Functional analysis of long non-coding RNAs in cancer

Sammanfattning: For years the functional role of noncoding RNAs was greatly underestimated. Whole genome RNA sequencing projects that unraveled pervasive transcription emitting from large parts of the human genome changed those perspectives and prompted scientists to further look into the involvement of noncoding RNAs (ncRNAs) in development and disease. To date, we have significantly advanced our understanding of ncRNAs, however only a fraction of them has been functionally characterized. The subject of this thesis is to investigate the functional role of long noncoding RNAs (lncRNAs) and their involvement in cancer, particularly in cutaneous malignant melanoma. Previously, the tumor suppressor gene PTEN has been reported to be post-transcriptionally regulated by its pseudogene. In paper I, we investigated an antisense RNA (PTENpg1 asRNA) that is transcribed from the PTEN pseudogene (PTENpg1). We uncovered various PTENpg1 asRNA isoforms and designated two of them as α and β. The role of the PTENpg1 asRNA β isoform is to form an RNA:RNA duplex with the PTENpg1 transcript. This interaction stabilizes and assists the PTENpg1 transcript out to the cytoplasm. On the other hand, the PTENpg1 asRNA α has a very different function, namely mediating epigenetic changes by recruiting EZH2 and DNMT3a to the PTEN promoter. In paper II, we further sought out to understand the recruitment of PTENpg1 asRNA α to the PTEN promoter. We observed promoter-associated/5´UTR transcript emitting from PTEN, which binds and facilitates the recruitment of PTENpg1 asRNA α to the PTEN promoter. In return, PTENpg1 asRNA recruits DNMT3a to the promoter, which leads to epigenetic silencing of PTEN. In paper III we investigated the role of PTENpg1 asRNA in vemurafenib resistance of melanoma. We observed increased PTENpg1 asRNA expression and consequently low PTEN levels caused by enrichment of EZH2 and H3K27me3 at the PTEN promoter. Further, we found that C/EBPβ transcriptionally induced PTENpg1 asRNA in vemurafenib-resistant melanoma cell lines. In addition, manipulation of key components of the PTENpg1 asRNA network caused re-sensitization of the resistant melanoma cells to vemurafenib, and high PTENpg1 asRNA expression was found to correlate with shorter survival in melanoma patients. In paper IV, we investigated the effect of the C/EBPβ antisense (C/EBPβ-AS) transcript on transcriptional regulation of C/EBPβ. We found that C/EBPβ auto-regulates its own and also regulates C/EBPβ-AS expression. In return, C/EBPβ-AS inhibits C/EBPβ positive feedback loop by modulating epigenetic changes at the C/EBPβ promoter in melanoma cell lines. Interestingly, knockdown of C/EBPβ-AS caused re-sensitization to vemurafenib. This thesis highlights the dynamics of lncRNAs in epigenetic silencing and their involvement in cancer and therapy resistance.