Abstract 4461: New potent AMPK activators against colorectal cancer stem cells

Abstract

Abstract Colorectal (CRC) cancer is the second leading cause of cancer deaths in the United States in 2014. Patients often die due to metastasis of CRC rather than from the primary disease, and it is thought that cancer stem cells play a major role in the survival and metastasis of cancer cells after treatment. A new class of small molecules, fluorinated N,N-diarylureas (FNDs), have been formulated and shown to reduce viability of CRC cells through AMPK activation. The purpose of the present study was to evaluate whether FNDs inhibit growth and induce apoptosis of CRC metastatic cell lines and CRC stem cells in vitro and to identify an FND for development as a potential chemotherapeutic agent. METHODS. FNDs were identified in a high-throughput screen and selected for study based on their relative induction of AMPK activity. Compounds are arbitrarily designated as 4a, 4b, 4h, 4j, 4k, 4z, 4aa, 4bb. Metastatic CRC cell lines HT29, HTC116, and KM20, were purchased from American Type Culture Collection. Two CRC stem cell lines were purchased from Celprogen. Cell viability and IC50 concentrations were measured using a sulforhodamine B colorimetric (SRB) cytotoxicity assay. Western blots were analyzed with antibodies: PARP for apoptosis, cyclin D1 for cell cycle progression, pAMPK to confirm AMPK activation, and β-actin as a loading control. RESULTS. AMPK activation after FND treatment was confirmed by western blot in metastatic CRC and stem cells with a peak activation at 12 h. Furthermore, AMPK activation was noted at concentrations as low as 5 μM, compared to metformin (an AMPK activator drug for DMII) which is typically dosed at 5-20 mM for metastatic cell lines. CRC stem cells were more resistant to FND treatment than regular metastatic CRC cell lines, with an IC50 concentration of 50 μM for stem cells as compared to 10-25 μM for metastatic cell lines. Cyclin D1 expression was undetectable with FND drug treatments at IC50 concentrations in CRC stem cell lines. FND 4b stood out as the strongest inducer of apoptosis, but all of the FND compounds increased apoptosis compared to control. CONCLUSIONS. AMPK is a key regulator of energy metabolism in cells, and cancer cells effectively make use of this pathway for their unregulated proliferation. Activation of AMPK by all FND compounds successfully inhibited the cell cycle, inhibited subsequent cellular proliferation, and induced a remarkable level of apoptosis in select FNDs in CRC metastatic and stem cells. Additionally, these effects were observed at much lower doses than other AMPK activators such as metformin and AICAR. Therefore, FNDs have considerable promise in the treatment of metastatic colon cancer, especially by preventing progression and metastasis as a result of their inhibition of CRC stem cells. Citation Format: Dasha Kenlan, Piotr G. Rychahou, Vitaliy Sviripa, David Watt, B. Mark Evers. New potent AMPK activators against colorectal cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4461. doi:10.1158/1538-7445.AM2015-4461