Transcriptomic Response to Calcium in Normal Colon Organoids is Impacted by Colon Location and Sex.

Observational studies indicate that calcium supplementation may protect against colorectal cancer. Stratified analyses suggest that this protective effect may differ based on anatomic subsite and sex, but these hypotheses have been difficult to test experimentally. Here, we exposed 36 patient-derived organoid lines derived from normal colon biopsies (21 right colons, 15 left colons) of unrelated subjects (18 female, 18 male) to moderate (1.66 mmol/L) or high (5.0 mmol/L) concentrations of calcium for 72 hours. We performed bulk RNA-sequencing to measure gene expression, and cell composition was inferred using single-cell deconvolution in CIBERSORTx. We tested for significant differences in gene expression using generalized linear models in DESeq2. Exposure to higher levels of calcium was associated with changes in cell composition (P < 0.05), most notably increased goblet and reduced stem cell populations, and differential expression of 485 genes (FDR < 0.05). We found that 40 of these differentially expressed genes mapped to genomic loci identified through colorectal cancer genome-wide association studies, suggesting a potential biologic overlap between calcium supplementation and inherited colorectal cancer risk. Stratified analyses identified more differentially expressed genes in colon organoids derived from right sided colon and male subjects than those derived from left sided colon and female subjects. We confirmed the presence of a stronger right-sided effect for one of these genes, HSD17B2 using qPCR in a subset of matched right and left colon organoids (n = 4). By relating our findings to genetic data, we provide new insights into how nutritional and genetic factors may interact to influence colorectal cancer risk. PREVENTION RELEVANCE: A chemopreventive role for calcium in colorectal cancer is still unclear. Here, we identify mechanisms through which calcium supplementation may reduce risk. Calcium supplementation increased differentiation and altered expression of colorectal cancer-related genes in a large study of patient-derived colon organoids. These findings were influenced by colon location and sex.

DNA methylation analysis of normal colon organoids from familial adenomatous polyposis patients reveals novel insight into colon cancer development.

BACKGROUND: Familial adenomatous polyposis (FAP) is an inherited colorectal cancer (CRC) syndrome resulting from germ line mutations in the adenomatous polyposis coli (APC) gene. While FAP accounts for less than 1% of all CRC cases, loss of APC expression is seen in > 80% of non-hereditary CRCs. To better understand molecular mechanisms underlying APC-driven CRC, we performed an epigenome-wide analysis of colon organoids derived from normal-appearing colons of FAP patients versus healthy subjects to identify differentially methylated regions (DMRs) that may precede the onset of CRC. RESULTS: We identified 358 DMRs when comparing colon organoids of FAP patients to those of healthy subjects (FDR < 0.05, |mean beta difference| = 5%). Of these, nearly 50% of DMRs were also differentially methylated in at least one of three CRC tumor and normal adjacent tissue (NAT) cohorts (TCGA-COAD, GSE193535 and ColoCare). Moreover, 27 of the DMRs mapped to CRC genome-wide association study (GWAS) loci. We provide evidence suggesting that some of these DMRs led to significant differences in gene expression of adjacent genes using quantitative PCR. For example, we identified significantly greater expression of five genes: Kazal-type serine peptidase inhibitor domain 1 (KAZALD1, P = 0.032), F-Box and leucine-rich repeat protein 8 (FBXL8, P = 0.036), TRIM31 antisense RNA 1 (TRIM31-AS1, P = 0.036), Fas apoptotic inhibitory molecule 2 (FAIM2, P = 0.049) and (Collagen beta (1-0)galactosyltransferase 2 (COLGALT2, P = 0.049). Importantly, both FBXL8 and TRIM31-AS1 were also significantly differentially expressed in TCGA-COAD tumor versus matched NAT, supporting a role for these genes in CRC tumor development. CONCLUSIONS: We performed the first DNA methylome-wide analysis of normal colon organoids derived from FAP patients compared to those of healthy subjects. Our results reveal that normal colon organoids from FAP patients exhibit extensive epigenetic differences compared to those of healthy subjects that appear similar to those exhibited in CRC tumor. Our analyses therefore identify DMRs and candidate target genes that are potentially important in CRC tumor development in FAP, with potential implications for non-hereditary CRC.

A risk variant for Barrett's esophagus and esophageal adenocarcinoma at chr8p23.1 affects enhancer activity and implicates multiple gene targets.

Nineteen genetic susceptibility loci for esophageal adenocarcinoma (EAC) and its precursor Barrett's esophagus (BE) have been identified through genome-wide association studies (GWAS). Clinical translation of such discoveries, however, has been hindered by the slow pace of discovery of functional/causal variants and gene targets at these loci. We previously developed a systematic informatics pipeline to prioritize candidate functional variants using functional potential scores, applied the pipeline to select high-scoring BE/EAC risk loci and validated a functional variant at chr19p13.11 (rs10423674). Here, we selected two additional prioritized loci for experimental interrogation: chr3p13/rs1522552 and chr8p23.1/rs55896564. Candidate enhancer regions encompassing these variants were evaluated using luciferase reporter assays in two EAC cell lines. One of the two regions tested exhibited allele-specific enhancer activity - 8p23.1/rs55896564. CRISPR-mediated deletion of the putative enhancer in EAC cell lines correlated with reduced expression of three candidate gene targets: B lymphocyte kinase (BLK), nei like DNA glycosylase 2 (NEIL2) and cathepsin B (CTSB). Expression quantitative trait locus (eQTL) mapping in normal esophagus and stomach revealed strong associations between the BE/EAC risk allele at rs55896564 (G) and lower expression of CTSB, a protease gene implicated in epithelial wound repair. These results further support the utility of functional potential scores for GWAS variant prioritization, and provide the first experimental evidence of a functional variant and risk enhancer at the 8p23.1 GWAS locus. Identification of CTSB, BLK and NEIL2 as candidate gene targets suggests that altered expression of these genes may underlie the genetic risk association at 8p23.1 with BE/EAC.

Prioritization and functional analysis of GWAS risk loci for Barrett's esophagus and esophageal adenocarcinoma.

Genome-wide association studies (GWAS) have identified ~20 genetic susceptibility loci for esophageal adenocarcinoma (EAC), and its precursor, Barrett's esophagus (BE). Despite such advances, functional/causal variants and gene targets at these loci remain undefined, hindering clinical translation. A key challenge is that most causal variants map to non-coding regulatory regions such as enhancers, and typically, numerous potential candidate variants at GWAS loci require testing. We developed a systematic informatics pipeline for prioritizing candidate functional variants via integrative functional potential scores (FPS) consolidated from multi-omics annotations, and used this pipeline to identify two high-scoring variants for experimental interrogation: chr9q22.32/rs11789015 and chr19p13.11/rs10423674. Minimal candidate enhancer regions spanning these variants were evaluated using luciferase reporter assays in two EAC cell lines. One of the two variants tested (rs10423674) exhibited allele-specific enhancer activity. CRISPR-mediated deletion of the putative enhancer region in EAC cell lines correlated with reduced expression of two genes-CREB-regulated transcription coactivator 1 (CRTC1) and Cartilage oligomeric matrix protein (COMP); expression of five other genes remained unchanged (CRLF1, KLHL26, TMEM59L, UBA52, RFXANK). Expression quantitative trait locus mapping indicated that rs10423674 genotype correlated with CRTC1 and COMP expression in normal esophagus. This study represents the first experimental effort to bridge GWAS associations to biology in BE/EAC and supports the utility of FPS to guide variant prioritization. Our findings reveal a functional variant and candidate risk enhancer at chr19p13.11 and implicate CRTC1 and COMP as putative gene targets, suggesting that altered expression of these genes may underlie the BE/EAC risk association.

A Functional Variant on 9p21.3 Related to Glioma Risk Affects Enhancer Activity and Modulates Expression of CDKN2B-AS1.

Genome-wide association studies have identified SNPs associated with glioma risk on 9p21.3, but biological mechanisms underlying this association are unknown. We tested the hypothesis that a functional SNP on 9p21.3 affects activity of an enhancer, causing altered expression of nearby genes. We considered all SNPs in linkage disequilibrium with the 9p21.3 sentinel SNP rs634537 that mapped to putative enhancers. An enhancer containing rs1537372 exhibited allele-specific effects on luciferase activity. Deletion of this enhancer in GBM cell lines correlated with decreased expression of CDKN2B-AS1. Expression quantitative trait loci analysis using non-diseased brain samples showed rs1537372 to be a consistently significant eQTL for CDKN2B-AS1. Additionally, our analysis of Hi-C data generated in neural progenitor cells showed that the bait region containing rs1537372 interacted with the CDKN2B-AS1 promoter. These data suggest rs1537372, a SNP at the 9p21.3 risk locus, is a functional variant that modulates expression of CDKN2B-AS1.

SNPs associated with colorectal cancer at 15q13.3 affect risk enhancers that modulate GREM1 gene expression.

Several genome wide association studies of colorectal cancer (CRC) have identified single nucleotide polymorphisms (SNPs) on chromosome 15q13.3 associated with CRC risk. To identify functional variant(s) underlying this association, we investigated SNPs in linkage disequilibrium with the risk-associated SNP rs4779584 that overlapped regulatory regions/enhancer elements characterized in colon-related tissues and cells. We identified several SNP-containing regulatory regions that exhibited enhancer activity in vitro, including one SNP (rs1406389) that correlated with allele-specific effects on enhancer activity. Deletion of either this enhancer or another enhancer that had previously been reported in this region correlated with decreased expression of GREM1 following CRISPR/Cas9 genome editing. That GREM1 is one target of these enhancers was further supported by an expression quantitative trait loci correlation between rs1406389 and GREM1 expression in the transverse but not sigmoid colon in the Genotype-Tissue Expression dataset. Taken together, we conclude that the 15q13.3 region contains at least two functional variants that map to distinct enhancers and impact CRC risk through modulation of GREM1 expression.

A functional variant on 20q13.33 related to glioma risk alters enhancer activity and modulates expression of multiple genes.

Genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs) associated with glioma risk on 20q13.33, but the biological mechanisms underlying this association are unknown. We tested the hypothesis that a functional SNP on 20q13.33 impacted the activity of an enhancer, leading to an altered expression of nearby genes. To identify candidate functional SNPs, we identified all SNPs in linkage disequilibrium with the risk-associated SNP rs2297440 that mapped to putative enhancers. Putative enhancers containing candidate functional SNPs were tested for allele-specific effects in luciferase enhancer activity assays against glioblastoma multiforme (GBM) cell lines. An enhancer containing SNP rs3761124 exhibited allele-specific effects on activity. Deletion of this enhancer by CRISPR-Cas9 editing in GBM cell lines correlated with an altered expression of multiple genes, including STMN3, RTEL1, RTEL1-TNFRSF6B, GMEB2, and SRMS. Expression quantitative trait loci (eQTL) analyses using nondiseased brain samples, isocitrate dehydrogenase 1 (IDH1) wild-type glioma, and neurodevelopmental tissues showed STMN3 to be a consistent significant eQTL with rs3761124. RTEL1 and GMEB2 were also significant eQTLs in the context of early CNS development and/or in IDH1 wild-type glioma. We provide evidence that rs3761124 is a functional variant on 20q13.33 related to glioma/GBM risk that modulates the expression of STMN3 and potentially other genes across diverse cellular contexts.

Interleukin-1beta released by microglia initiates the enhanced glutamatergic activity in the spinal dorsal horn during paclitaxel-associated acute pain syndrome.

Patients receiving paclitaxel for cancer treatment often develop an acute pain syndrome (paclitaxel-associated acute pain syndrome, P-APS), which occurs immediately after paclitaxel treatment. Mechanisms underlying P-APS remain largely unknown. We recently reported that rodents receiving paclitaxel develop acute pain and activation of spinal microglial toll like receptor 4 (TLR4) by paclitaxel penetrating into the spinal cord is a critical event in the genesis of P-APS. Our current study dissected cellular and molecular mechanisms underlying the P-APS. We demonstrated that bath-perfusion of paclitaxel, at a concentration similar to that found in the cerebral spinal fluid in animals receiving i.v. paclitaxel (2 mg/kg), resulted in increased calcium activity in microglia instantly, and in astrocytes with 6 min delay. TLR4 activation in microglia by paclitaxel caused microglia to rapidly release interleukin-1ß (IL-1ß) but not tumor necrosis factor α, IL-6, or interferon-γ. IL-1ß release from microglia depended on capthepsin B. IL-1ß acted on astrocytes, leading to elevated calcium activity and suppressed glutamate uptake. IL-1ß also acted on neurons to increase presynaptic glutamate release and postsynaptic AMPA receptor activity in the spinal dorsal horn. Knockout of IL-1 receptors prevented the development of acute pain induced by paclitaxel in mice. Our study indicates that IL-1ß is a crucial molecule used by microglia to alter functions in astrocytes and neurons upon activation of TLR4 in the genesis of P-APS, and targeting the signaling pathways regulating the production and function of IL-1ß from microglia is a potential avenue for the development of analgesics for the treatment of P-APS.

FTY-720 induces apoptosis in neuroblastoma via multiple signaling pathways.

Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor. High-risk NB is difficult to treat due to the lack of response to current therapies and aggressive disease progression. Despite novel drugs, alternative treatments and multi-modal treatments, finding an effective treatment strategy for these patients continues to be a major challenge. The current study focuses on examining the effects of FTY-720 or fingolimod, a drug that is FDA-approved for refractory multiple sclerosis, in NB. The results showed that FTY-720 regulates multiple pathways that result in various effects on calcium signaling, ion channel activation and cell survival/death pathways. FTY-720 rapidly inhibits TRPM7 channel activity, and inhibited TRPM7 kinase activity, modulates calcium signaling, induces a loss of mitochondrial membrane potential and opening of the mitochondrial permeability transition pore, and ultimately leads to cell death. Interestingly, the data also showed that low concentrations of FTY-720 sensitized drug-resistant NB cells to antineoplastic drugs. These results suggest that FTY-720 may be an attractive alternative for the treatment of NB.