LAUSR

Bisphenol AF (BPAF), a BPA analog, is widely used in the manufacturing of plastics, and has been detected in the environment and in human urine samples. Since BPAF may exhibit more potent endocrine-disrupting activities than BPA, the potential effect of BPAF exposure on breast cancer risk is a health concern. Previous reports highlight the interaction of BPAF with estrogen receptors (ERs), but the molecular pathways of BPAF-mediated cellular activities remain unclear. Therefore, we investigated BPAF-mediated cellular responses in MCF-7 and T47D ER+ breast cancer cells. Results from MTT and clonogenic assays indicated that BPAF (0.01 – 5 µM) induces cell proliferation in a concentration-dependent manner. Cell cycle analysis showed that BPAF (0.5 – 1 µM) significantly increased the percentage of cells in S phase and decreased cells in G0/G1 phase. Data from luciferase reporter assays indicated that BPAF potently activated estrogen response element (ERE)-mediated transcription, which was accompanied by the upregulation and activation/phosphorylation of ERα and ERβ. These data demonstrate that BPAF is a potent endocrine disruptor. Importantly, we also found that BPAF induces ErbB3 phosphorylation, and significantly activates downstream PI3K/Akt and MAPK pathways in both cell lines, as indicated by increased Akt, Erk1/2, and Src phosphorylation. We next examined the mRNA levels of key regulators downstream of ER and receptor tyrosine kinase (RTK) pathways, including AREG, TFF1, MYC, IGF1R/2R, NRG1, EGFR, ERRB3, ESR1/2, and CCND1, in BPAF-treated cells. We found that the most significantly upregulated gene by BPAF was AREG (Amphiregulin), an ER-targeted gene that encodes an RTK ligand protein. Concurrent ER and RTK signaling activation with AREG upregulation suggests that BPAF induces ER-RTK crosstalk through AREG-mediated signaling. To demonstrate the impact of ER signaling on BPAF-induced ER-RTK crosstalk, we blocked estrogen signaling with ICI-182,780 (Fulvestrant) in BPAF-treated cells, and found that estrogen signaling inhibition significantly suppressed BPAF-associated cell growth, ERE-mediated transcriptional activity, and signaling in ER and RTK pathways. We next examined the effect of AREG knockdown on the signaling in both RTK and ER pathways to determine the role of AREG in BPAF-induced ER-RTK crosstalk. Our data indicated that AREG knockdown not only markedly inhibited BPAF-induced proliferation and RTK signaling, but also significantly downregulated ER-mediated transcription. Overall, we demonstrated that ER-RTK crosstalk is essential for BPAF-induced oncogenic cellular responses. Importantly, we identified AREG as a critical mediator of the BPAF-induced signaling interactions. These novel findings highlight the potential clinical implications of BPAF-associated ER+ breast cancer risk and underscore the necessity to further evaluate BPAF as an endocrine disruptor. Citation Format: Qingxia Zhao, Amanda B. Parris, Erin W. Howard, Ying Xing, Xiaohe Yang. Bisphenol AF promotes ER+ breast cancer through amphiregulin-mediated ER-RTK signaling crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5046.