Abstract 3868: FND 4b decreases proliferation and increases apoptosis of triple negative breast cancer through AMPK activation

Abstract

Abstract Introduction. Triple negative breast cancer (TNBC), which comprises 15-20% of all breast cancers, is the deadliest and most aggressive subtype. TNBC affects a younger patient population and is associated with an increased risk of distant recurrence, higher rates of metastases, earlier time to recurrence, and worse prognosis after recurrence. AMP-activated protein kinase (AMPK) is a major cellular energy regulator that has recently been implicated in cancer progression. AMPK can act as a tumor suppressor by decreasing anabolism and inducing cell cycle arrest. FND 4b is a novel AMPK activator that has been shown to inhibit growth and induce apoptosis in colon cancer, but its effects have not been studied in breast cancer. The purpose of this project is to test the effects of FND 4b on AMPK activation, cellular proliferation, and apoptosis in several types of breast cancer—with a particular emphasis on TNBC. Methods. (i) To assess signaling pathways, estrogen-receptor positive (ER+) breast cancer cells (MCF-7 and T-47D), TNBC cells (MDA-MB-231 and HCC-1806), and breast cancer stem cells were treated with FND 4b (0, 1, 2.5, 5, 10, and 20 µM) for 24 h. Immunoblot analysis assessed phosphorylated and total forms of AMPK, acetyl CoA carboxylase (ACC), and ribosomal protein S6 as well as cyclin D1 and cleaved PARP. (ii) Growth assays with sulforhodamine B (SRB) were performed by treating ER+ breast cancer cells and TNBC cells with FND 4b (0, 2.5, 5, 10, and 20 µM) for 72 h and then quantifying cellular protein content. Proliferation was also assessed by treating all cell lines with FND 4b (0 or 5 µM) for 72 h and then counting viable cells. (iii) Cell death was assessed by treating ER+ breast cancer cells and TNBC cells with FND 4b (0, 2.5, 5, and 10 µM) for 72 h and then quantifying fragmented DNA in the cytoplasm by ELISA. Results. (i) FND 4b treatment for 24 h increased AMPK activation with concomitant decreases in ACC activity, phosphorylated S6, and cyclin D1 in ER+ breast cancer, TNBC, and breast cancer stem cells. (ii) FND 4b treatment for 72 h (5 µM) decreased proliferation in all breast cancer cells, while dose-dependent decreases in growth were also observed in ER+ breast cancer and TNBC cells. (iii) Increases in apoptosis were found in ER+ breast cancer and one TNBC cell line with FND 4b treatment for 72 h. Conclusions. Our findings indicate that FND 4b can decrease proliferation for a variety of breast cancers by activating AMPK—with notable effects on TNBC. The reductions in growth were mediated through decreases in fatty acid synthesis (ACC), mTOR signaling (S6), and cell cycle flux (cyclin D1). ER+ breast cancer cells were more susceptible to FND 4b-induced apoptosis, but MDA-MB-231 cells still underwent apoptosis with higher dose FND 4b treatment. Further development of FND compounds—with extensions to animal studies—could result in a novel therapeutic agent in the treatment of TNBC. Citation Format: Jeremy Johnson, Piotr Rychahou, Vitaliy M. Sviripa, Heidi L. Weiss, David Watt, B. Mark Evers. FND 4b decreases proliferation and increases apoptosis of triple negative breast cancer through AMPK activation [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 3868.