The Role of Adenosine Monophosphate-Activated Kinase α2 as a Putative Tumor Suppressor in Breast Cancer.

Background: The evaluation of adenosine monophosphate-activated kinase (AMPK) in tumor cell survival has revealed a critical interface between metabolism and cancer biology. As a major metabolic regulator, AMPK protects the cell from damage when nutrients are restricted. Therapeutic activation of AMPK using metformin has been shown to reduce the growth of most breast cancer cells but also promote the growth and metastasis of ERα negative tumor models. These contradictory findings indicate multiple roles for AMPK in breast cancer initiation and progression. Materials and Methods: AMPKα1 and AMPKα2 protein expression was quantified by immunohistochemistry in forty patient-matched primary human breast cancer and non-tumor margin samples; in addition ten normal breast reduction samples were evaluated. AMPKα2 was overexpressed in the ERα negative MDA-MB-231 breast cancer cell line and stable knockdown of AMPKα2 was performed with shRNA in the ERα positive T47D breast cancer cell line. Proliferation, viability, and soft agar colony growth assays were performed on all cell lines. Mouse embryonic fibroblasts (MEFs) from AMPKα2 knockout or wild-type C57/B6 mice were transformed with retrovirus containing oncogenic v-Ras and analyzed for changes in proliferation, soft agar growth, and tumorigenesis in athymic nude mice. The methylation status of the human AMPKα2 promoter region was investigated using quantitative methylation-specific PCR (qMSP) in MCF-7 and MDA-MB-231 breast cancer cell lines and primary breast tumors. Cell lines were treated with hypomethylation agents and evaluated for induced AMPKα2 expression by immunoblot. Results: AMPKα2 is the predominantly expressed alpha isoform in normal breast ductal epithelial tissue. In primary breast cancer, however, AMPKα2 was significantly repressed while AMPKα1 remained unchanged. Transient overexpression of AMPKα2 in breast cancer cell lines induced apoptosis within 48h. Silencing AMPKα2 revealed little change in proliferation rate in monolayer culture but exhibited increased soft agar colony growth. The tumorigenic capacity of v-Ras transformed AMPKa2 null MEFs was demonstrated by increased proliferation rates in vitro , soft agar growth, and tumor growth in nude mice. The promoter region of primary human breast cancer and breast cancer cell lines with low endogenous AMPKα2 levels were found to be methylated using qMSP. The same cell lines were able to re-establish AMPKα2 expression following treatment with the DNMT1 inhibitor, 5-aza-2-deoxycytidine. Discussion: AMPKα2 suppression in primary breast cancer indicates that its loss may provide a growth advantage for cancer cells. Overexpression of AMPKα2 in breast cancer cell lines induces apoptosis while the knockdown and knockout of the protein promotes tumorigenesis in vitro and in vivo . The loss of AMPKα2 function likely permits cell cycle progression under metabolic stress. The lack of proliferation control by AMPKa2 is hypothesized to increase the likelihood of acquiring genomic damage and mutational events. These results suggest that AMPKα2 may function as a tumor suppressor in human breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3153.