Maarit Tiirikainen

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

Smokers with the CHRNA Lung Cancer–Associated Variants Are Exposed to Higher Levels of Nicotine Equivalents and a Carcinogenic Tobacco-Specific Nitrosamine

A locus at 15q24/15q25.1, which includes the nicotinic acetylcholine receptor A subunits 3 and 5 (CHRNA3 and CHRNA5) genes, has recently been associated with lung cancer risk, self-reported number of cigarettes smoked per day, and a nicotine dependence scale. It is not clear whether the association with lung cancer is direct or mediated through differences in smoking behavior. We used urinary biomarkers to test whether two linked lung cancer risk variants in CHRNA3 (rs1051730) and CHRNA5 (rs16969968) are associated with intensity of smoking and exposure to a tobacco-specific carcinogenic nitrosamine per cigarette dose. We studied 819 smokers and found that carriers of these variants extract a greater amount of nicotine (P = 0.003) and are exposed to a higher internal dose of 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (P = 0.03) per cigarette than noncarriers. Thus, smokers who carry the CHRNA3 and CHRNA5 variants are expected to be at increased risk for lung cancer compared with smokers who do not carry these alleles even if they smoked the same number of cigarettes. Number of cigarettes per day, even if it could be accurately assessed, is not an adequate measure of smoking dose.

Variants on 9p24 and 8q24 Are Associated with Risk of Colorectal Cancer: Results from the Colon Cancer Family Registry

Recent publications have reported that common variants on 8q24 are associated with both prostate and colorectal cancers (CRC). In addition, one of these studies (the ARCTIC study) initially observed an association with a single nucleotide polymorphism (SNP) on 9p24 that was not confirmed in some of their validation data sets. In the research described here, we conducted a case-unaffected sibling analysis using population- and clinic-based discordant sibships (N = 1,567 sibships) from the Colon Cancer Family Registry (Colon CFR) to investigate the associations between common variants at 9p24 and 8q24 and risk of CRC. We also evaluated whether these associations differed by age, family history, and tumor characteristics, including microsatellite instability and tumor site. Associations were estimated using conditional logistic regression, treating sibship as the matching factor. Analyses were adjusted for age and sex, and stratified by ascertainment source (population versus clinic). We observed an association between a SNP on 9p24 (rs719725) and risk of CRC in the population-based series (AA versus CC: odds ratios, 1.46; 95% confidence interval, 1.06-2.02; AC versus CC: odds ratios, 1.50; 95% confidence interval, 1.14-1.98; P = 0.011 on 2 df). In the population-based series, we also detected statistically significant associations between two SNPs on 8q24, rs10505477 and rs6983267, and risk of CRC (P = 0.005 and P = 0.002, respectively). There was no evidence of statistically significant heterogeneity by age at diagnosis, family history of CRC, microsatellite instability, or tumor site at either locus and no evidence of interaction between SNPs on 8q24 and 9p24. These data suggest that common variants may play important roles in the risk of CRC.

Elucidating the Landscape of Aberrant DNA Methylation in Hepatocellular Carcinoma

Background Hepatocellular carcinoma (HCC) is one of the most common cancers and frequently presents with an advanced disease at diagnosis. There is only limited knowledge of genome-scale methylation changes in HCC. Methods and Findings We performed genome-wide methylation profiling in a total of 47 samples including 27 HCC and 20 adjacent normal liver tissues using the Illumina HumanMethylation450 BeadChip. We focused on differential methylation patterns in the promoter CpG islands as well as in various less studied genomic regions such as those surrounding the CpG islands, i.e. shores and shelves. Of the 485,577 loci studied, significant differential methylation (DM) was observed between HCC and adjacent normal tissues at 62,692 loci or 13% (p<1.03e-07). Of them, 61,058 loci (97%) were hypomethylated and most of these loci were located in the intergenic regions (43%) or gene bodies (33%). Our analysis also identified 10,775 differentially methylated (DM) loci (17% out of 62,692 loci) located in or surrounding the gene promoters, 4% of which reside in known Differentially Methylated Regions (DMRs) including reprogramming specific DMRs and cancer specific DMRs, while the rest (10,315) involving 4,106 genes could be potential new HCC DMR loci. Interestingly, the promoter-related DM loci occurred twice as frequently in the shores than in the actual CpG islands. We further characterized 982 DM loci in the promoter CpG islands to evaluate their potential biological function and found that the methylation changes could have effect on the signaling networks of Cellular development, Gene expression and Cell death (p = 1.0e-38), with BMP4, CDKN2A, GSTP1, and NFATC1 on the top of the gene list. Conclusion Substantial changes of DNA methylation at a genome-wide level were observed in HCC. Understanding epigenetic changes in HCC will help to elucidate the pathogenesis and may eventually lead to identification of molecular markers for liver cancer diagnosis, treatment and prognosis.

Transforming Growth Factor-β Receptor Inhibition Enhances Adenoviral Infectability of Carcinoma Cells via Up-Regulation of Coxsackie and Adenovirus Receptor in Conjunction with Reversal of Epithelial-Mesenchymal Transition

Expression of the Coxsackie and Adenovirus Receptor (CAR) is frequently reduced in carcinomas, resulting in decreased susceptibility of such tumors to infection with therapeutic adenoviruses. Because CAR participates physiologically in the formation of tight-junction protein complexes, we examined whether molecular mechanisms known to down-regulate cell-cell adhesions cause loss of CAR expression. Transforming growth factor-β (TGF-β)–mediated epithelial-mesenchymal transition (EMT) is a phenomenon associated with tumor progression that is characterized by loss of epithelial-type cell-cell adhesion molecules (including E-cadherin and the tight junction protein ZO-1), gain of mesenchymal biochemical markers, such as fibronectin, and acquisition of a spindle cell phenotype. CAR expression is reduced in tumor cells that have undergone EMT in response to TGF-β. This down-regulation results from repression of CAR gene transcription, whereas altered RNA stability and increased proteasomal protein degradation play no role. Loss of CAR expression in response to TGF-β is accompanied by reduced susceptibility to adenovirus infection. Indeed, treatment of carcinoma cells with LY2109761, a specific pharmacologic inhibitor of TGF-β receptor types I and II kinases, resulted in increased CAR RNA and protein levels as well as improved infectability with adenovirus. This was observed in cells induced to undergo EMT by addition of exogenous TGF-β and in those that were transformed by endogenous autocrine/paracrine TGF-β. These findings show down-regulation of CAR in the context of EMT and suggest that combination of therapeutic adenoviruses and TGF-β receptor inhibitors could be an efficient anticancer strategy. (Cancer Res 2006; 66(3): 1648-57)

Plasma Levels of B Vitamins and Colorectal Cancer Risk: The Multiethnic Cohort Study

B vitamins, such as folate, vitamin B6, and vitamin B12, play an important role as coenzymes in one-carbon metabolism and may affect colorectal cancer risk. We aimed to comprehensively investigate the relationships of plasma folate, pyridoxal-5′-phosphate (PLP, the active form of vitamin B6), vitamin B12, methylmalonic acid, homocysteine, and cysteine with colorectal cancer risk, accounting for suspected modifiers (alcohol intake, MTHFR C677T genotype, and plasma C-reactive protein) and potential confounders. We conducted a case-control study nested within the Multiethnic Cohort study and analyzed prospectively collected blood samples from 224 incident colorectal cancer cases and 411 controls matched on age, sex, race/ethnicity, study site, date/time of blood draw, and hours of fasting. We found an inverse association between plasma PLP levels and colorectal cancer, with odds ratios (95% confidence intervals) for increasing quartiles of 1.00, 0.84 (0.51-1.40), 0.62 (0.37-1.03), and 0.49 (0.29-0.83), with P trend = 0.009. This association was not explained by an association with plasma folate, seemed to be stronger at low levels of alcohol intake and among individuals with the MTHFR 677TT genotype, and was independent of plasma C-reactive protein levels. An inverse association with plasma folate was also observed among individuals with a low level of alcohol intake. These data suggest an independent role for vitamin B6 in reducing colorectal cancer risk. (Cancer Epidemiol Biomarkers Prev 2009;18(8):2195–201)

cis-Expression QTL analysis of established colorectal cancer risk variants in colon tumors and adjacent normal tissue.

Genome-wide association studies (GWAS) have identified 19 risk variants associated with colorectal cancer. As most of these risk variants reside outside the coding regions of genes, we conducted cis-expression quantitative trait loci (cis-eQTL) analyses to investigate possible regulatory functions on the expression of neighboring genes. Forty microsatellite stable and CpG island methylator phenotype-negative colorectal tumors and paired adjacent normal colon tissues were used for genome-wide SNP and gene expression profiling. We found that three risk variants (rs10795668, rs4444235 and rs9929218, using near perfect proxies rs706771, rs11623717 and rs2059252, respectively) were significantly associated (FDR q-value ≤0.05) with expression levels of nearby genes (<2 Mb up- or down-stream). We observed an association between the low colorectal cancer risk allele (A) for rs10795668 at 10p14 and increased expression of ATP5C1 (q = 0.024) and between the colorectal cancer high risk allele (C) for rs4444235 at 14q22.2 and increased expression of DLGAP5 (q = 0.041), both in tumor samples. The colorectal cancer low risk allele (A) for rs9929218 at 16q22.1 was associated with a significant decrease in expression of both NOL3 (q = 0.017) and DDX28 (q = 0.046) in the adjacent normal colon tissue samples. Of the four genes, DLGAP5 and NOL3 have been previously reported to play a role in colon carcinogenesis and ATP5C1 and DDX28 are mitochondrial proteins involved in cellular metabolism and division, respectively. The combination of GWAS findings, prior functional studies, and the cis-eQTL analyses described here suggest putative functional activities for three of the colorectal cancer GWAS identified risk loci as regulating the expression of neighboring genes.

Continuous Exposure to Chrysotile Asbestos Can Cause Transformation of Human Mesothelial Cells via HMGB1 and TNF-α Signaling

Malignant mesothelioma is strongly associated with asbestos exposure. Among asbestos fibers, crocidolite is considered the most and chrysotile the least oncogenic. Chrysotile accounts for more than 90% of the asbestos used worldwide, but its capacity to induce malignant mesothelioma is still debated. We found that chrysotile and crocidolite exposures have similar effects on human mesothelial cells. Morphological and molecular alterations suggestive of epithelial-mesenchymal transition, such as E-cadherin down-regulation and β-catenin phosphorylation followed by nuclear translocation, were induced by both chrysotile and crocidolite. Gene expression profiling revealed high-mobility group box-1 protein (HMGB1) as a key regulator of the transcriptional alterations induced by both types of asbestos. Crocidolite and chrysotile induced differential expression of 438 out of 28,869 genes interrogated by oligonucleotide microarrays. Out of these 438 genes, 57 were associated with inflammatory and immune response and cancer, and 14 were HMGB1 targeted genes. Crocidolite-induced gene alterations were sustained, whereas chrysotile-induced gene alterations returned to background levels within 5 weeks. Similarly, HMGB1 release in vivo progressively increased for 10 or more weeks after crocidolite exposure, but returned to background levels within 8 weeks after chrysotile exposure. Continuous administration of chrysotile was required for sustained high serum levels of HMGB1. These data support the hypothesis that differences in biopersistence influence the biological activities of these two asbestos fibers.

Urinary Isothiocyanates; Glutathione S-Transferase M1, T1, and P1 Polymorphisms; and Risk of Colorectal Cancer: The Multiethnic Cohort Study

Although an association between diet, especially cruciferous vegetables, and colorectal cancer has been hypothesized, recent studies have been inconsistent with their findings. One possibility for the discrepant results is that the interaction with related genes has not generally been considered. The present study examined the associations among urinary isothiocyanates, glutathione S-transferase (GST) polymorphisms, and colorectal cancer risk in a case-control study nested within the Multiethnic Cohort Study, based in Hawaii and Los Angeles, California. We measured prediagnositic urinary isothiocyanate levels adjusted for creatinine and analyzed GSTM1, GSTT1, and GSTP1 polymorphisms in 173 cases and 313 matched controls, with biospecimens collected between 2001 and 2006. Conditional logistic regression was used to compute odds ratios and 95% confidence intervals (95% CI). A detectable amount of urinary isothiocyanates was associated with a 41% decrease in colorectal cancer risk (95% CI, 0.36-0.98). No significant, main-effect associations were seen with a homozygous deletion of the GSTM1 or GSTT1 polymorphism, or with the AG or GG genotypes for GSTP1 rs1695. There was a weak suggestion that for individuals with the GSTP1 AG or GG genotype, a detectable amount of isothiocyanates further decreases ones risk of colorectal cancer compared with those with the GSTP1 AA genotype, but the interaction term was not statistically significant (P = 0.09). This is only the second study published on the association between urinary isothiocyanates and colorectal cancer risk. The results suggest that further studies, with larger numbers, examining a possible interaction with the GSTP1 polymorphisms are warranted. (Cancer Epidemiol Biomarkers Prev 2009;18(1):314–20)

Measurement of Circulating Cell‐Free DNA in Relation to 18F‐Fluorocholine PET/CT Imaging in Chemotherapy‐Treated Advanced Prostate Cancer

Purpose: To examine the effects of chemotherapy on circulating cell‐free DNA (cfDNA) composition in relation to investigational whole‐body measurement of tumor activity by fluorine‐18 fluorocholine (FCH) positron emission tomography/computed tomography (PET/CT) in hormone‐refractory prostate cancer (HRPC).