Introduction: The major clinical problem for HER2 breast cancer target therapies is the acquisition of resistance. Trastuzumab has been used for the treatment of HER2-positive early stage and metastatic breast cancers. Notoriously, up to 62% patients that respond to the initial treatment develop resistance within a year. Different cellular and molecular alterations may contribute to the development of Trastuzumab resistance, including epigenetic changes. The aim of our study has been the evaluation of epigenetic differences between Trastuzumab-sensitive and -resistant breast cancer cell lines to determine whether they might be used as potential biomarkers to help in the treatment assessment and monitoring.
Experimental procedures: To understand the epigenetic changes that may be associated with Trastuzumab resistance we compared the DNA methylation status of ∼450000 CpG sites from a DNA methylation microarray performed in Trastuzumab-sensitive and resistant cells. To identify the differentially methylated CpGs we considered differences between methylation groups of ≥60%. The functions associated to genes were analysed using the Gene Ontology and were used to select a small number of candidate genes. Their differences in promoter methylation status were validated by expression techniques (qPCR) and subsequently confirmed by demethylation agent (5-aza-DC) treatment followed by qPCR. The Trastuzumab resistant HER2-positive cell line (SKTR) had been previously developed in our laboratory by exposing SKBr3 cells to Trastuzumab, starting with 1μM for three months of exposure and increasing up to 2 μM for a 12 months period.
Summary of the new, unpublished data: The DNA methylation microarray data showed that different 5’-CpG island sites had altered methylation profile when comparing Trastuzumab-resistant (SKTR) and -sensible SKBr3 cells. We selected 4 candidate genes with a hypermethylation profile in -resistant vs. -sensible cells: TGFβ1 (Transforming Growth Factor Beta 1), BCL6 (B-Cell CLL/Lymphoma 6), KILLIN (P53-Regulated DNA Replication Inhibitor) and CTSZ (Cathepsin Z). The hypermethylation of these four candidate genes in SKTR was in agreement with their expression downregulation observed by PCR analysis. SKTR treatment with the demethylation agent 5-aza-2’-deoxycytidine restored the expression levels of all four genes to the levels of Trastuzumab-sensitive cells. Although previously related to breast cancer, TGFβ1, BCL6, KILLIN and CTSZ have not earlier been associated with the development of Trastuzumab resistance in breast cancer or in other cancer types and may be further studied as potential biomarkers.
Conclusions: These results provide a basis for future studies to validate the hypermethylation status of these genes as a predictive or monitoring biomarker of Trastuzumab resistance in HER2+ breast cancer patients.
Citation Format: Sonia Palomeras, Angel Diaz-Lagares, Sarrats Ariadna, Crujeiras Ana Belen, Giro-Perafita Ariadna, Juan Sandoval, Marc Rabionet, Manel Esteller, Teresa Puig. Epigenetic silencing of TGFβ1, BCL6, KILLIN and CTSZ is related to trastuzumab resistance in HER2-positive breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2130A.