IQGAP1 promotes anoikis resistance and metastasis through Rac1-dependent ROS accumulation and activation of Src/FAK signalling in hepatocellular carcinoma

Abstract

Background: Hepatitis B virus (HBV) has a crucial role in the progression of hepatocellular carcinoma (HCC). Tumour cells must develop anoikis resistance in order to survive before metastasis. This study aimed to investigate the mechanism of IQGAP1 in HBV-mediated anoikis evasion and metastasis in HCC cells.

Methods: IQGAP1 expression was detected by immunohistochemistry, real-time PCR and immunoblot analysis. Lentiviral-mediated stable upregulation or knockdown of IGAQP1, immunoprecipitation, etc. were used in function and mechanism study.

Results: IQGAP1 was markedly upregulated in HBV-positive compared with HBV-negative HCC cells and tissues. IQGAP1 was positively correlated to poor prognosis of HBV-associated HCC patients. IQGAP1 overexpression significantly enhanced the anchorage-independent growth and metastasis, whereas IQGAP1-deficient HCC cells are more sensitive to anoikis. Mechanistically, we found that HBV-induced ROS enhanced the association of IQGAP1 and Rac1 that activated Rac1, leading to phosphorylation of Src/FAK pathway. Antioxidants efficiently inhibited IQGAP1-mediated anoikis resistance and metastasis.

Conclusions: Our study indicated an important mechanism by which upregulated IQGAP1 by HBV promoted anoikis resistance, migration and invasion of HCC cells through Rac1-dependent ROS accumulation and activation of Src/FAK signalling, suggesting IQGAP1 as a prognostic indicator and a novel therapeutic target in HCC patients with HBV infection.

Fig. 1. IQGAP1 is upregulated in HBV-associated HCC cells and tissues.

a, b The qRT-PCR and immunoblot analysis of IQGAP1 levels in HBV-positive HCC cells (HepG2.2.15 and HepAD38-Tet) and HBV-negative cells (HepG2 and HepAD38+Tet). c IQGAP1 levels in Huh7 cells transfected with HBV1.3 or vector plasmid were determined by qPCR and immunoblot analysis. d Representative images of IQGAP1 expression in clinical normal and HCC tissues obtained by immunohistochemical analysis. e Immunoblot analysis of IQGAP1 protein levels in adjacent normal, HBV-negative and HBV-positive tissues of HCC patients. f The overall survival curves of HBV-associated HCC patients with low- or high-IQGAP1 expression levels. Data were mean ± standard deviation (SD) from at least three independent experiments. **P < 0.01 and ***P < 0.001.

Fig. 2. IQGAP1 promotes anoikis resistance, migration and invasion of HCC cells.

a The qRT-PCR and western blotting analysis of IQGAP1 levels in attached or detached HCC cells treated with or without Matrigel. b The efficiency of stable upregulation of IQGAP1 in HepG2 cells and stable downregulation of IQGAP1 in HepG2.2.15 cells was validated by qRT-PCR and western blotting analysis. c The anchorage-independent growths of indicated HCC cells were determined by soft agar colony formation assays. d The cell death of indicated HCC cells under suspension condition was determined by trypan blue exclusion assay. e Indicated HCC cells were placed on poly-HEMA-coated plates to prevent cell adhesion, and then followed by western blotting analysis with indicated antibodies. f Indicated HCC cells were subjected to transwell migration and invasion assays. The results were expressed as mean ± SD from at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 3. ROS are required for IQGAP1-mediated anoikis resistance and metastasis in HCC cells.

a, b The effect of IQGAP1 on ROS production in HCC cells was determined by DCF and DHE assay. c The mitochondrial ROS levels in indicated HCC cells were examined by MitoSOX Red staining assay. d–f The indicated HCC cells were maintained in suspension condition, and then intracellular ATP (d), reduced GSH (e) and NADPH (f) levels were measured. g Indicated HepG2 and HepG2.2.15 cells were exposed to 20 mM NAC or 10 μM H₂O₂, respectively, and then subjected to soft agar colony formation assays. h Indicated HCC cells were maintained in suspension condition, treated as in g, and then followed by trypan blue assay. i Indicated HCC cells were maintained, treated as in g, and analysed by western blotting with indicated antibodies. j Indicated HCC cells were treated as in g, and then subjected to transwell migration and invasion assays. The results were expressed as mean ± SD from at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 4. Rac1 acivation is essential for IQGAP1-mediated ROS production, anoikis resistance and metastasis in HCC cells.

a, b Indicated HCC cells were transfected with EGFP-Rac1T17N (dominant-negative Rac1 mutant) or EGFP-Rac1Q61L (dominant-active Rac1 mutant) plasmid, respectively. The production of intracellular ROS was determined by DCF and DHE assay. c The mitochondrial ROS levels in indicated HCC cells were examined by MitoSOX Red staining assay. d–f Indicated HCC cells were transfected, maintained in suspension condition and then intracellular ATP (d), reduced GSH (e) and NADPH (f) levels were measured. g Indicated HCC cells were transfected with EGFP-Rac1T17N or EGFP-Rac1Q61L plasmid, respectively. Soft agar colony formation assays were performed. h Indicated HCC cells were transfected as in g, maintained in suspension condition, and then followed by trypan blue assay. i Indicated HCC cells were transfected, maintained as in g, and analysed by western blotting with indicated antibodies. j Indicated HCC cells were transfected as in g, and then followed by transwell assays. The results were expressed as mean ± SD from at least three independent experiments. ***P < 0.001.

Fig. 5. IQGAP1 activates Src/FAK signalling pathway.

a Indicated HCC cells maintained in suspension condition were exposed to 20 mM NAC or 10 μM H₂O₂, respectively. After incubation for 48 h, immunoblot analysis was performed with indicated antibodies. b Indicated HCC cells were transfected with EGFP-Rac1T17N or EGFP-Rac1Q61L plasmid, respectively, and then analysed by western blotting with indicated antibodies. c HepG2 and HepG2.2.15 stably expressing IQGAP1 were treated with Y15 or PP2, and then followed by soft agar colony formation assays. d Indicated HCC cells were maintained in suspension condition, treated as in c and then followed by trypan blue assay. e Indicated HCC cells were maintained, treated as in d and analysed by western blotting with indicated antibodies. f Indicated cells treated with Y15 or PP2 were subjected to transwell migration and invasion assays. The results were expressed as mean ± SD from at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 6. IQGAP1 enhances anoikis resistance and metastasis of HCC cells in vivo.

a Indicated HCC cells transfected with Rac1T17N or Rac1Q61L plasmid were intraperitoneally injected into nude mice. The GFP-positive HCC cells were collected from ascites fluid and sorted by flow cytometer. The apoptotic rates of the tumour cells were counted with the trypan blue exclusion assay. b Effect of PP2 or Y15 on IQGAP1-mediated anoikis resistance in mouse peritoneal cavities model. c Indicated HCC cells were transfected as in a, and i.v. injected via tail veins of BALB/c nude mice. Photographs show H&E staining of dissected lung tissues 8 weeks after inoculation. d Effect of PP2 or Y15 on IQGAP1-mediated anoikis resistance in experimental mouse lung metastasis model. The results were expressed as mean ± SD from at least three independent experiments. ***P < 0.001.