Abstract 268: Circadian rhythms of glioma stem cells and progenitor cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The circadian timing system generates and maintains daily rhythms in physiological processes throughout the body. Disruption of circadian timing is associated with higher cancer incidence and more malignant cancers, although the interactions between cancer and circadian rhythms remain unclear. Embryonic stem cells appear to lack endogenous circadian rhythms until they differentiate. We asked whether cancer stem cells (CSCs) also lack persistent circadian oscillations at the molecular level. An inability to generate circadian rhythms may be linked to the undifferentiated state of CSCs, and treatments that can induce clock gene expression might help to initiate rhythms and promote differentiation. Because CSCs appear to evade standard cancer therapies that are effective against non-CSCs new methods to force their differentiation would be useful. To examine the circadian properties of CSCs, we selected the C6 rat glioma cell line because it expresses a strong circadian rhythm in gene expression. C6 CSCs have been characterized through flow cytometry by the side-population cell sorting method that relies on the ability of CSCs to efflux the fluorescent dye Hoechst 33342. We developed a Hoechst dye-exclusion method to identify putative CSCs in C6 monolayer cultures. This cell subpopulation failed to show a circadian rhythm in nuclear transport of a fusion protein of GFP and the circadian clock component mPER2, while the remaining cells were rhythmic. Evidence that these non-rhythmic cells are CSCs was shown by their proliferation in a medium that promotes stem cell growth and their prevalence in neurospheres grown from the C6 line that are masses of CSCs and partially differentiated cells including progenitor cells. The spheres showed immunostaining for the stem cell surface marker CD-133 and they could be induced to form astrocyte and neuron-like cells according to the cell-specific markers GFAP and beta III-tubulin, respectively. To determine whether the neurospheres are capable of generating circadian rhythms, C6 cells expressing a firefly luciferase and mPER2 fusion protein under control by the mPer2 gene promoter were imaged after they formed neurospheres. Circadian rhythms in bioluminescence persisted when the cells were synchronized with a 2-hour pulse of forskolin that induces clock genes but not when the spheres were imaged without forskolin treatment. We interpret these results as indicating a lack of circadian clocks in CSCs but circadian clocks functioning in progenitor cells of neurospheres that require synchronization for coordinated circadian activity. Because CSCs in the microenvironment of tumors may also be arrhythmic, efforts to manipulate circadian clock genes should be examined when treating gliomas and possibly other cancers. Citation Format: Vishal P. Sharma, Michael E. Geusz. Circadian rhythms of glioma stem cells and progenitor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 268. doi:10.1158/1538-7445.AM2013-268