Dna methylome in her2-positive resistant breast cancer

Resumo

Approximately 15% to 20% of patients with breast cancer overexpress the human epidermal growth factor receptor 2 (HER2). Although the development of anti-HER2 treatments has significantly improved breast cancer outcomes, a large percentage of patients display primary or acquired resistance to the drug. Several efforts have focused on identifying and understanding the molecular and cellular mechanisms involved in treatment resistance. DNA methylation is the most well-characterized epigenetic modification in humans and is involved in regulating the expression of a great variety of critical genes in cancer. For this reason, DNA methylation status has emerged as one of the most promising epigenetic biomarkers for several types of cancer. This epigenetic marker can be useful in detecting tumors earlier or identifying patients with an increased risk of cancer, as well as evaluating disease progression or predicting the response to anticancer drugs. In the last few years some significant genes that are inactivated by promoter methylation in breast cancer have been identified, including BRCA1 and RASSF1A. In this thesis, we have focused on the DNA methylation analysis in HER2+ breast cancer resistant to current therapies (trastuzumab and lapatinib). In a first approach, global DNA methylation pattern was analyzed in trastuzumab and lapatinib-sensitive and - resistant models (SK, SKTR, SKLR and SKTLR) using the Infinium HumanMethylation450 BeadChip (450k array). Each sensitive and resistant model was clearly differentiated according to their methylation pattern, demonstrating DNA methylation involvement in trastuzumab and lapatinib resistance. Based on the global analysis, our study focused on identifying potential biomarkers for trastuzumab and lapatinib resistance. To this purpose, we performed an integrative analysis of promoter and island methylation and expression pattern (RNA-Seq) comparing our sensitive and resistant models. In each comparison different genes with methylation and expression pattern correlations were identified as potential biomarkers for trastuzumab, lapatinib and trastuzumab plus lapatinib resistance. Unfortunately, due to the difficulties in obtaining tumor samples similar to our in vitro models from human patients, some of the potential biomarkers identified could not be validated in subsequent studies. For this reason, we focused our study on analyzing trastuzumab resistance biomarkers; the basis of anti-HER2 therapies. Although four epigenetically regulated genes were identified (TGFBI, KILLIN, CXCL2, and SLC38A1), TGFBI was the only gene validated in the two trastuzumab-sensitive (SK and AU) and -resistant (SKTR and AUTR) HER2+ breast cancer models analyzed. Furthermore, functional analyses revealed that TGFBI reexpression induced greater sensitivity to trastuzumab in our SKTR model, probably through its integrin-binding domains (EPDIM, NKDIL, YH and RGD). Independent of integrin-binding domains, TGFBI overexpression showed activation of some protein of HER pathway similar to the SK cells. Finally, the in vivo results corroborated the previous in vitro results. When compared to their pre-treatment samples, a significant increase in TGFBI methylation levels was identified in the post-treatment samples from patients who had developed resistance to neoadjuvant anthracycline-taxane-based chemotherapy plus trastuzumab treatment. Moreover, TGFBI methylation analysis in pre-treatment samples of responder and non-responder patients, as well as in normal breast samples, allowed us to suggest the role TGFBI plays as a treatment response biomarker in HER2+ breast cancer. In summary, we demonstrated the role DNA methylation has in trastuzumab- and lapatinib-resistant HER2+ breast cancer. Furthermore, we identify the epigenetic inactivation of the TGFBI gene by promoter CpG island hypermethylation in trastuzumab resistance in vitro and in small cohort of HER2+ human samples. Our results suggest for the first time TGFBI hypermethylation as a biomarker for trastuzumab response in HER2+ breast cancer. Although further studies are required to identify the specific role TGFBI plays in trastuzumab resistance, the combination of TGFBI hypermethylation analysis with standard clinical markers may help stratify HER2+ patients according to the response to the trastuzumab treatment.