High neighborhood deprivation impacts DNA methylation and gene expression in cancer-related genes (RNA-Seq)

Background: Low socioeconomic status (SES) has been linked to chronic stress and poorer health outcomes for marginalized communities in the U.S. The biological processes mediating the impact of SES on health to promote chronic diseases, such as cancer, remain poorly understood. Here, we aim to determine how changes in DNA methylation are linked to neighborhood-level socioeconomic deprivation, thereby promoting cancer initiation and progression through cancer- and immune-related pathways. Methods: We extracted DNA from 402 fresh frozen tissues from 289 women in the NCI-Maryland Breast Cancer Cohort, including 185 tumor samples, 113 additional paired adjacent normal samples, and 104 normal tissues from reduction mammoplasty. Census-tract level socioeconomic deprivation was measured via a Neighborhood Deprivation Index (NDI) using the geocoded addresses from our study participants. DNA methylation values were acquired using the Illumina Infinium MethylationEPIC 850K Beadchip. We conducted a differentially methylated probe (DMP) analysis between tumor and adjacent normal tissue to characterize methylation profiles by NDI status. We also used methylCIBERSORT to estimate immune cell subpopulation differences by tissue type and NDI status. Results: NDI was significantly associated with self-reported race in our cohort in both normal (p<0.0001, 95% CI -4.32, -2.58) and tumor (p<0.0001, 95% CI -4.22, -2.79) tissue. Our DMP analysis showed more significant methylation events (hypo- or hyper- methylation of the tumor tissue compared to adjacent normal tissue) in the NDI high group compared to the NDI low group. We also identified five CpG sites that were significantly hypomethylated in the tumors of the NDI high group, including two tumor suppressor genes, LRIG1 (p=1.99 x10-10) and WWOX (p=6.47 x 10-9). These methylation changes translated to an inverse relationship between gene expression for these tumor suppressor genes and NDI. We also identified significantly lower neutrophils in the NDI high tumors compared to NDI low tumors (p=0.001, 95% CI -0.112, -0.028). Conclusions: Overall, our analysis provides evidence that high neighborhood deprivation can lead to pro-tumorigenic changes in DNA methylation and gene expression that may impact the immune microenvironment, breast cancer progression, and overall survival. RNA was isolated from frozen breast tumors using TRIzol method. The mRNA profiles of the above breast tumors were generated by deep sequencing using Illumina NovaSeq S2.

研究背景：美国边缘群体的低社会经济地位（socioeconomic status, SES）已被证实与慢性应激及较差的健康结局相关。但介导SES影响健康、进而促进癌症等慢性病发生的生物学过程仍未被充分阐明。本研究旨在明确DNA甲基化（DNA methylation）变化与社区层面社会经济剥夺的关联，及其如何通过癌症与免疫相关通路推动癌症发生与进展。
研究方法：本研究从NCI-马里兰乳腺癌队列（NCI-Maryland Breast Cancer Cohort）的289名女性受试者中提取了402份新鲜冷冻组织的DNA，其中包含185份肿瘤样本、113份配对癌旁正常组织样本，以及104份来自缩乳术的正常乳腺组织。研究通过受试者的地理编码地址，采用社区剥夺指数（Neighborhood Deprivation Index, NDI）评估普查区域层面的社会经济剥夺程度。采用Illumina Infinium MethylationEPIC 850K微珠芯片检测DNA甲基化水平。本研究开展了肿瘤与癌旁正常组织间的差异甲基化探针（differentially methylated probe, DMP）分析，以基于NDI状态刻画甲基化谱特征；同时使用methylCIBERSORT评估不同组织类型及NDI状态下的免疫细胞亚群差异。
研究结果：在本队列的正常乳腺组织（p<0.0001, 95%置信区间：-4.32, -2.58）与肿瘤组织（p<0.0001, 95%置信区间：-4.22, -2.79）中，NDI均与受试者自我报告的种族存在显著关联。差异甲基化探针分析显示，相较于NDI低分组，NDI高分组中肿瘤组织相较于癌旁正常组织的甲基化异常事件（低甲基化或高甲基化）更为显著。本研究还在NDI高分组的肿瘤中鉴定出5个显著低甲基化的CpG位点，其中包含2个抑癌基因：LRIG1（p=1.99×10^-10）与WWOX（p=6.47×10^-9）。这些甲基化变化与上述抑癌基因的表达水平及NDI呈负相关关系。此外，相较于NDI低分组的肿瘤，NDI高分组的肿瘤组织中中性粒细胞含量显著更低（p=0.001, 95%置信区间：-0.112, -0.028）。
研究结论：综上，本研究分析证实，高水平的社区剥夺可引发DNA甲基化与基因表达的促肿瘤发生变化，这些变化可能影响免疫微环境、乳腺癌进展及患者总生存期。本研究采用TRIzol法从冷冻乳腺肿瘤组织中分离RNA，并通过Illumina NovaSeq S2平台进行深度测序，获取了上述乳腺肿瘤的mRNA表达谱。