FUNCTIONAL STUDIES OF microRNAs IN DEVELOPMENT AND CANCER

Sammanfattning: MicroRNAs (miRNAs) COMPRISE a large family of small (~23 nucleotide in length), endogenous RNAs that regulate gene expression at the posttranscriptional level. Functional studies have indicated that miRNAs participate in the regulation of nearly all cellular processes investigated so far, including differentiation, apoptosis, and proliferation. Further, the deregulation of miRNA expression greatly contributes to human diseases, and is associated with many human pathologies, such as cancer. The studies in this thesis have focused on miRNA expression and regulation in various forms of malignancies. Specifically, we wanted to provide mechanistic insights into the role of miRNAs in tumorigenesis. In parallel, we hoped to discover new therapeutic targets that could be exploited clinically to treat childhood and adult cancer. In the work presented, we describe the functional consequences of miRNA perturbations in three distinct neoplasias: (1) chronic lymphocytic leukemia (CLL), the second most common type of blood cancer in adults; (2) neuroblastoma (NB), an embryonal malignancy of the sympathetic nervous system that is derived from primordial neural crest cells and occurs almost exclusively in infants and young children; and, (3) basal cell carcinoma (BCC), a basal cell-derived malignancy of the epidermis, which ranks as the most commonly diagnosed human cancer among fair-skinned individuals. Our CLL studies revealed that the DLEU2 transcript functions as a regulatory host gene for the miRNAs miR-15a and miR-16-1. These miRNAs were shown to target the G1 cyclins D1 and E1 for translational repression, resulting in a prominent cell cycle arrest. Further, ectopic expression of DLEU2 inhibited the colony-forming capacity of tumor cell lines, suggesting a tumor-suppressive function for miR-15a and miR-16-1. We also demonstrate that DLEU2 is transcriptionally regulated by the oncoprotein c-MYC, providing a novel mechanism by which MYC can regulate the G1 cyclins in a posttranscriptional manner. Functional loss of DLEU2 may thus constitute an important step in CLL tumorigenesis and various c-MYC-dependent cancers. In our analysis of MYCN-amplified neuroblastoma (NB), we investigated the molecular consequences and functional outcome of abnormal miRNA regulation and discovered that miR-17~92 cluster-derived miRNAs potentiate the tumorigenic behavior of this childhood cancer. Importantly, we could show that miR-18a and miR-19a target and repress the expression of estrogen receptor-? (ESR1), a ligand-inducible transcription factor implicated in neuronal differentiation. We propose that ESR1 represents a previously undescribed MYCN target in NB and demonstrate a unique oncogenic circuitry in which the repression of ESR1 through MYCN-regulated miRNAs may play a fundamental role in NB tumorigenesis. Finally, based on our genome-wide miRNA expression analysis of a non-melanoma skin cancer, we found that the skin-specific miRNA, miR-203, is preferentially lost in BCC. Functional analyses demonstrated that the inappropriate activation of the Hedgehog and MAPK pathways in BCCs may contribute to cancer progression via severely reduced expression of miR-203, which dramatically facilitates the misexpression of genes involved in the regulation of cell proliferation and cell cycle, including c-JUN and c-MYC. In this respect, miR-203 constitutes a gatekeeper miRNA controlling keratinocyte proliferation. The molecular reconstitution of miR-203 could therefore serve as a novel therapeutic strategy in the treatment of BCC tumors.