Targeting autophagy in cancer

Abstract

Autophagy is a mechanism by which cellular material is delivered to lysosomes for degradation, leading to the basal turnover of cell components and providing energy and macromolecular precursors. Autophagy has opposing, context-dependent roles in cancer, and interventions to both stimulate and inhibit autophagy have been proposed as cancer therapies. This has led to the therapeutic targeting of autophagy in cancer to be sometimes viewed as controversial. In this Review, we suggest a way forwards for the effective targeting of autophagy by understanding the context-dependent roles of autophagy and by capitalizing on modern approaches to clinical trial design.

Figure 1. Timeline of the Major Discoveries Leading to the Successful Targeting of Autophagy in Cancer

De Duve first coined the term ‘autophagy’ during a lysosomal conference in 1963. Since then key discoveries have been made elucidating the mechanisms of the process from yeast to cultured cell lines, into mice, and finally culminating in successful clinical trials and case studies in patient tumors. The timeline concludes with the Nobel Prize awarded to Yoshinori Ohsumi for Physiology or Medicine in 2016, emphasizing the impact of his work as well as that of many others along the way. CQ, chloroquine; HCQ, hydroxychloroquine 1963 - Christian de Duve coins the term “autophagy” 1970 - Bedoya shows that the anti-malarial drug, CQ can inhibit tumour cell growth in vitro 1997 - The Ohsumi group clone the first autophagy specific gene, ATG1, from Saccharomyces cerevisiae^, 1998 - Mizushima and colleagues identify the first autophagy specific gene in higher eukaryotes 1998 - Murakami and colleagues observe that CQ can inhibit autophagy 1999 - The Levine group suggest that Beclin 1 is a tumor suppressor gene 2003 - Briceno et al. show that CQ has anti-tumour affects in patients with glioblastoma 2004 - The Mizushima group create the first autophagy deficient mouse (Atg5^−/−), implicating autophagy in development 2007 - Amaravadi and colleagues show that inhibition of autophagy has a combinatory effect with other anti-cancer drugs 2013 - A number of groups show that genetic knock out of autophagy-related genes in tumour cells decreases tumour growth in vivo, e.g. ^,, 2014 - The White group show that systemic genetic inhibition of autophagy in tumour-bearing mice can create a therapeutic window to treat lung cancer 2014 - Multiple groups publish data from Phase I/II clinical trials using HCQ to selectively target autophagy in cancer patients^– 2016 - Yoshinori Ohsumi is awarded the Nobel Prize for Physiology or Medicine for his work discovering the mechanisms of autophagy 2017 - Autophagy inhibition can overcome resistance to kinase inhibitors in tumour cells and in patients

Figure 2. Autophagy can be inhibited at multiple stages

The process of autophagy is divided into five distinct stages: initiation, vesicle nucleation, vesicle elongation, vesicle fusion and cargo degradation. Nonspecific macroautophagy is initiated by upstream activation through either nutrient starvation or growth factors. Under starvation conditions, a drop in glucose transport results in a release of mTOR inhibition of the ULK1 complex, allowing for the progression of autophagy. The ULK1 complex (comprising ULK1, ULK2, FIP200, ATG101 and ATG13) induces vesicle nucleation which is then mediated by a class III PI3K complex consisting of multiple proteins. Beclin 1, a BCL-2 homology (BH)-3 domain only protein, is phosphorylated by ULK1 and acts as an overall scaffold for the PI3K complex, facilitating localization of autophagic proteins to the phagophore. BCL-2 and BCL-XL interact with Beclin 1 at the BH3 domain to decrease the pro-autophagic activity of Beclin 1 by interrupting the Beclin 1–VPS34 complex formation and decreasing the interaction of Beclin 1 with UVRAG. Additional negative regulation of this process occurs with the phosphorylation of VPS34 (also known as PIK3C3), which decreases its interaction with Beclin 1. In contrast, AMBRA binds Beclin 1 and stabilizes the PI3K complex. ATG14 and UVRAG also bind Beclin 1 to promote interactions between Beclin 1 and VPS34 and phagophore formation. VPS15 is required for optimal VPS34 function by enhancing VPS34 interaction with Beclin 1. The growing double membrane undergoes vesicle elongation to eventually form an autophagasome: a process mediated by two ubiquitin-like conjugation systems. The first system involves the conjugation of phosphatidylethanolamine (PE) to cytoplasmic LC3-I to generate the lipidated form, LC3-II which is facilitated by the protease, ATG4B, and the E1-like enzyme, ATG7, whereby LC3-II is incorporated into the growing membrane. The second conjugation system is mediated again by ATG7 as well as the E2-like enzyme, ATG10, resulting in an ATG5-ATG12 conjugate. Subsequently, the SNARE protein, syntaxin 17 (STX17) facilitates autophagosome fusion with the lysosome, resulting in an autophagolysosome. The low pH of the lysosome results in degradation of the autophagosome contents. This process can be targeted pharmacologically upstream by means of direct ULK1, VPS34, or Beclin 1 inhibition. It can also be targeted by wortmannin and 3-methyladenine (3-MA) which act as PI3K inhibitors. Downstream targets include direct ATG4B inhibitors as well as chloroquine or hydroxychloroquine and bafilomycin, which act to prevent autophagosome fusion with the lysosome. PE, phosphatidylethanolamine.

Figure 3. Molecular Mechanism of Autophagy Dependence

Pre-clinical and clinical models have indicated that the tumor microenvironment, for example pancreatic stellate cells in the case of pancreatic cancer, p53 status, RAS family status, activation of JAK–STAT and PI3K signaling may all play roles in the determination of autophagy dependence within cancer cells, both in vitro and in patients. These pathways have all been shown to affect autophagy either positively or negatively and many participate in cross-pathway signaling. Signaling through p53 can both promote and inhibit autophagy and may interact with other proteins activated by mutations to enhance autophagy dependence, especially in pancreatic cancer. Activation of EGFR via amplification or mutation leads to the downstream up-regulation of the PI3K-AKT-mTOR pathway as well as activation of STAT3 and the RAS pathway. Although autophagy inhibition can occur through mTOR activation, these downstream effects collectively result in stimulation of autophagy and an increase in autophagy dependence. Mutations or alterations in the RAS family (specifically KRAS) have been shown to promote autophagy, enhancing tumor growth and therapy resistance. Specific mutations in RAF such as BRAF^V600E promote autophagy dependence in multiple tumors including central nervous system (CNS) tumors and melanoma. Finally, autophagy regulation of JAK-STAT signaling through IL-6 has been identified as a mechanism of autophagy dependence in breast cancer. All of these pathways are complex and interact on multiple levels. Identification of tumors with these pathways and as of yet to be identified pathways will provide methods of detection of autophagy dependent tumors.