Research in the Cancer Stem Cell Project is focused on deciphering the mechanisms that establish and maintain cancer stem cells and to develop a novel approach targeting cancer stem cells. In particular, the Project studies the molecular links between 1) telomerase and cancer stem cells and 2) functional non-coding RNA and cancer.

Stem cells as well as cancer stem cells are defined as cells, with the ability to both extensively self-renew and differentiate into progenitor cell types. Although embryonic stem cells and organ-specific somatic stem cells both share the biological properties of self-renewal and differentiation, the mechanism(s) of preservation of these potentials of stem cells are largely obscure. Accumulating evidence indicates that embryonic stem cells as well as undifferentiatedhighly- proliferating progenitor cells and germline cells express high levels of telomerase activity. As these cells differentiate into mature cells, the telomerase activity decreases. It is widely believed that this constitutive expression of telomerase is merely important for maintaining telomere homeostasis and that this function of telomerase may contribute to maintenance of the self-renewal potential of stem cells. However, while the telomerase activity and self-renewal potential appear to be correlated, more recent evidence shows that telomere homeostasis and the self-renewal potential may not necessarily be linked. These observations suggest that telomerase expression in stem cells may play an important role in preserving the self-renewal potential of stem cells through mechanisms beyond those involved in telomere maintenance. The research in this Project is focused on studying the potential functional role(s) of telomerase/TERT in maintaining the self-renewal potential of cancer stem cells. To purify the telomerase holoenzyme, HeLa cells stably expressing an aminoterminal FLAG- HA-tagged hTERT were established. A protein complex containing hTERT was purified from the cells by tandem affinity purification (TAP) using FLAG-M2 and HA antibodies. Following purification, proteins within the complex were separated by SDS-PAGE and identified by peptide sequencing using mass spectrometry. To date, using this system, several important molecules associated with stem cell maintenance were identified in the hTERT complex by mass spectrometry and each protein was reconfirmed as being found in the telomerase holoenzyme complex by co-immunoprecipitation with specific antibodies. The identification of associated proteins may provide insight as to the biochemical mechanisms responsible for the maintenance of stem cell properties by hTERT.

Accumulating evidence now indicates that functional non-coding RNA is widely involved in physiology of organisms, including in stem cell regulation. The research in this Project is focused on studying the molecular basis of maintenance of the genome by RNAs, especially by non-coding RNAs such as siRNAs, miRNAs and snoRNAs. Moreover, the long-term goal of this research is to decipher the molecular mechanisms of involvement of functional non-coding RNAs in the carcinogenic process. For this mission, the research is focused on identifying the mammalian homologue of RNA-dependent RNA polymerase. RNA-dependent RNA polymerase (RdRp) is a critical enzyme responsible for chromatin maintenance mediated by RNAs. While the existence of a functional mammalian homologue of RdRp has been predicted, such an enzyme has not yet been identified. Until date, a putative RdRp complex has been identified and the biological significance of this enzyme is being studied. Moreover, screening for an inhibitory compound for the enzyme is also expected to be launched.