Circadian rhythm modulation of breast cancer gene expression depends on stem cell properties induced in culture

It is important to know whether breast cancer cells within tumors have functional circadian clocks. If so, they may benefit from the ability provided by the clock to predict when important daily events occur such as nutrient availability in blood following meals or elevated immune cell surveillance at night. If instead the circadian clock is nonfunctional, then clinical treatments could be directed towards activating the clock because evidence indicates circadian rhythm loss leads to more aggressive tumor growth. Here, we examined the role of circadian clocks in metastasis-related events, focusing on epithelial-mesenchymal transition (EMT), including cancer stem cell (CSC) generation, and mesenchymal-epithelial transition (MET). Because of their differing circadian properties two tumor-forming cell lines were compared, MCF-7 human breast cancer and C6 rat glioma cells. C6 monolayer and tumorsphere cultures express distinct circadian rhythms in gene expression. In contrast, monolayer MCF-7 cell cultures are reported to be arrhythmic and so appear to lack a functional circadian clock. Nevertheless, MCF-7 cells may contain inducible circadian oscillators that fail to generate circadian rhythms under standard culture conditions but may function in tumor microenvironments. Tumorspheres simulate the three-dimensional tumor structure and were used here to test for favorable conditions enabling circadian rhythms in cancer cells. Circadian rhythms in EMT, MET, mitosis, migration, and cell death were evaluated through time-lapse imaging over several days. Bioluminescence imaging and quantitative RT-PCR were used to test for circadian rhythms in expression of the core circadian clock gene Per2 in MCF-7 cells. Following treatment with serum-free culture medium designed for stem cells, EMT and CSC-related protein markers of MCF-7 and C6 cells were confirmed by immunofluorescence: MSI1 and CD133 in MCF-7; OCT4, TWIST1, ZEB1, vimentin in C6. Morphometry showed distinct changes in cell shape following EMT as shown by nearly all MCF-7 cells having an average roundness of 0.815 ±0.09 SD and a C6 roundness of 0.855 ±0.029. This medium increased the number of MCF-7 tumorspheres expressing circadian rhythms by 31.5%, and average amplitude of rhythms increased 11.6-fold. MET was then induced by returning cells to serum-containing medium, which produced cells with a flattened cell morphology indicating epithelial or pseudoepithelial differentiation. A circadian clock in C6 cells appeared to gate MET events after the return to serum (95% confidence limit of period: 20.8-26.9 hrs, p<0.05). The circadian timing properties of MCF-7 tumorspheres suggest that the circadian clock may also be present in tumors and that cell-cell interactions or paracrine factors could sustain otherwise poorly performing circadian clocks in breast cancer. Citation Format: Arpan De, Vishal Premdev Sharma, Dilshan Harshajith Beligala, Angelia Marie Lee, Michael Eric Geusz. Circadian rhythm modulation of breast cancer gene expression depends on stem cell properties induced in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1888.