Oncogenic and Circadian Effects of Small Molecules Directly and Indirectly Targeting the Core Circadian Clock

Circadian rhythms are essential for controlling the cell cycle, cellular proliferation, and apoptosis, and hence are tightly linked to cell fate. Several recent studies have used small molecules to affect circadian oscillations; however, their concomitant cellular effects were not assessed, and they have not been compared under similar experimental conditions. In this work, we use five molecules, grouped into direct versus indirect effectors of the circadian clock, to modulate periods in a human osteosarcoma cell line (U2OS) and determine their influences on cellular behaviors, including motility and colony formation. Luciferase reporters, whose expression was driven via Bmal1- or Per2-promoters, were used to facilitate the visualization and quantitative analysis of circadian oscillations. We show that all molecules increase or decrease the circadian periods of Bmal1 and Per2 in a dose-dependent manner, but period length does not correlate with the extent of cell migration or proliferation. Nonetheless, molecules that affected circadian oscillations to a greater degree resulted in substantial influence on cellular behaviors (ie, motility and colony formation), which may also be attributable to noncircadian targets. Furthermore, we find that the ability and extent to which the molecules are able to affect oscillations is independent of whether they are direct or indirect modulators. Because of the numerous connections and feedback between the circadian clock and other pathways, it is important to consider the effects of both in assessing these and other compounds.