The faithful maintenance of DNA methylation homeostasis indispensably requires DNA methyltransferase 1 (DNMT1) in cancer progression. We previously identified DNMT1 as a potential candidate target for oral squamous cell carcinoma (OSCC). However, how the DNMT1- associated global DNA methylation is exploited to regulate OSCC remains unclear. The shRNA-specific DNMT1 knockdown was employed to target DNMT1 on oral cancer cells in vitro, as was the use of DNMT1 inhibitors. A xenografted OSCC mouse model was established to determine the effect on tumor suppression. High-throughput microarrays of DNA methylation, bulk and single-cell RNA sequencing analysis, multiplex immunohistochemistry, functional sphere formation and protein immunoblotting were utilized to explore the molecular mechanism involved. Analysis of human samples revealed associations between DNMT1 expression, global DNA methylation and collaborative molecular signaling with oral malignant transformation. We investigated DNMT1 expression boosted steadily during oral malignant transformation in human samples, and its inhibition considerably minimized the tumorigenicity in vitro and in a xenografted OSCC model. DNMT1 overexpression was accompanied by the accumulation of cancer-specific DNA hypomethylation during oral carcinogenesis; conversely, DNMT1 knockdown caused atypically extensive genome-wide DNA hypomethylation in cancer cells and xenografted tumors. This novel DNMT1-remodeled DNA hypomethylation pattern hampered the dual activation of PI3K-AKT and CDK2-Rb and inactivated GSK3β collaboratively. When treating OSCC mice, targeting DNMT1 achieved greater anticancer efficacy than the PI3K inhibitor, and reduced the toxicity of blood glucose changes caused by the PI3K inhibitor or combination of PI3K and CDK inhibitors as well as adverse insulin feedback. Targeting DNMT1 remodels a novel global DNA hypomethylation pattern to facilitate anticancer efficacy and minimize potential toxic effects via balanced signaling synergia. Our study suggests DNMT1 is a crucial gatekeeper regarding OSCC destiny and treatment outcome.

中文翻译：

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DNMT1靶向重塑整体DNA低甲基化以增强口腔鳞状细胞癌的肿瘤抑制并规避毒性

在癌症进展中，DNA 甲基化稳态的忠实维持不可或缺地需要 DNA 甲基转移酶 1 (DNMT1)。我们之前将 DNMT1 确定为口腔鳞状细胞癌 (OSCC) 的潜在候选靶点。然而，如何利用 DNMT1 相关的全局 DNA 甲基化来调节 OSCC 仍不清楚。与使用 DNMT1 抑制剂一样，在体外采用 shRNA 特异性 DNMT1 敲低来靶向口腔癌细胞上的 DNMT1。建立异种移植 OSCC 小鼠模型以确定其对肿瘤抑制的效果。利用DNA甲基化的高通量微阵列、批量和单细胞RNA测序分析、多重免疫组织化学、功能球形成和蛋白质免疫印迹来探索所涉及的分子机制。对人类样本的分析揭示了 DNMT1 表达、整体 DNA 甲基化以及协同分子信号传导与口腔恶性转化之间的关联。我们研究了人类样本口腔恶性转化过程中 DNMT1 表达的稳定增强，并且其抑制作用大大降低了体外和异种移植 OSCC 模型中的致瘤性。 DNMT1过度表达伴随着口腔癌发生过程中癌症特异性DNA低甲基化的积累；相反，DNMT1 敲低会导致癌细胞和异种移植肿瘤中非典型广泛的全基因组 DNA 低甲基化。这种新型 DNMT1 重塑的 DNA 低甲基化模式阻碍了 PI3K-AKT 和 CDK2-Rb 的双重激活，并协同灭活了 GSK3β。在治疗OSCC小鼠时，靶向DNMT1比PI3K抑制剂具有更好的抗癌功效，并降低了PI3K抑制剂或PI3K和CDK抑制剂组合引起的血糖变化的毒性以及胰岛素不良反馈。靶向 DNMT1 重塑了一种新型的全局 DNA 低甲基化模式，以提高抗癌功效，并通过平衡的信号协同作用最大限度地减少潜在的毒性作用。我们的研究表明 DNMT1 是 OSCC 命运和治疗结果的关键看门人。