Peter E. Zage

SPARC expression is associated with impaired tumor growth, inhibited angiogenesis and changes in the extracellular matrix

Secreted protein, acidic and rich in cysteine (SPARC), is a multifunctional matricellular glycoprotein. In vitro, SPARC has antiangiogenic properties, including the ability to inhibit the proliferation and migration of endothelial cells stimulated by bFGF and VEGF. Previously, we demonstrated that platelet‐derived SPARC also inhibits angiogenesis and impairs the growth of neuroblastoma tumors in vivo. In the present study, we produced rhSPARC in the transformed human embryonic kidney cell line 293 and show that the recombinant molecule retains its ability to inhibit angiogenesis. Although 293 cell proliferation was not affected by exogenous expression of SPARC in vitro, growth of tumors formed by SPARC‐transfected 293 cells was significantly impaired compared to tumors comprised of wild‐type cells or 293 cells transfected with a control vector. Consistent with its function as an angiogenesis inhibitor, significantly fewer blood vessels were seen in SPARC‐transfected 293 tumors compared to controls, and these tumors contained increased numbers of apoptotic cells. Light microscopy revealed small nests of tumor cells surrounded by abundant stromal tissue in xenografts with SPARC expression, whereas control tumors were comprised largely of neoplastic cells with scant stroma. Mature, covalently cross‐linked collagen was detected in SPARC‐transfected 293 xenografts but not in control tumors. Our studies suggest that SPARC may regulate tumor growth by inhibiting angiogenesis, inducing tumor cell apoptosis and mediating changes in the deposition and organization of the tumor microenvironment.

Association of epigenetic inactivation of RASSF1A with poor outcome in human neuroblastoma

Purpose: To investigate the prevalence and potential clinical significance of epigenetic aberrations in neuroblastoma (NB). Experimental Design: The methylation status of 11 genes that are frequently epigenetically inactivated in adult cancers was assayed in 13 NB cell lines. The prevalence of RASSF1A and TSP-1 methylation was also analyzed in 56 NBs and 5 ganglioneuromas by methylation-specific PCR. Associations between the methylation status of RASSF1A and TSP-1 and patient age, tumor stage, tumor MYCN status, and patient survival were evaluated. Results: Epigenetic changes were detected in all 13 NB cell lines, although the pattern of gene methylation varied. The putative tumor suppressor gene RASSF1A was methylated in all 13 cell lines, and TSP-1 and CASP8 were methylated in 11 of 13 cell lines. Epigenetic changes of DAPK and FAS were detected in only small numbers of cell lines, whereas none of the cell lines had methylation of p16, p21, p73, RAR-β2, SPARC, or TIMP-3. RASSF1A was also methylated in 70% of the primary NB tumors tested, and TSP-1 methylation was detected in 55% of the tumors. RASSF1A methylation was significantly associated with age >1 year (P < 0.01), high-risk disease (P < 0.016), and poor survival (P < 0.001). In contrast, no association between TSP-1 methylation and prognostic factors or survival was observed. Conclusions: Our results suggest that epigenetic inactivation of RASSF1A may contribute to the clinically aggressive phenotype of high-risk NB.

Multiple receptor tyrosine kinases regulate HIF-1α and HIF-2α in normoxia and hypoxia in neuroblastoma: implications for antiangiogenic mechanisms of multikinase inhibitors

Novel treatment approaches are needed for children with advanced neuroblastoma. Studies with neuroblastoma cells have indicated the presence of a hypoxia-driven vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-1 autocrine loop modulating hypoxia-inducible factor-1alpha (HIF-1α). Whether other receptor tyrosine kinases (RTKs) are capable of modulating HIF-1α levels and whether RTKs can regulate HIF-2α as well is largely unknown. We evaluated neuroblastoma cell lines for expression of various RTKs. Although cell lines were heterogeneous in the expression of VEGFR-1, -3, c-Kit and RET, most cells expressed PDGFR-α and -β. Ligand-induced activation of multiple RTKs upregulated HIF-1α levels, whereas activation of VEGFR-1 alone upregulated HIF-2α. Multitargeted tyrosine kinase inhibitor sunitinib reduced hypoxia-induced rises in HIF-1α and HIF-2α through mechanisms involving effects on both mRNA levels and protein stability. In addition, sunitinib and sorafenib had direct effects on tumor cell viability in vitro. In a neuroblastoma xenograft model, tumor growth inhibition by sunitinib was associated with inhibition of angiogenesis and reduced HIF-1α levels. These findings show that multiple RTKs may regulate the HIF axis in normoxia and hypoxia and suggest that multikinase inhibitors may exert antiangiogenic effects not only by direct effects on endothelial cells, but also by blocking compensatory hypoxia- and ligand-induced changes in HIF-1α and HIF-2α.

Outcomes of the POG 9340/9341/9342 trials for children with high-risk neuroblastoma: a report from the Children's Oncology Group.

From 1993 to 1995, the Pediatric Oncology Group (POG) enrolled patients with high‐risk neuroblastoma on three sequential, conjoined studies: a phase II induction window (9340), followed by intensive multiagent induction chemotherapy (9341), and subsequent myeloablative therapy with autologous stem cell rescue (9342). We report here the outcomes of patients treated on these studies.

Identification and preclinical characterization of AZ-23, a novel, selective, and orally bioavailable inhibitor of the Trk kinase pathway

Tropomyosin-related kinases (TrkA, TrkB, and TrkC) are receptor tyrosine kinases that, along with their ligands, the neurotrophins, are involved in neuronal cell growth, development, and survival. The Trk-neurotrophin pathway may also play a role in tumorigenesis through oncogenic fusions, mutations, and autocrine signaling, prompting the development of novel Trk inhibitors as agents for cancer therapy. This report describes the identification of AZ-23, a novel, potent, and selective Trk kinase inhibitor. In vitro studies with AZ-23 showed improved selectivity over previous compounds and inhibition of Trk kinase activity in cells at low nanomolar concentrations. AZ-23 showed in vivo TrkA kinase inhibition and efficacy in mice following oral administration in a TrkA-driven allograft model and significant tumor growth inhibition in a Trk-expressing xenograft model of neuroblastoma. AZ-23 represents a potent and selective Trk kinase inhibitor from a novel series with the potential for use as a treatment for cancer. [Mol Cancer Ther 2009;8(7):1818–27]

The selective Trk inhibitor AZ623 inhibits brain-derived neurotrophic factor-mediated neuroblastoma cell proliferation and signaling and is synergistic with topotecan

TrkB expression is associated with poor prognosis for patients with neuroblastoma. AZ623 is a novel potent and selective inhibitor of the Trk family of tyrosine kinases. The authors hypothesized that AZ623 would inhibit TrkB‐mediated signaling in neuroblastoma tumor cells and would be synergistic when combined with chemotherapy.

Cross-Talk between Schwann Cells and Neuroblasts Influences the Biology of Neuroblastoma Xenografts

Neuroblastoma (NB) tumors with abundant schwannian stroma have a differentiated phenotype, low vascularity, and are associated with a favorable prognosis. These observations suggest that cross-talk between Schwann cells and neuroblasts may influence tumor biology. To test this hypothesis, we developed a novel NB xenograft model with infiltrating mouse Schwann cells. Human SMS-KCNR NB cells were injected intrafascicularly (sciatic nerve-engrafted NB, n = 19) or outside the sciatic nerve (control, n = 12). Xenografts were harvested 4 to 12 weeks after tumor cell inoculation for histological studies. Schwann cells were immunostained with S-100 and species-specific p75(NGFR), major histocompatibility complex, and human leukocyte antigen antibodies. The number of proliferating cells, infiltrating Schwann cells, apoptotic cells, differentiated neuroblasts, and blood vessels in the sciatic nerve-engrafted NB tumors were compared to controls. Significantly more Schwann cells were detected in the sciatic nerve-engrafted NB xenografts than controls (P < 0.001). The infiltrating Schwann cells were S-100-positive and reacted with anti-mouse major histocompatibility complex class Ib and p75(NGFR) but not anti-human p75(NGFR) and human leukocyte antigen class I antibodies. The sciatic nerve-engrafted tumors also had lower numbers of proliferating neuroblasts, higher numbers of differentiated neuroblasts and apoptotic cells, and decreased vascular density compared to controls. Our results indicate that infiltrating Schwann cells of mouse origin are capable of promoting human neuroblast differentiation, inducing apoptosis, and inhibiting proliferation and angiogenesis in vivo.

Notch pathway activation induces neuroblastoma tumor cell growth arrest

Notch pathway signaling has critical roles in differentiation, proliferation, and survival, and has oncogenic or tumor suppressor effects in a variety of malignancies. The goal of this study was to evaluate the effects of Notch activation on human neuroblastoma cells.

Retinoic acid induces REST degradation and neuronal differentiation by modulating the expression of SCF(β-TRCP) in neuroblastoma cells.

The repressor element‐1 silencing transcription factor (REST) is a repressor of neuronal genes. Its expression is associated with poor neuronal differentiation in many neuroblastoma patient samples and cell lines. Because retinoic acid promotes neuronal differentiation, the authors postulated that it involves modulation of REST expression.

Signaling of ERBB receptor tyrosine kinases promotes neuroblastoma growth in vitro and in vivo

ERBB receptor tyrosine kinases can mediate proliferation, migration, adhesion, differentiation, and survival in many types of cells and play critical roles in many malignancies. Recent reports suggest a role for EGFR signaling in proliferation and survival of neuroblastoma, a common form of pediatric cancer that often has an extremely poor outcome.