Circadian function in cancer: regulating the DNA damage response.

P roper circadian clock function is essential for the coordination of cellular functions within an organism in response to light and dark cycles. Although epidemiological studies have linked altered circadian rhythms to cancer susceptibility (1, 2), the molecular mechanisms tying the circadian clock to cancer are poorly understood. In PNAS, Lee and Sancar (3) demonstrate that loss of the core circadian clock proteins Cry1 and Cry2 can sensitize tumor cells to DNA damage-induced apoptosis. This effect is shown to be particularly relevant in cells that have lost p53 (TP53), a key tumor suppressor and mediator of chemosensitivity, which is mutated or deleted in the majority of human cancers. These findings imply unique roles for circadian rhythm in regulating the DNA damage response and point to a potential avenue for treating refractory p53deficient cancers. The central oscillator of the circadian clock is made up of the transcriptional activators Clock and Bmal1, which heterodimerize to form an active complex. This induces transcription of a large suite of genes controlling multiple physiological processes, such as the cell cycle and metabolism (4). In addition, the Clock/Bmal1 complex activates the cryptochrome (Cry1 and Cry2) and period (Per1, Per2, and Per3) genes, which form a negative feedback loop capable of suppressing Clock/ Bmal1-mediated transcription. This socalled transcription–translation feedback loop results in cyclical expression over a 24-h period, with the Clock/Bmal1 complex activity generally highest during daylight hours and Cry-Per inhibitory activity peaking during the night. A previous report by Sancar and colleagues (5) demonstrated that germline disruption of cryptochrome in p53deficient mice, which are highly tumor prone, increased their tumor-free survival. Notably, this effect was associated with an increase in DNA damage sensitivity of the Cry1/Cry2/p53-deficient tumor cells compared with those lacking p53 alone. This was a provocative finding given the crucial role of p53 as a cellular executioner after DNA damage. When cells undergo irreparable DNA damage due to exposure to genotoxins such as UV radiation, the p53 protein activates transcription of proapoptotic target genes, effectively removing the damaged cells from the organism. Consequently, loss of p53 in both humans and mice allows cells to persist and acquire new mutations necessary for cancer progression, yielding tumors that are refractory to our most common therapies, including ionizing radiation and chemotherapy. This previous report therefore suggested that loss of cryptochrome could reverse the DNA damage resistance induced by loss of p53. Killing such p53-deficient tumor cells is clearly one of the major challenges in cancer therapy. In their new article, Lee and Sancar offer insight into the mechanism by which cryptochrome deficiency sensitizes cells lacking p53 to apoptosis. Remarkably, they demonstrate that cryptochromes are involved in the regulation of the p53related gene p73 (TP73), which like p53 functions as a tumor suppressor and helps maintain genomic integrity (6–8). Transcription of TAp73, a p73 isoform with strong structural and functional similarity to p53, is enhanced in Cry1/Cry2-deficient cells in response to DNA damage. Loss of Cry1/Cry2 relieves repression of Clock/ Bmal1, thereby leading to Egr1 up-regulation and recruitment to the TAp73 promoter. Activation of TAp73 after Cry1/ Cry2 loss also requires the DNA damageinduced removal of the repressor C-EBPα from the TAp73 promoter. Thus, TAp73 is both temporally regulated by the circadian clock and acutely regulated in response to DNA damage (Fig. 1). These findings may have particular implications for the field of cancer chronotherapy, which seeks to determine whether the effectiveness and tolerability of chemotherapy can be linked to the time of day treatment is given. Indeed, it has been found that treatment schedule can impact both long-term survival and nonspecific toxicity (9, 10). However, the widespread application of such observations to standard clinical practice has been hampered by a lack of insight into how the circadian cycle influences the response to specific chemotherapeutic agents. The new study demonstrates that p53-deficient tumor cells with compromised circadian function exhibit increased apoptosis and slower tumor growth in vivo after treatment with oxaliplatin (3). The finding that this effect is linked to p73 supports previous studies showing that platinum agents induce apoptosis at least in part through phosphorylationdependent activation of TAp73 (11, 12) and that TAp73 levels are a key determinant of chemosensitivity (6, 13). It will therefore be of interest to determine whether the cryptochromeand TAp73-dependent response observed by Lee and colleagues is specific to treatment with platinum or will be seen with other common chemotherapeutic agents. In addition to enhancing tumor chemosensitivity, these findings might also provide hope for approaches to limit the toxicity of chemotherapy for normal tissues. The model proposed herein suggests that in normal cells, the circadian cycling may result in a window when the amount of Egr1 bound to the TAp73 promoter is low, potentially blunting the apoptotic response of normal cells to chemotherapy. Interestingly, it has been found that tumor cells in patients do not cycle with the same kinetics as their Fig. 1. Circadian clock-dependent sensitization of p53-deficient cells to DNA damage. (A) In normal cells with functional p53, the DNA damage response is mediated primarily by p53. (B) Tumor cells that have lost p53 are resistant to DNA damage-inducing chemotherapy. (C) Tumor cells lacking both p53 and circadian clock proteins Cry1 and Cry2 have high Egr1 levels. Egr1 bound to the TAp73 promoter allows for increased activation of TAp73 after DNA damage, resulting in tumor cell death.