Slowing Down Tumors by Fixing Clock Genes

A new approach that restores circadian rhythms in cells

Clock Genes & Circadian Rhythms

Human cells have clock genes, that when working properly, prevent cancer. Cancer is defined as the uncontrolled division of abnormal cells in the body. The clock genes control our body’s circadian rhythms, which hold power over our cortisol hormones, blood pressure, melatonin, growth hormones, testosterone, prolactin, temperature, and cell replication. And when functional they also slow down tumors. Slowing down tumors can provide time for other treatments to work. “In the majority of cancer cell lines, circadian rhythms are absent or poorer compared to normal cells,” said Dr. Nicolas Cermakian, a researcher at McGill University.

The circadian clock consists of two clock genes, called per and cry. They interact with each other to generate oscillations of gene expressions called, BMAL1 and CLOCK, transcription factors. They operate on an auto-regulatory feedback loop, where transcription factors produce a negative feedback on their own expression.

A Study on Clock Genes

A study done in 2010 showed how the clock gene transcription factor BMAL1 played a role in regulating tumor cell apoptosis, cell cycle progression, DNA damage response, and homeostasis regulation. Scientists from McGill University, the University of California, and the University of Montreal conducted a similar study in 2017 that resulted in the discovery of the cellular mechanisms involved between activating tumor clock genes, restoring circadian rhythms, and slowing down the replication of cells in tumors.

The 2017 study investigated circadian clocks in tumor cells and tumor cell proliferation before and after Dexamethasone (DEX) treatment. DEX is a glucocorticoid that is both a clock gene activator and synchronizer. Taking into account circadian rhythms display oscillations of about 24 hours, they were able to calculate circadian variations based on data from their experiments. The researchers examined levels of gene and protein expressions in tumor cells before and after clock gene activation. They did this to understand the roles each played in the cell cycle, in response to the treatment. The levels were examined by extracting and incubating cells with antibodies and then analyzing the cell suspensions with flow cytometry. They looked for cell cycle genes — c-MYC, Cyclin E, CDK2, and p21 — and the clock gene transcription factor BMAL1.

Each gene has a specific function, c-MYC activates Cyclin E, Cyclin E complexes with CDK2 to start the cell cycle, and p21 inhibits CDK2. Transcription factors BMAL1 and CLOCK complex together repress Cyclin E, which causes cell replication to slow down. And, they found that treatment with DEX returned cell cycle genes and BMAL1 generation to a 24-hour rhythm.

Clock gene activator slowing down tumors

They tested DEX’s effect on the distribution of melanoma cells at several cell cycle stages. They did this by collecting, staining, and analyzing cells at different points in time with flow cytometry. The researchers found that fewer cells, consequently, entered the DNA replication phase 24 hours after treatment. Also, a single DEX treatment significantly reduced cell numbers after 50 hours, and a second treatment further reduced the cell count after 96 hours.

“It is hard to tell whether the treatment reduced the proliferation speed down to normal speeds because we did not have a control cell type. B16 cells (non-cancerous metastasizing tumor cells) were used, and different cell types have different division times,” said Dr. Cermakian.

Clock Gene Activation Verified

They were able to verify the activation of the tumor clock by DEX using two methods. First, they injected the tumors of mice with DEX every 48 hours and conducted an Immuno-histochemistry test on the tumor slices. Their analysis revealed a significant rhythm of BMAL1 protein levels in DEX-treated tumors. Second, researchers removed the melanoma cell’s circadian rhythm genes, applied DEX, and found DEX had no effect. They did this by knocking down their BMAL1 clock transcription factor with RNA and a lentiviral vector. Those cells were injected back into the mice, now lacking a functional clock, and DEX was administered. What they found was that removing the circadian clock transcription factor prevented the restoration of circadian rhythms by DEX in the tumors.