Circadian pathway genetic variation and cancer risk: evidence from genome-wide association studies

The circadian clock is a time-tracking rhythmic biological system (internal timing machine) with a periodicity of about 24 h that enables organisms to anticipate environmental changes (such as food availability) and allows them to modify their behaviour and physiological functions (e.g. sleep and wakefulness, basal metabolism, body temperature, blood pressure, hormone production and immunity) in the most efficient way [1]. This system consists of two components: the central clock, located in the suprachiasmatic nucleus of the brain, and the peripheral clocks, which are present in virtually all body tissues. Circadian rhythms are controlled by what are called circadian pathway genes [2], which have been discovered in all studied species: remarkably, the disruption of these rhythms has been linked to the risk of different diseases such as insomnia, depression, jet leg, stomach ailments, heart attack and cancer [3]. As regards the latter, a growing wealth of evidence supports the potential tumour suppressor role of the biological clock [4]. In particular, single germline variations of circadian genes have been associated with the predisposition of some tumour types such as breast carcinoma [5], although the evidence is not conclusive due to the scarcity of data in this recent field of research.

Germline DNA variation has been long recognized as a key component of the individual risk to develop cancer, and recently the discovery rate of susceptibility loci is being greatly accelerated by genome-wide association studies (GWASs) which can test up to one million single nucleotide polymorphisms (SNPs) in thousands of subjects at a time [6]. However, the proportion of genetic susceptibility to complex traits (such as cancer) explained by single locus analysis still remains small, whereas it is increasingly recognized that multiple locus analysis — such as gene and gene set (or pathway) analysis — is more powerful for dissecting the genetic architecture of complex diseases according to the principles of systems genetics [7]. In fact, a single SNP can have an effect too small to be detected by the single locus approach, whereas gene/pathway analysis, which jointly tests multiple SNPs from the same gene/pathway, can more likely identify the association between the outcome and the basic functional unit involved in disease development [8‐10].

In this work, we intended to investigate whether germline genetic variation of the circadian pathway is associated with the risk of cancer by analysing publicly available data from GWASs. To this aim we chose to focus on the three most common tumour types, i.e. lung, breast and prostate carcinomas, which account for up to 40% of all cancer incident cases and observed deaths [11].

In this study we found that germline genetic variation in the circadian pathway is associated with the risk of developing breast, prostate and lung carcinoma in a large cohort of cases (n = 42,068) and controls (n = 47,646). This association was also maintained in subgroup analyses for estrogen receptor negative breast cancer, aggressive prostate cancer and both squamous carcinoma and adenocarcinoma lung cancer. To the best of our knowledge, this is the first time that ARTP-based gene and pathway analysis has been applied to the relationship between circadian genes’ germline variation and cancer susceptibility. Thus far, molecular epidemiology studies have investigated only single variants of single circadian genes in relationship with some tumour types (such as breast, pancreatic and prostate carcinomas) [5, 20]. In particular, according to our recent systematic review and meta-analysis of the published literature on the subject [5], out of 687 SNPs (located in 14 circadian genes) only 10 SNPs located in five genes (NPAS2 rs10165970, rs895520, rs17024869 and rs7581886; CLOCK rs3749474 and rs11943456; RORA rs7164773 and rs10519097; RORB rs7867494; and PER3 rs1012477) resulted in being significantly associated with the predisposition to only one tumour type, that is, breast carcinoma. Moreover, none of the SNPs investigated in the three GWAS meta-analyses included in the present study reached statistical significance after adjustment for multiple testing [17‐19]. In contrast, pathway analysis enabled us to link with high statistical significance (pathway P values always lower than 1 × 10^–5) the circadian pathway variation to the susceptibility not only of breast cancer but also to that of other two most common malignancies such as prostate and lung carcinoma. This relationship was sustained by 15 statistically significant genes out of 17 genes investigated, with only CSNK1D and TIMELESS being excluded from the association (see Tables 1 and 2).

The implication of most circadian genes in all three tumour types (as well as all their subtypes) indicates that variation of this pathway could actually be involved in the predisposition to cancer in general, which still requires more investigation to be demonstrated in patients affected with malignancies other than those considered in this study. On the other hand, our results point out that the germline variation of some genes (ARNTL, CLOCK, RORA and RORB) is shared by all three tumour types, whereas the polymorphisms of other genes might be more specific to one or two malignancies (see the Venn diagram in Fig. 2). This finding suggests that some circadian genes might be more relevant than others in terms of cancer predisposition. In particular, it is noteworthy that all the above-mentioned four shared genes belong to the positive loop of the circadian pathway (that is, the stimulatory component of the biological clock circuit; see Fig. 1) and that RORA is the most significant gene associated with all tumour types (except for prostate carcinoma, where it ranks second) and subtypes (see Tables 1 and 2). However, the biological meaning of these observations requires dedicated studies to be elucidated. For instance, it is known that the CLOCK gene product activity can affect both estrogen [21] and androgen pathways [22], which is concordant with the relationship between circadian pathway perturbation and the risk of hormone-driven malignancies such as breast and prostate cancer, respectively; however, the association with lung carcinoma remains less intuitive and warrants further investigation on the cascade of molecular events underlying the link between the biological clock and this type of tumour.

Overall, our data underscore the fact that a biological relationship undetected by single polymorphisms can be unveiled by pathway analysis, confirming the power of this multi-SNP and multi-gene approach [8‐10, 23].

In particular, our results support the pre-clinical evidence regarding the candidate role of the circadian pathway as a tumour suppressor circuit acting through the transcriptional control of (or the direct interaction with) key regulators of cell proliferation, apoptosis and DNA repair (and thus genomic stability) and metabolism, such as Ciclin-D1, c-Myc, Mdm2, p53, Gadd45-alpha, Atm, Chk1, Nampt and Sirt-1 [4, 24, 25], which are well known to play a pivotal role in carcinogenesis.

In a Mendelian randomization perspective (that is, using variation in genes of known function to examine the causal effect of a given environmental exposure/behaviour on disease, reasonably assuming that genes are not themselves associated with any confounding factors) [26, 27], our data also support the hypothesis that the disruption of the physiological internal clock — as in sleep deprivation, insomnia, work shifting and jet leg — might lead to an increased risk of cancer, as suggested by some classical epidemiology studies [28‐31] and confuted by others [32‐35], with most of them focusing on breast cancer.

Certainly, we cannot draw any definitive conclusion on this subject, as dedicated studies of fine mapping are needed to systematically investigate the relationship between germline variation of the circadian pathway molecular components and cancer risk. Moreover, functional experiments are required to fully dissect the actual link between circadian pathway polymorphisms and the molecular mechanisms underlying cancer development. Finally, a pathway-based polygenic risk score [36, 37] should be tested to translate genetic information into clinically valuable risk prediction. In fact, pathway analysis only provides evidence of association between a given biological circuit and the predisposition to a studied disease; it does not provide any clue to the magnitude of the risk linked to a specific (that is, individual) genetic signature. We hope that this study can represent a decisive step forward towards the personalization of cancer risk prediction, with potentially important implications in terms of screening programs [38].

In conclusion, our results — based on pathway analysis of the largest series ever analysed in this research field — strengthen the already-existing (but discordant) clinical epidemiology data and genetic evidence (based on single polymorphisms) supporting the link between the genetic control of the circadian pathway and the development of cancer, which prompts further investigation in this promising area of cancer research.