P> The focus of the CMBS is pediatric neuroectodermal tumors including neuroblastoma, brain tumors and Ewings sarcoma. RETINOIDS and 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 and 9cisRA in inhibiting growth and inducing differentiation, whether this compound has clinical utility remains to be evaluated. Previously we identified that retSDR1 a gene localized to chromosome 1p and lost in N-myc amplified NB(NB) is a retinal alcohol dehydrogenase is involved in retinoid metabolism. Current studies are aimed at evaluating retinoid metabolism in NB cells and evaluating whether alterations in retinoid levels contribute to tumorigenesis. N-myc and cell cycle We have published 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 its phosphorylation by cyclinE-dependent kinases which 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. Recently we published that NGF activation of TrkA causes a transcriptional decrease in N-myc levels via activation of the MAPkinase path. Furthermore the decrease in N-myc levels causes a decrease in cell cycle progression that is mediated by decreases in Cyclin E dependenet kinases and E2F transcription factors as well as an increase in p27. 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 blocks the effects of chemotherapy in low TrkB-expressing cells while low does of BDNF 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 Recent studies have further mapped the signaling molecules mediating BDNF/TrkB rescue of NB cells from chemotherapy and used genetic means to find that AKT can substitute for rescuing NB cells from death induced by chemotherapy and that a dominant negative AKT can block BDNF's ability to rescue cells. We have used a novel phosphatidylinositol ether lipid analogue,PIA, which targets and block the activity of AKT and demonstrated an ability of PIA6 to overcome BDNF induced resistance to chemotherapy. Pre-clinical Trials- Our previous studies on the differentiative effects of Interferon gamma (INFg)as well as a clinical trial using INFg to increase Class I MHC expression led to our finding that INFg increased caspase 8 expression. Caspase 8 is the apical caspase in the Death Receptor mediated path. We have found that INFg does induce caspase 8 in 8/15 NB cell lines examined yet this only restored TRAIL sensitivity in 1/8 cell lines. After a careful examination of a large number of intermediaries in the Death Receptor path, we identified that a lack of membrane TRAIL receptors, DR1/2 was the lesion that correlated with TRAIL resistance. A histopathologic examination of TRAIL receptors in 16 NB tumor tissues (in collaboration with Dr. M. Tsokos) indicated that none express DR1 and only 2/16 express DR2. Treatment of NB cells with INFg and TRAIL induced apoptosis in NB cells transfected with DR1 but not a control vector. The DR1 promoter contains a p53 response element such that treatment with chemotherapy increases expression of DR1.We showed that by treating cells with chemotherapy you can increase expression of surface membrane TRAIL receptors and in combination with INFg this can render cells sensitive to TRAIL induced cell death. Furthermore we identified that systemic treatment of NB patients with INFg could lead to increases in caspase 8 in their tumors. The importance of this finding was the identification of an additional lesion in the TRAIL Death Receptor Path in NB cells and formulation of a clinically feasible strategy to reconstitute TRAIL sensitivity in NB cells. Our previous studies indic 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-kit 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.
