Neuroblastoma is the most common extracranial solid tumor of childhood with an incidence of 9.6/million caucasians and 7.0/million blacks in the US. Spontaneous differentiation and regression of some tumors occurs in vivo; yet long term survival of children with neuroblastoma is only about 30%. Preliminary Data from our laboratory suggests a role for somatostatin in regulation of neuroblastoma cell growth. This proposal seeks to further understand the etiology of neuroblastoma by examining the role of the anti-growth factor, somatostatin, in proliferation and differentiation of this neural crest tumor. Initial experiments will characterize somatostatin receptor expression in neuroblastoma cell lines using RT-PCR to identify active transcription of receptor genes (SSR1, SSR2, and SSR3). Competitive binding techniques will be used to identify high affinity binding sites as a measure of functional receptor protein. Cytofluorometry with anti-receptor antibodies will be used to quantify membrane receptors. The expression of receptor mRNA and functional recaptor protein will be correlated with effects of various somatostatin analogs on proliferation rate of neuroblastoma cell lines in culture. These studies will thus determine whether somatostatin receptor expression can be used to define molecular phenotypes. Further experiments will examine the effect of differentiation agents, including retinoic acid, phorbol ester, forskolin, and nerve growth factor on expression of somatostatin receptors in various neuroblastoma cell lines in order to identify metabolites which might upregulate somastostatin receptor expression. Molecular techniques will also be utilized to upregulate SSR1, SSR2, and SSR3 expression; plasmid or retroviral constructs will be developed for stable expression of somatostatin receptor in neuroblastoma cell lines. A neuroblastoma cell line which lacks high affinity binding of somatostatin will be transfected and somatostatin receptor gene expression verified by both reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and high affinity binding of somatostatin. Down-regulation of SSR1, SSR2, and SSR3 will be attempted using antisense oligodeoxyribonucleotides. Functional experiments will examine the effect of somatostatin and stable analogs of somatostatin on growth of these transformed neuroblastoma cell lines in culture. These experiments will compare the effect of somatostatin on growth of neuroblastoma cells which constitutively express somatostatin receptors with growth of receptor negative cells. Finally, the results of these experiments will be translated into studies of primary neuroblastoma tumors surgically excised before therapy. Somatostatin receptor expression will be examined by both RT-PCR and high affinity binding; results will be correlated with Stage of disease and survival. Human neuroblastoma tumors will be propagated in a xenograft model in which somatostatin receptor expression will be upregulated followed by therapeutic administration of somatostatin analogs to control tumor growth. We anticipate this basic research will ultimately translate into a molecular classification of neuroblastoma and used to design more effective therapies for this pediatric tumor.
