Circadian clock regulation of epithelial-mesenchymal and mesenchymal-epithelial transitions in glioma and breast cancer cells

Circadian rhythms control cancer cell behavior in tumors and in vitro. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are two key events of metastasis that lead to cancer progression and aggressiveness. Studies suggest that genes controlling circadian rhythms also regulate EMT, which generates mesenchymal cells (M-cells) with cancer stem cell (CSC) properties through dedifferentiation. MET enables post-EMT cells to differentiate back to epithelial cells at metastatic sites and initiate secondary tumors. If a circadian clock can be identified in M-cells, then it could be manipulated pharmacologically to more effectively target the cells with novel anticancer agents specific to CSCs or cause their differentiation into more easily treated and less aggressive cells before metastasis occurs. We tested for any role of circadian clocks in EMT and MET events by using cancer cell lines from two different tissues. We compared C6 rat glioma cells that have a well-established circadian clock with MCF-7 human breast tumor cells that are considered lacking a functional clock. EMT was induced in cell cultures by exchanging standard serum-containing medium (SM) with stem cell medium (SCM), a non-serum medium containing specific growth factors promoting CSC survival. Single-cell behavior and morphological states were quantified microscopically through time-lapse imaging. Expression of EMT markers ZEB1 and TWIST, mesenchymal markers vimentin and PDGFRA, and stem cell markers OCT4, nestin, MSI1 and CD133 were validated by immunocytochemistry. Both C6 and MCF-7 cultures showed circadian oscillations in the population size of post-EMT M-cells. MET was then induced by returning the cultures to SM from SCM. MET events observed in glioma CSCs clustered significantly at a particular phase of the circadian cycle. The cellular microenvironment also influenced migration properties of C6 cells with SM promoting faster closure in a standard wound healing assay than SCM. We tested the feasibility of interrupting the circadian clock in C6 cells with RNA interference for later exploration of clock gene connections with EMT and MET. At least 98% of cells could be loaded with fluorescent random-sequence oligonucleotides, which persisted for four days and did not interfere with EMT. This study supports use of circadian rhythms in cancer cells to more effectively time treatments controlling pre-metastatic events