Cancer stem cell generation during epithelial-mesenchymal transition is temporally gated by intrinsic circadian clocks
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RESEARCH PAPER
Cancer stem cell generation during epithelial‑mesenchymal transition is temporally gated by intrinsic circadian clocks Arpan De1,2 · Dilshan H. Beligala1,3 · Vishal P. Sharma1,4 · Christian A. Burgos1 · Angelia M. Lee1 · Michael E. Geusz1 Received: 13 January 2020 / Accepted: 13 July 2020 © Springer Nature B.V. 2020
Abstract Epithelial-mesenchymal transition (EMT) is a key event preceding tumor cell metastasis that increases cell invasiveness and cancer stem cell (CSC) populations. Studies suggest that genes used in generating circadian rhythms also serve in regulating EMT. To test the role of circadian clocks in cellular EMT events two cancer cell lines were compared, one that has a wellestablished circadian clock, C6 from rat glioma, and one that does not, MCF-7 from human breast tumor. MCF-7 tumorsphere cultures were tested for evidence of circadian rhythms because of previously reported circadian rhythm enhancement in C6 tumorspheres shown by elevated rhythm amplitude and increased expression of circadian clock gene Per2. Bioluminescence imaging of Per2 gene expression in MCF-7 tumorspheres revealed a previously unconfirmed circadian clock in this important cancer research model. Inducing CSC generation through EMT in C6 and MCF-7 monolayer cultures revealed circadian oscillations in the size of the post-EMT CSC population, confirming that circadian rhythms are additional processes controlling this stage of cancer progression. EMT was verified by distinct cellular morphological changes and expression of stem cell proteins OCT4, nestin, MSI1, and CD133 along with EMT-related proteins ZEB1, vimentin, and TWIST. Quantifying single-cell events and behaviors through time-lapse imaging indicated the post-EMT population size was determined largely by circadian rhythms in epithelial-like cancer cells undergoing EMT. We then identified a specific phase of the circadian rhythm in Per2 gene activation as a potential target for therapeutic treatments that may suppress EMT, minimize CSCs, and limit metastasis. Keywords Epithelial-mesenchymal transition · Cancer stem cell · Tumorsphere · OCT4 · Circadian rhythm Abbreviations ANOVA Analysis of variance BLI Bioluminescence imaging CCD Charge-coupled device Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10585-020-10051-1) contains supplementary material, which is available to authorized users. * Michael E. Geusz [email protected] 1
Department of Biological Sciences, Bowling Green State University, 217 Life Science Bldg., Bowling Green, OH 43403, USA
2
Present Address: Department of Neurosurgery, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
3
Present Address: Department of Molecular Biology and Biotechnology, Faculty of Science, University of Peradeniya, Peradeniya 20400, Sri Lanka
4
Present Address: Celsee, Inc., Ann Arbor, MI 48108, USA
CSC Cancer stem cell DAPI 4′,6-Diamidino-2-phenylindole DMEM Dulbecco’s modified eagle medium E-cell Epithelial cancer cel
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