As confusion mounts over RNA isoforms involved in phenotypic plasticity, aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity (ITH). Protease serine 3 (PRSS3), possessing four splice variants (PRSS3-SVs; PRSS3-V1–V4), is an indispensable trypsin that shows paradoxical effects on cancer development. Here, we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer, exhibiting opposing functions and clinical outcomes, namely, oncogenic PRSS3-V1 and PRSS3-V2 versus tumor-suppressive PRSS3-V3, by targeting different downstream genes. We identified an intragenic CpG island (iCpGI) in PRSS3. Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1 (MZF1) to regulate PRSS3 transcription. The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2′-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression. Epigenetic silencing of PRSS3-V3 via iCpGI methylation (iCpGIm) in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease. Thus, UHRF1/DNMT1–MZF1 axis-modulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs, conferring nongenetic functional ITH, with implications for early detection of lung cancer and targeted therapies.

随着对参与表型可塑性的 RNA 亚型的混淆越来越多，异常 CpG 甲基化介导的选择性剪接破坏越来越被认为是肿瘤内异质性 (ITH) 的驱动因素。蛋白酶丝氨酸 3 (PRSS3) 具有四种剪接变体（PRSS3-SV；PRSS3-V1 – V4），是一种不可或缺的胰蛋白酶，对癌症发展显示出矛盾的作用。在这里，我们发现PRSS3转录本及其亚型在肺癌中表达不同，表现出相反的功能和临床结果，即致癌 PRSS3-V1 和 PRSS3-V2与肿瘤抑制 PRSS3-V3，通过靶向不同的下游基因。我们鉴定了一个基因内 CpG 岛 (iCpGI)PRSS3。iCpGI 的超甲基化由 UHRF1/DNMT1 复合物介导，干扰髓样锌指 1 (MZF1) 的结合以调节PRSS3转录。大蒜衍生化合物二烯丙基三硫化物与 5-aza-2'-脱氧胞苷协同作用，通过位点特异性 iCpGI 去甲基化作用在肺腺癌细胞中发挥抗肿瘤作用，特别是允许 MZF1 上调 PRSS3-V3表达。在 BALF 和肿瘤组织中通过 iCpGI 甲基化 (iCpGIm) 对 PRSS3-V3进行表观遗传沉默与肺癌患者的早期临床进展有关，但与鳞状细胞癌或炎症性疾病患者无关。因此，UHRF1/DNMT1–MZF1 轴调制位点特异性 iCpGIm 调节PRSS3-SV，赋予非遗传功能性 ITH，对肺癌的早期检测和靶向治疗具有重要意义。