Abstract 858: The circadian clock of glioma cells undergoing epithelial-mesenchymal transition
Authors: De Arpan , Beligala Dilshan Harshajith , Sharma Vishal Premdev , Fry Benjamin Ryan , Geusz Michael Eric 
Published: July 2017

Highly invasive gliomas contain many cells that have changed to an enhanced migratory state through an epithelial-mesenchymal transition (EMT). These cells have the phenotype of glioma stem cells (GSCs). Similar to GSCs, glioma cells undergoing EMT show phenotypic heterogeneity, altered gene expression, and resistance to anticancer drugs along with increased invasiveness. Circadian rhythms in tumor cells influence the progression and severity of cancer and appear to regulate cell division cycles. Increased cancer incidence and progression have often been linked to disruption or deregulation of the molecular mechanism of the circadian clock. One of the major clock proteins, PER2, has been shown to play a regulatory role as a tumor and EMT suppressor in metastatic breast cancer cell lines. GSCs in tumorsphere cultures contain circadian clocks that may regulate their cancer properties. The possibility of a regulatory role for circadian rhythms in EMT was examined here. We used a standard method to induce EMT in the C6 rat glioma cell line that has known circadian rhythms in gene expression generated by the circadian clocks within its cells. EMT was induced by exchanging the cell culture medium with a serum-free stem cell medium (SCM) containing growth factors (EGF, FGF2, PDGF-alpha-beta). Cell cultures were imaged continuously with a microscope and digital camera in a cell incubator to monitor cell shape, cell death, migration, and apoptosis. During EMT, cells changed from an extended flat state to rounded and spindle shapes, ceased proliferating, and expressed EMT markers ZEB1 and vimentin. At the end of two days in SCM, 33.4% of the cells were ZEB1-positive and only 1.26% were GFAP-positive (n=3 cultures). Cell diameters after EMT were within the size range of C6 GSCs described as Hoechst-negative cells positive for stem cell markers nestin and CD133. Following EMT, small tumorspheres began to form. After initiating EMT, the rounded cells were counted at hourly intervals for up to four days after the medium exchange. As the number of post-EMT cells increased, the population size oscillated, and when examined by Lomb-Scargle periodogram analysis, four cultures had a significant period within the circadian range, 19-29 hours, (average 22.20 ±2.45 SD, p&lt;0.05). One had a 16-hour period and one lacked a significant rhythm. Rhythms were present even when circadian clocks in the cultures were not synchronized with an initial forskolin treatment. We conclude that EMT may be timed by endogenous circadian oscillators in gliomas that favor larger numbers of post-EMT cells at a particular time of day. These results suggest that pharmacological treatments that suppress EMT or target GSCs would be more effective when delivered at particular times during the circadian cycle of the tumor.

Citation Format: Arpan De, Dilshan Harshajith Beligala, Vishal Premdev Sharma, Benjamin Ryan Fry, Michael Eric Geusz. The circadian clock of glioma cells undergoing epithelial-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 858. doi:10.1158/1538-7445.AM2017-858