Clock Genes and Cancer

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1 Clock Genes and Cancer Patricia A. Wood, MD, PhD, 1 Xiaoming Yang, PhD, 1 and William J. M. Hrushesky, MD 1 Integrative Cancer Therapies 8(4) The Author(s) 2009 Reprints and permission: sagepub.com/journalspermissions.nav DOI: / Abstract Period genes (Per2, Per1) are essential circadian clock genes. They also function as negative growth regulators. Per2 mutant mice show de novo and radiation-induced epithelial hyperplasia, tumors, and an abnormal DNA damage response. Human tumors show Period gene mutations or decreased expression. Other murine clock gene mutations are not associated with a tumor prone phenotype. Shift work and nocturnal light exposure are associated with circadian clock disruption and with increased cancer risk. The mechanisms responsible for the connection between the circadian clock and cancer are not well defined. We propose that circadian disruption per se is not uniformly tumor promoting and the mechanisms for tumor promotion by specific circadian clock disturbances will differ dependent upon the genes and pathways involved. We propose that Period clock gene mutations promote tumorigenesis by unique molecular pathways. Per2 and Per1 modulate β-catenin and cell proliferation in colon and non-colon cancer cells. Per2 mutation increases intestinal β-catenin levels and colon polyp formation. Per2 mutation also increases Apc Min/+ -mediated intestinal and colonic polyp formation. Intestinal tumorigenesis per se may also alter clock function as a result of increased β-catenin destabilizing PER2 protein. Levels and circadian rhythm of PER2 in Apc Min/+ mouse intestine are markedly decreased, and selective abnormalities in intestinal clock gene and clock-controlled gene expression are seen. We propose that tumor promotion by loss of PERIOD clock proteins is unique to these clock genes as a result of altered β-catenin signaling and DNA damage response. PERIOD proteins may offer new targets for cancer prevention and control. Keywords circadian, neoplasm, colon cancer, polyps, Period genes, b-catenin Background The molecular mechanisms of 24-hour timekeeping and circadian rhythm generation in the central clock of the brain and in the peripheral tissues are based on interactive positive and negative transcriptional-translational feedback loops generated by at least 9 core circadian clock genes (Figure 1). 1 Up to 10% of the genome of cells is expressed, at the RNA level, in a circadian-coordinated manner, with each tissue displaying unique tissue-specific expression profiles. 2 The circadian clock controls cellular processes such as proliferation, apoptosis, DNA repair, metabolism, detoxification, and the DNA damage response. 3,4-12 Disruption of the circadian clock or alterations in clock genes might disrupt this time-of-day control, thus, resulting in abnormalities in cell proliferation, apoptosis, DNA damage response, and metabolism, which could contribute to tumor promotion (Figure 2). Period Genes and Cancer The Per2 mutant mouse is the only clock mutant mouse that is associated with a tumor-prone phenotype (Table 1). 13 Other clock mutant mice are not tumor prone. Sequencing of human breast and colorectal tumors has detected Per1 and Per2 mutations. 14 Per2 mutant mice show de novo and radiationinduced tumor formation with abnormal levels of clock genes and oncogenes, and resistance to radiation-induced apoptosis and p53 protein induction. 13 Period RNA and protein levels have been reported to be depressed in several tumor types, including acute leukemia, breast, colon, endometrial, lung, and pancreatic cancer Abnormalities in Period promoter methylation and histone acetylation status have also been reported in human tumors. 18 Therefore, both genetic and epigenetic changes in Period genes are found in human tumors. Overexpression of Per1 or Per2 also inhibits cancer cell proliferation and clonogenicity. 15,19-21 Polymorphisms in several human clock genes are associated epidemiologically with 1 Medical Chronobiology Laboratory, WJB Dorn VA Medical Center, School of Medicine, University of South Carolina, Columbia, SC, USA Corresponding Author: Patricia A. Wood, Medical Chronobiology Laboratory, WJB Dorn VA Medical Center, School of Medicine, University of South Carolina, 6439 Garners Ferry Rd, Columbia, SC 29209, USA patricia.wood2@va.gov

2 304 Integrative Cancer Therapies 8(4) Figure 1. The mammalian circadian clockwork of a self-sustaining transcriptional-translational feedback loop of core clock genes (Clock, Bmal1, Per, Cry, NPAS2) that takes 24 hours to complete. The clock output affects tissue-specific functions through clockcontrolled genes Figure 2. Downregulation of PER2 or PER1 in a murine breast cancer cell line (MTCL) increases b-catenin and cell growth increased cancer risk; however, the molecular basis for these associations is not known (Table 1) PER2 as a Promoter of Intestinal Tumorigenesis We have studied the effect of the Per2 mutation on intestinal tumor formation in the Apc Min/+ mouse model. We have shown that homozygous Per2 mutation (backcrossed 8-10 generations on a C57BL/6 background) on an Apc Min/+ mouse background (backcrossed 8-10 generations on a C57BL/6 background) doubles the number of both small intestinal polyps and colonic polyps. 26 The Per2 mutation also increases the degree of anemia, splenomegaly, and extramedullary hematopoiesis that is seen in Apc Min/+ mice. We have also shown that tissue-specific Per2 downregulation within a tumor, by shrna, can accelerate in vivo breast tumor growth. 36 Therefore, loss of PER2 accelerates tumorigenesis in both intestinal and breast cancer models. If the Per2 mutation and the Apc Min/+ mutation act together to potentiate intestinal tumorigenesis, how might this occur? The Apc mutation, in humans or rodents, is associated with accelerated b-catenin signaling. Therefore, could the Per2 mutation result in abnormalities in b-catenin signaling that would then further potentiate Apc Min/+ mediated tumorigenesis? Alternatively, or in addition, the Per2 mutation has known abnormalities in clock function and clock output. Could the Apc Min/+ mutation result in abnormalities in clock function that would then further potentiate Per2-mediated tumorigenesis? Loss of PER2 Increases b-catenin and b-catenin Signaling We have shown in human colon cancer cells that downregulation of PER2 increases b-catenin protein. 26 Cyclin D is circadian regulated and is a b-catenin target gene. Following downregulation of PER2, cyclin D protein increases in these cell lines, as does cell proliferation. When b-catenin protein induction is prevented by

3 Wood et al 305 Table 1. Clock Genes and Cancer Mouse Human Clock Gene Gene Abnormality Cancer Phenotype Gene Abnormality Cancer Phenotype Negative loop Per2 Per2 m/m26,27, Per2 -/- Tumors (late onset): lymphoma, Tumor mutation Breast cancer teratoma, salivary hyperplasia 26,13 XRT promotes tumors Intestinal polyps Per1 Per1 -/-? Tumor mutation Breast cancer, colon cancer Per3? Polymorphism 28 Breast cancer Cry1 Cry1 -/- Cry2 -/-29,30 No tumors XRT does not promote tumors Cross p53 mutant fewer tumors Cry2? Polymorphism 31,32 Prostate cancer, lymphoma Positive loop Clock Clock m/m,33 No tumors (dominant negative) XRT does not promote tumors NPAS2 NPAS2 -/-34? Polymorphism 32,24 Prostate cancer, lymphoma, breast cancer Bmal1 Bmal1 -/-35 No tumors (die early) b-catenin sirna, PER2 downregulation no longer increases cyclin D protein or accelerates cell growth. PER2 downregulation is associated with increased b-catenin/tcf (transcription factor) transcriptional activity as measured by TOPFLASH promoter assay. Therefore, PER2 alters cell growth and cyclin D, and these effects are dependent on b-catenin. Our data support the hypothesis that PER2 loss during intestinal tumorigenesis may, in part, act through upregulation of b-catenin, increasing intestinal b-catenin signaling and cell proliferation (Figure 2). 26 Loss of PER1 Results in Similar Effects We have shown that PER2 downregulation also increases cell growth and b-catenin in noncolon cell lines with normal Apc-b-catenin signaling pathways such as HeLa cells. 26 This is also the case in a murine breast cancer cell line, MTCL. 37 In this MTCL cell line, downregulation of either PER2 or PER1 increases b-catenin and increases cell growth (Figure 3). Downregulation of PER1 in tumors, similar to the results with PER2, also increases breast cancer growth in vivo. 38 Therefore, it appears that both PERIOD proteins, PER2 and PER1, can modulate b-catenin and consequently alter cell growth, and these effects are not dependent on an underlying, aberrant Apcb-catenin pathway. Loss of PER2 In Vivo We have shown that mice with a homozygous Per2 mutation (Per2 m/m, 15 weeks old, backcrossed 8-10 generations on a C57BL/6 background) have increased colonic polyp numbers and frequency. 26 Small-intestinal mucosa b-catenin protein in 15-week-old Per2 m/m mice is 2-fold higher compared with that in wild type mice, when measured in late activity. 26 This increase in small-intestinal mucosa b-catenin in Per2 m/m mice is associated with an increase in c-myc protein, a b-catenin target gene. Therefore, Per2 m/m mice show an increase in colon polyp formation and increased mucosal b-catenin and b-catenin target genes, such as c-myc. How this altered b-catenin expression effects intestinal epithelial cell proliferation is not known. Similar studies in Per1 knockout mice and their intestinal tissues have not been done. The Apc Min/+ Mutation Is Associated With an Intestinal PER2 Deficiency The majority of colorectal cancers and Apc Min/+ polyps show elevated b-catenin, b-catenin signaling, and consequently an upregulation of the F box protein b-trcp. 39 b-trcp is part of one of the ubiquitin E3 ligase complexes that controls protein proteosomal degradation. The F box protein determines the binding specificity of the complex and, hence, which specific proteins are targeted for degradation. b-trcp binds many

4 306 Integrative Cancer Therapies 8(4) Figure 3. Role of PERIOD proteins in circadian clock and tumor suppression. PERIOD proteins regulate cell cycle progression through circadian gated expression of cell cycle proteins (WEE1, cyclins, p21, p53), control of cell cycle regulators (b-catenin, vascular endothelial growth factor [VEGF], cyclin D, c-myc), control of the DNA damage response, and through modulation of other clock genes (Per1, Bmal1). It is proposed that Per2 mutation, in part, promotes colonic tumorigenesis through disruption of the circadian control of b-catenin-dependent pathways and alterations in the DNA damage response. Core circadian clock genes are underlined. Sine wave indicates daily rhythms. Arrow indicates stimulation; T-bar, inhibition; dashed line, indirect effects proteins, including b-catenin, and it also binds PER1 and PER2 proteins We asked whether PERIOD proteins might be lower in colorectal cancers as a result of upregulation of b-trcp. We have shown that downregulation of b-catenin decreases PER2 and b-trcp proteins in cancer cells, and this is associated with increased stability of PER2 protein. 43 Conversely, overexpression of wild type or mutant b-catenin protein decreases the stability of PER2 protein, and this PER2 instability is reversed when the induction of b-trcp is prevented. Therefore, accelerated b-catenin expression is associated with PER2 protein instability and lower PER2 levels as a result of increased b-trcp protein levels. In Apc Min/+ intestinal mucosa, the expected large (6-fold) daily rhythm in PER2 protein is damped, and the levels are markedly depressed (6-fold lower at some times of day) compared with wild-type mice. 43 The exact mechanisms for this intestinal PER2 deficiency are yet to be determined but could include alterations in b-trcp and/or other mechanisms. The intestinal mucosa of Apc Min/+ mice also demonstrate abnormalities in clock function. Some clock gene RNA rhythms and levels are depressed (eg, Per2), whereas other clock genes show that the peak timing is shifted (eg, Per1), and still others are not altered (eg, Bmal1, Rev-erba). The circadian rhythms and levels of some of the clock output genes (eg, Dbp, Wee1) are also damped, with the peak times remaining unchanged. Behaviorally, Apc Min/+ mice show normal circadian activity rhythms (amplitude, peak timing, mean levels) measured by 24-hour wheel running, compared with wild-type mice, and a normal circadian period length. Therefore, the circadian gene expression abnormalities in these mice appears to be selective and not simply the result of an overall circadian disturbance or sick behavior of these mice. Summary We propose that circadian disruption per se may not uniformly be tumor promoting and that the mechanisms for tumor promotion by specific circadian clock disturbances will differ depending on the gene(s) and pathway(s) involved. We propose that Period clock gene mutations promote tumorigenesis by unique molecular pathways that result in increased b-catenin and b-catenin signaling, which promote cell proliferation. Intestinal tumorigenesis per se may also alter PER proteins and clock function as a result of increased b-catenin signaling causing PER2 protein destabilization. Our results support the role of the Per2 clock gene as a tumor suppressor. We propose that tumor promotion by loss of PERIOD clock proteins is unique to Period genes, compared with the loss of non-period clock proteins. Loss of PERIOD protein may also have similar effects in promoting other epithelial cancers. PERIOD clock proteins may offer new targets for cancer prevention and control. Other forms of circadian disruption that are associated with tumor promotion, such as light at night and shift work, may cause tumor promotion by distinctly different

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