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Epidemiological studies indicate that disruption of circadian rhythm by shift work increases the risk of breast and prostate cancer. Our studies demonstrated that carcinogens disrupt the circadian expression of circadian genes (CGs) and circadian-controlled genes (CCGs) during the early stages of rat mammary carcinogenesis. A chemopreventive regimen of methylselenocysteine (MSC) restored the circadian expression of CGs and CCGs, including PERIOD 2 (PER2) and estrogen receptor β (ERS2), to normal. The present study evaluated whether changes in CG and CCG expression in whole blood can serve as indicators of circadian disruption in shift workers.Fifteen shift workers were recruited to a crossover study. Blood samples were drawn before (6 PM) and after (8 AM) completing a night shift after at least seven days on floating night-shift rotation, and before (8 AM), during (1 PM), and after (6 PM) completing seven days on day shift. The plasma melatonin level and messenger RNA (mRNA) expression of PER2, nuclear receptor subfamily 1, group d, member 1 (NR1D1), and ERS2 were measured, and the changes in levels of melatonin and gene expression were evaluated with statistical analyses.The mRNA expression of PER2 was affected by shift (p = 0.0079); the levels were higher in the evening for the night shift, but higher in the morning for the day shift. Increased PER2 expression (p = 0.034) was observed in the evening on the night versus day shifts. The melatonin level was higher in the morning for both day shifts (p = 0.013) and night shifts (p 0.0001).Changes in the level of PER2 gene expression can serve as a biomarker of disrupted circadian rhythm in blood cells. Therefore, they can be a useful intermediate indicator of efficacy in future MSC-mediated chemoprevention studies.

Introduction Epidemiological studies indicate that disruption of circadian rhythm by shift work is associated with increased risk of breast and prostate cancers. Rat studies demonstrated that carcinogens also disrupt the rhythmic expression of circadian and circadian controlled genes (CCG). More importantly, chemopreventive methylselenocysteine (MSC) resets the expression levels and phases of circadian and CCG genes, including estrogen receptor β (ER β ) (Fang, Zhang & Zarbl Cancer Prev. Res. 3: 640–652, 2010). Unpublished findings indicate that MSC resets rhythmic oscillations in histone and Bmal1 acetylation on the Per2 gene promoter, suggesting a mechanism for MSC- mediated resetting of circadian rhythm. The goal of the present study was to determine if changes in circadian and CCG expression in white blood cells can serve as biomarkers of circadian disruption in shift workers, and to assess the ability of MSC to reset the circadian rhythm in peripheral cells. Materials and methods Phase I: Fifteen hospital interns and residents (age 21–34) were recruited to a cross-over biomarker study. Blood samples were drawn at 8 AM, noon and 8 PM after completing at least one week on the day shift, and at 8 AM and 8 PM, after completing one week of service on the night shift. Serum melatonin and expression of circadian genes (Per2, NR1D1) and CCG (ER β ) were measured in peripheral blood samples. Descriptive statistics and mixed linear models evaluated changes in gene expression. Phase II: Results of this study were used to design a blinded randomized placebo-controlled intervention trial to explore the ability of a dietary supplement of MSC to reset normal circadian and circadian controlled gene expression. One hundred healthy volunteers, who work predominantly at night, were randomized to a placebo or MSC interventions groups (50 subjects per group). Blood samples were collected prior to the intervention to determine baseline levels of biomarkers. Subjects were asked to take a daily supplement containing MSC (3 ppm Se) or a placebo for a period of 30 days, while keeping a sleep log. Biomarkers are reassessed after termination of the intervention. Results Results showed that when working the day-shift, the average Per2 mRNA levels were lower at night vs morning; however, when working the night-shift, per2 mRNA was significantly higher at night vs morning (p = 0.01), indicating a change in Per2 mRNA expression with a change from day-to night-shift. We also observed a significant increase in Per2 mRNA expression (p = 0.034) during night- vs day-shift based on the samples collected in the evening. However, NR1D1 and ER β mRNA expression, very low at these time points, were not significantly affected by schedule changes. These findings formed the basis for a MSC interventional trial in 100 shift workers. The interventional study has been completed and the circadian biomarker analysis is in progress. Conclusion These results indicate that night- vs day-shift has a significant impact on Per2 gene expression, suggesting that it can be used as a biomarker to indicate disrupted circadian rhythm in shift workers. Per2 expression shows promise as a useful in longitudinal studies of MSC chemoprevention of cancer in shift workers. Acknowledgements These studies are supported by a Translational Research Grant from the V Foundation for Cancer Research, and NIH grants U19ES011387 (H. Zarbl, P.I.) and P30ES005022 (H. Zarbl, P.I.) from the National Institute for Environmental Health Sciences. We thank Kathy Kelly McNeil, Michelle Robertson, Susan Goodin and Benjamin Crabtree for contributions to this study.

Rat strains differ dramatically in their susceptibility to mammary carcinogenesis. On the assumption that susceptibility genes are conserved across mammalian species and hence inform human carcinogenesis, numerous investigators have used genetic linkage studies in rats to identify genes responsible for differential susceptibility to carcinogenesis. Using a genetic backcross between the resistant Copenhagen (Cop) and susceptible Fischer 344 (F344) strains, we mapped a novel mammary carcinoma susceptibility (Mcs30) locus to the centromeric region on chromosome 12 (LOD score of ∼8.6 at the D12Rat59 marker). The Mcs30 locus comprises approximately 12 Mbp on the long arm of rat RNO12 whose synteny is conserved on human chromosome 13q12 to 13q13. After analyzing numerous genes comprising this locus, we identified Fry, the rat ortholog of the furry gene of Drosophila melanogaster, as a candidate Mcs gene. We cloned and determined the complete nucleotide sequence of the 13 kbp Fry mRNA. Sequence analysis indicated that the Fry gene was highly conserved across evolution, with 90% similarity of the predicted amino acid sequence among eutherian mammals. Comparison of the Fry sequence in the Cop and F344 strains identified two non-synonymous single nucleotide polymorphisms (SNPs), one of which creates a putative, de novo phosphorylation site. Further analysis showed that the expression of the Fry gene is reduced in a majority of rat mammary tumors. Our results also suggested that FRY activity was reduced in human breast carcinoma cell lines as a result of reduced levels or mutation. This study is the first to identify the Fry gene as a candidate Mcs gene. Our data suggest that the SNPs within the Fry gene contribute to the genetic susceptibility of the F344 rat strain to mammary carcinogenesis. These results provide the foundation for analyzing the role of the human FRY gene in cancer susceptibility and progression.

Using Quantitative Trait Locus (QTL) mapping in a backcross between susceptible Fischer 344 and resistant Copenhagen rat strains, we identified Fry, the rat ortholog of the Drosophila melanoganster furry gene, as a mammary carcinoma susceptibility (Mcs) gene. The Fischer 344 Fry allele harbors two single nucleotide polymorphisms that alter amino acid residues that are highly conserved from yeast to humans. One of these SNPs replaces an Alanine with a Serine residue, creating a de novo concensus sequence for several cancer-associated protein kinases. In the lower eucaryoyes, the furry orthologs are implicated in diverse cell processes including epithelial cell polarization, morphogenesis, proliferation, cytokinesis, and regulation of the NDR family of kinases, some of which are putative human tumor suppressor genes. Consistent with its function as a tumor suppressor gene, ectopic expression of the FRY gene was reduced in 3 of 4 human breast cancer cell lines. Ectopic expression of the wildtype allele significantly reduced the tumorigenicity of the human MDA-MB-231 mammary carcinoma cell line. In contrast to the parental MDA-MB-231 cells, which grew randomly in Matrigel® matrix, those expressing Fry (231CFry) formed spherical colonies of differentiated cells, closely resembling those formed by MCF-10A normal mammary epithelial cell line. When xenographed into nude mice, the 231CFry cells demonstrated a dramatic reduction in tumorigenicity (8x reduction in tumor volume at 15 days) and diminished invasiveness into surrounding tissues. Gene expression profiling suggested that in MDA-MB-231 cells, ectopic Fry induced the expression of gene networks involved in cell differentiation, proliferation, and polarization, consistent with a role for FRY in suppressing epithelial-mesenchymal transition (EMT). To investigate the role of human FRY in breast cancer, we examined FRY mRNA expression in >1,500 human breast cancers, representing distinct eight cohorts entered into the Oncomine 3.0 Cancer Profiling Database. Our analysis revealed that decreased FRY mRNA was highly correlated with poorly differentiated breast cancer phenotypes (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-131. doi:10.1158/1538-7445.AM2011-LB-131

sis after PSG. The PDSSwas not higher in theOSA group (n = 64, median obstructive apnea hypopnea index (OAHI) 7.4, PDSS 13) compared to BS group (n = 116, median OAHI 0.1, PDSS 12). No relationship was seen between the PDSS and markers of OSA severity (OAHI, respiratory arousal index, desaturation index), or sleep duration (total sleep time, sleep efficiency). Conclusion: The PDSS was consistent with the SDSC in measuring EDS in children as young as 5 years. In children aged 11–12.9 years the PDSS score was higher overall, and sleep restriction was associated with a higher PDSS score. This relationship with sleep deprivation was not seen the younger children and suggests an increased propensity for EDS in pre-adolescent children. Alternatively the PDSS may not be the right instrument to detect EDS in young children. There was no correlation between PSG markers of OSA severity and EDS.