The role of LIN28B and XRN2 on microRNA regulation in neuroblastoma.

Neuroblastoma is a pediatric cancer whose heterogeneous nature is reflected in clinically favorable and unfavorable tumor subtypes, with tumors having MYCN amplification or loss of chromosome l l q being associated with particularly poor patient survival. In particular, the severity of deregulation caused by the aberrant expression of MYCN means that it remains the most important genetic prognostic marker of the disease, despite advances in the field. This thesis examines signals up and down-stream of MYCN in an attempt to further elucidate its role in neuroblastoma pathogenesis.

Previously, microRNA profiling of a set of 145 primary neuroblastoma tumors identified a miRNA signature deregulated as a result of MYCN amplification and predictive of clinical outcome in neuroblastoma. Following on from this work it was decided to examine signals that could in turn regulate MYCN expression. This led to a refined analysis of the let-7 family of miRNAs, all of which were predicted to target the 3' UTR of MYCN mRNA.

Results of this analysis provide the first evidence that the down-regulation of several let-7 family members are significantly associated with poor patient EFS and OS in neuroblastoma. Multivariate analysis then demonstrated that a signature comprising the entire let-7 family was predictive of patient survival independent of patient age and disease stage. Moreover, ectopic up-regulation of let-7a significantly impeded cell viability of both MYCN amplified and non-amplified neuroblastoma cell lines. It was also determined that the let 7 family (with the exception of let-7e and miR-98) exhibit coordinate expression across the tumour cohort. This is likely a consequence of LIN28B, a known inhibitor of primary and/or pre-let-7 processing, which is up-regulated in tumors from patients with poor survival. Another key element in this signaling pathway was discovered after llqLOH tumours were identified as the tumour subtype with lowest expression of the let-7 family. The expression of mir-125b, which maps to the deleted region on l l q , was positively correlated with let-7 and inversely correlated with LIN28B. Validation of LIN28B as a direct target of miR-125b explained, at least in part, LIN28B up-regulation in llqLOH tumours. It is also of note that expression of the let-7 family was lower in MYCN tumours compared to all other tumours lacking llqLOH. Results strongly indicate MYCN is not regulating LIN28B but is in fact downstream of LIN28B. Therefore, it is the blocking of the let-7 family by LIN28B that causes an up-regulation of MYCN. We conclude from this work that let-7 family members act as tumor suppressors in MYCN amplified and l lq - neuroblastoma tumor subtypes, although each of the tumor subtypes appear to differ in their endogenous levels of these miRNAs.

Additionally, this thesis investigates the mechanism underlying the widespread down-regulation of miRNAs observed in MYCN amplified tumours. Previous work by Chartterjee et al., which demonstrated that XRN2 can degrade a specific subset of miRNAs in Caenorhabditis elegans, led to the examination of this exoribonuclease in neuroblastoma. The data demonstrates that XRN2 is highly expressed in high risk tumours, and significantly over-expressed in the MYCN amplified tumour subtype. Direct binding of MYCN to the promoter region of XRN2 was confirmed by ChlP-sequencing data, and positive regulation of XRN2 was demonstrated using the MYCN repressible SHEP-21N cell line. Subsequent miRNA profiling of XRN2 silenced cells demonstrated the up-regulation of a set of tumour suppressive miRNAs (miR-330, miR-143, miR-149, miR-148b, miR-140, and miR- 26a), which were down-regulated in MYCN amplified tumours. Consistent with this finding, silencing of XRN2 resulted in a significant decrease in cell proliferation in the MYCN amplified Kelly and in the non MYCN SKNAS cell lines. The data demonstrates that all-trans-retinoic acid (ATRA), a compound that causes differentiation in many neuroblastoma cell lines, reduces XRN2 protein levels. In contrast, XRN2 silencing produced a more differentiated phenotype in SHSY5Y, suggesting that XRN2 may be involved in the ATRA-induced differentiation of neuroblastoma cell lines.

This thesis hypothesises that the aberrant expression of LIN28B and XRN2 has contributed to the down-regulation of tumour suppressor miRNAs in neuroblastoma, each of them acting at different levels during miRNA biogenesis:

1. Over-expression of LIN28B in high-risk tumours is responsible for the inhibition of the processing into mature miRNA of the let-7 family members.

a. The expression of LIN28B is increased in llqLOH and MNA tumours. In llqLO H tumours, the loss of miR-125b contributes to increased expression of LIN28B as it directly regulates LIN28B protein expression.

b. In turn, over-expression of LIN28B contributes to enhanced MYCN expression through the down-regulation of the let-7 members as this family of miRNAs directly regulates MYCN expression posttranscriptionally.

c. Expression of LIN28B is a prognostic factor of survival independent of MYCN status, patient age and stage. Similarly, the down-regulation of a let-7 expression signature is predictive of poor survival in neuroblastoma tumours.

2. The 3'-5' exoribonuclease XRN2 is responsible for the degradation of a subset of mature miRNAs that might act as tumour suppressor miRNAs and be involved in differentiation:

a. MYCN promotes XRN2 expression

b. XRN2 knockdown results in the up-regulation of a subset of miRNAs that are significantly down-regulated in MNA tumours and are associated with poor survival.

This thesis hypothesizes that XRN2 is the effector of MYCN driven down-regulation of a specific subset of miRNAs.

The findings presented in this thesis demonstrate how deregulation of the LIN28-let-7-MYCN pathway contributes to the enhanced expression of MYCN and neuroblastoma progression. The elucidation of this pathway might offer new opportunities for pharmacological intervention and new therapeutic approaches for neuroblastoma. In addition, the identification of the 5'-3' exoribonuclease XRN2 as the causative agent for the decay of mature tumour suppressive miRNAs opens new perspectives in the field of miRNA stability and the mechanisms of posttranscriptional gene regulation, as well as a mechanism underlying the downregulation of some tumour suppressor miRNAs in neuroblastoma pathogenesis.