Influence of Daytime LED Light Exposure on Circadian Regulatory Dynamics of Metabolism and Physiology in Mice.

Light is a potent biologic force that profoundly influences circadian, neuroendocrine, and neurobehavioral regulation in animals. Previously we examined the effects of light-phase exposure of rats to white light-emitting diodes (LED), which emit more light in the blue-appearing portion of the visible spectrum (465 to 485 nm) than do broad-spectrum cool white fluorescent (CWF) light, on the nighttime melatonin amplitude and circadian regulation of metabolism and physiology. In the current studies, we tested the hypothesis that exposure to blue-enriched LED light at day (bLAD), compared with CWF, promotes the circadian regulation of neuroendocrine, metabolic, and physiologic parameters that are associated with optimizing homeostatic regulation of health and wellbeing in 3 mouse strains commonly used in biomedical research (C3H [melatonin-producing], C57BL/6, and BALB/c [melatonin-non-producing]). Compared with male and female mice housed for 12 wk under 12:12-h light:dark (LD) cycles in CWF light, C3H mice in bLAD evinced 6-fold higher peak plasma melatonin levels at the middark phase; in addition, high melatonin levels were prolonged 2 to 3 h into the light phase. C57BL/6 and BALB/c strains did not produce nighttime pineal melatonin. Body growth rates; dietary and water intakes; circadian rhythms of arterial blood corticosterone, insulin, leptin, glucose, and lactic acid; pO2 and pCO2; fatty acids; and metabolic indicators (cAMP, DNA, tissue DNA 3H-thymidine incorporation, fat content) in major organ systems were significantly lower and activation of major metabolic signaling pathways (mTOR, GSK3ß, and SIRT1) in skeletal muscle and liver were higher only in C3H mice in bLAD compared with CWF. These data show that exposure of C3H mice to bLAD compared with CWF has a marked positive effect on the circadian regulation of neuroendocrine, metabolic, and physiologic parameters associated with the promotion of animal health and wellbeing that may influence scientific outcomes. The absence of enhancement in amelatonic strains suggests hyperproduction of nighttime melatonin may be a key component of the physiology.

Epigenetic inhibition of the tumor suppressor ARHI by light at night-induced circadian melatonin disruption mediates STAT3-driven paclitaxel resistance in breast cancer.

Disruption of circadian time structure and suppression of circadian nocturnal melatonin (MLT) production by exposure to dim light at night (dLAN), as occurs with night shift work and/or disturbed sleep-wake cycles, is associated with a significantly increased risk of breast cancer and resistance to tamoxifen and doxorubicin. Melatonin inhibition of human breast cancer chemoresistance involves mechanisms including suppression of tumor metabolism and inhibition of kinases and transcription factors which are often activated in drug-resistant breast cancer. Signal transducer and activator of transcription 3 (STAT3), frequently overexpressed and activated in paclitaxel (PTX)-resistant breast cancer, promotes the expression of DNA methyltransferase one (DNMT1) to epigenetically suppress the transcription of tumor suppressor Aplasia Ras homolog one (ARHI) which can sequester STAT3 in the cytoplasm to block PTX resistance. We demonstrate that breast tumor xenografts in rats exposed to dLAN and circadian MLT disrupted express elevated levels of phosphorylated and acetylated STAT3, increased DNMT1, but reduced sirtuin 1 (SIRT1) and ARHI. Furthermore, MLT and/or SIRT1 administration blocked/reversed interleukin 6 (IL-6)-induced acetylation of STAT3 and its methylation of ARH1 to increase ARH1 mRNA expression in MCF-7 breast cancer cells. Finally, analyses of the I-SPY 1 trial demonstrate that elevated MT1 receptor expression is significantly correlated with pathologic complete response following neo-adjuvant therapy in breast cancer patients. This is the first study to demonstrate circadian disruption of MLT by dLAN driving intrinsic resistance to PTX via epigenetic mechanisms increasing STAT3 expression and that MLT administration can reestablish sensitivity of breast tumors to PTX and drive tumor regression.

Effects of Daytime Exposure to Light from Blue-Enriched Light-Emitting Diodes on the Nighttime Melatonin Amplitude and Circadian Regulation of Rodent Metabolism and Physiology.

Regular cycles of exposure to light and dark control pineal melatonin production and temporally coordinate circadian rhythms of metabolism and physiology in mammals. Previously we demonstrated that the peak circadian amplitude of nocturnal blood melatonin levels of rats were more than 6-fold higher after exposure to cool white fluorescent (CWF) light through blue-tinted (compared with clear) rodent cages. Here, we evaluated the effects of light-phase exposure of rats to white light-emitting diodes (LED), which emit light rich in the blue-appearing portion of the visible spectrum (465-485 nm), compared with standard broadspectrum CWF light, on melatonin levels during the subsequent dark phase and on plasma measures of metabolism and physiology. Compared with those in male rats under a 12:12-h light:dark cycle in CWF light, peak plasma melatonin levels at the middark phase (time, 2400) in rats under daytime LED light were over 7-fold higher, whereas midlight phase levels (1200) were low in both groups. Food and water intakes, body growth rate, and total fatty acid content of major metabolic tissues were markedly lower, whereas protein content was higher, in the LED group compared with CWF group. Circadian rhythms of arterial plasma levels of total fatty acids, glucose, lactic acid, pO2, pCO2, insulin, leptin, and corticosterone were generally lower in LED-exposed rats. Therefore, daytime exposure of rats to LED light with high blue emissions has a marked positive effect on the circadian regulation of neuroendocrine, metabolic, and physiologic parameters associated with the promotion of animal health and wellbeing and thus may influence scientific outcomes.

Daytime Blue Light Enhances the Nighttime Circadian Melatonin Inhibition of Human Prostate Cancer Growth.

Light controls pineal melatonin production and temporally coordinates circadian rhythms of metabolism and physiology in normal and neoplastic tissues. We previously showed that peak circulating nocturnal melatonin levels were 7-fold higher after daytime spectral transmittance of white light through blue-tinted (compared with clear) rodent cages. Here, we tested the hypothesis that daytime blue-light amplification of nocturnal melatonin enhances the inhibition of metabolism, signaling activity, and growth of prostate cancer xenografts. Compared with male nude rats housed in clear cages under a 12:12-h light:dark cycle, rats in blue-tinted cages (with increased transmittance of 462-484 nm and decreased red light greater than 640 nm) evinced over 6-fold higher peak plasma melatonin levels at middark phase (time, 2400), whereas midlight-phase levels (1200) were low (less than 3 pg/mL) in both groups. Circadian rhythms of arterial plasma levels of linoleic acid, glucose, lactic acid, pO2, pCO2, insulin, leptin, and corticosterone were disrupted in rats in blue cages as compared with the corresponding entrained rhythms in clear-caged rats. After implantation with tissue-isolated PC3 human prostate cancer xenografts, tumor latency-to-onset of growth and growth rates were markedly delayed, and tumor cAMP levels, uptake-metabolism of linoleic acid, aerobic glycolysis (Warburg effect), and growth signaling activities were reduced in rats in blue compared with clear cages. These data show that the amplification of nighttime melatonin levels by exposing nude rats to blue light during the daytime significantly reduces human prostate cancer metabolic, signaling, and proliferative activities.

PIWI-interacting RNA 021285 is involved in breast tumorigenesis possibly by remodeling the cancer epigenome.

Although PIWI-interacting RNAs (piRNAs) account for the largest class of the small non-coding RNA superfamily, virtually nothing is known of their function in human carcinogenesis. Once thought to be expressed solely in the germ line where they safeguard the genome against transposon-induced insertional mutations, piRNAs are now believed to play an active role in somatic gene regulation through sequence-specific histone modification and DNA methylation. In the current study, we investigate the role of piRNA-021285 (piR-021285) in the regulation of the breast cancer methylome. Genotypic screening of a panel of single-nucleotide polymorphism (SNP)-containing piRNAs revealed a significant association between SNP rs1326306 G>T in piR-021285 and increased likelihood for breast cancer in a Connecticut-based population (441 cases and 479 controls). Given nascent but compelling evidence of piRNA-mediated gene-specific methylation in the soma, a genome-wide methylation screen was then carried out using wild type (WT) and variant piR-021285 mimic-transfected MCF7 cells to explore whether the observed association could be attributed in part to piR-021285-induced methylation at cancer-relevant genes. We found significant methylation differences at a number of experimentally implicated breast cancer-related genes, including attenuated 5' untranslated region (UTR)/first exon methylation at the proinvasive ARHGAP11A gene in variant mimic-transfected cells. Follow-up functional analyses revealed both concurrent increased ARHGAP11A mRNA expression and enhanced invasiveness in variant versus WT piR-021285 mimic-transfected breast cancer cell lines. Taken together, our findings demonstrate the first evidence supporting a role of piRNAs, a novel group of non-coding RNA, in human tumorigenesis via a piRNA-mediated epigenetic mechanism, which warrants further confirmation and investigation.

The circadian gene CRY2 is associated with breast cancer aggressiveness possibly via epigenomic modifications.

Although the role of core circadian gene cryptochrome 2 (CRY2) in breast tumorigenesis has been demonstrated, the correlations of CRY2 with clinical parameters in breast cancer patients and its involvement in epigenetic processes such as DNA methylation remain relatively unexplored. In the current study, we first queried the Oncomine database and the Gene Expression-Based Outcome for Breast Cancer Online (GOBO) database to identify associations between CRY2 expression levels and clinical parameters in breast cancer patients. We then silenced CRY2 in vitro and performed a genome-wide methylation array to determine the epigenetic impact of CRY2 silencing. The Ingenuity Pathway Analysis software was used to further explore the genes exhibiting altered methylation identified using the array. We found that CRY2 was frequently down-regulated in breast cancer tissue compared to adjacent normal tissue or breast tissue from healthy controls. Lower CRY2 expression was associated with estrogen receptor (ER)-negativity (P < 0.0001), higher tumor grade (P < 0.0001), and shorter overall survival time in breast cancer patients (HR = 1.44, 95 % confidence interval (CI) 1.09-1.91). Genome-wide methylation analysis showed that a total of 515 CpG sites were hypermethylated following CRY2 knockdown, while 730 sites were hypomethylated. The pathway analysis revealed several cancer-relevant networks with genes exhibiting significantly altered methylation following CRY2 silencing. These findings suggest that the core circadian gene CRY2 is associated with breast cancer progression and prognosis, and that knockdown of CRY2 causes the epigenetic dysregulation of genes involved in cancer-relevant pathways, which provide further evidence supporting a role of the circadian system in breast tumorigenesis.

The influence of red light exposure at night on circadian metabolism and physiology in Sprague-Dawley rats.

Early studies on rodents showed that short-term exposure to high-intensity light (> 70 lx) above 600 nm (red-appearing) influences circadian neuroendocrine and metabolic physiology. Here we addressed the hypothesis that long-term, low-intensity red light exposure at night (rLEN) from a 'safelight' emitting no light below approximately 620 nm disrupts the nocturnal circadian melatonin signal as well as circadian rhythms in circulating metabolites, related regulatory hormones, and physi- ologic parameters. Male Sprague-Dawley rats (n = 12 per group) were maintained on control 12:12-h light:dark (300 lx; lights on, 0600) or experimental 12:12 rLEN (8.1 lx) lighting regimens. After 1 wk, rats underwent 6 low-volume blood draws via cardiocentesis (0400, 0800, 1200, 1600, 2000, and 2400) over a 4-wk period to assess arterial plasma melatonin, total fatty acid, glucose, lactic acid, pO2, pCO2, insulin, leptin and corticosterone concentrations. Results revealed plasma melatonin levels (mean ± 1 SD) were high in the dark phase (197.5 ± 4.6 pg/mL) and low in the light phase (2.6 ± 1.2 pg/mL) of control condi- tions and significantly lower than controls under experimental conditions throughout the 24-h period (P < 0.001). Prominent circadian rhythms of plasma levels of total fatty acid, glucose, lactic acid, pO2, pCO2, insulin, leptin, and corticosterone were significantly (P < 0.05) disrupted under experimental conditions as compared with the corresponding entrained rhythms under control conditions. Therefore, chronic use of low-intensity rLEN from a common safelight disrupts the circadian organization of neuroendocrine, metabolic, and physiologic parameters indicative of animal health and wellbeing.

Targetome profiling and functional genetics implicate miR-618 in lymphomagenesis.

Despite the voluminous body of observational evidence concerning the role of miRNAs in cancer, significant knowledge gaps remain concerning the molecular circumstances that underlie the miRNA-cancer connection. In this study, we employ a multidisciplinary approach to establish an association between miR-618 and non-Hodgkin lymphoma (NHL) in a human population and attempt to explicate this association at the molecular level. A high-throughput, transcriptome-wide RIP-Chip-based method was used to identify members of the miR-618 targetome, which were analyzed for functional relevance using a gene network-based approach. Findings were confirmed by genotyping a SNP (rs2682818) in the stem-loop sequence of miR-618 in a population-based case-control study of NHL (455 cases and 527 controls). Lastly, we analyzed the functional impact of rs2682818 on miR-618 expression and its consequent implications for the lymphomagenic process. A total of 128 miR-618 targets were identified, which were enriched for genes that have functional roles in lymphoma-relevant pathways. This is consistent with our finding of a significant association between rs2682818 G>T in the miR-618 stem-loop and follicular lymphoma (FL) (OR: 1.65, 95% CI: 1.05-2.60). In vitro analysis of rs2682818's functional impact revealed that the variant T allele resulted in reduced levels of mature miR-618, which in turn may lead to deregulation of miR-618-controlled pathways relevant to follicular lymphoma. Taken together, our findings implicate miR-618 in follicular lymphomagenesis, identify miR-618 as a potential risk biomarker for follicular lymphoma, and illuminate miR-618-regulated lymphomagenic pathways that can serve as therapeutic targets for follicular lymphoma.

Diabetes mellitus and colorectal cancer prognosis: a meta-analysis.

BACKGROUND: Diabetes mellitus is associated with an increased incidence of colorectal cancer, but the impact of diabetes mellitus on colorectal cancer prognosis is not clear. OBJECTIVE: We conducted a meta-analysis of observational studies to examine the association between preexisting diabetes mellitus and colorectal cancer all-cause mortality, cancer-specific mortality, and recurrence. DATA SOURCES: Medline and Embase were searched through August 22, 2012. STUDY SELECTION: We included studies reporting all-cause mortality, cancer-specific mortality, disease-free survival, or recurrence in patients who have colorectal cancer according to diabetic status. INTERVENTION: Meta-analyses were performed by the use of random-effects models. MAIN OUTCOME MEASURES: The primary outcomes measured were all-cause mortality, cancer-specific mortality, and disease-free survival. RESULTS: Twenty-six articles met our inclusion criteria. Patients with colorectal cancer who had diabetes mellitus had a 17% increased risk of all-cause mortality (relative risk, 1.17; 95% CI, 1.09-1.25) and a 12% increased risk of cancer-specific mortality (relative risk, 1.12; 95% CI, 1.01-1.24) in comparison with those who did not have diabetes mellitus. Those with diabetes mellitus also had poorer disease-free survival (relative risk, 1.54; 95% CI, 1.08-2.18) compared with their nondiabetic counterparts. In subgroup analyses, diabetes mellitus was associated with all-cause mortality in both rectal (relative risk, 1.24; 95% CI, 1.07-1.29) and colon cancer patients (relative risk, 1.17; 95% CI, 1.07-1.29). Sensitivity analyses including only patients with nonmetastatic disease identified stronger associations between diabetes mellitus and both all-cause (relative risk, 1.32; 95% CI, 1.21-1.44) and cancer-specific (relative risk, 1.27; 95% CI, 1.06-1.52) mortality. LIMITATIONS: Some studies had short follow-up or did not report mean or median follow-up. The included studies were heterogeneous in study population, diabetes mellitus diagnostic criteria, and outcome ascertainment. CONCLUSION: Patients with colorectal cancer who have diabetes mellitus are at greater risk for all-cause and cancer-specific mortality and have worse disease-free survival than those who do not have diabetes mellitus. Studies are warranted to determine whether the proper treatment could attenuate the excess mortality among patients with colorectal cancer who have diabetes mellitus.

Advanced sleep schedules affect circadian gene expression in young adults with delayed sleep schedules.

BACKGROUND: Human circadian rhythms are regulated by the interplay between circadian genes and environmental stimuli. The influence of altered sleep-wake schedules or light on human circadian gene expression patterns is not well characterized. METHODS: Twenty-one young adults were asked to keep to their usual sleep schedules and two blood samples were drawn at the end of the first week from each subject based on estimated time of dim light melatonin onset (DLMO); the first sample was obtained one and a half hours before the estimated DLMO and the second three hours later, at one and a half hours after the estimated DLMO. During the second week, participants were randomized into two groups, one that received a one hour blue-light (λmax=470 nm) exposure in the morning and one that received a comparable morning dim-light exposure. Two blood samples were obtained at the same clock times as the previous week at the end of the second week. RESULTS: We measured the expression of 10 circadian genes in response to sleep-wake schedule advancement and morning blue-light stimulation in the peripheral blood of 21 participants during a two-week field study. We found that nine of the 10 circadian genes showed significant expression changes from the first to the second week for participants in both the blue-light and dim-light groups, likely reflecting significant advances in circadian phase. CONCLUSIONS: This wholesale change in circadian gene expression may reflect considerable advances in circadian phase (i.e., advance in DLMO) from the first to the second week resulting from the advanced, daily personal light exposures.

Association of AMP-activated protein kinase with risk and progression of non-Hodgkin lymphoma.

BACKGROUND: Metabolic dysregulation has been identified as an "emerging hallmark" of cancer. The heterotrimeric AMP-activated protein kinase (AMPK) complex is a central regulator of the metabolic system and an important component of the mTOR pathway and the p53 axis, making it uniquely positioned to influence carcinogenesis through its canonical functions in the metabolic arena, as well as through more traditional mechanisms such as regulation of apoptosis and angiogenesis. METHODS: We conducted a population-based genetic association study to examine the impact of mutations in AMPK subunit genes on risk of non-Hodgkin lymphoma (NHL). We also analyzed public microarray data to determine the expression of AMPK in NHL cells and to assess the influence of AMPK expression on overall survival in patients with NHL. RESULTS: We identified an AMPK subunit haplotype, which was significantly associated with NHL [OR, 5.44, 95% confidence interval (CI), 2.15-13.75] in women with no family history of cancer. Haplotypes in two subunits, PRKAA2 and PRKAG3, were nominally associated with the follicular and diffuse large B-cell lymphoma histologic subtypes, respectively, although these associations did not retain statistical significance after correction for multiple comparisons. Further, both of these subunits were differentially expressed (P < 0.05) in one or more lymphoma cell type, and higher expression of two versions of the AMPK-ß subunit was significantly associated with increased 5-year survival among patients with NHL (P = 0.001 and P = 0.021). CONCLUSION: These results provide evidence for AMPK involvement in the pathogenesis and progression of NHL. IMPACT: These findings may lead to a novel area of research into NHL treatment and chemoprevention.

Melatonin enhances DNA repair capacity possibly by affecting genes involved in DNA damage responsive pathways.

BACKGROUND: Melatonin, a hormone-like substance involved in the regulation of the circadian rhythm, has been demonstrated to protect cells against oxidative DNA damage and to inhibit tumorigenesis. RESULTS: In the current study, we investigated the effect of melatonin on DNA strand breaks using the alkaline DNA comet assay in breast cancer (MCF-7) and colon cancer (HCT-15) cell lines. Our results demonstrated that cells pretreated with melatonin had significantly shorter Olive tail moments compared to non-melatonin treated cells upon mutagen (methyl methanesulfonate, MMS) exposure, indicating an increased DNA repair capacity after melatonin treatment. We further examined the genome-wide gene expression in melatonin pretreated MCF-7 cells upon carcinogen exposure and detected altered expression of many genes involved in multiple DNA damage responsive pathways. Genes exhibiting altered expression were further analyzed for functional interrelatedness using network- and pathway-based bioinformatics analysis. The top functional network was defined as having relevance for "DNA Replication, Recombination, and Repair, Gene Expression, [and] Cancer". CONCLUSIONS: These findings suggest that melatonin may enhance DNA repair capacity by affecting several key genes involved in DNA damage responsive pathways.

Targetome profiling, pathway analysis and genetic association study implicate miR-202 in lymphomagenesis.

BACKGROUND: miRNAs have been implicated in numerous tumorigenic pathways, and previous studies have associated miR-202 dysregulation with various cancer types, including follicular lymphoma. METHODS: The miR-202 targetome was identified by ribonucleoprotein immunoprecipitation-microarray (RIP-Chip), and functional interactions among identified targets were investigated using the Ingenuity Pathway Analysis tool. We also conducted a population-based genetic association study of a polymorphism within the miR-202 stem-loop sequence and risk of non-Hodgkin lymphoma. In vitro gain-of-function experiments were further conducted to elucidate the functional significance of the variant. RESULTS: A total of 141 potential members of the miR-202 targetome were identified by a transcriptome-wide RIP-Chip assay. Functional interactions among identified targets suggested that miR-202-regulated genes are involved in biologic pathways relevant for hematologic function and cancer. Consistent with this, a genetic association analysis using human blood samples revealed a significant association between a germline mutation (rs12355840) in the miR-202 precursor sequence and follicular lymphoma risk. An in vitro functional assay further showed that the variant allele resulted in diminished miR-202 levels, possibly by altering precursor-processing efficiency. CONCLUSIONS: Taken together, our findings suggest that miR-202 is involved in follicular lymphomagenesis. IMPACT: These findings implicate miR-202 as a potential tumor suppressor in follicular lymphoma and warrant the investigation of miR-202 as a novel biomarker of follicular lymphoma risk.

Genetic and epigenetic associations of circadian gene TIMELESS and breast cancer risk.

Results from recent molecular epidemiologic studies suggest that the core circadian genes play a role in breast tumorigenesis, possibly by influencing hormone regulation or other pathways relevant to cancer. In order to further evaluate this hypothesis, we conducted a genetic and epigenetic association study of the circadian regulator TIMELESS in breast carcinogenesis. We detected significant associations between two tagging SNPs (rs2291738 and rs7302060) in the TIMELESS gene and breast cancer among 441 breast cancer cases and 479 cancer-free controls, with apparent effect modification by ER/PR status. The presence of the C allele of rs7302060 was found to be associated with reduced breast cancer risk (OR, 0.54; 95% CI, 0.54-0.99). In addition, both the G/G genotype of rs2291738 and the C/C genotype of rs7302060 were associated with reduced risk of breast cancer among ER- or PR-positive breast cancer cases (OR, 0.46; 95% CI, 0.22-0.97 and OR, 0.36; 95% CI, 0.17-0.78, respectively). We also observed a significant association between stage II, III, and IV breast cancers and TIMELESS promoter hypomethylation in peripheral blood lymphocytes (OR, 0.35; 95% CI, 0.13-0.96) in 80 breast cancer cases and 80 age-matched controls, which is corroborated by documented overexpression of TIMELESS in breast tumor tissue compared to adjacent normal tissue. Our findings support the hypothesized role of circadian genes in breast tumorigenesis, and identify a set of circadian biomarkers for breast cancer susceptibility.

Epigenetic impact of long-term shiftwork: pilot evidence from circadian genes and whole-genome methylation analysis.

Epigenetic association studies have demonstrated differential promoter methylation in the core circadian genes in breast cancer cases relative to cancer-free controls. The current pilot study aims to investigate whether epigenetic changes affecting breast cancer risk could be caused by circadian disruption through exposure to light at night. Archived DNA samples extracted from whole blood of 117 female subjects from a prospective cohort conducted in Denmark were included in this study. A polymerase chain reaction (PCR)-based method was used for detection of gene-promoter methylation, whereas genome-wide methylation analysis was performed using the Illumina Infinium Methylation Chip. Long-term shiftwork resulted in the same promoter hypomethylation of CLOCK and hypermethylation of CRY2, as was previously observed in breast cancer case-control studies. Genome-wide methylation analysis further discovered widespread methylation alterations in shiftworkers, including changes in many methylation- and cancer-relevant genes. Pathway analysis of the genes with altered methylation patterns revealed several cancer-related pathways. One of the top three networks generated was designated as "DNA replication, recombination, and repair, gene expression, behavior" with ESR1 (estrogen receptor α) featured most prominently in the network, underscoring the potential breast cancer relevance of the genes differentially methylated in long-term shiftworkers. These results, although exploratory, demonstrate the first evidence of the cancer-relevant epigenetic effects of night shiftwork, which warrant further investigation. Considering there are millions of shiftworkers worldwide, understanding the effects of this exposure may lead to novel strategies for cancer prevention and new policies regulating shiftwork.

Genetic and epigenetic association studies suggest a role of microRNA biogenesis gene exportin-5 (XPO5) in breast tumorigenesis.

Given strong evidence implicating an important role of altered microRNA expression in cancer initiation and progression, the genes responsible for microRNA biogenesis may also play a role in tumorigenesis. Exportin-5 (XPO5) is responsible for exporting pre-miRNAs through the nuclear membrane to the cytoplasm, and is thus critical in miRNA biogenesis. In the current study, we performed both genetic and epigenetic association studies of XPO5 in a case control study of breast cancer. We first genotyped two missense SNPs in XPO5, rs34324334 (S241N) and rs11544382 (M1115T), and further analyzed methylation levels in the XPO5 promoter region for blood DNA samples from a breast cancer case-control study. We found the variant genotypes of rs11544382 to be associated with breast cancer risk (OR=1.59, 95% CI: 1.06 -2.39), compared to the homozygous common genotype. When stratified by menopausal status, the variant alleles of both rs11544382 (OR=1.82, 95% CI: 1.09-3.03) and rs34324334 (OR=1.76, 95% CI: 1.10-2.83) were significantly associated with breast cancer risk in post-menopausal women. The methylation analysis showed that the "high" and combined "high/middle" tertiles of methylation index were associated with reduced risk of breast cancer (OR=0.34, 95% CI:0.15-0.81 and OR=0.47, 95% CI:0.24-0.94, respectively; P(trend)=0.015). These results were corroborated by data from a publicly available tissue array, which showed lower levels of XPO5 expression in healthy controls relative to tumor or adjacent tissues from breast cancer patients with tumor tissue exhibiting the highest expression levels. These findings support the hypothesis that variations in components of the miRNA biogenesis pathway, in this case XPO5, may affect an individual's risk of developing breast cancer.

Phenotypic effects of the circadian gene Cryptochrome 2 on cancer-related pathways.

BACKGROUND: Circadian genes continue to gain attention as important transcriptional regulators with the potential to influence a variety of biological pathways, including many cancer-related processes. The core circadian gene cryptochrome 2 (CRY2) is essential for proper circadian timing, and is a key component of the negative arm of the circadian feedback loop. As such, aberrant expression of CRY2 may influence carcinogenic processes and thereby impact cancer susceptibility. METHODS: We silenced CRY2 in breast cancer cell lines (MCF-7) using small-interfering oligos (siRNA) and measured the impact of CRY2 knockdown on a number of cancer-relevant parameters. Cell cycle distribution, cell viability, and apoptotic response were measured in CRY2 knockdown (CRY2-) and normal (CRY2+) cell populations using flow cytometry in cells with and without exposure to a mutagen challenge. DNA damage accumulation was measured using the single cell gel electrophoresis (comet) assay, and damage was quantified using the Olive tail moment, which considers the amount and distance of DNA migration away from the nucleus, indicative of DNA strand breaks. Expression changes in cancer-relevant transcripts were measured by whole genome microarray. The Student's t-test was used for statistical comparisons, and P-values obtained from the microarray were adjusted for multiple comparisons using the false discovery rate correction, in order to obtain an adjusted Q-value for each observation. RESULTS: The comet assay results indicated that upon exposure to the same dose of chemical mutagen, CRY2- cells accumulate significantly more unrepaired DNA damage than CRY2+ cells (P = 0.040), suggesting that CRY2 may be important for DNA repair. In addition, a number of transcripts with relevance for DNA damage repair displayed altered expression following CRY2 silencing. These included BCCIP (Q = 0.002), BCL2 (Q = 0.049), CCND1 (Q = 0.009), CDKN1A (Q < 0.001), GADD45A (Q = 0.002), HERC5 (Q < 0.001), MCM5 (Q = 0.042), PPP1R15A (Q < 0.001), SUMO1 (Q < 0.001), and UBA1 (Q = 0.023). However, no significant influence of CRY2 knockdown on cell cycle distributions, cell cycle checkpoints in response to mutagen challenge, or apoptotic response was detected. CONCLUSIONS: In total, these data suggest a limited, but potentially important role for CRY2 in the regulation of DNA damage repair and the maintenance of genomic stability. Future investigations may focus on identifying the mechanisms by which CRY2 may regulate the expression of transcripts with known relevance for carcinogenesis.

The core circadian gene Cryptochrome 2 influences breast cancer risk, possibly by mediating hormone signaling.

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 single-nucleotide polymorphisms in CRY2 were identified for genotyping in a case-control population (n = 441 cases and n = 479 controls), and three single-nucleotide polymorphisms (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 (95% 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 several breast cancer-relevant genes that 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.

CLOCK in breast tumorigenesis: genetic, epigenetic, and transcriptional profiling analyses.

The transcription factors responsible for maintaining circadian rhythm influence a variety of biological processes. Recently, it has been suggested that the core circadian genes may play a role in breast tumorigenesis, possibly by influencing hormone regulation or other pathways relevant to cancer. To evaluate this hypothesis, we conducted a genetic and epigenetic association study, as well as a transcriptional profiling array and a pathway-based network analysis. We report significant correlations between single nucleotide polymorphisms associated with the central circadian regulator CLOCK and breast cancer risk, with apparent effect modification by estrogen receptor/progesterone receptor status. We also found that hypermethylation in the CLOCK promoter reduced the risk of breast cancer, and lower levels of CLOCK expression were documented in healthy controls relative to normal or tumor tissue from patients with breast cancer. Finally, we silenced CLOCK in vitro and performed a whole-genome expression microarray and pathway analysis, which identified a cancer-relevant network of transcripts with altered expression following CLOCK gene knockdown. Our findings support the hypothesis that circadian genes influence tumorigenesis, and identify a set of circadian gene variants as candidate breast cancer susceptibility biomarkers.

The circadian gene NPAS2 is a novel prognostic biomarker for breast cancer.

Mounting evidence suggests that neuronal PAS domain protein 2 (NPAS2) and other circadian genes are involved in tumorigenesis and tumor growth, possibly through their control of cancer-related biologic pathways. A missense polymorphism in NPAS2 (Ala394Thr) has been shown to be associated with risk of human tumors including breast cancer. The current study further examined the prognostic significance of NPAS2 in breast cancer by genotyping the Ala394Thr polymorphism and measuring NPAS2 expression. DNA extracted from 348 breast cancer tissue samples was analyzed for NPAS2 genotype using the TaqMan allelic discrimination assay. Of these, 287 also had total RNA available for use in real-time PCR assays to determine NPAS2 expression. NPAS2 genotypes and expression levels were analyzed for associations with prognostic outcomes, as well as correlations with clinical characteristics. A high level of NPAS2 expression was strongly associated with improved disease free survival (AHR = 0.43, 95% CI: 0.21-0.86, P trend = 0.022) and overall survival (AHR = 0.42, 95% CI: 0.19-0.96, P trend = 0.036). In addition, there was a borderline, but nonsignificant association between the NPAS2 genotype corresponding to Thr394Thr and disease free survival (AHR = 1.82, 95% CI: 0.96-3.46). The Ala/Ala, Ala/Thr, and Thr/Thr genotypes were also differentially distributed by tumor severity, as measured by TNM classification (chi (2) (6df, N = 344) = 14.96, P = 0.020). These findings provide the first evidence suggesting prognostic significance of the circadian gene NPAS2 in breast cancer.