Anti-Breast Cancer Activity of a Novel Strategy of Targeting Autophagy Induced by Pan-HDAC Inhibitor and Acetylated Hsp70.

Abstract During the in vivo growth of breast cancers, hypoxia, deprivation of nutrients, acidosis and increased reactive oxygen species collectively induce the metabolic and protein-misfolding/denaturing stress. This increases the levels of heat shock proteins (hsp) and induces autophagy to avoid 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 protein re-folding and prevent protein aggregation. Increase in hsp70 levels are also known to inhibit both intrinsic and extrinsic pathway of apoptosis. In our present studies, we have determined that the in vitro stress induced by nutrient withdrawal or treatment with pan-HDAC inhibitor (HDI) panobinostat (PS) (Novartis Pharmaceuticals) results in hyperacetylation of hsp70, which induces autophagy of 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 form 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, that mediates vesicle nucleation. We have determined that hyperacetylated hsp70 significantly enhances the stability and activity of VPS34, which leads to binding of VPS34 to Beclin1 (ATG6). Hyperacetylated hsp70 also recruits the PHD domain containing E3 ligase for sumoylation, KAP1, to induce 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 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 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, the in vivo role of hyperacetylated hsp70 in autophagy induced during growth of the orthotopically implanted MB-231 xenografts in the mammary fat pads of NOD/SCID mice was also established. In cohorts of mice, following growth to a size of 100, 200, 500 and 1000 cu mm, tumor cell-lysates demonstrated tumor size-dependent increase in the intracellular levels of hyperacetylated hsp70, Beclin1 and LC3 II. Collectively, these novel findings suggest that, stress-induced 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 co-treatment with a pan-HDAC inhibitor and an inhibitor of autophagy to eliminate breast cancer cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3124.