On ATG4B as Drug Target for Treatment of Solid Tumours-The Knowns and the Unknowns

Abstract

Autophagy is an evolutionary conserved stress survival pathway that has been shown to play an important role in the initiation, progression, and metastasis of multiple cancers; however, little progress has been made to date in translation of basic research to clinical application. This is partially due to an incomplete understanding of the role of autophagy in the different stages of cancer, and also to an incomplete assessment of potential drug targets in the autophagy pathway. While drug discovery efforts are on-going to target enzymes involved in the initiation phase of the autophagosome, e.g., unc51-like autophagy activating kinase (ULK)1/2, vacuolar protein sorting 34 (Vps34), and autophagy-related (ATG)7, we propose that the cysteine protease ATG4B is a bona fide drug target for the development of anti-cancer treatments. In this review, we highlight some of the recent advances in our understanding of the role of ATG4B in autophagy and its relevance to cancer, and perform a critical evaluation of ATG4B as a druggable cancer target.

Keywords: ATG4; autophagy; biomarker; drug screening; pancreatic ductal adenocarcinoma (PDAC); screening assay; small molecule compound.

Figure 1

Key components in the autophagy pathway. Autophagy is initiated by the ULK protein kinase complex, leading to activation of the Beclin-1–VPS34 complex and generation of phosphatidyl-inositol-3-phosphate (PI3P). PI3P serves as a recognition domain in the phagophore membrane for recruitment and assembly of other protein complexes such as the WD-repeat protein interacting with phosphoinositides (WIPI) proteins. These early events lead to activation of a series of ubiquitin-like conjugation reactions, including ATG12-5 formation and LC3/GABARAP processing. Recruitment of cargo to the phagophore and closure of the autophagosome is followed by fusion with the lysosome and degradation of cargo in the autolysosome.

Figure 2

LC3/GABARAP protein processing. The LC3/GABARAP pro-peptide pro-LC3/GABARAP is first cleaved at the C-terminus by ATG4B, generating LC3/GABARAP-I in the cytoplasm. LC3/GABARAP-I is then conjugated to phosphatidylehtanolamine (PE) by ATG7/ATG3 and the ATG12-5–16L1 complex. LC3/GABARAP-PE can be deconjugated by ATG4 proteins for recycling. The different C-terminal ends of pro-LC3/GABARAP, LC3/GABARAP-I, and LC3/GABARAP-II are depicted in the bottom panels.

Figure 3

Biochemical and cell-based assays to monitor ATG4B activity. Biochemical assays such as the in vitro GST–LC3 processing assay (top left) that relies on poly-acrylamide gel electrophoresis are often used to monitor cleavage of a LC3–GST pseudo-substrate, but are not suitable for high-throughput screening applications. The LC3–FRET assay (top right) has been used in large-scale screening applications in vitro. The luciferase release assay (bottom left) that relies on ATG4B-dependent release of an N-termially truncated Gaussia luciferase (ΔNGLuc) enzyme and export into supernatants of cells is suitable for cell-based high-throughput screening. The tandem autophagy flux reporter (bottom right) that monitors ATG4B-dependent cleavage of a tandem GFP–LC3–RFP–LC3dC (C-terminal truncation) is suitable for cell-based high-content screening and in vivo. For more detail on each assay see text.