WOMEN IN CANCER THEMATIC REVIEW: Circadian rhythmicity and the influence of ‘clock’ genes on prostate cancer

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   Figure 1

   Relationship between the various clock genes. Input signals such as sunlight activate the 8 known core clock genes: period (Per1–Per3), casein kinase Iε (CKIε), Clock/NPAS2, Bmal1/ARNTL and cryptochrome (Cry1–2). Clock and Bmal1 are two basic helix-loop-helix (bHLH) transcription factors (each containing a bHLH region and a DNA-binding domain – DBD), which bind the E-box sequence (CACGTG) in the promoter of target genes as a heterodimer. Target genes include Per, Cry and NR1D1, which produces an orphan nuclear receptor protein REV-ERBAα. When REV-ERBAα is phosphorylated by GSK3β, a downstream target of Akt, it transcriptionally represses target genes such as Bmal1 and Cry. In this way, REV-ERBAα balances against the effects of another orphan nuclear receptor RORα, which transcriptionally upregulates Bmal1. Similarly, Per1 is regulated by phosphorylation by CKIε, which is itself regulated by ERK kinases. Activated Per dimerizes with Cry to form a complex that disrupt the Clock-Bmal1 complex and prevent transcription. P denotes protein phosphorylation.
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   Figure 2

   General structure of core clock genes Per and Cry. The three Per genes encode proteins that contain the PER–ARNT–SIM (PAS) and the related PAC motifs and form oligomers with the Cry proteins to form functional DNA-binding complexes. Per1 contains a BTRC interaction domain at the N terminal end. In addition, Per proteins also contain a phosphorylation domain where casein kinase 1 binds and phosphorylates these proteins. The very C terminus contains a specific domain that allows oligomerization with the Cry proteins. In addition, Per2 contains a PPAR gamma-binding site between the CKIε- and CRY-binding sites that is not seen in Per1 or Per3. Per2 also has a stabilization domain upstream of the PAS motifs not seen in other Per proteins. All Per proteins, however, express nuclear localization and export sequences that allows them to enter and leave the nucleus as needed. The structure of the Cry proteins is much less complex. Both Cry1 and Cry2 contain a cryptochrome (flavoproteins that are sensitive to blue light) interaction domain, as well as a domain that allow them to interact with and inhibit the CLOCK/BMAL1 dimers. In addition, Cry1 also contain a domain that allows interaction with TIMELESS (which negatively regulates CLOCK/BMAL-1 interactions), but this domain is not found in Cry2. (Based on http://www.uniprot.org/.)
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   Figure 3

   24-h cycle of the circadian clock. The expression of Clock itself does not significantly vary during the 24-h cycle, but it is only one of few genes in this group that does not. Major regulation of the circadian clock is mediated by the orphan nuclear receptors RORα and REV-ERBAα, which determine the expression of Bmal1 (also known as ARNTL), the key binding partner of Clock. During the day, the Clock-Bmal1 dimer transcribes genes that result in the synthesis of Per and Cry, as well as REV-ERBAα. As the day progresses, Per and Cry dimerizes and forms a stable complex that translocates to the nucleus. Any free Per remaining in the cytoplasm is phosphorylated by CKIε, which causes it to be ubiquitinated and degraded. As night falls, the Per/Cry oligomer complex in the nucleus disrupts the formation of the Clock-Bmal1 heterodimer, which then stops producing Per and Cry. In addition, REV-ERBAα is phosphorylated by GSK3β, which stabilizes the protein, and allow it to transcriptionally repress the expression of Bmal1. Bmal1 levels are also suppressed when melatonin levels increase in the circulation, which inhibits the expression of RORα that positively regulates Bmal1 expression. Decrease in Bmal1 further disrupts the Clock-Bmal1 complex and prevents the transcription of target genes, including Per and Cry. As REV-ERBAα levels in the cell, and melatonin levels in the circulation, decrease early in the morning, Bmal1 synthesis is resumed. Further, Per and Cry levels in the nucleus also fall off due to decreased synthesis, and Clock-Bmal1 complexes resume, which then allows the circadian clock to move forward. P denotes protein phosphorylation, U denotes protein ubiquitination.
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   Figure 4

   Circadian regulation of testosterone production in young and old men. Healthy young men demonstrate high levels of testosterone production in early mornings that decline as the day progresses leading to lower levels by the evening. During the day, Per complexes promote testosterone production, as did Bmal1; the produced testosterone binds to and activates the androgen receptor (AR), but as night falls, activated AR suppresses Per levels, thereby decreasing testosterone production and deactivates the AR. This in turn suppressed Bmal1 levels, further preventing Per expression. As Per levels increase once more the next morning, along with Bmal1 levels, testosterone production is renewed. However, several clock genes (Bmal1, Per1, Per2, Per3 and Rev-ErbAα) are reduced in older males compared with younger ones, resulting in the loss of circadian rhythmicity in testosterone synthesis observed in ageing males. T, testosterone.
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   Figure 5

   Negative feedback loop between melatonin and the androgen receptor. Testosterone (T) is converted to dihydrotestosterone (DHT), which in the prostate, binds to the androgen receptor (AR) in the cytoplasm of prostate-derived cells, including prostate cancer. DHT binding activates the AR, which then translocates to the nucleus and binds to androgen response elements (ARE) in the target genes, thereby stimulating their transcription. AR transcriptional activity represses the expression of the melatonin receptors MT1 and MT2, thereby preventing their binding to melatonin. This prevents melatonin’s ability to induce growth arrest and apoptosis, and to prevent oxidative stress. In turn, melatonin and its receptors inhibit AR translocation to the nucleus, not only by preventing testosterone conversion to DHT but also by mechanisms involving PKCα or RORα.

• © 2016 Society for Endocrinology