BACKGROUND Acquired resistance to trastuzumab is a major clinical problem in the treatment of HER2-positive (HER2+) breast cancer patients. The selection of trastuzumab-resistant patients is a great challenge of precision oncology. The aim of this study was to identify novel epigenetic biomarkers associated to trastuzumab resistance in HER2+ BC patients. METHODS We performed a genome-wide DNA methylation (450K array) and a transcriptomic analysis (RNA-Seq) comparing trastuzumab-sensitive (SK) and trastuzumab-resistant (SKTR) HER2+ human breast cancer cell models. The methylation and expression levels of candidate genes were validated by bisulfite pyrosequencing and qRT-PCR, respectively. Functional assays were conducted in the SK and SKTR models by gene silencing and overexpression. Methylation analysis in 24 HER2+ human BC samples with complete response or non-response to trastuzumab-based treatment was conducted by bisulfite pyrosequencing. RESULTS Epigenomic and transcriptomic analysis revealed the consistent hypermethylation and downregulation of TGFBI, CXCL2, and SLC38A1 genes in association with trastuzumab resistance. The DNA methylation and expression levels of these genes were validated in both sensitive and resistant models analyzed. Of the genes, TGFBI presented the highest hypermethylation-associated silencing both at the transcriptional and protein level. Ectopic expression of TGFBI in the SKTR model suggest an increased sensitivity to trastuzumab treatment. In primary tumors, TGFBI hypermethylation was significantly associated with trastuzumab resistance in HER2+ breast cancer patients. CONCLUSIONS Our results suggest for the first time an association between the epigenetic silencing of TGFBI by DNA methylation and trastuzumab resistance in HER2+ cell models. These results provide the basis for further clinical studies to validate the hypermethylation of TGFBI promoter as a biomarker of trastuzumab resistance in HER2+ breast cancer patients.

中文翻译：

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TGFBI的表观遗传沉默赋予人类乳腺癌患者对曲妥珠单抗的抗性。

背景技术获得的对曲妥珠单抗的耐药性是治疗HER2阳性（HER2 +）乳腺癌患者的主要临床问题。耐曲妥珠单抗的患者的选择是精确肿瘤学的巨大挑战。这项研究的目的是鉴定与HER2 + BC患者中曲妥珠单抗耐药性相关的新型表观遗传标记。方法我们进行了全基因组DNA甲基化（450K阵列）和转录组分析（RNA-Seq），比较了曲妥珠单抗敏感（SK）和曲妥珠单抗耐药（SKTR）HER2 +人乳腺癌细胞模型。分别通过亚硫酸氢盐焦磷酸测序和qRT-PCR验证了候选基因的甲基化和表达水平。通过基因沉默和过表达，在SK和SKTR模型中进行功能测定。通过亚硫酸氢盐焦磷酸测序对24种HER2 +人BC样品进行了完全响应或不响应曲妥珠单抗的甲基化分析。结果表观基因组和转录组学分析显示，与曲妥珠单抗耐药相关的TGFBI，CXCL2和SLC38A1基因始终存在高度甲基化和下调。这些基因的DNA甲基化和表达水平已在敏感和耐药模型中进行了验证。在这些基因中，TGFBI在转录和蛋白质水平上均表现出最高的与高甲基化相关的沉默。TGFBI在SKTR模型中的异位表达表明对曲妥珠单抗治疗的敏感性增加。在原发性肿瘤中，HER2 +乳腺癌患者的TGFBI高甲基化与曲妥珠单抗耐药性显着相关。结论我们的研究结果首次表明，在HER2 +细胞模型中，通过DNA甲基化产生的TGFBI的表观遗传沉默与曲妥珠单抗之间存在关联。这些结果为进一步临床研究奠定了基础，以验证TGFBI启动子的高甲基化作为HER2 +乳腺癌患者中曲妥珠单抗耐药性的生物标志物。