Analysis of Circadian Properties and Clock Regulation of Glioma and Breast Cancer Stem Cells

Increased cancer risk is linked to disruption of circadian rhythms. Cancer stem cells (CSCs) are a known cause of cancer aggressiveness, but their circadian properties have not been described. In this study we describe the circadian properties of C6 rat glioma tumorspheres and MCF-7 human breast CSCs. We discovered circadian rhythms in gene expression within C6 glioma tumorspheres enriched in CSCs and found that the circadian clock is particularly robust in medium lacking any growth factors. A method is introduced for identifying individual CSCs in culture for single-cell analysis. CSCs in monolayer, attached cell cultures failed to show a circadian rhythm in nuclear localization of mPER2 protein, suggesting that cell interactions or the tumor-like microenvironment within tumorspheres enable circadian timing. The MCF-7 cancer cell line, derived from an epithelial breast tumor, has been widely studied because of its aggressiveness and high percentage of CSCs. Although several cancer cell lines have distinct circadian rhythms in gene expression, the reason why many other lines apparently lack circadian clocks remains unclear. Similarly, circadian rhythms of cells within tumors are also often poorly organized or absent. Considering the cell heterogeneity of cancer cell lines, including CSCs within these lines, it seemed likely that some of the cells could retain a functional circadian clock. To test this idea, we probed the circadian properties of MCF-7 cultures with a reporter gene that expresses a functional mPER2 protein fused with firefly luciferase under the control of the mouse Per2 promoter. MCF-7 cells grew as small clusters in medium containing fetal bovine serum or one containing growth factors stimulating CSC proliferation. The percentage of clusters expressing the mPer2 gene was surprisingly high, and at least 60% of these expressed a circadian rhythm. Reporter gene expression and Per2 mRNA were elevated in response to growth factors that prevent CSC differentiation. Despite previous reports suggesting otherwise, these results indicate that circadian clocks could have a functional role in MCF-7 breast cancer cells and that these clocks were previously undetected. Alternatively, the mouse mPer2 transfection may have rescued MCF-7 cells from an arrhythmic state. The presence of circadian rhythms in C6 and MCF-7 cultures enriched in CSCs suggests that there may be links between circadian clock genes and genes that inhibit CSC differentiation. These interactions should be tested further and might be used to develop novel therapeutic approaches for suppressing tumor growth by targeting CSCs