3
`
`Commentary/Hypothesis
`
`Does ‘‘Clock’’ Matter in Prostate Cancer?
`
`Yong Zhu,1 Tongzhang Zheng,1 Richard G. Stevens,2 Yawei Zhang,1 and Peter Boyle3
`
`1Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut;
`2University of Connecticut Health Center, Farmington, Connecticut; and 3IARC, Lyon Cedex, France
`
`Abstract
`
`The ancient adaptation of a 24-hour circadian clock has
`profound effect on our daily biochemical, physiologic, and
`behavioral processes, including the monitoring of sex hor-
`mone levels. Although the disruption of the circadian cycle
`has been implicated in the etiology of hormone-related female
`breast cancer, few studies have been undertaken to determine
`if a link exists in the development of the most common cancer
`type among men whose etiology remains largely unknown:
`hormone-related prostate cancer. Here, we hypothesize that
`both altered-lighted environments and genetic variations in
`
`genes responsible for maintaining circadian rhythms may
`result in deregulation of clock-associated biological process-
`es, such as androgen expression, and consequently influence
`an individual’s risk of prostate cancer. There is also a potential
`for the interaction of genetic variants and exposures, such as
`evening shift work. Confirmation of this hypothesis will
`add to our understanding of the role of the circadian clock
`in prostate tumorigenesis and further facilitate the develop-
`ment of novel risk and prognostic biomarkers for prostate
`(Cancer Epidemiol Biomarkers Prev 2006;15(1):3 – 5)
`cancer.
`
`Introduction
`
`Prostate cancer is the most common and the second most
`fatal cancer among men in the industrialized world. Despite
`tremendous efforts to improve our understanding of this
`cancer, its etiology remains largely unknown. To date, the only
`well-established risk factors for prostate cancer are older age,
`family history of the disease, and race. Recent studies of
`another hormone-related cancer, breast cancer, have supported
`the hypothesis that circadian disruption may be a novel risk
`factor and that genetic determinants for circadian rhythms
`may play a role in breast tumorigenesis (1, 2). It is from these
`encouraging findings that we postulate that both circadian
`disturbance and genetic variations that alter functions of
`circadian genes are involved in hormone-related prostate
`tumorigenesis among men.
`
`Clock-Cancer Connection: Epidemiologic Evidence
`
`One fundamental component of our biological world is a
`universal 24-hour oscillation in biochemical, physiologic,
`and behavioral processes, which occurs in almost all living
`organisms due to an ancient adaptation to the rotation of the
`earth. Disruptions of
`these critical rhythms may have a
`profound influence on our health and might be involved in
`tumorigenesis (3).
`The clock-cancer connection has been investigated in studies
`of pilots, flight attendants, and shift workers who are more
`likely to have disrupted circadian cycles due to abnormal work
`hours. Observations from a Nordic pilots cohort showed that
`the relative risk of prostate cancer increased as the number of
`flight hours in long distance aircraft increased (4). A similar
`cohort of men working for Air Canada showed a significantly
`
`Received 8/15/05; revised 10/7/05; accepted 11/15/05.
`Grant support: NIH grants CA110937, CA62986, ES11659, and CA108369.
`The costs of publication of this article were defrayed in part by the payment of page charges.
`This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
`Section 1734 solely to indicate this fact.
`Requests for reprints: Yong Zhu, Department of Epidemiology and Public Health, Yale
`University School of Medicine, New Haven, CT 06520. Phone: 203-785-4844; Fax: 203-737-6023.
`E-mail: yong.zhu@yale.edu
`Copyright D 2006 American Association for Cancer Research.
`doi:10.1158/1055-9965.EPI-05-0631
`
`increased incidence rate of prostate cancer when compared
`with the respective Canadian population rates (5). These
`increased rates of prostate cancer may be due to circadian
`hormonal disturbances associated with long distance flights
`although confounding by other factors cannot be ruled out.
`Similarly, an increased risk of breast cancer among female
`flight attendants has also been observed (6). In addition, recent
`studies in female shift workers who work predominantly
`at night have reported an elevated risk for breast cancer
`(reviewed in ref. 7). This hypothesis was based on a light-at-
`night suppression of melatonin (8). Analogously, melatonin
`has been reported to be lower in men with prostate cancer than
`those without (9) and melatonin has been shown in vitro to
`modulate prostate cancer cell growth via androgen receptor
`interactions (10). The clock-cancer connection has gained some
`very limited but consistent support from previous studies.
`Other findings that may support a connection between
`circadian rhythms and cancer risk come from studies of the
`blind, who have a reduced or abolished sensitivity to
`environmental light exposures (11). In studies of totally blind
`men, prostate cancer risk was reduced (12, 13) although this
`reduction was not significant. A reduced risk of breast cancer
`has also been reported among blind women (13-15).
`These observations have suggested a role for circadian
`rhythms in the etiology of both prostate and breast cancers
`as previously proposed (16), which implies some common
`etiologic features shared by these two hormone-related cancer
`types. However,
`little is known about possible molecular
`mechanisms underlying this clock-cancer connection.
`
`Circadian Genes and Their Roles in Tumorigenesis
`
`Emerging areas of interest for circadian and cancer research are
`the roles of several newly identified clock genes in the master
`circadian pacemaker of the suprachiasmatic nucleus and in
`the peripheral cells and tissues, and how this clock apparatus
`controls the expression of a wide variety of genes. To date, there
`have been nine core circadian genes identified: Clock, casein
`kinase Ie (CKIe), cryptochrome 1 (Cry1), chryptochrome 2 (Cry2),
`Period1 (Per1), Period2 (Per2), and Period3 (Per3), Bmal1, and
`NPAS2. A model of transcription-translation feedback loops
`
`Cancer Epidemiol Biomarkers Prev 2006;15(1). January 2006
`
`
`Research.
`
`Downloaded from on March 3, 2016. © 2006 American Association for Cancercebp.aacrjournals.org
`
`MYLAN - EXHIBIT 1034
`
`

`
`4
`
`Does ‘‘Clock’’ Matter in Prostate Cancer?
`
`of these circadian genes has been proposed to explain the
`molecular clockwork (17) in which cell functions are regulated
`through the expression of clock-controlled genes. It has been
`estimated that 2% to 10% of all mammalian genes are clock-
`controlled genes,
`indicating an extensive circadian gene
`regulation (18).
`Recent findings suggest that circadian genes may function as
`tumor suppressors at the systemic, cellular, and molecular
`levels due to their involvement in cell proliferation, apoptosis,
`cell cycle control, and DNA damage response (reviewed in ref.
`3). In humans, the rhythmic expression of several cyclins and
`tumor suppressor p53 is regulated by the circadian clock (19). It
`has also been shown that the core circadian regulator, casein
`kinase I, functions in promoting cell proliferation and tumor-
`igenesis (20). A recent study showed that g radiation – induced
`apoptosis is circadian time dependent and disruption of the
`Per2 gene stops the response of all core circadian genes to g
`radiation (21). Moreover, disruption of circadian rhythms in
`mice is associated with accelerated growth of malignant tumors,
`suggesting that the host circadian clock may play an important
`role in endogenous control of tumor progression (22).
`
`Polymorphisms in Circadian Genes: Phenotypic Effects
`
`Several molecular epidemiologic studies have examined genetic
`polymorphisms in clock-related genes and corresponding
`phenotypes, such as morning/evening preference and depres-
`sive symptoms. A single nucleotide polymorphism located in
`the 3V flanking region of the human clock gene was reported
`to be a predictor of diurnal preference in a population-based
`randomly selected subjects (23). In addition, associations of Per3
`polymorphisms with delayed sleep phase syndrome or diurnal
`preference have recently been reported (24, 25). In the first study
`of a circadian gene variant and risk of cancer, we reported that
`the variant Per3 genotype was significantly associated with an
`increased risk of breast cancer among premenopausal women
`(1). Findings from this study and other association studies with
`other circadian gene variants require much more work to be
`confirmed. If associations of circadian gene variants with cancer
`risk exist, then an obvious question becomes how these variants
`might interact with exposures such as shift work to modify their
`effects on risk. For example, a variant which predisposes to
`morning preference may be particularly detrimental for night
`workers.
`
`Possible Mechanisms: Clock-Controlled Androgen
`Expression?
`
`The prostate is an androgen-dependent organ because andro-
`gens are essential for its normal growth and maintenance.
`Androgen may act as a carcinogen by increasing cellular
`proliferation and thereby increase the chance of random
`DNA copy errors. Evidence from animal studies has shown
`that large amounts of androgens given to rodents can induce
`prostate cancer (26). A human study also reported that long-
`term exposure to high levels of androgens is associated with
`prostate cancer risk (27). Levels of testosterone, a principle
`circulating androgen, have further been shown to play a
`pivotal role in the differentiation and maintenance of selected
`prostate cancer cells lines both in vitro and in vivo (28).
`The effect of circadian rhythms on the expression of
`androgens has been previously documented in animals (29).
`In men, a circadian pattern, with regard to the levels of serum
`total testosterone, has also been observed and this circadian
`rhythm becomes blunted with normal aging (30). Circadian
`rhythms in the plasma levels of cortisol, dehydroepiandroster-
`one, y4-androstenedione, testosterone, and dihydrotestoster-
`one have also been detected in healthy young men (31). These
`observations suggest that expression of these androgens might
`be controlled by circadian rhythms. Moreover, circadian
`genes are directly involved in the regulation of tumor-related
`genes in prostate tumorigenesis. For example, the expression
`of cyclin D1 is deregulated in Per2 mutant mice (32). Cyclin D1
`activates the cyclin-dependent kinase 6 that may bind to the
`androgen receptor and stimulate its transcriptional activity in
`the presence of dihydrotestosterone (33).
`The above findings suggest that androgens might play a role
`in prostate cancer development. Given the possible effect of the
`circadian rhythm on androgen expression, we postulate that
`circadian disruptions, caused by both genetic and environmen-
`tal factors, may affect androgen expression patterns and
`consequently contribute to prostate cancer development, off-
`ering a potential mechanism for prostate tumorigenesis (Fig. 1).
`
`Hypothesis
`
`We hypothesize that circadian disruption may explain some
`etiology of prostate cancer and may provide a novel panel
`of biomarkers to determine an individual’s associated risk.
`
`Figure 1. Proposed role of the circadian clock in prostate
`tumorigenesis. The circadian rhythms can be affected by either
`genetic variants in circadian genes or altered light exposure.
`Consequently, androgen expression and biological pathways
`regulated by circadian rhythms may contribute to prostate
`cancer development and progression. In addition, circadian
`genes are directly involved in the regulation of tumor-related
`genes (such as cyclin D1) in prostate tumorigenesis.
`
`Cancer Epidemiol Biomarkers Prev 2006;15(1). January 2006
`
`
`Research.
`
`Downloaded from on March 3, 2016. © 2006 American Association for Cancercebp.aacrjournals.org
`
`

`
`Cancer Epidemiology, Biomarkers & Prevention
`
`5
`
`Specifically, increased exposure to light at night will disrupt
`circadian rhythms and consequently elevate the risk of
`prostate cancer. In addition, genetic and epigenetic variations
`in circadian genes may result in deregulation of circadian
`rhythms and other biological processes regulated by these
`genes; we propose that genetic variations and methylation
`changes altering the expression and functionality of circadian
`genes will be potential biomarkers associated with an
`individual’s risk of prostate cancer.
`
`Testing the Hypothesis
`
`From an epidemiologic perspective, a well-designed, popula-
`tion-based case-control study would provide evidence for the
`role of circadian disruption in prostate cancer. In this type
`of study, the questionnaire for assessing environmental light
`exposures and sleeping patterns must be carefully designed to
`ensure revealing results. Additional studies involving noctur-
`nal occupational groups, such as shift workers, would also test
`the hypothesis in prostate cancer.
`Genetic and epigenetic analyses of the circadian genes
`will further illustrate the role of circadian genes in prostate
`tumorigenesis. Three approaches could be applied in this
`regard: (a) candidate genetic variation approach, to examine
`associations between genotypes and haplotypes of predicted
`functional single nucleotide polymorphisms in the circadian
`genes and prostate cancer risk; (b) whole gene approach, to
`investigate associations between haplotypes estimated from
`haplotype-tagging single nucleotide polymorphisms in the
`circadian genes and prostate cancer risk; and (c) epigenetic
`approach, to explore changes of methylation status in CpG
`islands of the circadian genes as biomarkers for prostate cancer.
`Analysis on the joint-effect of circadian biomarkers and
`environmental factors would also test the hypothesis that
`increased exposure to light at night may elevate the risk of
`prostate cancer for individuals with the putative high-risk
`genotypes and haplotypes of circadian genes. Furthermore,
`determining possible associations between shift work and
`level of androgens, such as testosterone, may help to illustrate
`potential effects of aberrant
`light exposures on androgen
`expressions and provide a likely molecular mechanism should
`an increased risk be observed between shift work and prostate
`cancer.
`
`Conclusions
`
`We propose that some prostate cancer cases could be
`explained by light-induced circadian disruptions and/or
`genetic/epigenetic variations in circadian genes and/or gene-
`environment interactions between these two factors. We also
`propose that the clock-regulated androgen expressions might
`be one potential mechanism involved in prostate tumorigen-
`esis. These hypotheses are testable in a molecular epidemio-
`logic study and could lead to the development of novel risk
`and prognostic biomarkers.
`
`Acknowledgments
`We thank Carly Guss (Yale University, New Haven, CT) and Derek
`Leaderer (Colby College, Waterville, ME) for their careful reading and
`comments on the manuscript.
`
`References
`1. Zhu Y, Brown HN, Zhang Y, Stevens RG, Zheng T. Period3 structural
`variation: a circadian biomarker associated with breast cancer in young
`women. Cancer Epidemiol Biomarkers Prev 2005;14:268 – 70.
`
`2.
`
`3.
`
`7.
`
`Stevens RG, Rea MS. Light in the built environment: potential role of
`circadian disruption in endocrine disruption and breast cancer. Cancer
`Causes and Control 2001;12:279 – 87.
`Fu L, Lee CC. The circadian clock: pacemaker and tumour suppressor. Nat
`Rev Cancer 2003;3:350 – 61.
`4. Pukkala E, Aspholm R, Auvinen A, et al. Cancer incidence among 10,211
`airline pilots: a Nordic study. Aviat Space Environ Med 2003;74:699 – 706.
`5. Band PR, Le ND, Fang R, et al. Cohort study of Air Canada pilots: mortality,
`cancer incidence, and leukemia risk. Am J Epidemiol 1996;143:137 – 43.
`6. Rafnsson V, Tulinius H, Jonasson JG, Hrafnkelsson J. Risk of breast cancer in
`female flight attendants: a population-based study (Iceland). Cancer Causes
`Control 2001;12:95 – 101.
`Stevens RG. Circadian disruption and breast cancer: from melatonin to clock
`genes. Epidemiology 2005;16:254 – 8.
`8. Blask DE, Dauchy RT, Sauer LA, Krause JA, Brainard GC. Growth and fatty
`acid metabolism of human breast cancer (MCF-7) xenografts in nude rats:
`impact of constant light-induced nocturnal melatonin suppression. Breast
`Cancer Res Treat 2003;79:313 – 20.
`9. Bartsch C, Bartsch H, Schmidt A, Ilg S, Bichler KH, Fluchter SH. Melatonin
`and 6-sulfatoxymelatonin circadian rhythms in serum and urine of primary
`prostate cancer patients: evidence for reduced pineal activity and relevance
`of urinary determinations. Clin Chim Acta 1992;209:153 – 67.
`10. Zisapel N. Benign and tumor prostate cells as melatonin target sites. The
`pineal gland and cancer: neuroimmunoendocrine mechanisms in malig-
`nancy. Berlin: Springer; 2001.
`11. Lockley SW, Skene DJ, Arendt J, Tabandeh H, Bird AC, Defrance R.
`Relationship between melatonin rhythms and visual loss in the blind. J Clin
`Endocrinol Metab 1997;82:3763 – 70.
`12. Feychting M, Osterlund B, Ahlbom A. Reduced cancer incidence among the
`blind. Epidemiology 1998;9:490 – 4.
`13. Pukkala E, Ojamo M, Rudanko SL, Stevens RG, Verkasalo PK. Does
`incidence of breast cancer and prostate cancer decrease with increasing
`degree of visual impairment. Cancer Causes Control. In press.
`14. Verkasalo PK, Pukkala E, Stevens RG, Ojamo M, Rudanko SL. Inverse
`association between breast cancer incidence and degree of visual impair-
`ment in Finland. Br J Cancer 1999;80:1459 – 60.
`15. Hahn RA. Profound bilateral blindness and the incidence of breast cancer.
`Epidemiology 1991;2:208 – 10.
`16. Stevens RG, Davis S, Thomas DB, Anderson LE, Wilson BW. Electric power,
`pineal function, and the risk of breast cancer. FASEB J 1992;6:853 – 60.
`17. Young MW, Kay SA. Time zones: a comparative genetics of circadian clocks.
`Nat Rev Genet 2001;2:702 – 15.
`18. Storch KF, Lipan O, Leykin I, et al. Extensive and divergent circadian gene
`expression in liver and heart. Nature 2002;417:78 – 83.
`19. Bjarnason GA,
`Jordan RC, Sothern RB. Circadian variation in the
`expression of cell-cycle proteins in human oral epithelium. Am J Pathol
`1999;154:613 – 22.
`20. Morin PJ. h-Catenin signaling and cancer. BioEssays 1999;21:1021 – 30.
`21. Fu L, Pelicano H, Liu J, Huang P, Lee C. The circadian gene Period2 plays an
`important role in tumor suppression and DNA damage response in vivo .
`Cell 2002;111:41 – 50.
`22. Filipski E, King VM, Li X, et al. Host circadian clock as a control point in
`tumor progression. J Natl Cancer Inst 2002;94:690 – 7.
`23. Katzenberg D, Young T, Finn L, et al. A CLOCK polymorphism associated
`with human diurnal preference. Sleep 1998;21:569 – 76.
`24. Ebisawa T, Uchiyama M, Kajimura N, et al. Association of structural
`polymorphisms in the human period3 gene with delayed sleep phase
`syndrome. EMBO Rep 2001;2:342 – 6.
`25. Archer SN, Robilliard DL, Skene DJ, et al. A length polymorphism in the
`circadian clock gene Per3 is linked to delayed sleep phase syndrome and
`extreme diurnal preference. Sleep 2003;26:413 – 5.
`26. Brown CE, Warren S, Chute RN, Ryan KJ, Todd RB. Hormonally induced
`tumors of the reproductive system of parabiosed male rats. Cancer Res
`1979;39:3971 – 6.
`27. Gann PH, Hennekens CH, Ma J, Longcope C, Stampfer MJ. Prospective
`study of sex hormone levels and risk of prostate cancer. J Natl Cancer Inst
`1996;88:1118 – 26.
`28. Soronen P, Laiti M, Torn S, et al. Sex steroid hormone metabolism and
`prostate cancer. J Steroid Biochem Mol Biol 2004;92:281 – 6.
`29. Francavilla A, Eagon PK, DiLeo A, et al. Circadian rhythm of hepatic
`cytosolic and nuclear estrogen and androgen receptors. Gastroenterology
`1986;91:182 – 8.
`30. Plymate SR, Tenover JS, Bremner WJ. Circadian variation in testosterone, sex
`hormone-binding globulin, and calculated non-sex hormone-binding
`globulin bound testosterone in healthy young and elderly men. J Androl
`1989;10:366 – 71.
`31. Guignard MM, Pesquies PC, Serrurier BD, Merino DB, Reinberg AE.
`Circadian rhythms in plasma levels of cortisol, dehydroepiandrosterone, y4-
`androstenedione, testosterone and dihydrotestosterone of healthy young
`men. Acta Endocrinol 1980;94:536 – 45.
`32. Lee CC. The circadian clock and tumor suppression by Mammalian period
`genes. Methods Enzymol 2005;393:852 – 61.
`33. Lim JT, Mansukhani M, Weinstein IB. Cyclin-dependent kinase 6 associates
`with the androgen receptor and enhances its transcriptional activity in
`prostate cancer cells. Proc Natl Acad Sci U S A 2005;102:5156 – 61.
`
`Cancer Epidemiol Biomarkers Prev 2006;15(1). January 2006
`
`
`Research.
`
`Downloaded from on March 3, 2016. © 2006 American Association for Cancercebp.aacrjournals.org