Chunli Yan

The 8q24 cancer risk variant rs6983267 shows long-range interaction with MYC in colorectal cancer

An inherited variant on chromosome 8q24, rs6983267, is significantly associated with cancer pathogenesis. We present evidence that the region harboring this variant is a transcriptional enhancer, that the alleles of rs6983267 differentially bind transcription factor 7-like 2 (TCF7L2) and that the risk region physically interacts with the MYC proto-oncogene. These data provide strong support for a biological mechanism underlying this non-protein-coding risk variant.

Functional Enhancers at the Gene-Poor 8q24 Cancer-Linked Locus

Multiple discrete regions at 8q24 were recently shown to contain alleles that predispose to many cancers including prostate, breast, and colon. These regions are far from any annotated gene and their biological activities have been unknown. Here we profiled a 5-megabase chromatin segment encompassing all the risk regions for RNA expression, histone modifications, and locations occupied by RNA polymerase II and androgen receptor (AR). This led to the identification of several transcriptional enhancers, which were verified using reporter assays. Two enhancers in one risk region were occupied by AR and responded to androgen treatment; one contained a single nucleotide polymorphism (rs11986220) that resides within a FoxA1 binding site, with the prostate cancer risk allele facilitating both stronger FoxA1 binding and stronger androgen responsiveness. The study reported here exemplifies an approach that may be applied to any risk-associated allele in non-protein coding regions as it emerges from genome-wide association studies to better understand the genetic predisposition of complex diseases.

Comprehensive Functional Annotation of 77 Prostate Cancer Risk Loci

Genome-wide association studies (GWAS) have revolutionized the field of cancer genetics, but the causal links between increased genetic risk and onset/progression of disease processes remain to be identified. Here we report the first step in such an endeavor for prostate cancer. We provide a comprehensive annotation of the 77 known risk loci, based upon highly correlated variants in biologically relevant chromatin annotations— we identified 727 such potentially functional SNPs. We also provide a detailed account of possible protein disruption, microRNA target sequence disruption and regulatory response element disruption of all correlated SNPs at . 88% of the 727 SNPs fall within putative enhancers, and many alter critical residues in the response elements of transcription factors known to be involved in prostate biology. We define as risk enhancers those regions with enhancer chromatin biofeatures in prostate-derived cell lines with prostate-cancer correlated SNPs. To aid the identification of these enhancers, we performed genomewide ChIP-seq for H3K27-acetylation, a mark of actively engaged enhancers, as well as the transcription factor TCF7L2. We analyzed in depth three variants in risk enhancers, two of which show significantly altered androgen sensitivity in LNCaP cells. This includes rs4907792, that is in linkage disequilibrium () with an eQTL for NUDT11 (on the X chromosome) in prostate tissue, and rs10486567, the index SNP in intron 3 of the JAZF1 gene on chromosome 7. Rs4907792 is within a critical residue of a strong consensus androgen response element that is interrupted in the protective allele, resulting in a 56% decrease in its androgen sensitivity, whereas rs10486567 affects both NKX3-1 and FOXA-AR motifs where the risk allele results in a 39% increase in basal activity and a 28% fold-increase in androgen stimulated enhancer activity. Identification of such enhancer variants and their potential target genes represents a preliminary step in connecting risk to disease process.

Comprehensive Functional Annotation of Seventy-One Breast Cancer Risk Loci

Breast Cancer (BCa) genome-wide association studies revealed allelic frequency differences between cases and controls at index single nucleotide polymorphisms (SNPs). To date, 71 loci have thus been identified and replicated. More than 320,000 SNPs at these loci define BCa risk due to linkage disequilibrium (LD). We propose that BCa risk resides in a subgroup of SNPs that functionally affects breast biology. Such a shortlist will aid in framing hypotheses to prioritize a manageable number of likely disease-causing SNPs. We extracted all the SNPs, residing in 1 Mb windows around breast cancer risk index SNP from the 1000 genomes project to find correlated SNPs. We used FunciSNP, an R/Bioconductor package developed in-house, to identify potentially functional SNPs at 71 risk loci by coinciding them with chromatin biofeatures. We identified 1,005 SNPs in LD with the index SNPs (r2≥0.5) in three categories; 21 in exons of 18 genes, 76 in transcription start site (TSS) regions of 25 genes, and 921 in enhancers. Thirteen SNPs were found in more than one category. We found two correlated and predicted non-benign coding variants (rs8100241 in exon 2 and rs8108174 in exon 3) of the gene, ANKLE1. Most putative functional LD SNPs, however, were found in either epigenetically defined enhancers or in gene TSS regions. Fifty-five percent of these non-coding SNPs are likely functional, since they affect response element (RE) sequences of transcription factors. Functionality of these SNPs was assessed by expression quantitative trait loci (eQTL) analysis and allele-specific enhancer assays. Unbiased analyses of SNPs at BCa risk loci revealed new and overlooked mechanisms that may affect risk of the disease, thereby providing a valuable resource for follow-up studies.

Identification and characterization of functional risk variants for colorectal cancer mapping to chromosome 11q23.1

Genome-wide association studies of colorectal cancer (CRC) have identified a number of common variants associated with modest risk, including rs3802842 at chromosome 11q23.1. Several genes map to this region but rs3802842 does not map to any known transcribed or regulatory sequences. We reasoned, therefore, that rs3802842 is not the functional single-nucleotide polymorphism (SNP), but is in linkage disequilibrium (LD) with a functional SNP(s). We performed ChIP-seq for histone modifications in SW480 and HCT-116 CRC cells, and incorporated ChIP-seq and DNase I hypersensitivity data available through ENCODE within a 137-kb genomic region containing rs3802842 on 11q23.1. We identified SNP rs10891246 in LD with rs3802842 that mapped within a bidirectional promoter region of genes C11orf92 and C11orf93. Following mutagenesis to the risk allele, the promoter demonstrated lower levels of reporter gene expression. A second SNP rs7130173 was identified in LD with rs3802842 that mapped to a candidate enhancer region, which showed strong unidirectional activity in both HCT-116 and SW480 CRC cells. The risk allele of rs7130173 demonstrated reduced enhancer activity compared with the common allele, and reduced nuclear protein binding affinity in electromobility shift assays compared with the common allele suggesting differential transcription factor (TF) binding. SNPs rs10891246 and rs7130173 are on the same haplotype, and expression quantitative trait loci (eQTL) analyses of neighboring genes implicate C11orf53, C11orf92 and C11orf93 as candidate target genes. These data imply that rs10891246 and rs7130173 are functional SNPs mapping to 11q23.1 and that C11orf53, C11orf92 and C11orf93 represent novel candidate target genes involved in CRC etiology.

An enhancer from the 8q24 prostate cancer risk region is sufficient to direct reporter gene expression to a subset of prostate stem-like epithelial cells in transgenic mice

SUMMARY Regions in the 8q24 gene desert contribute significantly to the risk of prostate cancer and other adult cancers. This region contains several DNA regions with enhancer activity in cultured cells. One such segment, histone acetylation peak 10 (AcP10), contains a risk single nucleotide polymorphism (SNP) that is significantly associated with the pathogenesis of colorectal, prostate and other cancers. The mechanism by which AcP10 influences cancer risk remains unknown. Here we show that AcP10 contains a sequence that is highly conserved across terrestrial vertebrates and is capable in transgenic mice of directing reporter gene expression to a subset of prostate lumenal epithelial cells. These cells include a small population of Nkx3.1-positive cells that persist even after androgen ablation. Castration-resistant Nkx3.1-positive (CARN) cells were shown by others to function both as stem cells and cells of origin of prostate cancer. Our results thus provide a mechanism by which AcP10 could influence prostate cancer risk.

Abstract A08: Widespread tobacco smoking-associated changes in DNA methylation and gene expression in lung tissue of smokers

Significance: Epigenome-wide association studies (EWAS) have identified thousands of CpGs showing smoking-associated DNA methylation changes in blood cell DNA of smokers. Several of these CpGs are strongly associated with lung cancer risk. Until recently, the relationship of such DNA methylation changes in the blood and epigenetic alterations in the lung, as well as their molecular link to lung cancer development, remained poorly understood. Using a limited EWAS of 237 lung tissue samples, we recently identified seven smoking-associated hypomethylated CpGs in lung tissue, five of which had been previously reported in blood, and one of which had been strongly associated with lung cancer risk. We showed that the latter CpG flanked a smoking-inducible enhancer element driving expression of a xenobiotic response gene. Here we expand these observations to identify tobacco-affected regulatory elements implicated in lung cancer. Methods: We performed an EWAS using Inifinium HumanMethylation450K BeadChip data from The Cancer Genome Atlas (TCGA) corresponding to 393 eligible lung adenocarcinoma tumor samples. We replicated our findings in 27 adjacent normal lung tissue samples, then examined epigenetic enhancer marks near the smoking-associated altered CpGs using epigenomic data from primary lung cells and lung adenocarcinoma cell lines. We next identified potential responsive target genes of these enhancers by examining smoking-associated changes in gene expression on the 1 megabase flanks of the enhancers. Results: 37,372 CpGs were significantly differentially methylated with respect to smoking in TCGA adenocarcinoma tumor samples after Benjamini Hochberg correction (p Conclusion: Many smoking-associated methylation changes in lung tissue are located in the vicinity of epigenetic regulators in lung tissue, and could be driving expression of smoking-associated genes that may affect lung cancer risk. Funding: R01 HL114094 to IALO, P30 CA014089, USC Provost’s fellowship and Roy E. Thomas Foundation Graduate Scholarship to DJM. Citation Format: Daniel J. Mullen, T. Ryan Stueve, Chunli Yan, Kimberly D. Siegmund, Ite A. Laird-Offringa. Widespread tobacco smoking-associated changes in DNA methylation and gene expression in lung tissue of smokers [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A08.

Abstract A37: Tobacco smoke increases lung adenocarcinoma risk by downregulating TGF-beta and AhR-regulated focal adhesion proteins involved in injury resolution

Background: The most prevalent type of lung cancer—lung adenocarcinoma—arises from the most distal reaches of the lung in the alveolar epithelium. The alveolar epithelium consists of two cell types: (i) thin type I alveolar epithelial cells (AT1) that facilitate gas exchange and constitute more than 90% of the alveolar surface, and (ii) cuboidal surfactant-producing type II (AT2) cells. Following injury, dynamic interactions between extracellular matrix components, integrins, focal adhesion proteins, and cytoskeletal components allow AT2 progenitor cells to migrate, spread, and transdifferentiate into AT1 cells to restore the denuded epithelial barrier. Methods: Utilizing primary human AT2 cells isolated from lung transplant remnants, our lab has established and extensively profiled an in vitro model of epithelial restitution, wherein AT2 cells differentiate over a period of six days into terminally differentiated AT1-like cells. Because cigarette smoking is the most recognized risk factor for lung adenocarcinoma (LUAD), and because the alveolar space is the primary target of tobacco smoke, we also compared the RNA-seq profiles of AT2>AT1 differentiation to those of A549 LUAD cells exposed to cigarette smoke condensate (CSC). Results: We find that CSC downregulates many factors that are upregulated in the AT2>AT1 restitution model, particularly integrins, extracellular matrix components, and novel focal adhesion proteins that are also downregulated in LUAD relative to adjacent normal tissue. We describe regulation of four such novel focal adhesion proteins, specifically by TGF-β and aryl hydrocarbon receptor (AhR)-mediated mechanisms. Conclusions: Our findings shed light on early mechanisms in the carcinogenic process, and support the notion that alveolar cells engaged in repeated cycles of injury and repair are critical actors in LUAD risk trajectories. Note: This abstract was not presented at the conference. Citation Format: Theresa Ryan Stueve, Chenchen Yang, Crystal N. Marconett, Chunli Yan, Beiyun Zhou, Zea Borok, Ite A. Laird-Offringa. Tobacco smoke increases lung adenocarcinoma risk by downregulating TGF-beta and AhR-regulated focal adhesion proteins involved in injury resolution [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A37.

The Functionality of Prostate Cancer Predisposition Risk Regions Is Revealed by AR Enhancers

Prostate Cancer (PCa) genetic risk has recently been defined in numerous genome-wide association studies (GWAS), which revealed more than 50 disease-associated single nucleotide polymorphisms (SNPs), known as tagSNPs, each at a different locus. More than 80% of these tagSNPs are located in noncoding regions of the genome for which functionality remains unknown. We and others hypothesize that at least some of these SNPs affect noncoding genomic regulatory signatures such as enhancers. Many research laboratories including ours have profiled the genomic distribution of androgen receptor (AR) and the dynamic state of the PCa genome for active chromatin regions (H3K9,14ac), open chromatin regions (DNaseI), enhancers (H3K4me1/2), and active/engaged enhancers (H3K27ac). In order to identify candidate functional SNPs, which may confer risk associated with PCa, we recently developed an open-source (R/Bioconductor) package, called FunciSNP (Functional Integration of SNPs), which systematically integrates SNPs from the 1000 genomes project with these biologically active chromatin features. Here we report several potential AR enhancers, defined by genome-wide data and from chromatin biofeatures, which may be functionally involved in PCa risk.

Abstract 1646: Integrative analysis identifies functional prostate cancer risk SNPs in genomic regulatory regions defined as enhancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Prostate Cancer (PCa) genetic risk has recently been defined in numerous genome-wide association studies (GWASs), which have revealed more than 50 disease-associated single nucleotide polymorphs (SNP), known as tagSNPs, each at a different locus. More than 80% of these tagSNPs are in intergenic regions of the genome for which functionality remains unknown. Taking the 1000 genomes data into consideration, more than 10,000 correlated SNPs are revealed, which each define risk due to linkage disequilibrium (correlated to the tagSNP with an r2>0.5). The above therefore makes the identification of functional and/or causal SNPs not a trivial task. Therefore, we hypothesize that at least some of these SNPs affect non-coding genomic regulatory signatures, such as enhancers, insulators or repressors. In order to reduce the number of candidate functional SNPs, we developed an open-source (R/Bioconductor) package, called FunciSNP (Functional Integration of SNPs), which systematically integrates the 1000 genomes SNP data with chromatin features of interest. To define functionality in non-coding DNA associated with PCa risk we have utilized chromatin features generated by next-generation sequencing technologies, which capture regions genome-wide for which both open chromatin states and enhancers are likely. The open chromatin state (DnaseI, H3K9,14ac), enhancers (H3K4me1), and active/engaged enhancers (H3K27ac) were generated either by our lab, or harvested from the ENCODE project, or retrieved from recent publications. All chromatin features were identified in the same prostate cancer cell line (LNCaP). Thus, we have identified 113 PCa risk correlated SNPs at androgen receptor occupied regions (AROR), 1,545 at DNase1 sensitivity sites and 403 at histone modified regions [H3K4me1 (160 SNPs), H3K9,14 ac (121 SNPs), H3k27ac (122 SNPs)]; all features excluded transcription start sites of known annotated genes. Interestingly, the majority of the identified correlated SNPs (69%) are located in either chromosome 8 or 10. ARORs coinciding with DNase1 sites revealed four novel SNPs correlated with four GWAS tagSNPs. Of the four novel surrogate SNPs, two are located 4kb upstream from KLK3, one is located within the 3′ UTR of NKX3.1 and one is located within an intron of RUVBL1. DNase1 sites coinciding with any histone modification provided 17 novel SNPs correlated with 10 GWAS tagSNPs. Of the 17 novel SNPs, four are located in 8q24 genomic region and each are more than 100kb away from a known annotated gene. Each one of above-mentioned novel SNPs is assessed for effects on potential transcription factor response elements. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1646. doi:1538-7445.AM2012-1646