GDF11 inhibits the malignant progression of hepatocellular carcinoma via regulation of the mTORC1‑autophagy axis

Abstract

Hepatocellular carcinoma (HCC) is a common malignant tumor, which is associated with a poor prognosis and high mortality rate. It is well known that growth differentiation factor 11 (GDF11) acts as a tumor suppressor in various types of cancer, including HCC. The present study aimed to determine the tumor-suppressive properties of GDF11 in HCC and to assess the intrinsic mechanisms. In the present study, the human hepatoma cell line Huh-7 was transfected with the GDF11 overexpression plasmid (Oe-GDF11) for gain-of-function experiments to investigate the effects of GDF11 on the biological behaviors of HCC cells, including proliferation, colony formation, apoptosis, cell cycle arrest, migration, invasion, epithelial-mesenchymal transition (EMT) and angiogenesis. The proliferation, colony formation, apoptosis, cell cycle, migration, invasion and angiogenesis of HCC cells were assessed by CCK-8, EdU staining, colony formation, flow cytometry, wound healing, Transwell and tube formation assays, respectively. Apoptosis-, cell cycle-, EMT-related key factors were also determined by western blot assay. Furthermore, Oe-GDF11-transfected Huh-7 cells were treated with the mammalian target of rapamycin (mTOR) activator MHY1485 for rescue experiments to explore whether GDF11 could exert antitumor effects against HCC via mediating the mTOR complex 1 (mTORC1)-autophagy axis. In the present study, GDF11 was verified to be lowly expressed in HCC cells. Overexpression of GDF11 inhibited the proliferation, colony formation, migration, invasion, EMT and angiogenesis of HCC cells, and facilitated the apoptosis and cell cycle arrest of HCC cells. Additionally, it was verified that overexpression of GDF11 inactivated the mTORC1 signaling pathway to enhance autophagy in HCC cells. Treatment with the mTOR activator MHY1485 partially reversed the tumor-suppressive effects of GDF11 overexpression on HCC. In conclusion, GDF11 may exert tumor-suppressive properties in HCC cells through inactivating the mTORC1 signaling pathway to strengthen autophagy.

Keywords: growth differentiation factor 11; hepatocellular carcinoma; mammalian target of rapamycin complex 1-autophagy axis.

Figure 1

GDF11 suppresses the mTORC1 signaling pathway and strengthens autophagy in hepatocellular carcinoma cells. (A) Differences in GDF11 expression in HHL-5 human normal hepatocytes and the human hepatoma cell lines SNU-449, Hep3B and Huh-7 were detected by RT-qPCR and western blotting. (B) Huh-7 cells were transfected with either Oe-GDF11 or Oe-NC, and transfection efficiency was validated by RT-qPCR and western blotting. (C) Huh-7 cells were transfected with either Oe-GDF11 or Oe-NC. LC3 expression was determined by immunofluorescence staining. (D) Huh-7 cells were transfected with either Oe-GDF11 or Oe-NC. p-mTOR, mTOR, p-p70 S6K T389, p70 S6K, p-S6, S6, LC3-I, LC3-II, Beclin-1 and p62 protein expression levels were detected by western blotting. ^**P<0.01, ^***P<0.001. GDF11, growth differentiation factor 11; mTORC1, mammalian target of rapamycin complex 1; RT-qPCR, reverse transcription-quantitative PCR; Oe, overexpression plasmid; NC, negative control; p, phosphorylated.

Figure 2

GDF11 strengthens autophagy in hepatocellular carcinoma cells by suppressing the mTORC1 signaling pathway. Huh-7 cells were transfected with either Oe-GDF11 or Oe-NC. Oe-GDF11-transfected Huh-7 cells were treated with the mTOR activator MHY1485. (A) LC3 expression was determined by immunofluorescence staining. (B) p-mTOR, mTOR, p-p70 S6K T389, p70 S6K, p-S6, S6, LC3-I, LC3-II, Beclin-1 and p62 protein expression levels were detected by western blotting. ^**P<0.01, ^***P<0.001. GDF11, growth differentiation factor 11; mTORC1, mammalian target of rapamycin complex 1; Oe, overexpression plasmid; NC, negative control; p, phosphorylated.

Figure 3

GDF11 inhibits the proliferation and colony-forming ability of hepatocellular carcinoma cells by suppressing the mTORC1 signaling pathway. Huh-7 cells were transfected with either Oe-GDF11 or Oe-NC. Oe-GDF11-transfected Huh-7 cells were treated with the mTOR activator MHY1485. (A) Cell viability was investigated using a Cell Counting Kit-8 assay. (B) Cell proliferative ability was investigated using EdU staining. (C) Cell colony-forming ability was investigated using a colony formation assay. ^*P<0.05, ^***P<0.001, vs. Oe-NC group. GDF11, growth differentiation factor 11; mTORC1, mammalian target of rapamycin complex 1; Oe, overexpression plasmid; NC, negative control.

Figure 4

GDF11 facilitates hepatocellular carcinoma cell apoptosis by suppressing the mTORC1 signaling pathway. Huh-7 cells were transfected with either Oe-GDF11 or Oe-NC. Oe-GDF11-transfected Huh-7 cells were treated with the mTOR activator MHY1485. (A) Cell apoptosis was investigated using flow cytometry. (B) Bcl-2, Bax, cleaved caspase-3 and caspase-3 protein expression levels were detected by western blotting. ^***P<0.001. GDF11, growth differentiation factor 11; mTORC1, mammalian target of rapamycin complex 1; Oe, overexpression plasmid; NC, negative control.

Figure 5

GDF11 induces hepatocellular carcinoma cell cycle arrest by suppressing the mTORC1 signaling pathway. Huh-7 cells were transfected with either Oe-GDF11 or Oe-NC. Oe-GDF11-transfected Huh-7 cells were treated with the mTOR activator MHY1485. (A) Cell cycle distributions were investigated using flow cytometry. (B) CDK4, CDK6 and cyclin D1 protein expression levels were detected by western blotting. ^*P<0.05, ^**P<0.01, ^***P<0.001. GDF11, growth differentiation factor 11; mTORC1, mammalian target of rapamycin complex 1; Oe, overexpression plasmid; NC, negative control.

Figure 6

GDF11 inhibits hepatocellular carcinoma migration, invasion, EMT and angiogenesis by suppressing the mTORC1 signaling pathway. Huh-7 cells were transfected with either Oe-GDF11 or Oe-NC. Oe-GDF11-transfected Huh-7 cells were treated with the mTOR activator MHY1485. (A) Cell migration was investigated using a wound healing assay. (B) Cell invasion was investigated using a Transwell invasion assay. (C) E-cadherin, N-cadherin, Snail and Vimentin protein expression levels were detected by western blotting. (D) HUVECs were incubated with the conditioned media of Huh-7 cells at 37˚C for 24 h. In vitro angiogenesis of HUVECs was investigated using a tube formation assay. ^*P<0.05, ^**P<0.01, ^***P<0.001. GDF11, growth differentiation factor 11; mTORC1, mammalian target of rapamycin complex 1; Oe, overexpression plasmid; NC, negative control; HUVECs, human umbilical vein endothelial cells.