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ATG4B Targeting Peptide as a Potential Inhibitor for Gastrointestinal Cancer

Authors
Chih-Wen Shu Ph.D.1, 2, 3, Wei-Lun Tsai M.D.4, Wan-Hsiang Hu M.D., Ph.D.5, 6, Paul Morgan M.D., Ph.D.7, Shih-Yi Yang1, Pei-Yu Jhou1, Pei-Chi Li1, Cheng-Hsin Lee8, Pei-Feng Liu Ph.D.8, 9, 10

Affiliations

  1. Institute of BioPharmaceutical Sciences, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
  2. Center of Excellence for Metabolic Associated Fatty Liver Disease, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
  3. Innovation Center for Drug Development and Optimization, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
  4. Department of Internal Medicine, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan
  5. Department of Colorectal Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83341, Taiwan
  6. Graduate Institute of Clinical Medical Science, College of Medicine, Chang Gung University, Kaohsiung, Taiwan
  7. Icahn School of Medicine at Mount Sinai, NY, NY, 10029, USA 8Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
  8. Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan
  9. Center for Cancer Research, Kaohsiung Medical University, Kaohsiung 80708, Taiwan

Corresponding Author
Chih-Wen Shu (cwshu@mail.nsysu.edu.tw)

Introduction
Autophagin-1 (ATG4B) is a class of cytosolic cysteine proteases in autophagy machinery. Elevated expression of ATG4B is strongly associated with gastrointestinal cancer development and poor outcome. The α-helix secondary structure is important for protein-protein interactions, suggesting helical peptides could be valuable scaffolds to design inhibitors against ATG4B to suppress gastrointestinal cancer cells.

Methods
Three α-helical peptides (H1, H2 and H3) derived from human ATG8 homologue GABARAPL2 (GBRL2) were synthesized to examine the effects on ATG4B proteolytic activity. The second helical peptide, GBRL2H2, fusion with cell permeable peptide Tat (Tat-GBRL2H2) were used to inspect its effects on cancer cell viability and tumor formation.

Results
The GBRL2H2 peptide sufficiently reduced proteolytic activity of ATG4B. Tat-GBRL2H2 significantly diminished cellular ATG4B activity and autophagic flux in gastrointestinal cancer cells. Constituently, Tat-GBRL2H2 decreased cell viability and colony formation of cancer cells. Tat-GBRL2H2 peptide also largely increased dead cell population of cancer cells along with elevated reactive oxygen species (ROS) and caspase-3/7 activation. The peptide-induced cell death can be rescued by ROS scavengers and apoptosis inhibitor zVAD, implying Tat-GBRL2H2 might trigger ROS to induce cancer cell apoptosis. In addition, Tat-GBRL2H2 diminished the tumorsphere formation of gastrointestinal cancer cells in vitro and in vivo.

Conclusion
Our results provide proof of concept for the utility of the peptide as a potent ATG4B inhibitor with therapeutic potential for gastrointestinal cancer.