Our circadian rhythm is vital for far more than just a good night's sleep—it plays a crucial role in almost all of our body's physiological and behavioral activities. In fact, “even single neurons and fibroblasts harbor a conserved, cell-autonomous circadian clock,” according to researchers in the Perelman School of Medicine at the University of Pennsylvania.
Disruption of this delicate internal clock can have significant negative effects and has been implicated in a number of disease states, including neurodegenerative disorders such as Huntington's and Parkinson's disease (Trends Endocrinol Metab 2016;27(4):192-203).
In addition, “disrupted rhythms, which can occur in many ways, such as shift work and jet lag, were previously shown to increase susceptibility to cancer,” according to Amita Sehgal, PhD, Professor of Neuroscience and Director of Penn's Chronobiology Program. Research shows dysregulated circadian rhythms increase the risk of many cancers, including lung, breast, skin, oral, and prostate (Cancer Res 2004; doi:10.1158/0008-5472.CAN-04-0674). The fact that many different tumors are affected suggests basic cellular changes, yet little is known about the mechanisms driving the association between dysregulated circadian rhythms and tumorigenesis (Int J Genomics 2018; doi:10.1155/2018/8576890).
Sehgal and her team recently used cell-based circadian desynchronization experiments to discover what exactly happens to basic cell function with circadian rhythm disruption (PLoS Biol 2019; doi: 10.1371/journal.pbio.3000228). They found chronic circadian desynchronization causes cell proliferation along several oncogenetic pathways. Their research also supports the potential efficacy of chronotherapy, or timed treatment. Both discoveries have lasting implications for researchers and practicing clinicians alike.
Circadian Rhythm Under The Microscope
“We were interested in cellular mechanisms impacted by circadian disruption, so a cellular model provided the best approach,” Sehgal explained. “We used a CDK4/6 inhibitor because our work showed that this pathway, implicated in cancer, was affected by circadian disruption.”
To begin, the study used dexamethasone, a synthetic glucocorticoid and potent clock synchronizer, to induce chronic circadian desynchronization in cells. Controls were exposed to the drug for 10 days at regular 24-hour intervals, while experimental cells received serial 8-hour advances of the treatment every 2 days. This model successfully mimicked jet lag, according to the researchers.
While testing for oxidative stress/senescence, metabolism, proteolysis, and apoptosis showed no differences between the control and experimental cells, RNA sequencing told a different story. The controls had significant cycles of expression of 44 transcripts, many of which were compromised or lost altogether in the experimental cells. Further analysis revealed upregulation of a number of genes in the jet-lagged cells compared with the controls, including SH3D21 (implicated in ataxia-telangiectasia and colorectal cancer), EXOC3L2 (Alzheimer's disease), DHX58 (mammary tumors), PCSK1N (skin carcinogenesis, Alzheimer's disease, Parkinsonism-dementia), and JAG2 (myeloma).
“A major consequence of circadian disruption is an increase in proliferation of cancer cells,” noted Sehgal. “Importantly, we took an unbiased approach towards this problem, so we assayed a wide range of cellular functions and found that the major change was in proliferation rate.”
After discovering that circadian disruption led to an enrichment of gene networks crucial for cellular growth, proliferation, and cancer, the team compared gene expression patterns and found 11 oncogenes in particular were induced: MAFB, JUND, MAFA, OLIG2, NFKB2, JUN, CCND3, HMGA1, ERBB2, MYC, and AKT1. They also noted significantly increased cell numbers in their jet-lagged samples compared with controls. This may be due to the fact that chronic circadian desynchronization caused upregulation of genes involved in the proliferation phase of the cell cycle, G1/S, more so than it does for genes responsible for other parts of the cell cycle, according to the researchers.
The circadian disruption model further revealed that cyclin D1 protein expression increased in cultured tumor cells. Interestingly, a similar increase of cyclin D1 protein was observed in the tumor tissue, but not normal liver tissue, of mice subjected to the chronic jet lag condition, suggesting the proteins in cancerous cells are more sensitive to chronic jet lag than those in normal cells. In addition, the retinoblastoma (RB) protein showed increased phosphorylation by cyclin-dependent kinase (CDK) 4/6—opening the door for a possible targeted treatment approaching using a known CDK4/6 inhibitor.
Timing Is Everything
Sehgal's findings shed significant light on the mechanisms behind chronic circadian desynchronization and its effect on tumorigenesis. With this better understanding of the cyclin D1-CDK4/6–RB pathway, already an important target for cancer chemotherapy, the team hypothesized timed treatments may affect efficacy.
To test the theory, the researchers treating controls and experimentally jet-lagged mice—injected with the MCA carcinogen—with palbociclib (a potent oral CDK4/6 inhibitor initially FDA approved for the treatment of metastatic breast cancer in combination with endocrine therapy) either in the morning or at night. Control mice treated in the morning had significantly reduced tumor growth, but those treated at night did not. This time-dependent antitumor activity was compromised in the jet-lagged mice, the researchers noted. The team found similar results with a mouse melanoma model as well.
“In conjunction with the cell-based data, our in vivo animal data indicate that circadian regulation of G1/S progression confers time-of-day sensitivity to antitumor agents that act at this step, but the rhythm of sensitivity is lost under conditions of circadian desynchrony,” the researchers wrote in the study.
Now that her research has uncovered the lasting effects of dysregulated circadian rhythms on a cellular level, Sehgal expects future researchers to pay closer attention to their study protocols and take this into account. The time of day in which they perform experiments and noting whether the experiment subjects have normal rhythms could significantly impact the study outcomes, she noted. “In addition to circadian regulation being an important variable, this could help interpretation of experiments. For example, sometimes experiments do not reproduce well because they are performed at different times of day.”
Based on these findings, the team now plans to screen other anti-cancer drugs for effects specific to the treatment timing and identify their mechanism of action. Doing so could significantly change their efficacy—just by altering the timing of each dose.
The data could have a lasting impact on clinical practice just as much as it could for research. The study authors surmise that environmental or physiological disruption of circadian rhythms, as seen with shift work, abnormal sleep timing, or irregular psychosociological stresses, may be a crucial underlying mechanism leading to inter-individual variability for both tumor growth and treatment response.
This is why Sehgal hopes her findings prompt clinicians to take circadian rhythms into account when treating patients. “Most drugs are expected to show time-of-day specificity as the targets are known to cycle. However, they are not prescribed as such,” she admitted. “Also, maintenance of robust circadian rhythms is expected to promote wellness and could provide a prophylactic way to reduce disease risk.”
Rebecca Hepp is a contributing writer.