N-myc amplification and deregulated expression plays a crucial role in determining the clinical behavior of neuroblastoma (NB). Similarly in many NB cell lines, tumorigenicity has been shown to directly correlate with N-myc expression levels. The differential levels of N-myc expression observed in the phenotypically distinct subclones of the NB cell line BBL-W (W-N and W-S), appears to be largely due to cellular disparities in N-myc mRNA turnover. Unlike c-myc and c-fos, little is known about N-myc mRNA metabolism. The turnover of other unstable mRNAs appears to be a consequence of interactions between RNA-binding proteins and 3' untranslated region (3'UTR) AU-rich elements. RNA binding studies performed with W-N and W-S cells demonstrate differential activity of a 40-kDa protein (p40) that binds with high specificity to AU-rich sequences within the N-myc 3'UTR. Immunoprecipitation experiments indicate that p40 is a member of the ELAV-like RNA-binding protein family. It is, therefore, possible that ELAV-like proteins regulate N-myc mRNA turnover, and thereby, modulate the steady-state levels of N-myc expression and NC phenotype. The focus of this study will be to understand the cellular control of N-myc mRNA degradation in NB, and examine the role ELAV-like proteins play in determining NB phenotype. The first specific aim of this proposal is to functionally characterize the N-myc 3'UTR AU-rich sequences that bind p40. Chimeric mRNAs I which N-myc 3' UTR sequences replace the 3'UTR of stable mRNAs will be expressed in NB cells. The decay kinetics ofmRNAs containing mutated N- myc 3'UTR binding sits will be compared to that of non-mutated mRNA, mRNA turnover will also be studied using chimeric transcripts generated by domain interchange between the well characterized c-fos 3'UTR instability elements (which are known to interact with ELAV-like proteins) and the N-myc 3'UTR binding sites. The second specific aim is to study N-myc mRNA decay using in vitro systems in the presence and absence of competitor N-myc RNA fragments. The third specific aim is to characterize the role of ELAV-like proteins in N-myc mRNA turnover and NB phenotype in vivo. ELAV-like protein expression will be altered in NB cells by exogenous gene transfer or by the addition of anti-sense ELAV mRNA or oligomers. N- myc mRNA half-life and tumor cell phenotype and growth potential will be examined in NB cells with enhanced or down- regulated ELAV-like protein expression. In addition, ELAV-like protein expression will be examined in primary NB tumors by Western blot analysis, and the relationship between level of expression and clinical behavior in vivo will be analyzed. A better understanding of N-myc mRNA degradation may lead to novel strategies by which N-myc expression and NB phenotype can be altered. Thus, ultimately, these studies may provide the clues necessary for the development of effective therapy for children with NB tumors that express high levels of N-myc and are clinically aggressive.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA074824-02
Application #
2712882
Study Section
Pathology B Study Section (PTHB)
Program Officer
Marks, Cheryl L
Project Start
1997-08-01
Project End
2001-05-31
Budget Start
1998-06-01
Budget End
1999-05-31
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Northwestern University at Chicago
Department
Pediatrics
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Manohar, Chitra F; Short, Marc L; Nguyen, Anthony et al. (2002) HuD, a neuronal-specific RNA-binding protein, increases the in vivo stability of MYCN RNA. J Biol Chem 277:1967-73
Katzenstein, H M; Salwen, H R; Nguyen, N N et al. (2001) Antiangiogenic therapy inhibits human neuroblastoma growth. Med Pediatr Oncol 36:190-3
Cohn, S L; London, W B; Huang, D et al. (2000) MYCN expression is not prognostic of adverse outcome in advanced-stage neuroblastoma with nonamplified MYCN. J Clin Oncol 18:3604-13
Huang, D; Rutkowski, J L; Brodeur, G M et al. (2000) Schwann cell-conditioned medium inhibits angiogenesis. Cancer Res 60:5966-71
Huang, D; Rutkowski, J L; Brodeur, G M et al. (2000) Schwann cell-conditioned medium inhibits angiogenesis in vitro and in vivo. Med Pediatr Oncol 35:590-2
Katzenstein, H M; Rademaker, A W; Senger, C et al. (1999) Effectiveness of the angiogenesis inhibitor TNP-470 in reducing the growth of human neuroblastoma in nude mice inversely correlates with tumor burden. Clin Cancer Res 5:4273-8