Functional analysis of miRNA in chemotherapy resistant neuroblastoma.

The acquisition of drug resistance is the principle obstacle to the successful treatment of many forms of cancer. This is particularly true for neuroblastoma, a highly heterogeneous tumor accounting for 15 % of all pediatric cancer deaths. Thus, the elucidation of mechanisms involving drug resistance in this tumor, along with the development of novel approaches for sensitizing tumor cells to therapy,

would be of great translational benefit. There are multiple mechanisms and genetic pathways involved in the acquisition of this phenotype, all of which are influenced by initiation treatment, often cisplatin in the case of neuroblastoma. The purpose of this work is to ascertain the contribution of genomic factors influencing drug resistance in neuroblastoma, specifically those induced by cisplatin. To this end, I have developed in vitro neuroblastoma models; SK-N-ASCis24, KellyCis32, KellyCis83 and CHP-212CislOO, significantly resistant to cisplatin and other agents and identified a novel panel of miRNA and chromosomal aberrations associated with the cisplatin drug-resistant phenotype.

A focal gain discovered on chromosome 5 of SK-N-ASCis24 contains a region encoding NAIP; the founding member of the inhibitors of apoptosis (IAP) family. Through modulation of NAIP I have confirmed a functional role in relation to drug resistance. I have also identified miR-520f, the down regulation of which correlates with up regulation of NAIP. The study further demonstrates that miR-520f directly targets the NAIP 3'UTR, and that up regulation of miR-520f results in a significant increase in sensitivity to cisplatin. In addition we show that lower levels of miR-520f expression are associated with tumours post-treatment. In conclusion, this work provides the first evidence that NAIP and miR-520f are functional constituents in a neuroblastoma drug-resistance model not influenced by the overexpression of MYCN. Additionally, it demonstrates for the first time a significant association between patients post-treatment and low expression of miR-520f.

In the MNA models KellyCis83 and CHP-212CislOO, enhanced proliferation rates were observed when grown in low doses of cisplatin. Through miRNA profiling and analysis a unique panel of miRNA were identified that were induced by low dose treatment and preliminary functional analysis indicates a role in proliferation. The observation of this drug inducible phenotype could have a significant impact in a clinical setting due to the fact that residual cisplatin can be detected in patients for long periods after treatment, suggesting cisplatin treatment could actually drive tumor growth. Although the precise mechanism involved remains to be fully elucidated, this thesis demonstrates for the first time, a link between miRNA and enhanced proliferation in response to low dose treatment.

Overall, this work provides important biological insight into neuroblastoma drug resistance which has significant implications for future development of novel therapeutics and the more efficient design of re-initiation treatment, sparing patients unnecessary rounds of chemotherapy and ultimately increasing survival.