Scaffolding Protein IQGAP1 Is Dispensable, but Its Overexpression Promotes Hepatocellular Carcinoma via YAP1 Signaling

Abstract

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffolding protein that is overexpressed in a number of cancers, including liver cancer, and is associated with protumorigenic processes, such as cell proliferation, motility, and adhesion. IQGAP1 can integrate multiple signaling pathways and could be an effective antitumor target. Therefore, we examined the role of IQGAP1 in tumor initiation and promotion during liver carcinogenesis. We found that ectopic overexpression of IQGAP1 in the liver is not sufficient to initiate tumorigenesis. Moreover, we report that the tumor burden and cell proliferation in the diethylnitrosamine-induced liver carcinogenesis model in Iqgap1^-/- mice may be driven by MET signaling. In contrast, IQGAP1 overexpression enhanced YAP activation and subsequent NUAK2 expression to accelerate and promote hepatocellular carcinoma (HCC) in a clinically relevant model expressing activated (S45Y) β-catenin and MET. Here, increasing IQGAP1 expression in vivo does not alter β-catenin or MET activation; instead, it promotes YAP activity. Overall, we demonstrate that although IQGAP1 expression is not required for HCC development, the gain of IQGAP1 function promotes the rapid onset and increased liver carcinogenesis. Our results show that an adequate amount of IQGAP1 scaffold is necessary to maintain the quiescent status of the liver.

Keywords: IQGAP1; MET; YAP; liver cancer; scaffold protein.

FIG 1

IQGAP1 does not promote β-catenin-driven HCC. (A) HepG2 cells were transfected with GFP or IQGAP1 constructs for 72 h. Expression for IQGAP1 was normalized to B2M. (B) HepG2, Huh7, and Hep3B cell lines were transfected with control, IQGAP1, or (S45Y) β-catenin expression constructs for 72 h. Wnt/β-catenin activity was measured by the TOPFlash luciferase reporter assay and corrected for renilla luciferase. (C) HepG2 cells were transfected with GFP, IQGAP1, or (S45Y) β-catenin expression constructs for 72 h. Additionally, cells were also transfected with IQGAP1 and (S45Y) β-catenin constructs together. Wnt/β-catenin activity was measured and analyzed as previously described. (D) Representative livers from nontreated, (S45Y) β-catenin, IQGAP1, or (S45Y) β-catenin plus IQGAP1 groups. (E) Liver weight to body weight (LW/BW) ratio. Scale bar is 2.5 cm. Graphs show means ± SEM and dots represent individual mice. Student’s t test was used to determine significance. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

FIG 2

IQGAP1 deletion does not inhibit liver tumor development. Male Iqgap1^+/+ (n = 13), Iqgap1^+/− (n = 24), and Iqgap1^−/− (n = 16) mice were treated intraperitoneally with 5 mg/kg diethylnitrosamine (DEN) in sterile PBS at 12 to 15 days of age. At 1 year, tumor burden was assessed. (A) Representative Ki-67 immunohistochemistry images of livers of P15 mice 24 h after DEN injection. Scale bar is 50 μm. (B) Quantification of Ki-67-positive cells per field (n = 5 Iqgap1^+/+, 7 Iqgap1^+/−, and 6 Iqgap1^−/− mice). (C) Representative photos of gross livers at one year. Tumor nodules are indicated by a dashed border. Scale bar is 1 cm. (D) Tumor incidence based on presence of visible liver nodules. (E) Tumor multiplicity was measured by counting the number of visible tumors per liver. (F) Liver weight normalized to body weight divided into low and high groups. (G) Gene expression of Iqgap1, Iqgap2, and Iqgap3 in tumor-adjacent liver tissue and tumor tissue normalized to Gapdh expression. Values are displayed as means ± SEM. For tumor incidence, χ² test was used to determine significance between all 3 groups. For tumor multiplicity and largest tumor size, one-way ANOVA with Bonferroni’s multiple-comparison test was used to determine significance between groups. For liver-to-body-weight ratio, two-way ANOVA with Bonferroni’s multiple-comparison test was used to determine significance between groups. For gene expression, two-way ANOVA with Tukey’s multiple-comparison test was used to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Hepatic gene expression, DEN-induced mutations, and epithelial-mesenchymal transition (EMT) are unaffected by Iqgap1 loss. (A) The table shows gene expression patterns correlating with molecular subtypes of HCC adapted from Boyault et al. (25). Red represents upregulated genes. Blue represents downregulated genes. Gene expression of Rrm2, Tgfbr1, Fasn, Crp, Pepck, Angpt2, and Glul in tumor-adjacent liver tissue and tumor tissue normalized to Gapdh expression. (B to E) DNA mutation frequency at select codons in tumors of mice 50 weeks after DEN injection (n = 7 Iqgap1^+/+, 7 Iqgap1^+/−, and 8 Iqgap1^−/− mice). (F) Immunoblot of EMT markers MMP2, N-cadherin, E-cadherin, and Cdc42 in tumors of Iqgap1^+/+, Iqgap1^+/−, and Iqgap1^−/− mice 50 weeks after DEN treatment (n = 4 mice per group). For gene expression, two-way ANOVA with Tukey’s multiple-comparison test was used to determine significance. One-way ANOVA with Bonferroni multiple-comparison test was used to compare groups in panels B to E. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

IQGAP1 knockdown enhances MET expression. (A) Representative images of anti-Ki-67 immunohistochemistry staining in liver and tumor tissue of Iqgap1^+/+, Iqgap1^+/−, and Iqgap1^−/− animals (n = 9, 8, and 12 mice per group, respectively). Scale bar is 100 μm. (B) Representative immunohistochemistry images of anti-P-ERK staining in normal liver tissue and tumor tissue classified as either P-ERK negative or P-ERK positive. Scale bar is 100 μm. The number of tumors with each molecular classification (n = 23, 18, and 28 Iqgap1^+/+, Iqgap1^+/−, and Iqgap1^−/− tumors, respectively). (C) Immunoblot of DEN-treated Iqgap1^+/+, Iqgap1^+/−, and Iqgap1^−/− tumor extracts. Each lane contains extracts from a single mouse and quantified by densitometry. (D) Snu-449 HCC cells were transfected with either Control or IQGAP1 siRNA for 72 h. Cells were then serum starved overnight and treated with or without HGF (50 ng/μl) and with or without EMD1214063 (10 nM) for 4 h prior to harvest. Whole-cell lysates were immunoblotted for phosphorylated and total forms of specific targets and normalized to GAPDH. Conditions are representative of 3 independent experiments pooled. Quantification of immunoblots with respect to only HGF⁺ conditions by densitometry. Values are displayed as means ± SEM. For ERK staining, two-way paired ANOVA with Tukey’s multiple-comparison test was used to assess differences between groups. One-way paired ANOVA with Tukey’s multiple-comparison test was used for all others. *, P < 0.05.

FIG 5

IQGAP1 overexpression increases HCC development in B+M transposon system. (A) Experimental design for Sleeping Beauty transposase groups injected with either B+M or B+M+I and harvested after 4 or 8.5 weeks. (B) Whole liver protein lysates isolated after 4 weeks and analyzed by Western blotting to detect epitope-tagged (HA, V5, or Myc) and total IQGAP1, MET, and β-catenin, normalized to GAPDH. (C) Gene expression of Iqgap2 and Iqgap3 in whole livers from 4- and 8.5-week samples from the transposon model. Gene expression was normalized to Gapdh. (D) Representative livers from nontreated, B+M, and B+M+I groups. Macroscopic disease is visible as black or white lesions. (E) Liver weight to body weight (LW/BW) ratio. (F) Afp expression corrected for Gapdh. (G) Serial liver sections from 4-week and 8.5-week mice stained with H&E and GS (brown) to identify β-catenin driven HCCs. Necrotic regions are marked by asterisks. (H) Gene expression for Rrm2, Tgfbr1, Fasn, Crp, Pepck, Angpt2, and Glul in whole livers from 4- and 8.5-week samples from the transposon model. Gene expression was normalized to Gapdh. Graphs show means ± SEM, and dots represent individual mice. Two-way paired ANOVA with Tukey’s multiple-comparison test was used to determine significance between groups in panel E, and Student’s t test was used for panel D. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Gross morphology scale bar (C) is 2.5 cm, and histology scale bars (F) are 100 μm.

FIG 6

IQGAP1 overexpression does not induce Wnt/β-catenin or MET signaling in vivo. (A) Cytosolic and nuclear proteins from whole livers (4-week NT, B+M, and B+M+I) were analyzed for IQGAP1 and β-catenin. Cytosolic protein was normalized to GAPDH and nuclear protein normalized to LaminB1. GAPDH and LaminB1 show purity of cytosolic or nuclear fractions, respectively. (B) Whole liver lysates from NT (n = 3), B+M (n = 5), or B+M+I (n = 4) were pooled and immunoprecipitated (IP) for β-catenin and then immunoblotted (IB) for E-cadherin, IQGAP1, or β-catenin. Sample inputs were probed for E-cadherin, β-catenin, or GAPDH to demonstrate equal amounts of protein from each group and were used for IPs. (C) Liver sections from mice under the HDTVI model for 4 weeks stained for V5 (green), HA (red), or myc (white) to identify nodules by immunofluorescence expressing MET, IQGAP1, and β-catenin, respectively. Tumors under the B+M condition are demarcated by a dashed line. Scale bar is 50 μm. Graphs show means ± SEM, and dots represent individual mice. (D) Expression of Ctnnb1 and Wnt/β-catenin target genes Birc5, Lect2, Ccnd1, and Axin2 in whole livers from 4- and 8.5-week samples, normalized to Gapdh. (E) Whole-liver protein lysates analyzed by immunoblotting for total and phosphorylated tyrosine residues of MET (Y1234/1235), AKT-1 (S437), mTOR (S2448), and STAT3 (Y705). Phosphorylated protein is normalized to GAPDH and corrected for total respective protein; values are expressed relative to the NT control (set to 1). For panel D, two-way paired ANOVA with Tukey’s multiple-comparison test and all others compared via Student’s t test to determine significance. Graphs show means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 7

IQGAP1 overexpression drives YAP1 nuclear translocation and activity. (A) HepG2 cells were transfected with 6 μg GFP, 4 μg IQGAP1, or 2 μg (S127A) YAP1 constructs for 24 h. Expression for IQGAP1 or YAP1 was normalized to B2M. (B) HepG2 cells were transfected with 500, 1,000, or 1,500 ng GFP, 1,000 ng IQGAP1, or 500 ng (S127A) YAP1 expression constructs for 24 h. YAP1 activity was measured using the YAP1 luciferase reporter and corrected using renilla luciferase. (C) Liver sections from 2-week NT, B+M, or B+M+I mice stained with GS (brown) to identify normal pericentral (demarcated by white dashed line) or ectopic (demarcated by black dashed line) regions. Scale bar is 100 μm. (D) Serial liver sections from 2-week mice stained to mark YAP1 nuclei (top, defined by arrows) or GS (bottom). Scale bar is 50 μm. (E) Cytosolic and nuclear protein from whole livers (4-week pooled NT [n = 3], B+M [n = 5], and B+M+I [n = 4]) were analyzed for YAP1 or IQGAP1. Cytosolic protein was normalized to GAPDH and nuclear protein to LaminB1. GAPDH and LaminB1 show purity of cytosolic or nuclear fractions, respectively. (F) Expression of Yap1 and Hippo/YAP target genes Amotl2, Ccn1, Ccn2, and Jag1 in whole livers from 4- and 8.5-week samples, normalized to Gapdh. Graphs show means ± SEM, and dots represent individual mice. Data in panels A and B are representative of studies replicated in Snu-449 and Huh7 HCC cells. For panel F, two-way paired ANOVA with Tukey’s multiple-comparison test was used, and all others were compared using Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 8

NUAK2 expression is elevated in HCCs with IQGAP1 overexpression. (A) HepG2 cells were transfected with 6 μg GFP, 4 μg IQGAP1, or 2 μg (S127A) YAP1 constructs for 24 h. Expression for NUAK2 was normalized to B2M. (B) Whole-cell lysates were immunoblotted for IQGAP1, YAP1, and NUAK2 and normalized to GAPDH. Conditions presented are representative of 3 independent experiments pooled. (C) Huh7 HCC cells were transfected with 6 μg GFP, 4 μg IQGAP1, or 2 μg (S127A) YAP1 constructs for 24 h. Cells were pulsed with EdU for 30 min prior to fixation and staining for EdU (green) and Hoechst (blue). (D) Expression of Nuak2 in whole livers from 4- and 8.5-week NT, B+M, and B+M+I samples, normalized to Gapdh. (E) Whole-liver protein lysates from 4-week NT, B+M, and B+M+I mice analyzed by Western blotting to detect NUAK2 and YAP1, which are normalized to GAPDH. (F) Pie charts demonstrating the distribution of HCC cases with IQGAP1^High expression from the TCGA cohort. Distribution further breaks down the number of cases with IQGAP1^High that also contain NUAK2^High expression. (G) Overall survival for a subset of patients with IQGAP1^High/NUAK2^High expression compared to IQGAP1^Unaltered/NUAK2^Unaltered patients. (H) RNA-sequencing data from the TCGA samples with elevated IQGAP1/NUAK2 expression compared to those without was used for IPA analysis to determine molecular pathways that are activated/inhibited (left) with corresponding changes to the genes that regulate respective molecular pathways (right). Graphs show means ± SEM, and dots represent individual mice. Data in panels A to C are representative of studies replicated in multiple HCC cell lines. (G) Log-rank (Mantel-Cox) test was used to determine significance for survival curve, and Student’s t test was used for all others. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.