Circadian clocks and breast cancer

The global impact of breast cancer is large and growing. With 1.67 million cases reported in 2012, it is the second most commonly diagnosed cancer worldwide [1, 2]. The incidence of the disease is much higher in the developed world, with four times as many cases in Western Europe as in Middle Africa and Eastern Asia. This suggests that aspects of a modern western lifestyle may influence the onset and progression of breast cancer. One possibility is a disruption to our internal body clocks, known as circadian clocks [3].

Intrinsic circadian clocks are driven by environmental time cues such as the natural day/night cycle. Our bodies translate timing cues into molecular oscillations within individual cells, which then drive 24-hour rhythms in cellular processes in almost every tissue in the body [4‐6]. These cell-autonomous molecular oscillators make up the body’s internal timing system, and are synchronized by the master pacemaker, the suprachiasmatic nucleus (SCN) [7]. However, circadian clocks can become perturbed through irregular shift work, through repeated bouts of jet lag and during ageing. Weakened or damaged circadian clocks alter the susceptibility to certain diseases and directly drive others.

One of the processes regulated by the circadian clock is the cell cycle. Disruption of circadian rhythms can therefore be associated with abnormal cell divisions that occur in cancer [8]. Indeed, there are links between altered circadian clocks and tumorigenesis in metastatic colorectal cancer, osteosarcoma, pancreatic adenocarcinoma and, most notably, breast cancer [8].

The influence of altered circadian rhythm on breast cancer was first noted in the 1960s [9]. Since then, it has become clear that circadian disruption interrupts the complex multi-step molecular mechanisms underpinning breast cancer [1]. The indication that tumorigenesis is linked to circadian rhythms suggests that manipulating those rhythms might be a remedial approach for treating cancer. This could, for example, lead to more efficacious therapies, novel adjuvant strategies and, ultimately, improved breast cancer outcome [10].

In this review, we discuss the current understanding of links between circadian disruption and breast cancer risk.

Located in the anterior hypothalamus, the SCN is the central pacemaker that coordinates circadian rhythms with the solar day [11]. The bilateral SCN receives innervation directly from the retina via the retinohypothalamic tract. The majority of its ~20,000 densely packed neurons are ‘pacemaker cells’, with each neuron containing its own oscillatory machinery capable of producing a prolonged and robust circadian rhythm even in ex-vivo culture [12].

Light is a dominant synchronizing time-giver. However as well as light/dark cycles, the SCN is also responsive to changes in rest/activity cycles [5]. Non-photic stimuli, such as neuroendocrine signals and feeding behaviour, can also influence SCN pacemaking [5]. The SCN uses several neural and endocrine outputs to synchronize clocks of many peripheral organs [6]. One of these is the hormone melatonin [13], which is released rhythmically at night and relays information to peripheral organs [14].

The SCN is not actually required for peripheral organs to generate their own rhythms. Rather, it acts more like the conductor of an orchestra, guiding each organ to oscillate in the ideal phase for that specific tissue [15].

The basic genetic regulation of the circadian clock is highly conserved across the animal kingdom. Most mammalian clock genes were first identified via mutagenic studies in fruit flies [16]. The core molecular clock generates oscillations in protein levels via a series of auto-regulatory transcriptional/translational feedback loops [17]. Several clock genes encode transcription factors, with the molecular clock driving rhythmic expression of downstream clock-controlled genes [18].

The major components involved in this cellular clock network include the transcriptional activators Circadian Locomotor Output Cycles Kaput (CLOCK) and Brain and muscle Arnt-like protein-1 (BMAL1). CLOCK has a paralogue, Neuronal PAS domain protein 2 (NPAS2), which compensates for loss of CLOCK in the SCN and peripheral oscillators [19, 20]. The other main components are Period (PER1 and PER2) and Cryptochrome (CRY1 and CRY2), which form the negative arm of a feedback loop [17]. Additional regulatory systems such as nuclear hormone receptors and epigenetic mechanisms have also been identified [21, 22].

Circadian gene expression is the result of a series of transcription and translation events. This leads to the expression of a different set of proteins that switches them off at the end the day. The process then begins again with timely removal of the repressors (PERs and CRYs).

At the beginning of the circadian day, Bmal1 expression is driven by RORα, allowing the formation of CLOCK/BMAL1 heterodimers (Fig. 1). This transcription complex binds to CACGTG E-box sequences in the promoters of the Per and Cry genes, increasing their expression. The CLOCK/BMAL1 complex also increases expression of Rev-erbα, which suppresses Bmal1 transcription [23]. The CLOCK/BMAL1-mediated increase in Per and Cry expression allows for the accumulation of PER in the cytosol, where it is phosphorylated by casein kinase 1ε and 1δ. Phosphorylated PER becomes ubiquitinated and is readily degraded, but CRY accumulation allows the formation of a stable PER/CRY/CK1 complex [7]. This complex inhibits the transcriptional capacity of CLOCK/BMAL1, preventing further expression of Per and Cry, and also Rev-erbα. Eventually phosphorylated PER and CRY are lost, de-repressing Bmal1 transcription and allowing for higher levels of BMAL1 that start the next circadian day [18].

Collectively these cycles ensure that the circadian clock oscillates with a period of 24 hours, driven by the appropriate environmental time cues (Fig. 1).

Peripheral tissues have their own intrinsic self-sustained circadian oscillations, but are dependent on the central clock and tissue-specific factors for synchronization. Local cellular oscillators regulate specific circadian programmes of gene expression that vary according to tissue function, with typically 5–10 % of all genes being transcribed rhythmically [24].

Clock target genes can be controlled directly by the CLOCK/BMAL1 complex, or indirectly via the circadian expression of transcription factors. In this way, the circadian clock confers a rhythmic aspect to a wide range of tissues. In the breast, for example, the expression of nearly 600 genes is controlled in a circadian manner.

The circadian regulation of gene expression impacts upon many cellular processes and complex behaviours. This is exemplified by SIRT1 and TIMELESS, proteins that independently intersect the clock and the cell cycle machines. For example, SIRT1 reduces proliferation by deacetylating β-catenin [25] and binding p53 [26], whereas TIMELESS advances the circadian clock in response to DNA damage [27, 28].

The circadian system is therefore well integrated with other physiologies. If the clock becomes disrupted, however, this can lead to disease [5].

There is growing evidence that circadian clock gene expression plays a role in breast biology. In mice, mutations of specific circadian genes disrupt behavioural and molecular rhythms. Such mutations also reveal the involvement of cellular clocks in the initiation of carcinogenesis.

Breast tissue contains a network of branched epithelial ducts surrounded by a basement membrane, outside which is a fibroblast-rich and adipose-rich stromal extracellular matrix [29]. Clocks have been discovered within breast epithelium using real-time bioluminescent imaging of mammary explants from PER2::Luciferase [30] mice. There are daily rhythmic variations in the expression of core clock proteins BMAL1 and PER2. Moreover, during the development of the gland from virgin through to full lactation, tissue isolated at the same time of day reveals that Bmal1 and Per1 mRNA levels increase in late pregnancy and lactation, while Per2 falls [31, 32].

The levels of clock gene expression are also controlled by the breast tissue microenvironment. For example, we have found that a stiff extracellular matrix such as is found in ageing and cancer leads to suppression of core clock rhythms. Intriguingly, local stiffness of the breast stroma and extracellular matrix adjacent to breast epithelium has a major role in determining cancer outcome [33, 34]. The mechanisms linking the cell exterior to clock control are not yet known, but could involve the cytoskeleton and/or nuclear envelope proteins, one of which regulates transcription of the Bmal1 gene [35].

The molecular clock is also crucial for regulating the survival of stem cells. Indeed the self-renewal capacity of mammary progenitor stem cells becomes compromised in mice that have a defective CLOCK/BMAL1 complex, revealing a circadian influence on breast function [36]. Studies in other tissues have demonstrated similar links between the circadian clock and stem cells. For example, undifferentiated neural stem cell cultures from BMAL1^−/− mice have an increased propensity to differentiate into glia over neurons [37]. In addition, circadian oscillations in the release of hematopoietic stem cells (HSCs), through chemokine Cxcl12 and GSK3β, could result in a coordinated release of cells and thereby repopulation of the bone marrow stem cell niche [38].

Clock genes are expressed in normal breast. However, their levels are variable and controlled both by the cellular microenvironment and by the developmental stage of the tissue. Disruption of clock gene expression can increase breast cancer risk (Table 1).

Over the last 20 years, epidemiological evidence has corroborated a link between altered clocks and breast cancer. Women who work irregular shift patterns, such as nurses, have a higher frequency of hormone-related breast cancer [63‐69], and the higher levels of cancer may be greater for women who started night shifts before a first pregnancy [70]. Elevated breast cancer risk also occurs in women exposed to high levels of ambient light at night [71]. Irregular shift work refers to working a mix of nights and days during a week. In these individuals, the circadian clock has too little time to entrain to the new shift pattern before it is changed again. Notably, however, working consistently at night causes far less disruption to the circadian rhythm.

Mice subjected to a simulated shift-work paradigm have a significantly increased risk of acquiring mammary tumours [72]. By inverting the light cycle every week, mice acquired tumours faster than age-matched littermates housed under normal light/dark cycles. Disruption of sleep patterns, and thereby the internal body clock, of shift workers therefore directly influences their physiology and the rate of cancer development. Moreover, working night shifts three or more times per month elevates the risk of breast cancer in humans. This risk increases with age and with more hours per week of night-shift work [63, 66, 67, 73].

Constant light exposure during the night disturbs circadian pacemaker activity in the mammalian SCN. This ultimately disrupts circadian rhythms throughout the body. One mechanism may be through altered production of the hormone melatonin. Night-time light exposure reduces the rhythmic secretion of melatonin from the pineal gland [74]. This hormone has oncostatic activity in experimental animals with mammary tumours, and also in human breast cancer cells in culture [75] (Fig. 2). Melatonin promotes genomic stability, and may also have anti-invasive and anti-metastatic activity [76]. For example, in endocrine-responsive cancer, melatonin reduces the expression and activity of aromatase, which normally converts testosterone to oestrogen. The oncostatic action of melatonin on hormone-dependent mammary tumours is mainly based on its anti-estrogenic actions, both reducing biosynthesis of oestrogen from androgens and neutralizing the cellular effects of oestrogen. Rats exposed to small amounts of light at night, rather than no light, had disrupted melatonin profiles and faster-growing mammary tumours that were tamoxifen resistant; this could be reversed by melatonin supplementation [77]. In humans, reduction of nocturnal melatonin levels may increase the effects of oestrogen and thereby contribute to breast cancer risk—and indeed, make tumours insensitive to steroid therapy. Epidemiological studies in nurses also reveal that shift work induces a significant increase in circulating oestradiol levels, which could further disrupt mammary oestrogen signalling and thereby promote cancer [78].

Another potential mechanism linking altered light exposure at night-time to breast cancer risk could be via the circadian regulation of micro-RNA (miRNA) expression. Several miRNAs known to be involved with breast cancer risk show fluctuations during the day. For example, the levels of miR-150-5p and miR-133a-3p are dramatically altered by circadian disruption [79]. Some of these miRNAs are interconnected with the expression of proteins known to have roles in breast cancer, such as NFkB and Stat3. The levels of some transcripts that encode proteins involved with cancer are also altered in circadian-disrupted mice [80].

Night-time light exposure can also happen as a result of loss of rhythmic behaviour, such as in SCN-lesioned animals. This both increases the risk of tumour formation and enhances progression of pre-existing tumours. For example, in SCN-ablated mice that are inoculated with implants of osteosarcoma or pancreatic adenocarcinoma, tumours grow two to three times faster than in sham-operated comparators [8]. In the breast, a robust circadian rhythm is associated with slow-growing tumours, while altered rhythms are linked to faster growing tumours [81, 82].

Together the evidence suggests that disrupting circadian clocks via night-time light exposure has an adverse role on tumour progression. This applies to those working irregular shift patterns, although it is still controversial whether there are links in flight attendant jet lag [83] or whether the widespread use of electric lighting at night might contribute to breast cancer [84].

The mechanisms by which altered light exposure contributes to cancer are not yet understood fully. However, the link between circadian disruption and breast cancer may have profound societal importance.

Tissue-based oscillators within the breast are coordinated by the SCN, and are influenced by photic, endocrine, neural and metabolic cues. Analysis of the strong circadian component within the breast has highlighted its influence on the cell cycle, which impacts on tumour progression and carcinoma aggression.

In-vivo evidence has revealed the role of the circadian molecular clockwork in tumour suppression, highlighting that breast cancer should no longer be treated as a local disorder but rather can result from systemic defects in tissue control.

Mouse models have demonstrated the importance of core clock genes within the breast, whether that is in progenitor cell renewal, tumour incidence, or interaction with the oestrogen pathway. Collectively, this indicates that there is an oncostatic role of some clock genes.

Together with the epidemiological data linking an increased incidence of breast cancer in shift workers, this has serious implications for the demands of modern society. Circadian disruption imposes a major public health issue that has yet to receive the recognition it deserves. Nocturnal lifestyles perturb the normal patterns of circadian behaviour, resulting in detrimental effects on clock-controlled physiological and metabolic pathways. Increased public awareness of the circadian influences on breast cancer risk could enhance lifestyle choices, as well as improve the alignment of physiological systems with the daily body clock.

There is still a fair way to go in characterizing the molecular basis of how the circadian system influences breast cancer, and which of the different genetic types of breast cancer are most influenced by altered clocks. Further delineation of the mechanisms linking clock genes to cancer is needed in order to fully understand how an altered circadian system and how rotating shift work influence the disease. This knowledge will help to improve future therapeutic interventions.

BMAL1, Brain and muscle Arnt-like protein-1; CLOCK, Circadian Locomotor Output Cycles Kaput; CRY, Cryptochrome; EMT, epithelial-mesenchymal transition; ER, oestrogen receptor; miRNA, micro-RNA; NPAS2, Neuronal PAS domain protein 2; PER, Period; SCN, suprachiasmatic nucleus; SNP, single nucleotide polymorphism