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Combined CRISPR and proteomics screening reveal a cohesin-CTCF-bound allele contributing to increased expression of RUVBL1 and prostate cancer progression [RNA]

ABSTRACT: Introduction Genome-wide association studies along with expression quantitative trait loci (eQTL) have identified hundreds of single nucleotide polymorphisms (SNPs) and their target genes in prostate cancer (PrCa). Although these genetic associations to PrCa have been widely reported, functional characterization of these risk loci remains challenging. Methods To screen for regulatory SNPs, we designed a library containing 9133 guide RNAs (gRNAs) to target 2,166 candidate SNP sites implicated in PrCa. We performed negative screening in dCas9-KRAB stable prostate cell lines and applied the RIGOR program to identify the essential SNPs for cell proliferation. We further characterized the regulatory role of a selected single nucleotide polymorphim (SNP, rs60464856) using luciferase reporter assay, ChIP-qPCR, and xCas9 base editing in prostate cells. Finally, we investigated the biological impact of the SNP-regulated gene RUVBL1 on cell proliferation and tumor growth via gene knockdown using in vitro and in vivo assays. Results From interference of 2,166 candidate SNPs via CRISPR interference screening, the RIGOR program identified 117 SNPs that could regulate genes to promote growth advantage in prostate cancer cell lines. Compared to unselected SNPs, the 117 candidates tended to reside near 5 kb flanking the transcription start sites (p = 0.01). To characterize the regulatory role of these SNPs, we selected one SNP (rs60464856) for detailed analysis. This SNP was covered by multiple gRNAs significantly depleted in the screening (FDR<0.05). Pooled SNP association analysis in the PRACTICAL cohort showed significantly higher PrCa risk for the G allele (pvalue=1.2E-16). eQTL analysis showed that the G allele is associated with an increased expression of RUVBL1 in multiple datasets. To further validate the CRISPR interference effect, we transfected a gRNA targeting the rs60464856 site in the dCas9 stable cell lines and observed significant inhibition of the RUVBL1 expression. We also applied the xCas9 adenine base editor to convert the rs60464856 A into G allele and observed an increased RUVBL1 expression in subclones carrying the rs60464856 G allele in prostate cell lines. To test if any protein showed allele-specific binding at rs60464856, we used SILAC-based DNA pull-down proteomics and observed that cohesin subunits (including SMC3) preferred the A allele. ChIP qPCR assays showed significant enrichment of CTCF and SMC3 signals at the rs60464856 site with preferential binding to the A allele. To evaluate the potential role of this locus in maintaining long-range chromatin structure, we analyzed a HiC dataset and found that the rs60464856 locus enriched consistent chromatin interactions in prostate cell lines. To determine the potential role of the rs60464856 target gene, we knocked down the RUVBL1 via shRNA and observed significant proliferation inhibition in prostate cell lines. We also tested PC3 cells with RUVBL1 knockdown in nude mice xenografts and observed reduced tumorigenesis. Gene set enrichment analysis showed that RUVBL1 expression was associated with the enrichment of cell cycle related pathways in both cell line and TCGA prostate cancer cohorts. Lastly, we showed that an increased RUVBL1 expression and its relevant pathway activation were associated with poor survival. Conclusion We applied the CRISPR interference screening at selected prostate cancer risk loci and identified over a hundred regulatory SNPs essential for prostate cell proliferation. Further analysis confirmed the important role of rs60464856 and its target gene RUVBL1 in prostate cell growth and tumorigenesis. To validate one of our candidate, we knocked down RUVBL1 gene in BPH1 and PC3 cell lines and observed the transcriptome alterations with RNA-seq

ORGANISM(S): Homo sapiens

PROVIDER: GSE224646 | GEO | 2023/07/05

REPOSITORIES: GEO

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Project description:Introduction Genome-wide association studies along with expression quantitative trait loci (eQTL) have identified hundreds of single nucleotide polymorphisms (SNPs) and their target genes in prostate cancer (PrCa). Although these genetic associations to PrCa have been widely reported, functional characterization of these risk loci remains challenging. Methods To screen for regulatory SNPs, we designed a library containing 9133 guide RNAs (gRNAs) to target 2,166 candidate SNP sites implicated in PrCa. We performed negative screening in dCas9-KRAB stable prostate cell lines and applied the RIGOR program to identify the essential SNPs for cell proliferation. We further characterized the regulatory role of a selected single nucleotide polymorphim (SNP, rs60464856) using luciferase reporter assay, ChIP-qPCR, and xCas9 base editing in prostate cells. Finally, we investigated the biological impact of the SNP-regulated gene RUVBL1 on cell proliferation and tumor growth via gene knockdown using in vitro and in vivo assays. Results From interference of 2,166 candidate SNPs via CRISPR interference screening, the RIGOR program identified 117 SNPs that could regulate genes to promote growth advantage in prostate cancer cell lines. Compared to unselected SNPs, the 117 candidates tended to reside near 5 kb flanking the transcription start sites (p = 0.01). To characterize the regulatory role of these SNPs, we selected one SNP (rs60464856) for detailed analysis. This SNP was covered by multiple gRNAs significantly depleted in the screening (FDR<0.05). Pooled SNP association analysis in the PRACTICAL cohort showed significantly higher PrCa risk for the G allele (pvalue=1.2E-16). eQTL analysis showed that the G allele is associated with an increased expression of RUVBL1 in multiple datasets. To further validate the CRISPR interference effect, we transfected a gRNA targeting the rs60464856 site in the dCas9 stable cell lines and observed significant inhibition of the RUVBL1 expression. We also applied the xCas9 adenine base editor to convert the rs60464856 A into G allele and observed an increased RUVBL1 expression in subclones carrying the rs60464856 G allele in prostate cell lines. To test if any protein showed allele-specific binding at rs60464856, we used SILAC-based DNA pull-down proteomics and observed that cohesin subunits (including SMC3) preferred the A allele. ChIP qPCR assays showed significant enrichment of CTCF and SMC3 signals at the rs60464856 site with preferential binding to the A allele. To evaluate the potential role of this locus in maintaining long-range chromatin structure, we analyzed a HiC dataset and found that the rs60464856 locus enriched consistent chromatin interactions in prostate cell lines. To determine the potential role of the rs60464856 target gene, we knocked down the RUVBL1 via shRNA and observed significant proliferation inhibition in prostate cell lines. We also tested PC3 cells with RUVBL1 knockdown in nude mice xenografts and observed reduced tumorigenesis. Gene set enrichment analysis showed that RUVBL1 expression was associated with the enrichment of cell cycle related pathways in both cell line and TCGA prostate cancer cohorts. Lastly, we showed that an increased RUVBL1 expression and its relevant pathway activation were associated with poor survival. Conclusion We applied the CRISPR interference screening at selected prostate cancer risk loci and identified over a hundred regulatory SNPs essential for prostate cell proliferation. Further analysis confirmed the important role of rs60464856 and its target gene RUVBL1 in prostate cell growth and tumorigenesis. To discover proliferative essential SNP loci, we used CRISPRi screening technology and DNA-seq to determine those functional candidate from previously published eQTL variants

Project description:Full title: Comprehensive Characterization of Three-Dimensional Models for Prostate Cancer Growth and Invasion in Laminin-rich Extracellular Matrix Prostate Cancer (PrCa) cells undergo acinar morphogenesis and spheroid formation in three-dimensional (3D) culture, supported by laminin-rich extracellular matrix (lrECM, Matrigel). We developed miniaturized 3D model systems that facilitate investigation of morphogenesis and invasion of normal and PrCa cell lines in lrECM. Primary and non-transformed cell lines formed round structures with strong cell-cell contacts and epithelial polarization, lumen and a complete basal lamina (BL). In contrast, most PrCa cell lines formed either defective, “mass” spheroids with incomplete BL, or invasive “stellate” structures. The bioinformatic analyses of genome-wide mRNA expression data revealed massive alteration of key functional and signaling pathways in 3D cultures, with lipid and steroid metabolism, epigenetic reprogramming, and differentiation-related transcription factors induced across all cell lines by lrECM. In invasive cells, AKT, PI3Kinase, mTOR, and hedgehog signaling pathways were most highly activated, validated by small molecule inhibitors compounds specifically targeting key regulatory molecules. Compounds against AKT and PI3kinase pathways were significantly more effective in invasive cells, compared to mass or round/normal phenotype spheroids, and monolayer culture. A severe morphologic conversion was observed in PC-3 and PC-3M cells, transforming initially round, normal-appearing epithelial spheroids into rapidly invading cell masses. Markers for EMT (epithelial-mesenchymal transition) were highly expressed already in early stage, round spheroids prior to invasive conversion, and were not further increased in invasive cells. This indicates that PrCa cells can display extraordinary plasticity. EMT may be involved in providing a metastable genotype that allows morphological transformation, but is not be required for invasive processes themselves. Total RNA was obtained from non-transformed prostate epithelial cells and prostate cancer cells cultured in monolayer and three-dimensional laminin-rich extracellular matrix (growth factor-reduced Matrigel).

Project description: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 r^2≥0.5. Greater than 88% of the 727 SNPs fall within putative enhancers, many of which 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 in the identification of these enhancers, we performed genomewide ChIP-seq for H3K27-acetylation, a mark of actively engaged enhancer regions, 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 (r^2=0.91) 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. ChIP-seq analysis of H3K27Ac in LNCaP charcoal-stripped serum, H3K27Ac in LNCaP charcoal-stripped serum +DHT, TCF7L2 in LNCaP

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Combined CRISPR and proteomics screening reveal a cohesin-CTCF-bound allele contributing to increased expression of RUVBL1 and prostate cancer progression [RNA]

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