While a period effect that is likely due to advancements in diagnostic techniques and increased medical detection of small thyroid nodules may explain some of the observed increase in the incidence, we speculate that birth cohort-related changes in environmental exposures (such as increased exposure to diagnostic X-rays and polybrominated diphenyl ethers) have also contributed to the observed increase in papillary thyroid cancer during the past decades.

Circadian genes have the potential to influence a variety of cancer-related biological pathways, including immune regulation, which may influence susceptibility to non-Hodgkin’s lymphoma (NHL). However, few studies have examined the role of circadian genes in lymphomagenesis. The current study examined Cryptochrome 2 (CRY2), a core circadian gene and transcriptional repressor, as a potential circadian biomarker for NHL. We first performed genetic association analyses of tagging SNPs in CRY2 and NHL risk using DNA samples from a population-based case-control study (N= 455 cases and 527 controls). Three SNPs were found to be significantly associated with risk of NHL when combining all subtypes (dbSNP IDs, odds ratios (ORs), and 95% confidence intervals: rs11038689, OR=2.34 (1.28-4.27), P=0.006; rs7123390, OR=2.40 (1.39-4.13), P=0.002; and rs1401417, OR=2.97 (1.57-5.63), P=0.001). Each of these associations remained significant when restricting the analysis to B-Cell cases and when further restricting to follicular lymphomas. An analysis of CRY2 diplotypes confirmed these significant findings. To further determine the functional impact of CRY2, we silenced the gene in vitro and performed a whole genome expression microarray. A pathway-based analysis showed that genes significantly altered by CRY2 knockdown formed networks associated with immune response and hematological system development. In addition, these genes were predicted to have significant impacts on several disease processes, including cancer (B-H P-value=3.75E-9) and hematological disease (B-H P=8.01E-8). In conclusion, both genetic association and functional analyses suggest that the circadian gene CRY2 may play an important role in NHL development.

Apart from regulating sleep and wakefulness, the circadian system may play an important role in other biological processes, including pathways involved in tumorigenesis. Two genetic association studies recently conducted by our lab have shown that a missense mutation in neuronal PAS domain protein 2 (NPAS2), a core circadian gene and transcriptional regulator, is significantly associated with risk of breast cancer and non -Hodgkin's lymphoma. Our current functional analyses provide the first in vitro evidence further demonstrating that cells with RNA interference -mediated depletion of NPAS2 fail to exhibit the expected cell cycle delay in response to mutagen treatment. DNA repair capacity, as measured by the comet assay, is also impaired. Moreover, a pathway-based PCR expression array of genes important for DNA damage signaling showed that knockdown of NPAS2 significantly represses the expression of several cell cycle and DNA repair genes. Thus, NPAS2 may play a role in tumorigenesis by affecting expression of cancer-related genes and could be considered a novel tumor suppressor.

As transcriptional regulators, circadian genes have the potential to influence a variety of biological pathways, including many cancer-related processes. Cryptochrome 2 (CRY2) is essential for proper circadian timing, and is a key component of the circadian regulatory feedback loop. Here, we report findings from genetic, epigenetic, loss-of-function, and transcriptional profiling analyses of CRY2 in breast cancer. Six SNPs in CRY2 were identified for genotyping in a case-control population (N=441 cases and N=479 controls), and three SNPs (rs11038689, rs7123390, and rs1401417) were significantly associated with postmenopausal breast cancer risk, with significant effect modification by menopausal status (dominant model for rs11038689: odds ratio (OR) = 0.71, 95% confidence interval (CI): 0.51–0.99, P for trend = 0.028; homozygous variants for rs7123390: OR = 0.44, 95% CI: 0.22–0.86, P for trend = 0.028; and rs1401417, OR=0.44, 95% CI: 0.21–0.92, P for trend = 0.017). Interestingly, this association was only evident in women with estrogen and progesterone receptor (ER/PR) negative breast tumors, but not with ER/PR positive tumors. Breast cancer patients also had significantly higher levels of CRY2 promoter methylation relative to controls, which is consistent with tissue array data showing lower levels of CRY2 expression in tumor tissue relative to adjacent normal tissue. Furthermore, in vitro analyses identified a number of breast cancer-relevant genes which displayed altered expression following CRY2 knockdown. These findings suggest a role for CRY2 in breast tumorigenesis, and provide further evidence that the circadian system may be an important modulator of hormone-related cancer susceptibility.