14-3-3ζ binds to and stabilizes phospho-beclin 1S295 and induces autophagy in hepatocellular carcinoma cells

J Cell Mol Med. 2020 Jan;24(1):954-964. doi: 10.1111/jcmm.14806. Epub 2019 Nov 11.

Abstract

Data from The Cancer Genome Atlas (TCGA) indicate that the expression levels of 14-3-3ζ and beclin 1 (a key molecule involved in cellular autophagy) are up-regulated and positively correlated with each other (R = .5, P < .05) in HCC tissues. Chemoresistance developed in hepatoma cancer cells is associated with autophagy initiation. This study aimed to explore 14-3-3ζ's role in regulating autophagy in HCC cells, with a focus on beclin 1. The co-localization of 14-3-3ζ and beclin 1 was detectable in primary HCC tissues. To simulate in vivo tumour microenvironment (hypoxia), CSQT-2 and HCC-LM3 cells were exposed to 2% oxygen for 24 hours. The protein levels of 14-3-3ζ and phospho-beclin 1S295 peaked at 12 hours following hypoxia. Meanwhile, the strongest autophagy flux occurred: LC3II was increased, and p62 was decreased significantly. By sequencing the coding area of BECN 1 gene of CSQT-2 and HCC-LM3 cells, we found that the predicted translational products of BECN 1 gene contained RLPS295 VP (R, arginine; L, leucine; P, proline; S, serine; V, valine), a classic 14-3-3ζ binding motif. CO-IP results confirmed that 14-3-3ζ bound to beclin 1, and this connection was markedly weakened when S295 was mutated into A295 (alanine). Further, 14-3-3ζ overexpression prevented phospho-beclin 1S295 from degradation and enhanced its binding to VPS34, whilst its knockdown accelerated the degradation. Additionally, 14-3-3ζ enhanced the chemoresistance of HCC cells to cis-diammined dichloridoplatium by activating autophagy. Our work reveals that 14-3-3ζ binds to and stabilizes phospho-beclin 1S295 and induces autophagy in HCC cells to resist chemotherapy.

Keywords: 14-3-3ζ; autophagy; beclin 1; chemotherapy-resistant; hepatocellular carcinoma; portal vein tumour thrombosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / metabolism*
  • Autophagy*
  • Beclin-1 / chemistry*
  • Beclin-1 / metabolism*
  • Carcinoma, Hepatocellular / metabolism
  • Carcinoma, Hepatocellular / pathology*
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Liver Neoplasms / metabolism
  • Liver Neoplasms / pathology*
  • Phosphorylation
  • Serine / chemistry
  • Serine / metabolism*
  • Tumor Cells, Cultured

Substances

  • 14-3-3 Proteins
  • Beclin-1
  • Serine