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RNA-seq analysis revealed the upregulation of p21 gene expression in neuroblastoma cells treated with FTY720 and Fenretinide
Mei-Hong Li, PhD1, Carol Bult, PhD2, Kathy Herbst, MS3, Linda Shapiro, PhD1, Fernando Ferrer, MD3.
1University of Connecticut Health Center, Farmington, CT, USA, 2The Jackson Laboratory, Bar Harbor, ME, USA, 3Connecticut Children's Medical Center, Hartford, CT, USA.

BACKGROUND: Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. Poor outcomes for children with advanced disease underscore the need for novel therapeutic strategies. Our prior study as well as others has shown that FTY720, a FDA-approved multiple sclerosis drug, has potent preclinical anti-cancer activity in various cancers including NB. However, FTY720 as a combination therapy with fenretinide (4-HPR), a prevalent anti-cancer drug in current clinical trial for NB, has not been reported. Herein for the first time we tested the combination therapy with FTY720 and 4-HPR in NB cells and determined the integral transcriptional changes triggered by these treatments in order to better understand how both drugs work in NB and further to develop novel molecular targets against NB.
METHODS: Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was performed to assess the effect of FTY720, 4-HPR and their combination on the cell viability in both SK-N-BE(2) and SK-N-AS NB cells. RNA from samples were isolated and sequenced to yield around 50 million reads per sample. Sequencing reads were checked for quality and those that passed quality filtering were aligned to the reference genome using RNA-Seq by Expectation-Maximization program. Differential gene expression was then estimated using the DESeq package followed by the Ingenuity Pathway Analysis (IPA), further confirmed by real-time PCR and western blot analysis.
RESULTS: FTY720 or 4-HPR alone significantly inhibited NB cell viability while their combination showed strong synergistic inhibitory effects in both NB cell lines. DESeq analysis from SK-N-BE(2) cells treated with FTY720 and 4-HPR showed that 309 out of ~23000 genes had the lowest adjusted p value (p=0) and the biggest induction/ reduction fold changes (Fold >5 or <0.2). Among them, IPA analysis indicated that the gene p21 and its related signaling pathway was one of the major potential targets that the combination therapy affected. Real-time PCR and western blot analysis further confirmed that p21 was significantly induced by either FTY720 or 4-HPR and the combination therapy further enhanced its expression in both NB cell lines.
CONCLUSIONS: Our data for the first time demonstrated that combination therapy with FTY720 and 4-HPR had strong anti-cancer activity in NB cells via induction of p21 expression, which suggests that p21 might be a potential anti-cancer target for NB.


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