Abstract P5-05-09: AMP≥2 Is a Putative Tumor Suppressor in Breast Cancer

Abstract

Abstract Background: Adenosine monophosphate-activated protein kinase (AMPK) is a major metabolic regulator responsible for maintaining cellular energy homeostasis. AMPK becomes activated under cellular stress conditions and phosphorylates target proteins to conserve energy consumption and restore available energy. Developing tumors need to overcome such metabolic restrictions due to their high metabolic rates and rapid cell division. Consequently, AMPK activity may be a critical interface between metabolism and cancer biology. The kinase activity for AMPK is mediated through two alpha catalytic isoforms, AMPK≥1 and AMPK≥2. Our previous studies have indicated that AMPK≥2 is the predominant isoform in normal breast tissue epithelium. However, we observed that AMPK≥2 appears to be selectively suppressed via an epigenetic mechanism in breast cancer cell lines and in primary breast tumors. Further, AMPK≥2 Knockout MEFs display increased tumorigenic potential when transformed by oncogenic v-Ras. These data support the hypothesis that breast cancer cells evade metabolic growth control through the loss of AMPK≥2 expression. Methods: To evaluate whether the AMPK≥2 isoform serves as a tumor suppressor, AMPK≥2-V5, AMPK≥1-V5, and GFP were stably overexpressed in the MCF-7 breast cancer cell line. We chose the MCF-7 cell line because it retains very low endogenous levels of AMPK≥2. Changes in cell growth were evaluated in vitro by direct proliferation and soft agar colony formation assays. AMPK activated signaling events were probed by culturing cells for 20hr in normoxic (21% O2) or hypoxic (1.5% O2) conditions and cell lysates prepared. The tumorigenicity of the established cell lines was examined in vivo by xenograft tumor formation in nude mice supplemented with timerelease estradiol pellets. Tumors were assessed by immunohistological, protein and RNA analysis. We also assessed the therapeutic advantage of re-expressing AMPK≥2 in conjunction with Metformin, a chemical AMPK activator that shows potential as an anti-cancer agent. Growth sensitivity of the established MCF-7 overexpression cell lines was evaluated by culturing cells as monolayers or in soft agar in the presence of 0.0mM, 0.5mM, 2.0mM, or 5.0mM Metformin. Results: In vitro stress assays show that reintroduction of AMPK≥2 inhibits cell proliferation and abrogates cyclin D1 and p38 signaling, thus supporting its role as a tumor suppressor. Contrary to this, AMPK≥2 overexpression might also bestow an initial survival response due to an observed increase in soft agar colony formation and VEGF expression. We see in vivo, however, that AMPK≥2 overexpression significantly reduces tumor xenograft size in nude mice, which is a consequence of reduced proliferation and not increased apoptosis. Interestingly, AMPK≥2 overexpression confers a significant increase in Metformin sensitivity at a low dose (0.5mM) and effectively inhibits cell proliferation and colony formation. Conclusions: These results suggest that AMPK≥2 functions as a tumor suppressor in human breast cancer and combination drug therapy targeting AMPK≥2 re-expression and activation could prove beneficial in clinical practice. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-09.