Antiangiogenic strategies are currently in use for the treatment of cancer, and the inhibition of angiogenesis could represent a powerful adjunct to traditional therapy of malignant tumors. Over the last 20 years, several reports have been published concerning the relationship between tumor progression and angiogenesis in neuroblastoma (NB) in experimental models in vitro and in vivo. Moreover, a high vascular index in NB correlates with poor prognosis, suggesting a dependence of aggressive tumor growth on active angiogenesis. Here, I present an overview of the literature concerning the antiangiogenesis approach in the treatment of NB.
KeywordsAngiogenesis, Antiangiogenesis, Neuroblastoma, Tumor growth
This work is dedicated to the memory of Dr. Vito Pistoia, a leading expert in this field.
Biological and Clinical Aspects of Neuroblastoma
Neuroblastoma (NB) belongs to a group of undifferentiated pediatric malignancies known as the small round-cell tumors of childhood. NB is the second most common solid tumor of infancy and childhood and arises from the primitive neuroepithelial cells of the neural crest. Thirty percent of NB tumors arise within the adrenal medulla, approximately 60% arise from the abdominal paraspinal ganglia, and the remaining is from the sympathetic ganglia in the chest, head/neck, and pelvis. NB can form relatively benign, localized and well-differentiated tumors successfully treated by surgical resection alone (stage I or II) or locally invasive (stage III) and metastatic (stage IV) tumors associated with a poor clinical outcome . FOX03-phosphorylation at threonine-32 (T32) and nuclear localization significantly correlate with stage IV disease .
However, in children less than 1 year of age, metastases limited to the bone marrow, liver or skin, but absent in bones (stage IV S) are associated with a favorable outcome. In some patients, the tumor may regress spontaneously, whereas, in other patients, it may differentiate into a benign ganglioneuroma after no minimal therapy . Genetic abnormalities, including amplification of the N-Myc oncogene, and other structural chromosomal rearrangements play a role in determining the tumor phenotype and predicting the outcome. The molecular and cytogenetic features of clinically aggressive NB differ from those observed in tumors associated with a good response to therapy.
The survival rate for patients with NB remains poor because the majority of children older than 1 year of age with advanced stage NB die from progressive disease and only 40% of children over 4 years old with NB survive for 5 years . Most patients with localized NB can be cured by surgery alone and most infants with an advanced-stage disease can be cured with moderate chemotherapy . However, for children older than 1 year with metastatic disease (high-risk cases), the outcome is usually fatal.
Angiogenesis in Neuroblastoma
Human NB cells induce angiogenesis in nude mouse . The vascularity of NB from patients with widely metastatic disease is significantly higher than in tumors from patients with local or regional disease . Cell suspensions from human NB cell lines stimulated in vitro endothelial cell proliferation and angiogenesis in vivo in the chick embryo chorioallantoic membrane (CAM) assay . Moreover, NB cells were frequently arranged in clusters and occupied a perivascular or intravascular position and also demonstrated immunohistochemically using a specific anti-human NB monoclonal antibody ( Fig. 5.1 ). Canete et al. in a retrospective study on 69 patients showed that tumor vascularity was not predictive of survival of patients and that neither disseminated nor local relapses were influenced by the angiogenic characteristics of the tumors. Vascular endothelial growth factor (VEGF) and VEGF receptors -1 and -2 (VEGFR-1 and -2) were expressed in human NB cell lines and primary human NB and VEGF expression was upregulated by hypoxia . Eggert et al. performed a systematic analysis of expression of angiogenic factors in 22 NB cell lines and in 37 tumor samples, and found that high expression levels of seven angiogenic factors [VEGF-A, VEGF-C, fibroblast growth factor-2 (FGF-2), angiopoietin-2 (Ang 2), transforming growth factor alpha (TGF-α) and platelet-derived growth factor-A (PDGF-A)], correlated strongly with the advanced stage of NB. The relationship of matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) and their specific inhibitor TIMP-2 expression pattern with clinical variables has been evaluated in 31 patients with NB by immunohistochemistry and increased expression of MMP-2 in stromal tissues of NB had significant association with advanced clinical stages . Decreased expression of TIMP-2 also had significant relationship with advanced clinical stages. The higher gelatinases activation ratio resulting from high expression of a membrane-type matrix metalloproteinase-1 (MT-MMP-1) on NB specimens was associated significantly with advanced stage and unfavorable outcome . Human NB cell lines secrete MMP-2 and MMP-9 and the extent of angiogenesis and the expression of the MMP-2 and MMP-9 were upregulated in advancing stages of human NB .
N-Myc amplification may also regulate the growth of NB vessels; N-Myc amplification or overexpression was associated with angiogenesis in experimental and clinical settings of NB. N-Myc amplification correlates with poor prognosis and enhanced vascularization of human NB, suggesting that the N-Myc oncogene could stimulate tumor angiogenesis and thereby allow NB progression. Moreover, N-Myc upregulates VEGF in NB cells , accordingly with the evidence showing a higher angiogenic response in vivo of N-Myc amplified NB specimens compare to non-amplified ones . Results from N-Myc amplified NB xenografts suggest that these tumors are relatively VEGF independent and upregulate alternative pro-angiogenic mechanisms, including the placental growth factor (PlGF) pathway . Zin et al. observed high PlGF expression in human advanced-stage NB, and PlGF downregulation significantly reduced NB growth in established NB xenografts by reducing cancer cell proliferation but did not suppress angiogenesis. VEGF-A and VEGF-D transcripts are moderately increased in the N-Myc amplified samples and soluble receptors sVEGFR1 and sVEGFR2 are downregulated .
TrkA expression inhibits tumor growth and down-regulates VEGF and FGF-2 in NB cell lines, whereas TrkB transfectants induced endothelial cell proliferation and migration . Immunohistochemical analysis showed that ανβ 3 integrin was expressed by 61% of microvessels in high-risk NB (stage IV and N-Myc amplified), but only by 18% of microvessels in low-risk tumors (stage I and II and non-N-Myc amplified) .
Tumor suppressor gene ΔNp63 promotes tumor growth in NB by promoting chronic proliferation and later on tumor angiogenesis by increasing secretion of interleukin-6 and -8 (IL-6 and IL-8) leading to elevated phosphorylation of STAT3 , Moreover, elevated phosphorylation of STAT3 induces stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), VEGF secretion .
STA-NB 15 tumors with ectopic FOX03 are angiogenic in vivo in the CAM assay and when xenografted into nude mice, a gene dosage-dependent effect of FOX03 in high stage STA-NB15 cells became evident: low-level activation increased tumor vascularization, whereas hyper-activation repressed tumor growth .
Antiangiogenesis in Neuroblastoma
Our knowledge on the mechanisms of angiogenesis in NB has provided the basis for a rational and targeted approach to the development of specific antiangiogenic approach in patients affected by NB.
TNP-470 . TNP-470, a synthetic analog of an antibiotic naturally secreted by Aspergillus fumigatus , induced a reduction in tumor growth rate, in microvascular density, and in the fraction of viable cells of a poorly differentiated human NB cell line xenografted in nude rats . TNP 470 treatment improved animal survival and reduced tumor growth of primary and metastatic murine NB . When TNP-470 was administered to animals with small tumors, the rate of growth reduced, while does not significantly altered the tumor growth rate when it was administered to animals with large tumors . Treatment with TNP-470 reduced the tumor growth, increased the tumor cell apoptotic fraction, and induced striking chromaffin differentiation of NB cells . Finally, TNP-470 significantly inhibited tumorigenicity when administered shortly after NB xenograft inoculation and when administered following cyclophosphamide .
Retinoids . Retinoic acid has been administered in stage IV patients after autologous stem cell transplantation as a maintenance treatment . The synthetic retinoid N-(4-hydroxyphenyl) retinamide (fenretinide, HPR) inhibits human NB cell growth through the induction of programmed cell death . HRP inhibited angiogenesis in vivo when VEGF- or FGF-2- loaded sponges or NB biopsies were implanted on the CAM surface ( Fig. 5.2 ). Moreover, the proliferative effect exerted on endothelial cells by NB cells-derived conditioned media was dose-dependently abolished when NB cells are preincubated in the presence of HPR . In children with advanced nonprogressive NB, 4HPR administration results in manageable toxicity and in drug plasma concentrations comparable with those that induce apoptosis in NB cell lines . More recently, Di Paolo et al. have designed a novel liposomal formulation to specifically deliver HRP to angiogenic vessels within the tumors, including NB. In this context, the Authors developed novel stabilized nanoliposomes decorated with NGR-motif peptides to enhance the antitumor activity of HRP on NB in vivo. The results showed that compared to the untargeted formulation, these vascular-targeted liposomes were more effective in triggering apoptosis of tumor cells, in reducing the number of tumor vessels ( Fig. 5.3 ), and in inducing a statistically significant increased mice lifespan .