Epidemiological studies have shown that humans with altered circadian rhythms have higher cancer incidence, with breast cancer being one of the most cited examples. To uncover how circadian disruptions may be correlated with breast cancer and its development, prior studies have assessed the expression of BMAL1 and PER2 core clock genes via RT-qPCR and western blot analyses. These and our own low-resolution data show that BMAL1 and PER2 expression are suppressed and arrhythmic. We hypothesized that oscillations persist in breast cancer cells, but due to limitations of protocols utilized, cannot be observed. This is especially true where dynamic changes may be subtle. In the present work, we generated luciferase reporter cell lines representing high-and low-grade breast cancers to assess circadian rhythms. We tracked signals for BMAL1 and PER2 to determine whether and to what extent oscillations exist and provide initial correlations of circadian rhythm alterations with breast cancer aggression. In contrast to previous studies, where no oscillations were apparent in any breast cancer cell line, our luminometry data reveal that circadian oscillations of BMAL1 and PER2 in fact exist in the low-grade, luminal A MCF7 cells but are not present in high-grade, basal MDA-MB-231 cells. To our knowledge, this is the first evidence of core circadian clock oscillations in breast cancer cells. This work also suggests that circadian rhythms are further disrupted in more aggressive/high tumor grades of breast cancer, and that use of real time luminometry to study additional representatives of breast and other cancer subtypes is merited.

The natural product nobiletin is a small molecule, widely studied with regard to its therapeutic effects, including in cancer cell lines and tumors. Recently, nobiletin has also been shown to affect circadian rhythms via their enhancement, resulting in protection against metabolic syndrome. We hypothesized that nobiletin's anti-oncogenic effects, such as prevention of cell migration and formation of anchorage independent colonies, are correspondingly accompanied by modulation of circadian rhythms. Concurrently, we wished to determine whether the circadian and anti-oncogenic effects of nobiletin differed across cancer cell lines. In this study, we assessed nobiletin's circadian and therapeutic characteristics to ascertain whether these effects depend on cell line, which here also varied in terms of baseline circadian rhythmicity. Three cell culture models where nobiletin's effects on cell proliferation and migration have been studied previously were evaluated: U2OS (bone osteosarcoma), which possesses robust circadian rhythms; MCF7 (breast adenocarcinoma), which has weak circadian rhythms; and MDA-MB-231 (breast adenocarcinoma), which is arrhythmic. We found that circadian, migration, and proliferative effects following nobiletin treatment were subtle in the U2OS and MCF7 cells. On the other hand, changes were clear in MDA-MB-231s, where nobiletin rescued rhythmicity and substantially reduced oncogenic features, specifically two-dimensional cell motility and anchorage-independent growth. Based on these results and those previously described, we posit that the effects of nobiletin are indeed cell-type dependent, and that a positive correlation may exist between nobiletin's circadian and therapeutic effects.

Macrophages are white blood cells that play disparate roles in homeostasis and immune responses. They can reprogram their phenotypes to pro-inflammatory (M1) or anti-inflammatory (M2) states in response to their environment. About 8–15% of the macrophage transcriptome has circadian oscillations, including genes closely related to their functioning. As circadian rhythms are associated with cellular phenotypes, we hypothesized that polarization of macrophages to opposing subtypes might differently affect their circadian rhythms. We tracked circadian rhythms in RAW 264.7 macrophages using luminescent reporters. Cells were stably transfected with Bmal1:luc and Per2:luc reporters, representing positive and negative components of the molecular clock. Strength of rhythmicity, periods and amplitudes of time series were assessed using multiple approaches. M1 polarization decreased amplitudes and rhythmicities of Bmal1:luc and Per2:luc, but did not significantly affect periods, while M2 polarization increased periods but caused no substantial alterations to amplitudes or rhythmicity. As macrophage phenotypes are also altered in the presence of cancer cells, we tested circadian effects of conditioned media from mouse breast cancer cells. Media from highly aggressive 4T1 cells caused loss of rhythmicity, while media from less aggressive EMT6 cells yielded no changes. As macrophages play roles in tumors, and oncogenic features are associated with circadian rhythms, we tested whether conditioned media from macrophages could alter circadian rhythms of cancer cells. Conditioned media from RAW 264.7 cells resulted in lower rhythmicities and periods, but higher amplitudes in human osteosarcoma, U2OS-Per2:luc cells. We show that phenotypic changes in macrophages result in altered circadian characteristics and suggest that there is an association between circadian rhythms and macrophage polarization state. Additionally, our data demonstrate that macrophages treated with breast cancer-conditioned media have circadian phenotypes similar to those of the M1 subtype, and cancer cells treated with macrophage-conditioned media have circadian alterations, providing insight to another level of cross-talk between macrophages and cancer.

We present a summary of our protocol for employment of small-molecule circadian rhythm modulators. The abilities of compounds to affect oscillations have received significant attention. We outline assessments of circadian changes using indirubin-3'-oxime (IO), 5-iodo-indirubin-3'-oxime (5I-IO), and 5-sulfonic acid-indirubin-3'oxime (5SA-IO), analyzing effects on Bmal1 and Per2 oscillations and oncogenic features.

Epidemiological studies have shown that humans with altered circadian rhythms have higher cancer incidence, with breast cancer being one of the most cited examples. To uncover how circadian disruptions may be correlated with breast cancer and its development, prior studies have assessed the expression of BMAL1 and PER2 core clock genes via RT-qPCR and western blot analyses. These and our own low-resolution data show that BMAL1 and PER2 expression are suppressed and arrhythmic. We hypothesized that oscillations persist in breast cancer cells, but due to limitations of protocols utilized, cannot be observed. This is especially true where dynamic changes may be subtle. In the present work, we generated luciferase reporter cell lines representing high-and low-grade breast cancers to assess circadian rhythms. We tracked signals for BMAL1 and PER2 to determine whether and to what extent oscillations exist and provide initial correlations of circadian rhythm alterations with breast cancer aggression. In contrast to previous studies, where the clock was deemed to be "broken" in breast cancer, our luminometry data reveal that circadian oscillations of BMAL1 and PER2 do in fact exist in the low-grade, luminal A cell line, MCF7 but are not apparent in high-grade, basal MDA-MB-231 cells.To our knowledge, this is the first evidence of core circadian clock oscillations in breast cancer cells. This work also suggests that circadian rhythms are increasingly disrupted with breast cancer tumor grade/aggressiveness, and that use of real time luminometry to study additional representatives of breast and other cancer subtypes is highly merited.

We present a summary of our protocol for employment of small-molecule circadian rhythm modulators. The abilities of compounds to affect oscillations have received significant attention. We outline assessments of circadian changes using indirubin-3’-oxime (IO), 5-iodo-indirubin-3’-oxime (5I-IO), and 5-sulfonic acid-indirubin-3’-oxime (5SA-IO), analyzing effects on Bmal1 and Per2 oscillations and oncogenic features.

The natural product nobiletin is a small molecule, widely studied with regard to its therapeutic effects, including in models of cancer. Recently, nobiletin has also been shown to affect circadian rhythms via their enhancement, resulting in protection against metabolic syndrome. We hypothesized that nobiletin's anti-oncogenic effects are correspondingly accompanied by modulation of circadian rhythms. Concurrently, we wished to determine whether the circadian and anti-oncogenic effects of nobiletin differed across cell culture models of cancer. In this study, we assessed nobiletin's circadian and therapeutic characteristics to ascertain whether these effects depend on cell line, which here also vary in terms of baseline circadian rhythmicity. Three cell culture models where nobiletin's anticancer effects have been studied previously were evaluated here: U2OS (bone osteosarcoma), which possesses robust circadian rhythms; MCF7 (breast adenocarcinoma), which has weak circadian rhythms; and MDA-MB-231 (breast adenocarcinoma), which is arrhythmic. We found that both circadian and anti-cancer effects following nobiletin treatment were subtle in the U2OS and MCF7 cells. On the other hand, changes were clear in MDA-MB-231s, where nobiletin rescued rhythmicity, and substantially reduced oncogenic features. Based on these results and those previously described, we posit that a positive correlation exists between nobiletin's circadian and therapeutic effects. Introduction: