5P - AMPKalpha1 restrains fibroblasts transformation and tumorigenesis in vivo

Abstract

Adenosine monophosphate-activated protein kinase (AMPK) is a critical metabolic sensor and redox modulator. Despite evidence linking AMPK tumor suppressor functions, the role of AMPK in chromosome instability and tumorigenesis is obscure. Murine embryo fibroblasts (MEF) were isolated from AMPK alpha1 knock out (AMPK&agr;1-KO), AMPK&agr;2-KO, and wild type (WT) C57BL/6J mouse embryos, and immortalized by employing standard 3T3 protocol to investigate the mechanisms of chromosomal instability and fibroblast-mediated angiogenesis. Chromatin proteins and aneuploidy were monitored in cultured MEFs by Western blot and metaphase spread. Athymic nude mice were employed to test tumorigenesis of fibroblasts in vivo. Deletion of AMPK&agr;1, but not AMPK&agr;2, exhibited a significant reduction in centromere-specific binding proteins C (CENP-C) and elevation of Polo-like kinase 4 (PLK4), a trigger of centriole biogenesis. Both CENP-C and PLK4 may be associated with the increased aneuploid (34%-66%) and micronucleus. Allograft model data demonstrated that knock out of AMPK&agr;1 enhanced the cellular proliferation of immortalized MEFs, vascularization, and tumor development in athymic nude mice in vivo. Mechanistically, it was shown that the protein level of noncanonical nuclear factor kappa B2 (NF-&kgr;B2)/p52 was elevated in AMPK&agr;1-KO MEFs and was responsible for induction of erythropoietin (Epo) expression. Lastly, the most conclusive evidence for AMPK&agr;1-dependent inhibition of fibroblast-mediated angiogenesis as well as tumor progression was that the allograft growth of the inoculated AMPK&agr;1-KO MEFs was attenuated by treatment with Epo neutralization antibody. Taken together, these data indicate that AMPK&agr;1 activation opposes tumor development and its loss fosters tumor progression in part by controlling chromosome stability and angiogenesis that support tumor growth.