Upregulation of ATG4D promotes anoikis resistance of tumor cells by autophagy enhancement

Anoikis resistance (AR) is a critical characteristic of metastatic tumor cells, and enhanced autophagy is an important contributor to AR. Autophagy-related 4D (ATG4D) is a key enzyme regulating autophagy. This study aims to investigate whether tumor cells promote AR by upregulating ATG4D expression to enhance autophagy and elucidate the underlying molecular mechanisms.

Hepatocellular carcinoma HepG2 cells and gastric cancer AGS cells were divided into an AR group and a control group. The AR group was cultured in ultra-low adhesion plates for 48 h, while the control group was cultured in conventional plates. Changes in the expression of autophagy markers LC3-Ⅱ and p62 were determined by Western blotting. Chloroquine (CQ, 30 μg/mL) was used to inhibit autophagy in control or AR tumor cells, followed by flow cytometry for apoptosis levels with an apoptosis detection kit. The expression of ATG4D at mRNA and protein levels were assessed by qRT-PCR and Western blotting, respectively. AR tumor cells were transfected with control siRNA or ATG4D siRNA using Lipofectamine. Subsequently, the protein expression levels of ATG4D, LC3-Ⅱ and p62 were detected by Western blotting, and apoptotic rates were analyzed with flow cytometry. A reporter plasmid containing the ATG4D promoter was constructed and transfected into control and AR tumor cells, respectively; after 24 h, luciferase reporter gene assay was employed to detect the changes in the activity of ATG4D promoter. To assess cell stability, the control or AR tumor cells were treated with actinomycin D (5 μg/mL) or cycloheximide (50 μg/mL), and then harvested at 0, 2, 4, 6, and 8 h after treatment. The mRNA and protein levels of ATG4D were determined by qRT-PCR and Western blotting, respectively.

Compared with the control tumor cells, the AR tumor cells showed significantly enhanced autophagy, with significantly elevated LC3-Ⅱ levels (P<0.01) and markedly reduced p62 levels (P<0.01). Inhibition of autophagy using CQ significantly increased apoptosis in AR tumor cells (P<0.01). qRT-PCR and Western blotting revealed significantly elevated ATG4D mRNA and protein levels in AR tumor cells compared with the control cells (P<0.01). ATG4D siRNA transfection significantly inhibited ATG4D expression, downregulated LC3-Ⅱ, upregulated p62, and promoted apoptosis in AR tumor cells (P<0.01). ATG4D promoter activity did not differ significantly between AR and control tumor cells. However, after actinomycin D treatment, ATG4D mRNA expression was significantly higher in AR tumor cells at all time points (P<0.001), indicating enhanced mRNA stability. In contrast, cycloheximide treatment showed no significant change in ATG4D protein levels over time in AR tumor cells, suggesting unaltered protein stability.

Enhanced autophagy caused by ATG4D upregulation promotes AR in tumor cells.