positive (P = 0.003) tumor cells was shown in marrow aspirates compared with the corresponding primary tumors. Conclusions: CXCR4 and c-Met are widely expressed in both rhabdomyosarcoma subtypes and, at higher levels, in isolated marrow-infiltrating tumor cells. High levels of expression are associated with unfavorable clinical features, tumor marrow involvement and, only for CXCR4, poor outcome. In rhabdomyosarcoma, CXCR4 and c-Met represent novel exploitable targets for disease-directed therapy.

Role of HMGB1 as a suitable biomarker of subclinical intestinal inflammation and mucosal healing in patients with inflammatory bowel disease.

Background:Noninvasive biomarkers of high- and low-grade intestinal inflammation and of mucosal healing (MH) in patients with inflammatory bowel disease are currently lacking. We have recently shown that fecal high mobility group box 1 (HMGB1) protein is a novel biomarker of gut inflammation. We aimed at investigating in a mouse model if HMGB1 was able to foresee both a clinically evident and a subclinical gut inflammation and if its normalization indicated MH. We also aimed at confirming the results in patients with Crohns disease (CD) and ulcerative colitis. Methods:C57BL6/J mice were treated with increasing doses of dextran sodium sulphate to induce colitis of different severity degrees; 28 with CD, 23 with ulcerative colitis, and 17 controls were also enrolled. Fecal HMGB1 was analyzed by enzyme-linked immunosorbent assay and immunoblotting. Results:Fecal HMGB1 increased by 5-, 11-, 18-, and 24-folds with dextran sodium sulphate doses of 0.25%, 0.50%, 1%, and 4%, respectively, showing that the protein detected a high-grade and a subclinical inflammation. After a recovery time of 4-week posttreatment, HMGB1 returned to control levels, paralleling MH. In patients, fecal HMGB1 significantly correlated with endoscopic indexes (Simple Endoscopic Score for Crohns Disease [SES-CD], endoscopic Mayo subscore), but not with the disease activity indexes (Crohns disease Activity Index, partial Mayo score). Conclusions:Fecal HMGB1 is a robust noninvasive biomarker of clinically overt and subclinical gut inflammation; it can also be a surrogate marker of MH. We suggest the use of fecal HMGB1 to monitor the disease course and assess therapy outcomes in inflammatory bowel disease.