Abstract A45: Regulatory network of the metastatic process in breast cancer

Abstract

Abstract Breast cancer (BC) is a heterogeneous disease composed of multiple subtypes with different molecular characteristics and clinical outcomes. In Brazil, this neoplasia is the first cause of cancer death in women, mainly due to the diagnosis in advanced stages, when the possibility of the development of metastases is greater. The metastatic process is related to expression of the epithelial-to-mesenchymal transition transcription factors (EMT-TFs) SNAIL, SLUG, SIP1, and TWIST1. Recently, we have described the role of NF-kB as a regulator of the gene expression of TWIST1, SLUG, and SIP1 factors in aggressive BC cells HCC-1954 (Her2/neu positive) and MDA-MB-231 (triple negative, TN). Through bioinformatics tools, we identified several binding sites for NF-kB in the promoter region of these genes that were confirmed by chromatin immunoprecipitation and luciferase reporter assays. In order to support our findings, we evaluated the expression of NF-kB, Twist, Slug, and Sip1 through RT-qPCR on breast tumor samples. A comparison among the breast cancer subtypes indicated that TN group expressed significantly more NF-kB/p65, Slug, and Sip1. We also observed elevated levels of Twist1 on HER2 than other subtypes. Then, for each tumor sample, mRNA levels for NF-kB/p65 were plotted versus mRNA levels for Slug, Twist, and Sip1. Statistical analysis using the Pearson method showed a clear positive correlation between NF-kB and Twist (r = 0.923; P < 0.0001), Slug (r = 0.786; P = 0.007), and Sip1 expression (r = 0.940; P < 0.0001). Therefore, the results confirmed the positive correlation between the levels of NF-kB and those EMT-TFs described as regulatory targets in this study. Among the EMT-TFs, Twist1 is described as the master regulator of this phenomenon in BC; however, its role during the evolution of different BC subtypes remains unclear. Therefore, the silencing of the Twist1 gene in the cell line HCC-1954 was performed through the specific interfering RNA (shTwist) and, concomitantly, we used the negative silencing control (Scramble). Twist1 silencing measured by RT-qPCR resulted in a 95% reduction of the relative expression of Twist1 after 48 h of transfection. For a high-throughput analysis of the effect of silencing, mRNA samples were subjected to large-scale microarray analysis through the GeneChip human exon array. We considered as differentially expressed the genes whose levels were 2-fold higher or lower than the Scramble. These results showed profound molecular alterations caused by the decrease in Twist1 expression, since 141 genes showed increased expression or decreased expression. An analysis using Metacore software grouped these genes according to molecular function, revealing numerous correlations between Twist1 with important biologic processes and signaling pathways such as blood coagulation, TGF-b/SMADs signaling, and interleukin-17. Our findings may contribute to a greater understanding of the metastatic process of this neoplasia and highlight NF-kB and Twist1 as a potential target for breast cancer treatment. Citation Format: Bruno R. B. Pires, Gerson M. Ferreira, Renata Binato, Eliana Abdelhay. Regulatory network of the metastatic process in breast cancer [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 A45.