The focus of the CMBS is pediatric neuroectodermal tumors including neuroblastoma, brain tumors and Ewings sarcoma. Retinoids & NB Retinoic Acid(RA) induced differentiation of neuroblastoma tumor cell lines continues to be our model for studies that define the signal transduction paths that mediate growth control, differentiation and cell death. Using a series of receptor selective retinoids ligands we found that RAR and RXR nuclear receptors are required to mediate maximal growth inhibiting and differentiating effects of retinoids. The most active combinations were ligands that activated RARbeta;RXR heterodimers followed by ligands activating RARalpha:RXR and RARgamma:RXR. We identified one agonist that was more potent than RA & 9cisRA in inhibiting growth and inducing differentiation, whether this compound has clinical utility remains to be evaluated. Differential display was utilized to identify genes that may be selectively activated by these agonists. One candidate differentially displayed gene is retSDR1, a retinal alcohol dehydrogenase which is involved in retinoid metabolism, and localizes to chromosome 1. We have found that one allele is deleted in Neuroblastoma cells with N-myc amplification. Our current working hypothesis is that the loss of this enzyme may render NB precursor cells insensitive to physiologic concentrations of retinoids and particularly compromise NB cells if physiologic concentrations are low as cells will have a redulced capacity to make storage forms of retinoids. Current studies are aimed at evaluating the metabolism of retinoids in NB cell lines and determining whether there are allelic polymorphisms or variations in retSDR1 that may compromise its activity. N-myc and cell cycle We have identified that N-myc and p27 levels are key in the ability of retinoids to arrest NB cell cycle in G1. We have determined that Nmyc over-expression increases cyclinE and cyclin E dependent kinases and decreases p27 levels. The decrease in p27 levels is due to cyclinE-dependent kinases that phophorylate p27 enabling it to be recognized by Skp2 in the SCF complex enabling its ubiquitination and degradation by the proteosome. Retionids cause a G1 arrest of cell growth in NB cells by inhibition of G1 cyclin-dependent kinases. The decrease in kinase activity is caused by an increase in p27kip and its binding to G1 cyclin-dependent kinases. The increase in p27 is not transcriptionally regulated. In R A treated NB cells as Nmyc decreases there is a decrease Cyclin E-dependent kinases and in phospohorylation of p27. Concurrently RA induces a decrease in SKP2 levels thus completely blocking the targeting of p27 to the proteosome. Thus during RA induced G1 arrest of NB cell cycle there is a N-myc dependent and N-myc independent changes in the targeting of p27 to the proteosome that enables levels to rise and block cell cycle progression. Trks and NB Biology In NB tumors, Trks serve as tumor markers; TrkA is expressed in good prognosis tumors and most poor prognosis tumor express TrkB. We have found that the differential activation of these signal transduction pathways in NB may alter their growth, invasiveness, chemosensitivity and cell survival. Neuroblastoma (NB) tumors expressing high levels of BDNF and TrkB are associated with poor 5-year survival outcomes. Our previous studies indicated that BDNF blocked the cytotoxic effects of vinblastine on NB cells. We evaluated the ability of BDNF to decrease the chemosensitivity of NB cells to a number of common chemotherapeutic agents and mapped the BDNF signaling system that mediates the chemoprotective effect. Two SH-SY5Y NB cell lines (TB3, TB8) expressing TrkB under the control of a tetracycline (Tet)-repressible promoter element were isolated, and used to assess apoptosis resulting from treatment with cisplatin (Cis), doxorubicin (Doxo), etoposide (Etop), and vinblastine (Vbl). BDNF treatment of high TrkB-expressing TB8 (Tet-) and TB3 (Tet-) cells blocked drug-induced cell death in a dose-dependent manner. Only high dose BDNF (100 ng/ml) blocked the effects of chemotherapy in low TrkB-expressing cells while low does of BDNF (33ng/ml) altered chemosensitivity in high TrkB-expressing cells. The inability of NGF to protect cells indicated that activation of p75 alone was not responsible for the chemoprotective effect. BDNF's ability to rescue the cells is TrkB dependent since it is blocked by the selective Trk tyrosine kinase inhibitor K252a. The PI3-kinase inhibitors LY294002 and Wortmannin but not the MEK inhibitor PD98059 or the PLC-gamma inhibitor U73122 block the ability of BDNF to rescue cells from chemotherapy indicating that downstream targets of PI3-kinase are required for BDNF rescue. BDNF also protected NGP and KCNR NB cells expressing endogenous TrkB receptors from chemotherapy induced death and inhibition of the PI3-kinase path could abrogate this effect. These results indicate that BDNF activation of TrkB via the PI3-kinase path protects NB cells from chemotherapy and that by specifically inhibiting the TrkB TK and/or PI3-kinase paths one may improve the chemosensitivity of NB cells. Pre-clinical Trials-MS-27-275 & STI571 A number of pediatric tumor have alterations in transcription factors. These mutant transcription factors may stimulate or repress genes important in controling cell growth and differentiation. We have studied an inhibitor of histone deacetylation, MS-27-275 and found it is active in a number of pediatric tumor cell lines in vitro. Ewing's sarcoma cells are characterized by a t(11;22) translocation that creates a chimeric transcription factor EWS/Fli. We have found that the EWS/Fli represses transcription of TGFB RII so that the TGFB signaling path is not active in these cells. We find that MS-27-275 increases TGFBRII and this may be due to the transcriptional relief of the EWS/Fli repression of the TGFBRII promoter. Thus MS-27-275 may target molecular alterations in Ewing's sarcoma.We have studied the activity of this drug in animal models and found that it is active in inhibiting the growth of sarcomas and neuroblastomas. In studying the biology of NB and Ewings' sarcoma tumors we have noted that the survival of selected tumor cell lines is dependent on an autocrine Stem Cell Factor/c-kt autocrine loop. Using the abl, c-kit, PDGFR tyrosine kinase inhibitor, STI571 we have found that the growth of cell lines derived from these tumors is significantly decreased. However IC50 dose required to inhibit cell growth was a log higher than the dose required to inhibit the c-kit or PDGFR kinase activity. This indicates that STI571 inhibits the growth of these tumor types by a novel mechanism. STI571 also inhibits the growth of established NB and EWS tumors in orthotopic xenograft models. Ulip/Tuc-4/CRMP-4 As part of a project to identify genes that marked differentiated NB cells that may have clinical utility as tumor markers, the CMBS has isolated a retinoid regulated gene 37G1 that has homology to unc-33 a C. elegans gene important in neuronal pathfinding. We published the cDNA sequence, mRNA and protein analysis and regulation in neuroblastoma. Antibodies to hUlip mark differentiated ganglionic cells in neuroblastoma tumors and brain tumors. HUlip also marks gemistocytic cells in astrocytic tumors and glioblastoma as well as all the cellular components of the subependymal giant cell astrocytoma (SEGA). Both the human and mouse genes have been isolated; the human gene maps to 5q31 and contains 14 exons spanning over 50kb. The murine and human promoters of Ulip have been isolated and characterized and contain a highly conserved set of transcription regulatory sites that are being tested for tissue specific expression. Using the murine gene, constructs for knocking out Ulip have been made and Ulip +/- mice have been made. We currently have 2 Ulip colonies of mice in the SJ/129 strain and into 6 generations on a C57Bl background. In both strains there have been no detectable alterations in expected breeding patterns. Animals do not have gross morphologic alterations. An analysis of Ulip-/- mice will lead to better understanding of the effects of loss of Ulip during development. Since the KO construct contains a B-galactosidase expression vector we have been able to study the developmental expression of Ulip during embryogenesis. We have found that although Ulip is widely expressed in neural cells fetal development in adults Ulip marks adult neural progenitor cells.
