Abstract
BackgroundAcquired 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.MethodsWe 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.ResultsEpigenomic 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.ConclusionsOur 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.
Highlights
Acquired resistance to trastuzumab is a major clinical problem in the treatment of human epidermal growth factor receptor 2 (HER2)-positive (HER2+) breast cancer patients
We took advantage of the 450K array methodology to characterize the DNA methylation profile associated with trastuzumab resistance in Breast cancer (BC), and compared a trastuzumab-sensitive (SK) and trastuzumab-resistant (SKTR) BC models (Fig. 1a)
We used more stringent criteria to determine the most differentially methylated CpGs in the promoters and islands, considering in SKTR the CpGs with a methylation level (β) in SK < 0.20 and in SKTR > 0.60 as hypermethylated, and in SKTR the CpGs with a methylation level (β) in SKTR < 0.20 and in SK > 0.60 as hypomethylated. This analysis revealed 184 differentially methylated CpGs corresponding to 152 genes which, according to a Gene Ontology analysis (GO), were significantly associated (FDR < 0.05) with several biological processes related to cancer, such as cell adhesion pathways (GO:0007155), regulation of transcription (GO:0006355), development (GO:0007275), and control of apoptosis (GO: 0043065) (Fig. 1d)
Summary
Acquired resistance to trastuzumab is a major clinical problem in the treatment of HER2-positive (HER2+) breast cancer patients. In the last few years, several studies have focused on identifying the molecular mechanisms of trastuzumab resistance, such as aberrant activation of downstream signaling pathways [7] or the HER2 carboxyl-terminal fragments (CTF), known as p95HER2, which are frequently found in HER2expressing BC cell lines and tumors [8]. Despite these efforts, the complete picture of the molecular mechanisms triggering trastuzumab resistance in BC remains unclear. Clarifying these mechanisms and identifying new resistance biomarkers is essential in the advance towards precision oncology in BC and the quest for new treatment options for those patients who do not respond to trastuzumab therapy
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