Combined SNPs sequencing and allele specific proteomics capture reveal functional causality underpinning the 2p25 prostate cancer susceptibility locus. Combined SNPs sequencing and allele specific proteomics capture reveal functional causality underpinning the 2p25 prostate cancer susceptibility locus

Genome wide association studies (GWASs) have identified numerous risk loci associated with prostate cancer, yet unraveling their functional significance remains elusive. Leveraging our high-throughput SNPs-seq method, we pinpointed rs4519489 within the multi-ancestry GWAS-discovered 2p25 locus as a potential functional SNP due to its significant allelic differences in protein binding. Here, we conduct a comprehensive analysis of rs4519489 and its associated gene, NOL10, employing diverse cohort data and experimental models. Clinical findings reveal a synergistic effect between rs4519489 genotype and NOL10 expression on prostate cancer prognosis and severity. Through unbiased proteomics screening, we reveal that the risk allele A of rs4519489 exhibits enhanced binding to USF1, a novel oncogenic transcription factor (TF) implicated in prostate cancer progression and prognosis, resulting in elevated NOL10 expression. Furthermore, we elucidate that NOL10 regulates cell cycle pathways, fostering prostate cancer progression. The concurrent expression of NOL10 and USF1 correlates with aggressive prostate cancer characteristics and poorer prognosis. Collectively, our study offers a robust strategy for functional SNP screening and TF identification through high-throughput SNPs-seq and unbiased proteomics, highlighting the rs4519489-USF1- NOL10 regulatory axis as a promising biomarker or therapeutic target for clinical diagnosis and treatment of prostate cancer. Overall design: To examine the potential enhancer functionality of the rs4519489 region, we performed chromatin immunoprecipitation sequencing (ChIP-seq) experiments for epigenetic markers in various cell lines and clinical specimens. Expanding ChIP-seq to include histone modifications in both normal and tumor prostate tissues from the CPGEA cohort confirmed enhancer/promoter activity at rs4519489, reinforcing its functional gene regulatory role. Subsequently, to assess the impact of NOL10 or USF1 knockdown on downstream gene expression, we performed RNA sequencing analysis to identify differentially expressed genes (DEGs) in LNCaP,DU145,PC3, and 22Rv1 cells treated with shRNA or siRNA targeting NOL10 or USF1.

全基因组关联研究（Genome Wide Association Studies, GWAS）已发现众多与前列腺癌相关的风险位点，但阐明这些位点的功能意义仍颇具挑战。本研究借助自主建立的高通量单核苷酸多态性测序（high-throughput SNPs-seq）方法，在多祖先GWAS发现的2p25位点中精准定位到rs4519489，因其在蛋白结合中存在显著的等位基因差异，该位点被鉴定为潜在功能性单核苷酸多态性。本研究针对rs4519489及其关联基因NOL10展开全面分析，整合多队列数据与多种实验模型。临床研究结果显示，rs4519489基因型与NOL10表达水平对前列腺癌的预后与疾病严重程度存在协同效应。通过无偏倚蛋白质组学筛选，本研究发现rs4519489的风险等位基因A与USF1的结合能力显著增强，USF1是一种参与前列腺癌进展与预后调控的新型致癌转录因子（transcription factor, TF），该结合事件可导致NOL10表达水平升高。此外，本研究阐明NOL10可通过调控细胞周期通路，促进前列腺癌的进展。NOL10与USF1的共表达与前列腺癌的侵袭性表型及不良预后显著相关。综上，本研究通过高通量SNPs-seq与无偏倚蛋白质组学技术，为功能性SNP筛选及转录因子鉴定提供了一套可靠的研究策略，并证实rs4519489-USF1-NOL10调控轴可作为前列腺癌临床诊断与治疗的潜在生物标志物或治疗靶点。

实验设计：为验证rs4519489区域的潜在增强子功能，本研究在多种细胞系与临床标本中开展了针对表观遗传标记的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）实验。将ChIP-seq拓展至覆盖CPGEA队列中正常与肿瘤前列腺组织的组蛋白修饰分析，证实rs4519489位点存在增强子/启动子活性，进一步明确了其基因调控的功能属性。随后，为评估NOL10或USF1敲低对下游基因表达的影响，本研究针对转染靶向NOL10或USF1的短发卡RNA（short hairpin RNA, shRNA）或小干扰RNA（small interfering RNA, siRNA）的LNCaP、DU145、PC3及22Rv1细胞开展RNA测序分析，以筛选差异表达基因（differentially expressed genes, DEGs）。