The observed connection between circadian disruption (e.g. shift work) and cancer risk in epidemiologic studies has led to the circadian gene hypothesis, which suggests that genetic variants in genes responsible for maintaining circadian rhythm may affect an individual’s susceptibility to human cancers. This hypothesis is supported by results from recent genetic association studies of breast cancer (28
), prostate cancer (24
), and NHL (5
). The findings from the current study of CRY2
provide more evidence demonstrating a role for the circadian genes in NHL susceptibility.
To the best of our knowledge, genetic variants in CRY2
have not been previously examined in NHL, and only one of the SNPs genotyped in the case-control portion of this analysis had been studied previously; in a population-based case-control study conducted in China, which showed a significant association between the variant allele of rs1401417 and increased risk of prostate cancer (24
). This finding is consistent with the significant association we observed in the NHL population, and further investigations into the nature of these relationships are warranted in order to determine whether CRY2
has a global impact on cancer susceptibility.
The observed associations between CRY2
and NHL risk provide additional molecular epidemiologic evidence supporting the proposed role of circadian genes as tumor suppressors (30
). Circadian genes have been shown to affect expression of 2–10% of mammalian genes (31
) including many cancer-related genes (30
). Emerging data from animal models have further demonstrated a substantial impact of circadian genes on tumor-related biological pathways such as cell proliferation, cell cycle control, DNA damage response, and apoptosis (30
). Mice with mutations in the circadian gene PER2
have deficiencies in DNA damage response and are more prone to tumorigenesis (32
). Altered expression of circadian genes also occurs in human tumors; as studies have shown that the period (PER
) genes fail to maintain daily rhythmic expression patterns in cancer cells (33
). Although CRY2
has also been shown to be involved in cell cycle regulation (34
), explicit mechanisms for its role in cancer susceptibility, especially NHL tumorigenesis, are currently unknown.
The microarray analysis, which implicated CRY2
as having the potential to influence gene expression in a number of pathways, including those with relevance for cancer and immune function, provides the first mechanistic evidence suggesting that CRY2
may be important for NHL susceptibility. The findings relative to IL-6 are of particular interest. It has been demonstrated that IL-6 can inhibit DNA synthesis by preventing cell cycle progression into S phase (35
). It has also been fairly well established that IL-6 plays an important role in B-cell proliferation (36
), and antibodies against IL-6 or its receptor have been explored as treatments for B-lymphoproliferative disorder (37
) and inflammatory autoimmune diseases (38
). In addition, a previous study has demonstrated that mice expressing an IL-6 transgene exhibit lymphoproliferation and plasmacytosis, and nearly a third of these mice developed follicular and diffuse large cell B-cell lymphomas (39
). Moreover, serum levels of IL-6 and VEGF have been shown to be of value in predicting clinical outcome in some patients with NHL (40
). The observation that silencing of CRY2
results in more than 8-fold induction of IL-6, and 6-fold induction of VEGFC, is therefore highly relevant in understanding the etiology of hematologic malignancies, and may have important implications for predicting NHL prognosis. Furthermore, cell signaling by IL-6 is partially mediated by its effects on the JAK/STAT3 pathway (42
). While JAK was unaffected following CRY2
silencing, STAT3 was upregulated more than twofold (B-H P=7.06E-5
). Since IL-6 has been shown to confer increased survival and chemotherapeutic resistance on lymphoma cells, which is at least partially mediated by STAT3; STAT3 has been proposed as a potential therapeutic target for patients with NHL (43
). As such, although the pleiotropic biological effects of CRY2 may make it a poor candidate to be directly targeted by therapeutic agents, its effects on both IL-6 and STAT3 may lead future investigations to consider circadian timing when administering cytokine-targeting chemotherapies.
Apart from IL-6, several other interleukins were also significantly altered in CRY2
knockdown cells. IL-18, which was upregulated by more than 10 fold, may play an important, but paradoxical role in cancer risk (44
). It has been implicated in cancer protection, through its role in activating immune cells to eliminate sporadic cancers, but may also promote tumor progression by encouraging angiogenesis, tumor growth and local invasion. Interestingly all three members of a newly described class of interferon lambdas (IL-28a, IL-28b, and IL-29) were significantly upregulated following CRY2
silencing. These immunoregulatory cytokines have antiviral and antitumor activity, and may also have potential as therapeutic agents in the treatment of cancer (45
Three members of the chemokine (C-C motif) ligand family (CCL3, CCL4, and CCL5) were also upregulated in CRY2
knockdown cells. These genes are clustered together on the long arm of chromosome 17, and are important for immune regulation and inflammation (46
). CCL3, which was upregulated more than 15 fold in CRY2
knockdown cells, has been shown to be elevated in patients with multiple myeloma (MM) and other hematologic cancers compared to healthy controls; and increased serum levels of CCL3 were associated with advanced MM stage (47
) and poorer prognosis in MM (48
). Of additional interest was the identification of four genes in the major histocompatibility complex, class I (HLA-A, HLA-B, HLA-C, and HLA-E), which were all significantly upregulated following CRY2
silencing. Apart from being central to immune regulation, these genes have been associated with several cancers, including Hodgkin’s disease (49
) and non-Hodgkin’s lymphoma (50
). Taken together, these results suggest that reductions in CRY2
have the potential to significantly impact processes relevant for lymphomagenesis, and while they represent a first step in understanding the mechanism by which SNPs in the CRY2
gene might influence NHL susceptibility.
It is important to recognize some of the study’s limitations. First, it is unclear how closely the effect of CRY2 silencing in vitro might mimic the conditions which would arise in vivo. At the level of the organism, circadian rhythmicity is made even more complex by environmental cues, such as light exposure, and hormonal pathways, which may influence circadian gene expression. In addition, since one aim of the study was to examine the influence of CRY2 silencing on immune response pathways, we chose not to use cells derived from lymphoma tissue, which may begin with some level of aberrant immune-related gene expression. As such, it will be necessary for future studies to confirm that the findings we observed are applicable to lymphocytes, and that the changes in gene expression following CRY2 silencing that we observed at the cellular level, are representative of those that would occur at the organismal level.
In summary, the findings from our case-control analysis suggest a novel association between the circadian gene CRY2 and risk of NHL; supporting the hypothesis that genetic variations in circadian genes may confer inherited susceptibility to NHL. The subsequent loss-of-function analysis and whole genome expression microarray suggest that the observed association could potentially be attributed to the impact of CRY2 on several genes important for cancer in general, as well as a number of genes with known relevance for hematological malignancies. Our findings provide a novel panel of promising biomarkers for NHL risk and prognosis, which warrant further investigation. In addition, since previous studies have suggested that circadian-related environmental exposures, such as light at night or rotating shift work, may influence cancer susceptibility, future investigations into potential interactions between circadian gene variants and environmental exposures may be of interest in developing cancer prevention strategies.