Studies on the mammalian piRNA pathway in cancer cells
Piwi-interacting RNAs (piRNAs) are a subset of noncoding RNAs that complex with members of the Argonaute protein family, the Piwi proteins. The Piwi/piRNA pathway is highly conserved in animals and is implicated in spermatogenesis, germline stem cell maintenance, and transposon suppression in the germline. In mammals, the latter occurs via de novo DNA methylation of transposons during male germ cell maturation. Available evidence suggests that the Piwi/piRNA pathway may also be active in somatic cells and cancer cells, but this has been little explored. Cancer cells are known to harbour many epigenetic defects, including the hypermethylation of CpG islands (CGI). The mechanism underlying aberrant CGI methylation, which often silences genes whose inactivation favours tumour growth, remains unknown. CGI hypermethylation occurs against a background of global hypomethylation, in which transposons have the potential to become active. In this thesis, I have tested the hypothesis that the Piwi/piRNA system is active in cancer cells and is responsible for directing the aberrant hypermethylation. This hypothesis predicts that mRNAs derived from genes are accidentally captured by the pathway and targeted for silencing in the same way transposons are in the germline. Survey of normal tissues and cancer cell lines revealed that the human Piwi mRNAs (Hiwi, Hili and Hiwi2) are expressed outside the germline. Hiwi2 was found to be ubiquitously expressed, and the protein was localised to the cytoplasm of MDAMB231 cancer cells. Deep sequencing of the small RNAs bound to Hiwi and Hiwi2 in MDAMB231 revealed a complex population of piRNAs derived from genes, and these piRNAs corresponded to unmethylated, rather than methylated CGIs. A prominent population of piRNAs derived from tRNAs was also identified. Mass spectrometry and polysome profiling demonstrated that both Hiwi and Hiwi2 associate with components of the translational machinery, along with other partner proteins known to bind Piwis in the germline. This thesis demonstrates that the Piwi/piRNA pathway is active in cancer, where it does not appear to have a role in transposon suppression or gene silencing. Rather, when taken together, the results of this thesis strongly suggest a role for the pathway in translational regulation in cancer.
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