Carcinogenesis is a multistep process (1). Cellular transformation can result from interactions between oncogenes, growth factors, growth factor receptors and growth suppressor genes (2). Autocrine production of growth factors, secondary to a loss of tumor growth suppressor gene activity, is one mechanism which allows malignant cells to maintain an unregulated proliferative state. One candidate growth factor, insulin-like growth factor II (IGF-II), can mediate autocrine growth of several malignancies (3,4), including Wilms tumor (5). Indeed, research on Wilms tumor has given insight to a potential mechanism underlying constitutive IGF-II production. The major fetal IGF-II promoter contains multiple binding domains for wt1, a zinc finger protein and member of the family of tumor growth suppressor genes (6). wt1, present in urogenital tissues (7), is a potent repressor of IGF-II transcription (8). Loss of IGF-II repression secondary to wt1 dysfunction is associated with the development of Wilms tumor (9,10). The objective of this proposal is to examine the role of IGF-II and potential IGF-II dysfunction in the growth regulation of a different cancer: human neuroblastoma (NBL). NBL is the second most common solid tumor in childhood (11) and accounts for 10% of all juvenile cancer (12). Histopathological examination of tumors reveals multiple neoplastic neural crest cell types, including neuroblasts, melanocytes, glial cells and chrondrocytes (13). Research on NBL has focused on the development of cell lines established from different NBL tumors (14). The SK-N-SH NBL cell line was derived from a 4 year old girl one month before her death (15). Analysis of clonal sublines of SK-N-SH reveal two distinct cell types: the SH-SY5Y clone consisting of N cells, or presumptive neuroblasts with small round cell bodies and neuritic processes (16) and the SHEP clone comprised of flattened substrate adherent cells with either glial, epithelial or melanocyte-like properties (16). SH-SY5Y cells exhibit serum independent growth, multiple in soft agar and form tumors in nude mice (16). In contrast cells, SHEP cells require serum and are unable to multiple in agar or form murine tumors(16). The current proposal focuses on the autocrine production of insulin-like growth factor-II (IGF-II) by NBL cells as one event in carcinogenesis . In this model, N cells (SH-SY5Y cells) produce IGF-II which acts as an autocrine N cell growth factor as well as a paracrine growth factor for S cells (SHEP cells), which do not produce IGF-II. These putative autocrine and paracrine loops are mediated via the type I IGF receptor and represent unregulated production of IGF-II secondary to loss of tumor growth suppressor gene activity. This proposal has three specific aims: 1. Determine if IGF-II, acting via the type I IGF receptor, can support the growth requirements of SHEP cells. 2. Determine whether deregulated expression of IGF-II alters the phenotype of SHEP cells. 3. Identify potential nervous system specific growth suppressor genes. Results gained from these studies are of definite clinical importance. Therapies aimed at interrupting the NBL IGF-II autocrine and paracrine loops may stop growth in all NBL cell types (17). Strategies include inhibiting both the ligand and receptor, using neutralizing antibodies, blocking antibodies, modified oligonucleotides or compounds which indirectly block IGF-II action, such as suramin (18). Clearly, anti-growth factor therapy, targeted at specific genes, has both theoretical and practical appeal in the treatment of NBL.
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