Active cancer-associated fibroblasts (CAFs) are major components of the tumor microenvironment, which promote carcinogenesis and modulate response to therapy. Therefore, targeting these cells or reducing their paracrine pro-carcinogenic effects could be of great therapeutic value. To this end, we sought to investigate the effect of eugenol, a natural phenolic molecule, on active breast CAFs. We have shown that decitabine (5-Aza-2′-deoxycytidine, DAC) and eugenol inhibit the expression of the DNA methyltransferase genes DNMT1 and DNMT3A at both the protein and mRNA levels in breast CAF cells. While the effect of eugenol was persistent, DAC had only a transient inhibitory effect on the mRNA level of both DNMT genes. Furthermore, eugenol and DAC suppressed the invasive/migratory and proliferative potential of CAF cells as well as their paracrine pro-carcinogenic effects both in vitro and in humanized orthotopic tumor xenografts. Interestingly, these inhibitory effects of decitabine and eugenol were mediated through E2F1 downregulation. Indeed, ectopic expression of E2F1 upregulated both genes and attenuated the effects of eugenol. Additionally, we provide clear evidence that eugenol, like DAC, strongly modulates the methylation pattern in active CAF cells, through methylating several oncogenes and demethylating various important tumor suppressor genes, which affected their mRNA expression levels. Importantly, the E2F1 promoter was also hypermethylated and the gene downregulated in response to eugenol. Together, these findings show that the active features of breast CAF cells can be normalized through eugenol-dependent targeting of DNMT1/DNMT3A and the consequent modulation in gene methylation.

中文翻译：

---

丁香酚通过 DNMT1/DNMT3A 的 E2F1 依赖性下调调节基因组甲基化并使乳腺癌相关成纤维细胞失活

活跃的癌症相关成纤维细胞 (CAF) 是肿瘤微环境的主要组成部分，可促进致癌作用并调节对治疗的反应。因此，靶向这些细胞或减少它们的旁分泌促癌作用可能具有很大的治疗价值。为此，我们试图研究丁香酚（一种天然酚类分子）对活性乳腺 CAF 的影响。我们已经证明地西他滨（5-Aza-2'-脱氧胞苷，DAC）和丁香酚在乳腺 CAF 细胞中抑制 DNA 甲基转移酶基因 DNMT1 和 DNMT3A 的蛋白质和 mRNA 水平的表达。虽然丁香酚的作用是持久的，但 DAC 对两个 DNMT 基因的 mRNA 水平只有短暂的抑制作用。此外，丁香酚和 DAC 抑制了 CAF 细胞的侵袭/迁移和增殖潜力以及它们在体外和人源化原位肿瘤异种移植物中的旁分泌促癌作用。有趣的是，地西他滨和丁香酚的这些抑制作用是通过 E2F1 下调介导的。事实上，E2F1 的异位表达上调了这两个基因并减弱了丁香酚的作用。此外，我们提供了明确的证据，表明丁香酚与 DAC 一样，通过甲基化几种致癌基因和去甲基化影响其 mRNA 表达水平的各种重要肿瘤抑制基因，强烈调节活性 CAF 细胞中的甲基化模式。重要的是，E2F1 启动子也被高度甲基化，并且该基因响应丁香酚而下调。一起，