Abstract A11: Gene expression analysis of Polycomb and Trithorax family members reveals putative role of ASXL2 in breast cancer subtypes

Abstract

Abstract Breast cancer (BC) is one of the largest public health issues in Brazil and worldwide, and it is categorized based on the expression of membrane receptors for estrogen, progesterone, and HER2, giving rise, respectively, to the molecular subtypes Luminal A, Luminal B, HER2, and triple negative (TN). The etiology of BC is related to genetic, epigenetic, and environmental factors. In fact, the new scenario in cancer studies has moved beyond observing genetic changes. In this context, epigenetics arises as a complex factor since its mechanisms, “not involving alterations in DNA sequence,” elevate the complexity of the query object. Among the epigenetic mechanisms, the antagonic protein families Polycomb (Pc) and Trithorax (Trx) have an important role in controlling chromatin conformation and gene expression. These families comprise different members, and alterations in their expression may lead to gene deregulation, since various genes can be activated or inactivated at once. This study aims to evaluate the expression of the main members of both families (Pc and Trx) in in vitro models of the BC molecular subtypes, as well as in a normal breast cell line. In order to achieve this goal, the cell lines HMEC (normal breast), MCF-7 (Luminal A), EVSA-T (Luminal B), HCC-1954 (HER2), and MDA-MB-231 (triple negative) were cultured under specified conditions, and their RNAs were obtained for analysis by real-time quantitative PCR. A total of 18 genes were evaluated: Polycomb Group (PRC2 Complex: EZH1, EZH2, EED, and SIRT1; PRC1 Complex: RING1, PHC3, BMI1, and YY1), Trithorax Group (SWI/SNF Complex: SMARCA2 and SMARCA4; NURF Complex: SMARCA1 and RBBP4; MLL Complex: KMT2A, WDR5, ASH2L, and PRMT6), and the genes ASXL1 and ASXL2 (not categorized into Pc or Trx). The GAPDH gene was used as constitutive control, and gene expression obtained in HMEC was applied in experiment normalization. On a first analysis, the evaluated genes were found overexpressed in the BC cell lines altogether compared to normal breast, suggesting that there is a deregulation in histone modifiers and/or chromatin remodelers in this disease. In relation to the cell lines, MCF-7, EVSA-T, and MDA-MB-231 presented overexpression of Pc and Trx genes in comparison to HMEC. We highlight some genes that were found overexpressed, such as PRMT6 and SMARCA4 (MCF7); EZH1, EZH2, SIRT1, and RBBP4 (EVSA-T), the latter being described as a participant in NURF, but with an expression correlated to the members of PRC2. In the HER2 in vitro model, we observed a heterogenous Pc and Trx expression, which presented itself as reduced, unaltered, or upregulated in a minor level. Furthermore, in the TN model, the genes SMARCA1 and ASXL2 presented a fold change of more than 20x. The ASXL2 gene has been described, in embryonic development, as a potential regulator and/or enhancer for Pc and Trx, a role still not investigated in BC. It has been suggested that the ASXL family can act in activation and repression of Pc and Trx activity, as well as in the transcriptional regulation of Pc and Trx genes and recruitment of Pc and Trx proteins to their targets in chromatin. In silico analysis showed that ASXL2 can interact with members from both families, ratifying its potential role in BC. Our results show that further studies should be undertaken in order to determine the relation between the expression of these genes and their function. Moreover, we observed that there is no antagonism in the expression of Pc and Trx in BC, suggesting that it is possible that both families are acting in the disease, which implies that an investigation of the regulation and location of these complexes in chromatin will allow a better comprehension of its operation and function in BC. Citation Format: Mariana Araujo, Eliana Abdelhay, Stephany Corrêa. Gene expression analysis of Polycomb and Trithorax family members reveals putative role of ASXL2 in breast cancer subtypes [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A11.