Development of Beclin 1-targeting stapled peptides as novel anti-proliferation strategy for Hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, accounting for around 90% of cases. HCC is a growing health problem globally and is now the third leading cause of cancer-related deaths. Unfortunately, the majority of HCC cases are detected at later stages when therapeutic options are limited. Due to liver dysfunction, many chemotherapy treatments cannot be used for HCC patients. Drugs like sorafenib or Lenvatinib, which are used as first-line treatments, only offer limited survival benefits of 2-3 months. The effectiveness of sorafenib is further reduced by the development of primary and acquired resistance after a period of treatment. Although a combination of monoclonal antibody drugs has shown little improvement in survival compared to targeted drugs, HCC patients exhibit a low response rate of around 20-30%. The overall 5-year survival rate for HCC patients is only about 18%. There is a pressing need for effective HCC therapies. Autophagy is an essential process of cellular metabolism that involves the degradation and recycling of intracellular macromolecular components in eukaryotic cells. It is a conserved process that is important for maintaining cellular homeostasis, eliminating damaged organelles, and protecting cell survival under stress. The liver is a major metabolic organ that is closely linked to autophagy. Autophagy also plays multiple roles in HCC tumorigenesis, promotion and drug resistance and autophagy-related proteins regulate various processes of HCC as suppressors or promoters can serve as prognostic biomarkers and therapeutic value. Beclin1 is an essential autophagy protein and is part of the Class III PI3K complex. It interacts with Atg14L and UVRAG in a mutually exclusive manner and forms Atg14L/UVRAG-containing Beclin1-Vps34 subcomplexes to promote autophagy and endolysosomal trafficking. In our previous work, the structure of the Beclin1-UVRAG coiled-coil complex was revealed to be strengthened by hydrophobic pairings and electrostatically complementary interactions. Using structure-based rational design, we developed hydrocarbon stapled peptides that bind specifically to Beclin1 to enhance the interaction between Beclin1 and UVRAG, further promoting Vps34-dependent autophagy and endolysosomal trafficking. Tat-SP4 is one of the leading Beclin1-targeting stapled peptides developed in our lab. In our project, Tat-SP4 was found to enhanced autophagy in several HCC cell lines. Interestingly, Tat-SP4 shows no further effect on EGFR endolysosomal degradation, while it was able to induce significant and rapid c-MET turnover. Additionally, Tat-SP4 had an anti-proliferative effect in several HCC cell lines and was able to override the adaptive sorafenib resistance effect in HCC cells. Mechanistic studies showed that Tat-SP4 induced cell death could be blocked by an inhibitor of autosis called digoxin. Tat-SP4 was also found to induce mitochondrial dysfunction. Tat-SP4 can disrupt intracellular calcium homeostasis, including the release of calcium ions from the endoplasmic reticulum (ER) and an increase in the levels of cytosolic and mitochondrial calcium ions. The presence of extracellular calcium ions protects against Tat-SP4-induced cell death and mitochondrial dysfunction by stabilizing intracellular ion homeostasis. In HCC patient-derived tumor xenograft (PDTX) mouse models, Tat-SP4 significantly reduced tumor volume at a dose of 40 mg/kg/day for approximately 30 days with no observable toxicity. As there is currently no effective targeted therapy for HCC, our results provide a promising new strategy for inhibiting HCC proliferation.