Abstract B21: Targeting autophagy induced by pan-HDAC inhibitor panobinostat and promoted by acetylated hsp70: A novel therapy for breast cancer

Abstract

Abstract Among the hallmarks of cancer is the stress phenotype, which is collectively induced by hypoxia, deprivation of nutrients, acidosis and increased reactive oxygen species, and exhibited as metabolic and proteinmisfolding/denaturing (proteotoxic) stress. This results in the constitutive activation of the heat shock response with elevated levels of heat shock proteins (hsp), and induction of autophagy, for avoiding cell death. Autophagy is a conserved catabolic pathway in which cytoplasmic macromolecules and organelles are sequestered in autophagosomes, which fuse with lysosomes for protein degradation and amino acid recycling. Elevated levels of hsp70 and hsp90 promote proper protein folding and inhibit both the intrinsic and extrinsic pathways of apoptosis. In our present studies, we have determined in vitro that the stress induced by nutrient withdrawal or treatment with pan-HDAC inhibitor (HDI) panobinostat (PS) (Novartis Pharmaceuticals) resulted in hyperacetylation of hsp70, which induced autophagy in the cultured breast cancer MB-231 and MCF-7 cells. The initial steps of autophagy include vesicle nucleation, vesicle expansion to form the phagophore, edges of which fuse to create the autophagosome. This process is triggered by a lipid kinase (PI3KC3) signaling complex, consisting of Vps34, Vps15, and autophagy-associated genes (ATG) 6 and ATG14, which mediate vesicle nucleation. We determined that hyperacetylated hsp70 significantly enhanced the stability and activity of VPS34, which led to binding of VPS34 to Beclin1 (ATG6). Hyperacetylated hsp70 also recruited the PHD domain containing E3 ligase for sumoylation, KAP1, and induced the sumoylation of VPS34 by SUMO-1. This was documented by confocal immunostaining with fluorescence-conjugated anti-VPS34 and anti-SUMO-1 antibodies, as well as by biochemical determination of the in vitro and in vivo sumoylation of VPS34. PS treatment also induced LC3II accumulation and formation of authophagosomes, the latter determined morphologically by electron microscopy. PS-induced, KAP-1 dependent sumoylation of VPS34 and downstream autophagosome formation were abrogated by siRNA to hsp70. Also, VPS34 expression was attenuated and the processing of LC3 was inhibited in the cells from the hsp70 knockout as compared to the control mice. Importantly, we also determined that, as compared to treatment with each agent alone, co-treatment with PS and the inhibitor of autophagy 3- methl-adenosine (3-MA) or chloroquine significantly enhanced cell death of breast cancer MB-231 and SUM159T cells. Furthermore, we determined the in vivo role of hyperacetylated hsp70 in autophagy. In cohorts of mice, following the growth of the orthotopically-implanted MB-231 xenografts in the mammary fat pads of NOD/SCID mice to a size of 100, 200, 500 and 1000 cu mm, tumor cell-lysates were obtained. Western blot analyses demonstrated size-dependent increase in the intracellular levels of hyperacetylated hsp70, Beclin1 and LC3II. Collectively, these findings suggest that stress phenotype associated heat shock response and increase in acetylated hsp70 promotes autophagy. Our findings also demonstrate that in established breast cancers, autophagy can be selectively targeted by cotreatment with the pan-HDAC inhibitor and an inhibitor of autophagy to eliminate breast cancer cells. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B21.