[Show abstract][Hide abstract] ABSTRACT: Genetic risk for breast cancer is conferred by a combination of multiple variants of small effect. To better understand how risk loci might combine, we examined whether risk-associated genes share regulatory mechanisms. We created a breast cancer gene regulatory network comprising transcription factors and groups of putative target genes (regulons) and asked whether specific regulons are enriched for genes associated with risk loci via expression quantitative trait loci (eQTLs). We identified 36 overlapping regulons that were enriched for risk loci and formed a distinct cluster within the network, suggesting shared biology. The risk transcription factors driving these regulons are frequently mutated in cancer and lie in two opposing subgroups, which relate to estrogen receptor (ER)(+) luminal A or luminal B and ER(-) basal-like cancers and to different luminal epithelial cell populations in the adult mammary gland. Our network approach provides a foundation for determining the regulatory circuits governing breast cancer, to identify targets for intervention, and is transferable to other disease settings.
[Show abstract][Hide abstract] ABSTRACT: Genome Wide Association Studies (GWAS) revealed SNP rs889312 on 5q11.2 to be
associated with breast cancer risk in women of European ancestry. In an attempt to
identify the biologically relevant variants, we analysed 909 genetic variants across
the 5q11.2 locus in 103,991 breast cancer cases and controls from 52 studies in the
Breast Cancer Association Consortium. Multiple logistic regression analyses
identified three independent risk signals: The strongest associations are with 15
correlated variants (iCHAV1) where the minor allele of best candidate, rs62355902,
associates with significantly increased risks of both estrogen receptor-positive (ER+:
OR=1.24, 95% CI 1.21-1.27; P-trend=5.7×10-44) and estrogen receptor-negative
tumors (ER-: OR=1.10, 95% CI 1.05-1.15; P-trend=3.0×10-4). After adjustment for
rs62355902, we found evidence for the association of a further 173 variants
(iCHAV2) containing three subsets with a range of effects, of which the strongest is
SNP rs113317823 (P-cond=1.61×10-5); and five variants comprising iCHAV3 (lead
rs11949391; ER+: OR=0.90, 95% CI 0.87-0.93; P-cond=1.4x10-4). Twenty six
percent of the prioritized candidate variants coincide with four putative regulatory
elements that interact with the MAP3K1 promoter through chromatin looping and
affect MAP3K1 promoter activity. Functional analysis indicates the cancer risk alleles
of four candidates increase MAP3K1 transcriptional activity: rs74345699 and
rs62355900 (iCHAV1); rs16886397 (iCHAV2a); and rs17432750 (iCHAV3).
Chromatin immunoprecipitation analysis revealed diminished GATA3 binding to the
minor (cancer protective) allele of rs17432750, indicating a mechanism for its action.
We propose that the cancer risk alleles act to increase MAP3K1 expression in vivo
and may promote breast cancer cell survival.
Full-text · Article · Dec 2014 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: The 10q26 locus in the second intron of FGFR2 is the locus most strongly associated with estrogen-receptor-positive breast cancer in genome-wide association studies. We conducted fine-scale mapping in case-control studies genotyped with a custom chip (iCOGS), comprising 41 studies (n = 89,050) of European ancestry, 9 Asian ancestry studies (n = 13,983), and 2 African ancestry studies (n = 2,028) from the Breast Cancer Association Consortium. We identified three statistically independent risk signals within the locus. Within risk signals 1 and 3, genetic analysis identified five and two variants, respectively, highly correlated with the most strongly associated SNPs. By using a combination of genetic fine mapping, data on DNase hypersensitivity, and electrophoretic mobility shift assays to study protein-DNA binding, we identified rs35054928, rs2981578, and rs45631563 as putative functional SNPs. Chromatin immunoprecipitation showed that FOXA1 preferentially bound to the risk-associated allele (C) of rs2981578 and was able to recruit ERα to this site in an allele-specific manner, whereas E2F1 preferentially bound the risk variant of rs35054928. The risk alleles were preferentially found in open chromatin and bound by Ser5 phosphorylated RNA polymerase II, suggesting that the risk alleles are associated with changes in transcription. Chromatin conformation capture demonstrated that the risk region was able to interact with the promoter of FGFR2, the likely target gene of this risk region. A role for FOXA1 in mediating breast cancer susceptibility at this locus is consistent with the finding that the FGFR2 risk locus primarily predisposes to estrogen-receptor-positive disease.
Full-text · Article · Nov 2013 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: TOX3 maps to 16q12, a region commonly lost in breast cancers and recently implicated in the risk of developing breast cancer. However, not much is known of the role of TOX3 itself in breast cancer biology. This is the first study to determine the importance of TOX3 mutations in breast cancers. We screened TOX3 for mutations in 133 breast tumours and identified four mutations (three missense, one in-frame deletion of 30 base pairs) in six primary tumours, corresponding to an overall mutation frequency of 4.5%. One potentially deleterious missense mutation in exon 3 (Leu129Phe) was identified in one tumour (genomic DNA and cDNA). Whilst copy number changes of 16q12 are common in breast cancer, our data show that mutations of TOX3 are present at low frequency in tumours. Our results support that TOX3 should be further investigated to elucidate its role in breast cancer biology.
[Show abstract][Hide abstract] ABSTRACT: The fibroblast growth factor receptor 2 (FGFR2) locus has been consistently identified as a breast cancer risk locus in independent genome-wide association studies. However, the molecular mechanisms underlying FGFR2-mediated risk are still unknown. Using model systems we show that FGFR2-regulated genes are preferentially linked to breast cancer risk loci in expression quantitative trait loci analysis, supporting the concept that risk genes cluster in pathways. Using a network derived from 2,000 transcriptional profiles we identify SPDEF, ERα, FOXA1, GATA3 and PTTG1 as master regulators of fibroblast growth factor receptor 2 signalling, and show that ERα occupancy responds to fibroblast growth factor receptor 2 signalling. Our results indicate that ERα, FOXA1 and GATA3 contribute to the regulation of breast cancer susceptibility genes, which is consistent with the effects of anti-oestrogen treatment in breast cancer prevention, and suggest that fibroblast growth factor receptor 2 signalling has an important role in mediating breast cancer risk.
[Show abstract][Hide abstract] ABSTRACT: Analysis of 4,405 variants in 89,050 European subjects from 41 case-control studies identified three independent association signals for estrogen-receptor-positive tumors at 11q13. The strongest signal maps to a transcriptional enhancer element in which the G allele of the best candidate causative variant rs554219 increases risk of breast cancer, reduces both binding of ELK4 transcription factor and luciferase activity in reporter assays, and may be associated with low cyclin D1 protein levels in tumors. Another candidate variant, rs78540526, lies in the same enhancer element. Risk association signal 2, rs75915166, creates a GATA3 binding site within a silencer element. Chromatin conformation studies demonstrate that these enhancer and silencer elements interact with each other and with their likely target gene, CCND1.
Full-text · Article · Mar 2013 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: The Gastro-Esophageal Malignancies in Northern Iran (GEMINI) research project is an example of recent progress in health research in Iran. The original aim of this project was to identify etiologic factors and prevention measures for upper gastrointestinal cancers in Northern provinces of Iran, but its achievements have gone much beyond this initial goal.
GEMINI consists of several projects including cancer registries, pilot studies, case-control studies, and the Golestan Cohort Study. GEMINI has been conducted through extensive collaborations between the Digestive Disease Research Center of Tehran University of Medical Sciences with other domestic and international health organizations. The achievements of GEMINI include producing new knowledge, introducing new research methods, developing and expanding health research and health care infrastructures, investing in human resources, and increasing the awareness and knowledge of policy makers and officials at all levels about the importance of chronic diseases in Iran's health priorities.
The success of GEMINI reveals the feasibility of large-scale health research studies in developing countries and serves as a successful model not only for health research in Iran, but also for similar research studies in other developing nations.
Full-text · Article · Jan 2013 · Archives of Iranian medicine
[Show abstract][Hide abstract] ABSTRACT: Although heat-shock protein 70 (HSP70), an evolutionarily highly conserved molecular chaperone, is known to be post-translationally modified in various ways such as phosphorylation, ubiquitination and glycosylation, physiological significance of lysine methylation has never been elucidated. Here we identify dimethylation of HSP70 at Lys-561 by SETD1A. Enhanced HSP70 methylation was detected in various types of human cancer by immunohistochemical analysis, although the methylation was barely detectable in corresponding non-neoplastic tissues. Interestingly, methylated HSP70 predominantly localizes to the nucleus of cancer cells, whereas most of the HSP70 protein locates to the cytoplasm. Nuclear HSP70 directly interacts with Aurora kinase B (AURKB) in a methylation-dependent manner and promotes AURKB activity in vitro and in vivo. We also find that methylated HSP70 has a growth-promoting effect in cancer cells. Our findings demonstrate a crucial role of HSP70 methylation in human carcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: A number of histone demethylases have been identified and biochemically characterized, yet their biological functions largely remain uncharacterized, particularly in the context of human diseases such as cancer. In this study, we describe important roles for the histone demethylase KDM3A, also known as JMJD1A, in human carcinogenesis. Expression levels of KDM3A were significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001), when assessed by real-time PCR. We confirmed that some other cancers including lung cancer also overexpressed KDM3A, using cDNA microarray analysis. Treatment of cancer cell lines with small interfering RNA targeting KDM3A significantly knocked down its expression and resulted in the suppression of proliferation. Importantly, we found that KDM3A activates transcription of the HOXA1 gene through demethylating histone H3 at lysine 9 di-methylation by binding to its promoter region. Indeed, expression levels of KDM3A and HOXA1 in several types of cancer cell lines and bladder cancer samples were statistically correlated. We observed the down-regulation of HOXA1 as well as CCND1 after treatment with KDM3A siRNA, indicating G(1) arrest of cancer cells. Together, our results suggest that elevated expression of KDM3A plays a critical role in the growth of cancer cells, and further studies may reveal a cancer therapeutic potential in KDM3A inhibition.
Full-text · Article · Aug 2012 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: It is well known that RB functions are regulated by posttranslational modifications such as phosphorylation and acetylation, but the significance of lysine methylation on RB has not been fully elucidated. Our expression analysis of SMYD2 by quantitative real-time polymerase chain reaction showed that expression levels of SMYD2 are significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (P < .0001), and its expression levels in tumor tissues were much higher than those of any other normal tissues. SMYD2 knockdown resulted in the suppression of cancer cell growth, and cell cycle analysis indicated that SMYD2 might play a crucial role in the G(1)/S transition. According to an in vitro methyltransferase assay, we found that SMYD2 methylates RB1 protein, and liquid chromatography-tandem mass spectrometry analysis revealed lysine 810 of RB1 to be methylated by SMYD2. Importantly, this methylation enhanced Ser 807/811 phosphorylation of RB1 both in vitro and in vivo. Furthermore, we demonstrated that methylated RB1 accelerates E2F transcriptional activity and promotes cell cycle progression. SMYD2 is an important oncoprotein in various types of cancer, and SMYD2-dependent RB1 methylation at lysine 810 promotes cell cycle progression of cancer cells. Further study may explore SMYD2-dependent RB1 methylation as a potential therapeutic target in human cancer.
Full-text · Article · Jun 2012 · Neoplasia (New York, N.Y.)
[Show abstract][Hide abstract] ABSTRACT: Although the physiologic significance of lysine methylation of histones is well known, whether lysine methylation plays a role in the regulation of nonhistone proteins has not yet been examined. The histone lysine methyltransferase SETD8 is overexpressed in various types of cancer and seems to play a crucial role in S-phase progression. Here, we show that SETD8 regulates the function of proliferating cell nuclear antigen (PCNA) protein through lysine methylation. We found that SETD8 methylated PCNA on lysine 248, and either depletion of SETD8 or substitution of lysine 248 destabilized PCNA expression. Mechanistically, lysine methylation significantly enhanced the interaction between PCNA and the flap endonuclease FEN1. Loss of PCNA methylation retarded the maturation of Okazaki fragments, slowed DNA replication, and induced DNA damage, and cells expressing a methylation-inactive PCNA mutant were more susceptible to DNA damage. An increase of methylated PCNA was found in cancer cells, and the expression levels of SETD8 and PCNA were correlated in cancer tissue samples. Together, our findings reveal a function for lysine methylation on a nonhistone protein and suggest that aberrant lysine methylation of PCNA may play a role in human carcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: Breast cancer is the most common cancer among women. To date, 22 common breast cancer susceptibility loci have been identified accounting for ∼8% of the heritability of the disease. We attempted to replicate 72 promising associations from two independent genome-wide association studies (GWAS) in ∼70,000 cases and ∼68,000 controls from 41 case-control studies and 9 breast cancer GWAS. We identified three new breast cancer risk loci at 12p11 (rs10771399; P = 2.7 × 10(-35)), 12q24 (rs1292011; P = 4.3 × 10(-19)) and 21q21 (rs2823093; P = 1.1 × 10(-12)). rs10771399 was associated with similar relative risks for both estrogen receptor (ER)-negative and ER-positive breast cancer, whereas the other two loci were associated only with ER-positive disease. Two of the loci lie in regions that contain strong plausible candidate genes: PTHLH (12p11) has a crucial role in mammary gland development and the establishment of bone metastasis in breast cancer, and NRIP1 (21q21) encodes an ER cofactor and has a role in the regulation of breast cancer cell growth.
[Show abstract][Hide abstract] ABSTRACT: In the past decade, a number of technologies to quantify allele-specific expression (ASE) in a genome-wide manner have become available to researchers. We investigate the application of single-nucleotide polymorphism (SNP) microarrays to this task, exploring data obtained from both cell lines and primary tissue for which both RNA and DNA profiles are available.
We analyze data from two experiments that make use of high-density Illumina Infinium II genotyping arrays to measure ASE. We first preprocess each data set, which involves removal of outlier samples, careful normalization and a two-step filtering procedure to remove SNPs that show no evidence of expression in the samples being analyzed and calls that are clear genotyping errors. We then compare three different tests for detecting ASE, one of which has been previously published and two novel approaches. These tests vary at the level at which they operate (per SNP per individual or per SNP) and in the input data they require. Using SNPs from imprinted genes as true positives for ASE, we observe varying sensitivity for the different testing procedures that improves with increasing sample size. Methods that rely on RNA signal alone were found to perform best across a range of metrics. The top ranked SNPs recovered by all methods appear to be reasonable candidates for ASE.
Analysis was carried out in R (http://www.R-project.org/) using existing functions.
[Show abstract][Hide abstract] ABSTRACT: Histone methyltransferases and demethylases are known to regulate transcription by altering the epigenetic marks on histones, but the pathologic roles of their dysfunction in human diseases, such as cancer, still remain to be elucidated. Herein, we show that the histone demethylase JMJD2B is involved in human carcinogenesis. Quantitative real-time PCR showed notably elevated levels of JMJD2B expression in bladder cancers, compared with corresponding nonneoplastic tissues (P < 0.0001), and elevated protein expression was confirmed by immunohistochemistry. In addition, cDNA microarray analysis revealed transactivation of JMJD2B in lung cancer, and immunohistochemical analysis showed protein overexpression in lung cancer. siRNA-mediated reduction of expression of JMJD2B in bladder and lung cancer cell lines significantly suppressed the proliferation of cancer cells, and suppressing JMJD2B expression lead to a decreased population of cancer cells in S phase, with a concomitant increase of cells in G(1) phase. Furthermore, a clonogenicity assay showed that the demethylase activity of JMJD2B possesses an oncogenic activity. Microarray analysis after knockdown of JMJD2B revealed that JMJD2B could regulate multiple pathways which contribute to carcinogenesis, including the cell-cycle pathway. Of the downstream genes, chromatin immunoprecipitation showed that CDK6 (cyclin-dependent kinase 6), essential in G(1)-S transition, was directly regulated by JMJD2B, via demethylation of histone H3-K9 in its promoter region. Expression levels of JMJD2B and CDK6 were significantly correlated in various types of cell lines. Deregulation of histone demethylation resulting in perturbation of the cell cycle, represents a novel mechanism for human carcinogenesis and JMJD2B is a feasible molecular target for anticancer therapy.
Full-text · Article · Sep 2011 · Cancer Prevention Research
[Show abstract][Hide abstract] ABSTRACT: Genetic mapping studies have identified multiple cancer susceptibility regions at chromosome 8q24, upstream of the MYC oncogene. MYC has been widely presumed as the regulated target gene, but definitive evidence functionally linking these cancer regions with MYC has been difficult to obtain. Here we examined candidate functional variants of a haplotype block at 8q24 encompassing the two independent risk alleles for prostate and breast cancer, rs620861 and rs13281615. We used the mapping of DNase I hypersensitive sites as a tool to prioritise regions for further functional analysis. This approach identified rs378854, which is in complete linkage disequilibrium (LD) with rs620861, as a novel functional prostate cancer-specific genetic variant. We demonstrate that the risk allele (G) of rs378854 reduces binding of the transcription factor YY1 in vitro. This factor is known to repress global transcription in prostate cancer and is a candidate tumour suppressor. Additional experiments showed that the YY1 binding site is occupied in vivo in prostate cancer, but not breast cancer cells, consistent with the observed cancer-specific effects of this single nucleotide polymorphism (SNP). Using chromatin conformation capture (3C) experiments, we found that the region surrounding rs378854 interacts with the MYC and PVT1 promoters. Moreover, expression of the PVT1 oncogene in normal prostate tissue increased with the presence of the risk allele of rs378854, while expression of MYC was not affected. In conclusion, we identified a new functional prostate cancer risk variant at the 8q24 locus, rs378854 allele G, that reduces binding of the YY1 protein and is associated with increased expression of PVT1 located 0.5 Mb downstream.
[Show abstract][Hide abstract] ABSTRACT: The emphasis in anticancer drug discovery has always been on finding a drug with great antitumor potential but few side-effects. This can be achieved if the drug is specific for a molecular site found only in tumor cells. Here, we find the enhancer of zeste homolog 2 (EZH2) to be highly overexpressed in lung and other cancers, and show that EZH2 is integral to proliferation in cancer cells. Quantitative real-time PCR analysis revealed higher expression of EZH2 in clinical bladder cancer tissues than in corresponding non-neoplastic tissues (P < 0.0001), and we confirmed that a wide range of cancers also overexpress EZH2, using cDNA microarray analysis. Immunohistochemical analysis showed positive staining for EZH2 in 14 of 29 cases of bladder cancer, 135 of 292 cases of non-small-cell lung cancer (NSCLC), and 214 of 245 cases of colorectal cancer, whereas no significant staining was observed in various normal tissues. We found elevated expression of EZH2 to be associated with poor prognosis for patients with NSCLC (P = 0.0239). In lung and bladder cancer cells overexpressing EZH2, suppression of EZH2 using specific siRNAs inhibited incorporation of BrdU and resulted in significant suppression of cell growth, even though no significant effect was observed in the normal cell strain CCD-18Co, which has undetectable EZH2. Because EZH2 expression was scarcely detectable in all normal tissues we examined, EZH2 shows promise as a tumor-specific therapeutic target. Furthermore, as elevated levels of EZH2 are associated with poor prognosis of patients with NSCLC, its overexpression in resected specimens could prove a useful molecular marker, indicating the necessity for a more extensive follow-up in some lung cancer patients after surgical treatment.
[Show abstract][Hide abstract] ABSTRACT: The research emphasis in anti-cancer drug discovery has always been to search for a drug with the greatest antitumor potential but fewest side effects. This can only be achieved if the drug used is against a specific target located in the tumor cells. In this study, we evaluated Minichromosome Maintenance Protein 7 (MCM7) as a novel therapeutic target in cancer.
Immunohistochemical analysis showed that MCM7 was positively stained in 196 of 331 non-small cell lung cancer (NSCLC), 21 of 29 bladder tumor and 25 of 70 liver tumor cases whereas no significant staining was observed in various normal tissues. We also found an elevated expression of MCM7 to be associated with poor prognosis for patients with NSCLC (P = 0.0055). qRT-PCR revealed a higher expression of MCM7 in clinical bladder cancer tissues than in corresponding non-neoplastic tissues (P < 0.0001), and we confirmed that a wide range of cancers also overexpressed MCM7 by cDNA microarray analysis. Suppression of MCM7 using specific siRNAs inhibited incorporation of BrdU in lung and bladder cancer cells overexpressing MCM7, and suppressed the growth of those cells more efficiently than that of normal cell strains expressing lower levels of MCM7.
Since MCM7 expression was generally low in a number of normal tissues we examined, MCM7 has the characteristics of an ideal candidate for molecular targeted cancer therapy in various tumors and also as a good prognostic biomarker for NSCLC patients.
[Show abstract][Hide abstract] ABSTRACT: A number of histone demethylases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human disease like cancer have not been well understood. Here, we demonstrate important roles of lysine-specific demethylase 1 (LSD1) in human carcinogenesis. Expression levels of LSD1 are significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001). cDNA microarray analysis also revealed its transactivation in lung and colorectal carcinomas. LSD1-specific small interfering RNAs significantly knocked down its expression and resulted in suppression of proliferation of various bladder and lung cancer cell lines. Concordantly, introduction of exogenous LSD1 expression promoted cell cycle progression of human embryonic kidney fibroblast cells. Expression profile analysis showed that LSD1 could affect the expression of genes involved in various chromatin-modifying pathways such as chromatin remodeling at centromere, centromeric heterochromatin formation and chromatin assembly, indicating its essential roles in carcinogenesis through chromatin modification.
Full-text · Article · Feb 2011 · International Journal of Cancer