Supplementary Tables 1-3 from Clock-Cancer Connection in Non–Hodgkin's Lymphoma: A Genetic Association Study and Pathway Analysis of the Circadian Gene Cryptochrome 2

Supplementary Tables 1-4 from CLOCK in Breast Tumorigenesis: Genetic, Epigenetic, and Transcriptional Profiling Analyses

Supplementary Tables 1-2 from The Core Circadian Gene Cryptochrome 2 Influences Breast Cancer Risk, Possibly by Mediating Hormone Signaling

Supplementary Tables 1-5 from microRNA miR-196a-2 and Breast Cancer: A Genetic and Epigenetic Association Study and Functional Analysis

Neonatal Dieulafoy lesion is a rare but severe condition that can be life-threatening if not intervened upon in a timely fashion. In the general population, the majority of lesions are successfully treated with endoscopic or angiographic intervention. Surgery is usually reserved for cases that fail endoscopic or angiographic intervention. We present a case of neonatal Dieulafoy lesion that occurred less than 24 h after delivery with hematemesis. The patient required large volume resuscitation and massive transfusion of blood products for acute blood loss. The lesion was successfully treated with surgical ligation after a failed attempt at endoscopic intervention.

BACKGROUND:The circadian clock and cell cycle are two global regulatory systems that have pervasive behavioral and physiological effects on eukaryotic cells, and both play a role in cancer development. Recent studies have indicated that the circadian and cell cycle regulator, TIMELESS, may serve as a molecular bridge between these two regulatory systems.METHODS:To assess the role of TIMELESS in tumorigenesis, we analyzed TIMELESS expression data from publically accessible online databases. A loss-of-function analysis was then performed using TIMELESS-targeting siRNA oligos followed by a whole-genome expression microarray and network analysis. We further tested the effect of TIMELESS down-regulation on cell proliferation rates of a breast and cervical cancer cell line, as suggested by the results of our network analysis.RESULTS:TIMELESS was found to be frequently overexpressed in different tumor types compared to normal controls. Elevated expression of TIMELESS was significantly associated with more advanced tumor stage and poorer breast cancer prognosis. We identified a cancer-relevant network of transcripts with altered expression following TIMELESS knockdown which contained many genes with known functions in cancer development and progression. Furthermore, we observed that TIMELESS knockdown significantly decreased cell proliferation rate.CONCLUSIONS:Our results suggest a potential role for TIMELESS in tumorigenesis, which warrants further investigation of TIMELESS expression as a potential biomarker of cancer susceptibility and prognostic outcome.

Eisenmenger syndrome (ES) is a known complication of congenital heart disease associated with unrepaired systemic to pulmonary shunts. Evidence for use of targeted pulmonary arterial hypertension therapy in ES is limited. The early experience using ambrisentan was evaluated in a cohort of consecutive patients with ES who initiated ambrisentan at Columbia University's Pulmonary Hypertension Center from January 1, 2007, to August 1, 2008. Effects of ambrisentan on rest and exercise systemic arterial oxygen saturation (S(a)O(2)), exercise capacity, functional status, hemoglobin levels, and hemodynamics were evaluated and compared using paired Student's t tests. Seventeen patients were evaluated at short-term (mean 163 ± 57 days) and longer term (mean 2.5 ± 0.5 years) follow-up. At short-term follow-up, there was an improvement in exercise capacity (6-minute walking distance 389 ± 74 vs 417 ± 77 m, p=0.03, n=11) and maintenance of rest S(a)O(2) (89 ± 7% vs 89% ± 6%, p=0.75, n=15), exercise S(a)O(2) (75 ± 15% vs 77% ± 15%, p=0.33, n=11), functional class (improvement in 2 patients, no change in 13), and hemoglobin (16.5 ± 2.8 vs 15.8 ± 1.8 g/dl, p=0.11, n=14). At longer term follow-up compared to baseline and short-term follow-up, there was stability of exercise capacity, S(a)O(2), functional class, and hemoglobin. In conclusion, in this single-center cohort of patients with ES, ambrisentan was safe and was associated with increasing exercise capacity at short-term follow-up, with patients maintaining S(a)O(2), functional class, and hemoglobin, and with no significant evidence of clinical deterioration at longer term follow-up. Additional studies are required to further assess the efficacy of ambrisentan in patients with ES.

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. © 2011 Wiley Periodicals, Inc.

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.

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.

Increasing evidence has suggested that microRNAs (miRNAs) play an important role in tumorigenesis. As transcriptional regulators, altered miRNA expression may affect many cancerrelated biological pathways, indicating that miRNAs can function as tumor suppressors and/or oncogenes. We first performed a genetic association analysis by screening genetic variants in 15 microRNA genes and detected that a common sequence variant in hsa-miR-196a-2 (rs11614913, C→T) was significantly associated with decreased breast cancer risk (for homozygous variant: OR=0.44, 95% CI, 0.28-0.70). Hypermethylation of a CpG island upstream (-700 bp) of the miR-196a-2 precursor was also associated with reduced breast cancer risk (OR=0.35, 95% CI, 0.15-0.81). By delivering expression vectors containing either wild-type or mutant precursors of miR-196a-2 into breast cancer cells, we demonstrated that this variant led to less efficient processing of the miRNA precursor to its mature form, as well as diminished capacity to regulate target genes. A whole genome expression microarray was preformed and a pathway-based analysis identified a cancer-relevant network formed by genes significantly altered following enforced expression of miR-196a-2. Mutagenesis analysis further showed that cell cycle response to mutagen challenge was significantly enhanced in cells treated with variant miR-196a-2 compared to cells treated with the wild-type. Taken together, our findings suggest that miR-196a-2 might have a potentially oncogenic role in breast tumorigenesis, and the function genetic variant in its mature region could serve as a novel biomarker for breast cancer susceptibility.

AbstractThe 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. Cancer Res; 70(4); 1459–68

Abstract 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. Cancer Prev Res; 3(4); 539–48. ©2010 AACR.

Abstract Increasing evidence has suggested that microRNAs (miRNA) play an important role in tumorigenesis. As transcriptional regulators, altered miRNA expression may affect many cancer-related biological pathways, indicating that miRNAs can function as tumor suppressors and/or oncogenes. We first performed a genetic association analysis by screening genetic variants in 15 miRNA genes and detected that a common sequence variant in hsa-miR-196a-2 (rs11614913, C→T) was significantly associated with decreased breast cancer risk (for homozygous variant: odds ratio, 0.44; 95% confidence interval, 0.28-0.70). Hypermethylation of a CpG island upstream (-700 bp) of the miR-196a-2 precursor was also associated with reduced breast cancer risk (odds ratio, 0.35; 95% confidence interval, 0.15-0.81). By delivering expression vectors containing either wild-type or mutant precursors of miR-196a-2 into breast cancer cells, we showed that this variant led to less efficient processing of the miRNA precursor to its mature form as well as diminished capacity to regulate target genes. A whole-genome expression microarray was done and a pathway-based analysis identified a cancer-relevant network formed by genes significantly altered following enforced expression of miR-196a-2. Mutagenesis analysis further showed that cell cycle response to mutagen challenge was significantly enhanced in cells treated with variant miR-196a-2 compared with cells treated with the wild-type. Taken together, our findings suggest that miR-196a-2 might have a potentially oncogenic role in breast tumorigenesis, and the functional genetic variant in its mature region could serve as a novel biomarker for breast cancer susceptibility. [Cancer Res 2009;69(14):5970–7]

Abstract Circadian genes have the potential to influence a variety of cancer-related biological pathways, including immunoregulation, 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 single nucleotide polymorphisms (SNP) 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 effect 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 hematologic system development. In addition, these genes were predicted to have significant effects on several disease processes, including cancer (B-H P = 3.75E−9) and hematologic 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. [Cancer Res 2009;69(8):3605–13]

The transcription factor NPAS2 is one of nine human core circadian genes that influence a variety of biological processes by regulating the 24-h circadian rhythm. Recently, it has been shown that NPAS2 is a risk biomarker in human cancers and plays a role in tumorigenesis by affecting cancer-related gene expression, and relevant biological pathways. However, it is difficult to study the biological involvement of NPAS2 in cancer development, as little is known about its direct transcriptional targets. The aim of the current study is to create a transcriptional profile of genes regulated by NPAS2, using a human binding ChIP-on-chip analysis of NPAS2 in MCF-7 cells. This genome-wide mapping approach identified 26 genes that contain potential NPAS2 binding regions. Subsequent real-time PCR assays confirmed 16 of these targets, and 9 of these genes (ARHGAP29, CDC25A, CDKN2AIP, CX3CL1, ELF4, GNAL, KDELR1, POU4F2, and THRA) have a known role in tumorigenesis. In addition, a networking analysis of these validated NPAS2 targets revealed that all nine genes, together with REN, are involved in a “Cancer, Cell cycle, Neurological Disease” network. These results report the first list of direct transcriptional targets of NPAS2 and will shed light on the role of circadian genes in tumorigenesis.

Bioinformatic tools are widely utilized to predict functional single nucleotide polymorphisms (SNPs) for genotyping in molecular epidemiological studies. However, the extent to which these approaches are mirrored by epidemiological findings has not been fully explored. In this study, we first surveyed SNPs examined in case–control studies of lung cancer, the most extensively studied cancer type. We then computed SNP functional scores using four popular bioinformatics tools: SIFT, PolyPhen, SNPs3D, and PMut, and determined their predictive potential using the odds ratios (ORs) reported. Spearman's correlation coefficient (r) for the association with SNP score from SIFT, PolyPhen, SNPs3D, and PMut, and the summary ORs were r = −0.36 (p = 0.007), r = 0.25 (p = 0.068), r = −0.20 (p = 0.205), and r = −0.12 (p = 0.370), respectively. By creating a combined score using information from all four tools we were able to achieve a correlation coefficient of r = 0.51 (p < 0.001). These results indicate that scores of predicted functionality could explain a certain fraction of the lung cancer risk detected in genetic association studies and more accurate predictions may be obtained by combining information from a variety of tools. Our findings suggest that bioinformatic tools are useful in predicting SNP functionality and may facilitate future genetic epidemiological studies.

Three known non-synonymous polymorphisms (Ala394Thr, Ser471Leu and Pro690Ala) in the largest circadian gene, Neuronal PAS domain protein 2 (NPAS2), were genotyped in a breast cancer case-control study conducted in Connecticut, USA (431 cases and 476 controls). We found that women with the heterozygous Ala394Thr genotype were significantly associated with breast cancer risk compared to those with the common homozygous Ala394Ala (OR = 0.61, 0.46-0.81, P = 0.001). This is the first evidence demonstrating a role of the circadian gene NPAS2 in human breast cancer, suggesting that genetic variations in circadian genes might be a novel panel of biomarkers for breast cancer risk.