Investigation of Cell Growth and Differentiation Pathways in Neuroblastoma

Neuroblastoma (NB) is a pediatric malignancy of the sympathetic nervous system and is derived from neural crest stem cells (NCSC). Over half of all patients present with metastatic disease with less than 50% surviving, despite aggressive multi-modal treatments including retinoic acid (RA) differentiation therapy. In my thesis studies, I used two approaches to identify novel genes and pathways involved in NB cell growth and differentiation. First, I identified genes that are differentially expressed between NCSCs and differentiated cells resident to the embryonic rat dorsal root ganglia (DRG), and prioritized those that correlated with patient outcome for further study. The expression of Kruppel-Like Factor 5 (KLF5) is increased in differentiated cells of the DRG and high expression of KLF5 correlates with favorable prognosis in NB patients. KLF5 was expressed in a panel of NB cell lines, was post-translationally modified selectively in cells with amplification of the MYCN oncogene, modified KLF5 protein forms were found in the nucleus and shRNA-mediated knockdown of KLF5 expression reduced apoptosis and increased proliferation in these cells. The second part of my thesis used an in-silico approach to identify small molecules that induced a gene expression profile similar to RA. I identified 4-hydroxytamoxifen (4HT), the active metabolite of the breast cancer therapeutic, tamoxifen, and demonstrated that it reduced NB cell growth and induced apoptosis in NB cell lines. 4HT also reduced NB xenograft tumour growth and increased survival relative to vehicle control. I further demonstrate that 4HT inhibits the transcriptional activity of the estrogen related receptor gamma (ERR) and shRNA mediated knockdown of ERR reduces NB cell growth in vitro. 4HT also induces the expression of cholesterol biosynthesis pathway enzymes, cellular cholesterol, and combination with the cholesterol biosynthesis inhibitor, fluvastatin, further reduces NB cell growth in vitro, identifying novel drug combinations for the treatment of NB.Ph.D.2019-11-19 00:00:0