Development of novel therapeutic agents for neuroblastoma through iron and cholesterol metabolisms study Sang Y. Lee, Ph.D. Assistant Professor Department of Neurosurgery Penn State College of Medicine

Neuroblastoma Neuroendocrine tumor arising from any neural crest element of the sympathetic nervous system Approximately 10% of all childhood cancers. Each year, approximately 650-700 new cases of NB occur in the US The second most common solid cancer in children The most common cancer in infants (20~50% of infant cancers) Account for 15% of childhood cancer mortality

Treatment of Neuroblastoma Based on risk categories Low risk patients --- frequently be observed w/o any treatment at all or cured with surgery alone Intermediate risk patients --- surgery and chemotherapy High risk patients --- intensive chemotherapy, surgery, radiation therapy, bone marrow/hematopoietic stem cell transplantation, biological based therapy with 13-cis-retinoic acid (isotretinoin or Accutane), and monoclonal antibody therapy (chimeric 14.18 antibody) Cure rate Low risk patients --- 90% Intermediate risk patients --- 70-90% High risk patients --- 30-40%

Hypothesis & rationale Cancer cells need more iron and cholesterol than normal cells for their rapid growth and building block of cell membrane C282Y HFE expressing NB and drug resistant NB cells have altered iron and cholesterol concentrations, due to altered expression of proteins needed for their metabolism

HFE genotype of NB cell lines H63D C282Y Drug resistance BE(2)-C wildtype Yes IMR-32 SH-SY5Y SK-N-AS homo SK-N-DZ SK-N-F1 SK-N-MC CHLA-136 CHLA-171 Cell line claimed cell type Contaminating cell line Actual misidentification reported by SK-N-MC neuroblastoma unknown sarcoma (Ewing's) Staege et al. 2004

Iron level of NB cells A. B. C. ** ** *** ** *** HFE mutant/SH-SY5Y cells have higher iron B. *** C. CHLA-171 NB cells have higher iron at early times

Cholesterol level of NB cell lines A. B. ** *** *** ** CHLA-171 NB cells have higher cholesterol at early culture period C282Y/SH-SY5Y cells showed increased cholesterol 7

Effect of HFE siRNA on the cholesterol level CHLA-171 NB cells (1 day) ** *** off-target effect

Effect of cholesterol inhibitors in the NB cells * *

Effect of serum stress in the NB cells   10% or 20% FBS 1% FBS CHLA-171 cells are more sensitive to serum stress than other NB cells

Effect of simvastatin on the NB s.c. tumor model Control group Simvastatin treated group Tumor weight Average/SE: 2.70.7 gram Tumor weight Average/SE: 1.60.5 gram

Conclusions Iron and cholesterol in NB cells during the time they were cultured Iron and  cholesterol in HFE stably transfected NB cells (Association between HFE expression level and cholesterol level?) Iron level affects cholesterol level of NB cells Drug resistant NB cells (SK-N-AS, CHLA-171) are sensitive to simvastatin CHLA-171 cells are sensitive to the serum stress Mild anti-tumor effect at 20 mg/kg simvastatin (3x/week, oral gavage) in SK-N-AS sc NB tumor model