Role of IQGAP3 in metastasis and epithelial-mesenchymal transition in human hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide owing to its high rates of metastasis and recurrence. The oncogene IQ motif-containing GTPase activating protein 3 (IQGAP3) is ubiquitously overexpressed in several human cancers, including liver, ovary, lung, large intestine, gastric, bone marrow, and breast malignancies and is involved in the invasion and metastasis of cancer cells. Therefore, we aimed to determine the biological role and molecular mechanism of IQGAP3 in HCC.

Methods: We used 120 archived clinical HCC samples, 9 snap-frozen HCC tumor tissues, and 4 normal liver tissues. Expression of IQGAP3 mRNA and protein in HCC cell lines (Hep3B, SMMC-7721, HCCC-9810, HepG2, BEL-7404, HCCLM3, QGY-7701, Huh7, and MHCC97H) and normal liver epithelial cells LO2 was examined by western blot, quantitative polymerase chain reaction, and immunohistochemistry. In addition, wound-healing and transwell matrix penetration assays were used to assess the migratory and invasive abilities of HCC cells, respectively.

Results: Expression of the IQGAP3 was robustly upregulated in HCC cells and tissues. High expression of IQGAP3 in HCC correlated with aggressive clinicopathological features and was an independent poor prognostic factor for overall survival. Furthermore, ectopic expression of IQGAP3 markedly enhanced HCC cell migration, invasion, and epithelial-to-mesenchymal transition (EMT) in vitro and promoted metastasis of orthotopic hepatic tumors in nude mice. Conversely, silencing endogenous IQGAP3 showed an opposite effect. Mechanistically, IQGAP3 promoted EMT and metastasis by activating TGF-β signaling.

Conclusions: IQGAP3 functions as an important regulator of metastasis and EMT by constitutively activating the TGF-β signaling pathway in HCC. Our findings present new evidence of the role of IQGAP3 in EMT and metastasis, indicating its potential as a prognostic biomarker candidate and a therapeutic target against HCC.

Keywords: Epithelial–mesenchymal transition; Hepatocellular carcinoma; IQGAP3; Metastasis; TGF-β signaling.

Fig. 1

IQGAP3 expression is upregulated in HCC. IQGAP3 mRNA levels in liver cancer tissues are assessed by analyzing The Cancer Genome Atlas liver cancer mRNA data set in a normal (n = 50) and HCC (n = 371) tissues and b the 50 paired adjacent non-tumor tissues (NT) and HCC tissues (T). Lines represent mean ± SD. P < 0.0001, t test. c Expression of IQGAP3 mRNA and protein in HCC cell lines (Hep3B, SMMC-7721, HCCC-9810, HepG2, BEL-7404, HCCLM3, QGY-7701, Huh7, and MHCC97H) and normal liver epithelial cells LO₂, as examined by western blotting and quantitative polymerase chain reaction. d Western blot and real-time polymerase chain reaction analyses of IQGAP3 expression in 4 normal liver tissues and 9 liver tumor tissues from HCC patients. Glyceraldehyde 3-phosphate dehydrogenase is used as a loading control (left panel). Correlation analysis is used for IQGAP3 mRNA and protein expression (right panel). Each bar represents the mean ± SD of three independent experiments. *P < 0.05. HCC hepatocellular carcinoma, IQGAP3 IQ motif-containing GTPase activating protein 3, SD standard deviation

Fig. 2

Upregulation of IQGAP3 is correlated with poor prognosis in human HCC. a Kaplan–Meier overall survival curves for HCC patients with low and high levels of IQGAP3 expression (n = 120; log-rank test, p < 0.01). b Representative immunohistochemistry analyses with high and low levels of IQGAP3 expression in tumor tissues from patients with HCC (>5 years, n = 24 and ≤5 years, n = 96). p < 0.001. HCC hepatocellular carcinoma, IQGAP3 IQ motif-containing GTPase activating protein 3

Fig. 3

IQGAP3 modulates the growth and prognosis of HCC in vivo. a Representative images of tumors from each experimental group. Orthotopic hepatic tumors model in nude mice were constructed using HCCLM3 cells with IQGAP3 overexpression (HCCLM3–IQGAP3) or vector control (HCCLM3-Vector). b Volumes of in situ tumors in the IQGAP3-overexpressing and control groups were measured on indicated days. Data are presented as mean ± SD. c Tumor weights of each group. d IQGAP3 overexpression significantly increased the number of intrahepatic metastatic nodules as compared with the control group. Summary data of each group are shown (n = 6/group). e Kaplan–Meier overall survival curves show a short survival time in the overexpression group. f Western blot assay detects IQGAP3, Snail, Twist1, MMP2, and MMP9 expression in the indicated tumors. Scale bars, 20 μm. g Histological analyses of liver tumors by hematoxylin and eosin (H & E) staining. Representative images of H & E staining of liver tissue samples from different experimental groups (n = 6/group). h Western blot assay detects EMT marker expression in the indicated tumors. Scale bars, 20 μm Data are expressed as mean ± SD. HCC hepatocellular carcinoma, IQGAP3 IQ motif-containing GTPase activating protein 3, SD standard deviation

Fig. 4

IQGAP3 enhances the migration and invasion of HCC cells. a, b Representative micrographs of the wound-healing assay in HCC cells showing the motilities of IQGAP3-overexpressing cells and IQGAP3-silenced cells at 0 and 36 h (HepG2 cell) or 24 h (HCCLM3) compared with vector controls. c, d Representative micrographs of the invasiveness of IQGAP3-overexpressing cells and IQGAP3-silenced cells compared with vector control cells in the transwell matrix penetration assay. Error bars represent mean ± SD from three independent experiments, *P < 0.05. e Representative micrographs of IQGAP3-transduced and IQGAP3-silenced cells cultured in the 3D spheroid invasion assay. f Gene Set Enrichment Analysis results indicate that IQGAP3 expression is significantly correlated with the metastasis-associated gene signatures based on The Cancer Genome Atlas HCC mRNA data set. HCC hepatocellular carcinoma, IQGAP3 IQ motif-containing GTPase activating protein 3, SD standard deviation

Fig. 5

IQGAP3 induces EMT in HCC. a, b Western blot analysis of IQGAP3 and EMT marker expression in HCC cells with IQGAP3 overexpression or knockdown, and their vector control cells. c, d The immunofluorescence assay shows the relative expression of E-cadherin and vimentin in HCC cells with IQGAP3 overexpression or knockdown and their vector control cells. HCC hepatocellular carcinoma, EMT epithelial to mesenchymal transition, IQGAP3 IQ motif-containing GTPase activating protein 3

Fig. 6

IQGAP3 activates the TGF-β signaling pathway. a Gene Set Enrichment Analysis shows that IQGAP3 expression is positively correlated with TGF-β–activated gene signatures (COULOUARN_TEMPORAL_TGFB1_SIGNATURE_UP and COULOUARN_TEMPORAL_TGFB1_SIGNATURE_DN). b TGF-β–responsive luciferase activity is measured in the indicated cells after 48 h culture with or without TGF-β1 (5 ng/mL) for 20 h using the dual luciferase assay. Values are presented as mean ± SD of triplicate samples. * p < 0.05. c Western blot analysis of the phosphorylated and total levels of Smad2 and Smad3 in the indicated HepG2 and HCCLM3 cells. GAPDH is used as a loading control. d IQGAP3 regulates the expression levels of numerous well-known genes downstream of TGF-β, as shown by real-time polymerase chain reaction analysis. * p < 0.05. e Western blot analysis of Smad2 and Smad3 expression in the cytoplasm and nucleus of HCCLM3 cells. GAPDH and p84 are used as loading controls for the cytoplasmic and nuclear fractions, respectively. HCC hepatocellular carcinoma, IQGAP3 IQ motif-containing GTPase activating protein 3, SD standard deviation, GAPDH glyceraldehyde 3-phosphate dehydrogenase, TGF-β transforming growth factor-β

Fig. 7

TGF-β signaling activation is required for IQGAP3-induced metastasis in HCC. a TGF-β–responsive luciferase activity induced by IQGAP3 is suppressed by depletion of Smad3 or treatment with TGF-β inhibitor SB431542 (10 μM) for 24 h in the indicated cells. Promotion of b invasion and c migration of HCC cells induced by IQGAP3 is inhibited by Smad3 silencing or SB431542 treatment (10 μM) for 24 h, as indicated by wound-healing and transwell matrix penetration assays. Error bars represent mean ± SD; *P < 0.05. HCC hepatocellular carcinoma, IQGAP3 IQ motif-containing GTPase activating protein 3, SD standard deviation, TGF-β transforming growth factor-β