Abstract 1881: Role of E2F activators and mitotic regulators controlled by E2Fs in EMT, invasion, and metastasis of breast cancer

Abstract The E2 Promoter Binding Factors (E2Fs) are a group of eight transcription factors that are highly deregulated in breast cancer and control several cellular processes, including proliferation and centrosome duplication. Broadly, E2Fs can be divided into transcriptional activators and repressors based on their role in gene transcription. In addition to their traditional cell cycle functions, it is known that deregulated Rb/E2F pathway signals the epithelial-to-mesenchymal transition (EMT); yet the underlining mechanism of how E2Fs drive EMT in breast cancer remains unknown. Published data from our laboratory demonstrates that overexpression of activating E2Fs leads to increased expression of mitotic regulators such as BUBR1, MPS1 (TTK), and SGO1, among others. Furthermore, published data from our laboratory shows that TTK promotes mesenchymal signaling through several mechanisms in breast cancer. Hence, we are investigating to what extent deregulation of E2F activators promotes EMT, invasion, and metastasis through the remaining mitotic regulators. Our hypothesis is that the overexpression of E2F activators contributes to tumorigenesis by promoting EMT through the induction of the transcription of specific mitotic regulators, thus leading to increased invasion and metastasis. To test our hypothesis, E2Fs (1, 2 and 3a) were knockdown individually or in combination of two or three E2F activators simultaneously using specific siRNAs sequences in MDA-MB-231 cells. After 48 hours, western blot analysis was performed to confirm the knockdown and to evaluate the effects of knockdown of these E2Fs on protein levels of mitotic regulators and EMT markers. Real-time PCR was performed to evaluate EMT mRNA expression levels. Additionally, a wound healing assay was conducted to evaluate cell migration, as an indicator of metastasis. All experiments were performed in triplicates and a non-parametric Mann Whitney test was used to evaluate statistical significance. Our results show that knockdown of E2F1 and the combination of all activating E2Fs decreased the expression of mitotic regulators and EMT proteins. Likewise, knockdown of E2Fs decreased the expression of certain EMT genes (but not all) and knockdown of E2F1 decreased cell migration. These results suggest that E2F1 has a prominent role in breast cancer EMT and cell migration. Currently, we are exploring the effects of knockdown of E2F-controlled mitotic regulators in breast cancer EMT and cell migration. Citation Format: Shirley Jusino, Melanie J. Hidalgo-Vargas, Jaya Padmanabhan, Srikumar P. Chellapppan, Harold I. Saavedra. Role of E2F activators and mitotic regulators controlled by E2Fs in EMT, invasion, and metastasis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1881.