Interplay between promoter methylation and chromosomal loss in gene silencing at 3p11-p14 in cervical cancer

Loss of 3p11-p14 is a frequent event in epithelial cancer and a candidate prognostic biomarker in cervical cancer. In addition to loss, promoter methylation can participate in gene silencing and promote tumor aggressiveness. We have performed a complete mapping of promoter methylation at 3p11-p14 in two independent cohorts of cervical cancer patients (n=149, n=121), using Illumina 450K methylation arrays. The aim was to investigate whether hypermethylation was frequent and could contribute to gene silencing and disease aggressiveness either alone or combined with loss. By comparing the methylation level of individual CpG sites with corresponding data of normal cervical tissue, 26 out of 41 genes were found to be hypermethylated in both cohorts. The frequency of patients with hypermethylation of these genes was found to be higher at tumor stages of 3 and 4 than in stage 1 tumors. Seventeen of the 26 genes were transcriptionally downregulated in cancer compared to normal tissue, whereof six genes showed a significant correlation between methylation and expression. Integrated analysis of methylation, gene dosage, and expression of the 26 hypermethylated genes identified three regulation patterns encompassing eight hypermethylated genes; a methylation driven pattern (<i>C3orf14, GPR27, ZNF717</i>), a gene dosage driven pattern (<i>THOC7, PSMD6</i>), and a combined methylation and gene dosage driven pattern (<i>FHIT, ADAMTS9, LRIG1</i>). In survival analysis, patients with both hypermethylation and loss of <i>LRIG1</i> had a worse outcome compared to those harboring only hypermethylation or none of the events. <i>C3orf14</i> emerged as a novel methylation regulated suppressor gene, for which knockdown was found to promote invasive growth in human papilloma virus (HPV)-transformed keratinocytes. In conclusion, hypermethylation at 3p11-p14 is common in cervical cancer and may exert a selection pressure during carcinogenesis alone or combined with loss. Information on both events could lead to improved prognostic markers.

3p11-p14区域缺失是上皮癌中的频发事件，同时可作为宫颈癌的候选预后生物标志物。除片段缺失外，启动子甲基化亦可参与基因沉默过程，并推动肿瘤侵袭性增强。本研究针对两独立宫颈癌患者队列（样本量分别为n=149、n=121），采用Illumina 450K甲基化芯片完成了3p11-p14区域启动子甲基化的全景绘制。本研究旨在探讨高甲基化是否频发存在，并可单独或联合片段缺失共同参与基因沉默及疾病侵袭性进展。通过将单个CpG位点的甲基化水平与正常宫颈组织的对应数据进行比对，本研究在两个队列中均发现41个基因中有26个存在高甲基化。上述基因发生高甲基化的患者比例，在3、4期肿瘤中显著高于1期肿瘤患者。与正常组织相比，26个基因中有17个在癌组织中呈现转录下调，其中6个基因的甲基化水平与表达量呈显著相关性。对26个高甲基化基因的甲基化状态、基因剂量及表达水平进行整合分析后，本研究鉴定出包含8个高甲基化基因的3种调控模式：甲基化驱动型模式（包含C3orf14、GPR27、ZNF717）、基因剂量驱动型模式（包含THOC7、PSMD6），以及甲基化与基因剂量联合驱动型模式（包含FHIT、ADAMTS9、LRIG1）。生存分析结果显示，同时存在LRIG1高甲基化与片段缺失的患者，其预后相较于仅存在LRIG1高甲基化或两者均无的患者更差。C3orf14被鉴定为一种新型甲基化调控抑癌基因，研究发现敲低该基因可促进人类乳头瘤病毒（human papilloma virus, HPV）转化的角质形成细胞的侵袭性生长。综上，3p11-p14区域的高甲基化在宫颈癌中十分常见，可单独或联合片段缺失在癌变过程中施加选择压力。同时涵盖这两类事件的检测信息，有望助力预后标志物的优化升级。