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

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 1^S295 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 1^S295 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 1^S295 and induces Autophagy in HCC cells to resist chemotherapy.