Abstract P4-08-01: AMPK facilitates breast cancer cell survival by modulating microenvironmental stress
Authors: Sullivan KL , Kopsiaftis S , Phoenix KN , Fox MM , Tsurutani N , Vella AT , Claffey KP 
Published: May 2016

Abstract
Recurrent and metastatic breast cancers are responsible for the majority of breast-cancer related deaths. These cancer cells are able to adapt to stressors within the tumor microenvironment including hypoxia, low nutrient levels, and chemotherapy-induced toxicities. Breast cancer cells can respond to these microenvironmental stressors through a variety of mechanisms, including cell cycle inhibition and metabolic alteration. Tumor cell survival is dependent on the ability to alter these mechanisms in response to stress. AMPK (AMP-activated protein kinase) is the main metabolic sensor of the cell, and both its expression and activity have been reported to be altered in breast cancer. Moreover, there are two isoforms of the catalytic subunit (α1 and α2), and differential functionality of these isoforms has been suggested. Using estrogen receptor-positive human breast cancer cell lines, we investigated the effect of differential AMPKα isoform expression on breast cancer cell survival. We found that over-expression of AMPKα2 in MCF-7 cells resulted in decreased ATP production in response to low glucose levels, while the knockdown of AMPKα2 in HCC1500 cells ablated this response to low glucose conditions. A similar difference in response was also seen when the cells were treated with a combination of nutrient stress and the estrogen receptor alpha (ERα) inhibitor, ICI182780. In response to this finding, we compared the glycolytic and oxygen consumption rates of our MCF-7 GFP and MCF-7 AMPKα2 cells. We found that in response to low glucose stress, AMPKα2 expressing MCF-7 cells maintained both a higher glycolytic rate and a higher oxygen consumption rate as compared to GFP cells. Furthermore, these cells seem to alter their cellular signaling in response to metabolic stress faster than GFP cells. To evaluate this differential response to microenvironmental stress in vivo, MCF-7 cells expressing either GFP or AMPKα2 were injected into athymic nude mice previously implanted with slow-release estradiol pellets. After one week, the estradiol pellets were removed to induce cellular dormancy for thirty days. Analysis of tumors at this time indicated that more of the AMPKα2 expressing cells survived estradiol deprivation than did the control cells. Analysis of proliferation by Ki67 staining indicated that the GFP cells maintained proliferation during deprivation, while AMPKα2 cells were largely negative for proliferation. ApoTag staining revealed a similar trend for apoptotic cells. This suggests that an inability to control cell cycle resulted in a decreased survival of the GFP cells under estradiol deprivation. Following the deprivation period, estradiol pellets were re-implanted and residual dormant tumors resumed growth. AMPKα2 tumors grew to roughly double the size of GFP tumors. Interestingly, AMPKα2 tumors had a higher number of mitotic events than did GFP tumors as visualized by Ki67 staining. This could be due to more viable cells being present following estradiol deprivation. We conclude that the expression of AMPKα2 promotes long-term breast cancer survival in estrogen-sensitive cells, due to their increased ability to sense and respond to changes in their microenvironment, which therefore increases their chances for survival.
Citation Format: Sullivan KL, Kopsiaftis S, Phoenix KN, Fox MM, Tsurutani N, Vella AT, Claffey KP. AMPK facilitates breast cancer cell survival by modulating microenvironmental stress. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-01.