Principles and current strategies for targeting autophagy for cancer treatment

Abstract

Autophagy is an evolutionarily conserved, intracellular self-defense mechanism in which organelles and proteins are sequestered into autophagic vesicles that are subsequently degraded through fusion with lysosomes. Cells, thereby, prevent the toxic accumulation of damaged or unnecessary components, but also recycle these components to sustain metabolic homoeostasis. Heightened autophagy is a mechanism of resistance for cancer cells faced with metabolic and therapeutic stress, revealing opportunities for exploitation as a therapeutic target in cancer. We summarize recent developments in the field of autophagy and cancer and build upon the results presented at the Cancer Therapy Evaluation Program (CTEP) Early Drug Development meeting in March 2010. Herein, we describe our current understanding of the core components of the autophagy machinery and the functional relevance of autophagy within the tumor microenvironment, and we outline how this knowledge has informed preclinical investigations combining the autophagy inhibitor hydroxychloroquine (HCQ) with chemotherapy, targeted therapy, and immunotherapy. Finally, we describe ongoing clinical trials involving HCQ as a first generation autophagy inhibitor, as well as strategies for the development of novel, more potent, and specific inhibitors of autophagy.

Figure 1. Anatomy of autophagy

Autophagy occurs through a multi-step process including 4 control points: 1) initiation, 2) nucleation, 3) maturation, and 4) delivery and degradation of AV contents. These steps occur irrespective of whether autophagy has been induced through stress/ubiquitinated substrate accumulation, or through starvation. During initiation, nascent AV membranes derived from multiple potential sources (including isolated membranes, ER or mitochondria outer membranes) form a cup-like structure onto which autophagosomal machinery, including LC3, dynamically associates. As the cup-like structure enlarges, it sequesters substrate, which includes ubiquitinated proteins or organelles in the case of stress/substrate induced autophagy, and soluble cytoplasm in the case of starvation induced autophagy. The double membrane comprising the nascent AV then closes to form the mature autophagic vesicle, which then targets and fuses with the lysosome. In the lysosome, hydrolytic enzymes digest the contents and inner membrane of the AV, with autophagic machinery (i.e., LC3) recycled through the cytoplasm for recruitment to other nascent autophagosomes.

Figure 2. Role of autophagy in suppressing liver damage and cell death

(A) Elevated p62, ubiquitin and accumulation of lipids in aged beclin1+/− mouse liver. Sections of liver from young (16 months old) and aged (> 24 months old) beclin1 +/+ and +/− mice and a representative spontaneous liver tumor from a beclin1+/− mouse were stained with H&E, and by immunohistochemistry for p62 and ubiquitin, and with BODIPY to indicate lipid droplet accumulation by fluorescence. (B) Autophagy promotes cell viability in metabolic stress. Representative images of immortal baby mouse kidney epithelial (iBMK) cells derived from atg7+/+ and −/− mice that were untreated, treated with metabolic stress (ischemia: no glucose and 1% oxygen or hypoxic conditions) and allowed to recover (35). Images generously provided by Dr. C. Karp and H.-Y. Chen from the White laboratory.

Figure 3. Network interactions between kinase signaling, protein metabolism and autophagy

Metabolic and therapeutic stresses (green) converge on mTORC1 and mediators of ER stress to induce autophagy. Autophagy serves a cytoprotective role in response to these stresses, by clearing damaged organelles, aggregated proteins, and recycling macromolecules to sustain survival. Arrows:activation; flat lines: inhibition. PI3K: phosphotidyl inositol-3 kinase IRS1: insulin receptor substrate 1; mTORC2: mammalian target of rapamycin complex 2; TSC: tuberous sclerosis complex; AMPK: AMP-activated protein kinase; REDD1: regulated in development and DNA damage 1; mTORC1: mammalian target of rapamycin complex 1; PERK: protein kinase-like endoplasmic reticulum kinase; eIF2α: eukaryotic initiation factor 2α: ER: endoplasmic reticulum; IRE-1: inositol requiring enzyme-1; JNK: Jun N-terminal kinase; HCQ: hydroxychloroquine; PI3K/mTORi: dual PI3K/mTOR inhibitors

Figure 4. Cancer Immunity and autophagy

Imbalance of autophagy leads to immune tolerance in cancer patients. Heightened autophagy in tumor cells prevents immune effector cell mediated cytotoxicity. In addition, Stress-induced release of the damage-associated molecular pattern molecule HMGB1 induces cytoprotective autophagy and once extruded into the extracellular matrix recruits regulatory T cells (Treg) resulting in anergy. Suppressed autophagy in dendrtic cells limits effective priming of antigen presentation that trains cytotoxic T cells. Simultaneous or sequential pharmacological induction of autophagy in dendritic cells, and autophagy inhibition in the tumor cell would ideally reverse this imbalance and enhance antitumor immunity. FAS: FS7-associated cell surface antigen; FASL: Fas ligand; MHC: major histocompatability complex; RAGE: receptor for advanced glycation endproducts

Figure 5. Pharmacodynamic assay for autophagy inhibition

Electron micrographs of peripheral blood mononuclear cells from a glioma patient enrolled on the phase I trial of temozolomide, radiation and hydroxychloroquine; (A) Pretreatment and (B) 3 weeks of combined therapy. Arrows: autophagic vesicles, scale bar 200 μm.