The Overexpression of IQGAP1 and β-Catenin Is Associated with Tumor Progression in Hepatocellular Carcinoma In Vitro and In Vivo

Abstract

The IQ-domain GTPase-activating protein 1 (IQGAP1) is a multifunctional scaffold protein, which interacts with diverse proteins to regulate cell adhesion and cell migration. The abnormal expression of IQGAP1 widely exists in many cancers, but biological roles of IQGAP1 cooperation with its interacting proteins to involve in tumorigenesis remain to clarify. In this study, we have found that IQGAP1 interacts with β-catenin and regulates β-catenin expression in hepatocellular carcinoma (HCC) cells. The expression levels of IQGAP1 and β-catenin and their associations have a positive correlation with cell metastasis ability in several HCC cell lines. The up-regulation of IQGAP1 and β-catenin improves cell proliferation and migration ability of HCC cells, whereas the knockdown of IQGAP1 by small interfering RNA can decrease β-catenin expression, which results in the reduction of cell proliferation and migration ability in vitro. In addition, a significantly higher expression of IQGAP1 and β-catenin also usually exists in human HCC tissues, especially their overexpression is clinicopathologically associated with tumor malignancy. Generally the overexpression and interactions of IQGAP1 and β-catenin contribute to HCC progression by promoting cell proliferation and migration.

Fig 1. IQGAP1 interacts with β-catenin and regulates β-catenin expression.

(A) Endogenous IQGAP1 interacts with β-catenin in HepG2 cells. 500ug of HepG2 cell lysate was immunoprecipitated with anti-IQGAP1 antibodies, and bound proteins were analyzed by immunoblotting with the indicated antibodies. 5% of total lysate was loaded as a control (input). (B) The IQGAP1 overexpression improved the expression of β-catenin. Transfection with pCMV6 empty plasmids was taken as a control and the untreated HepG2 cells were taken as a mock. flag-IQ: pFlag-IQGAP1 plasmids. (C) The IQGAP1 knockdown by IQGAP1 siRNA (si-IQ) reduced β-catenin expression. Transfection with non-target siRNA was taken as a control (si-NC), and untreated HepG2 cells were taken as a mock. (D)The expression of IQGAP1 and β-catenin was gradually increased in several HCC cell lines. LO2 was a normal liver cell line. 97L: MHCC-97L; 97H: MHCC-97H.

Fig 2. Different expression level of IQGAP1 and β-catenin in HCTs and PLTs.

The negative, weak, moderate and strong staining activity of IQGAP1 was respectively shown in HCTs (A-D) and PLTs (I-L). IQGAP1 located in cytoplasm and cell membrane in HCTs (D), and IQGAP1 was observed in cytoplasm in PLTs (L). The β-catenin with negative, weak, moderate and strong staining activity was respectively detected in HCTs (E-H) and PLTs (M-P). The expression of β-catenin located in cell membrane (G), cytoplasm and nucleus (H) in HCTs, while it mainly located in cell membrane (O) and cytoplasm (P) in PLTs. Scale bar represents 50 μm (original magnification×400). HCTs: hepatocellular carcinoma tissues. PLTs: para-cancerous liver tissues.

Fig 3. IQGAP1 activates β-catenin transcription and promotes its translocation to nucleus.

(A) IQGAP1 overexpression by transfecting with pFlag-IQGAP1 plasmids (flag-IQ) upregulated β-catenin mRNA level. Transfection with empty pCMV6 plasmids was taken as a control. And the mRNA level of c-myc, cyclin D1 and Axin2 upon IQGAP1 overexpression was shown in (D). (B) IQGAP1 knockdown, by specific siRNA for IQGAP1 (si-IQ), decreased β-catenin mRNA level. Transfection with non-target siRNA was taken as a control (si NC). (C) IQGAP1 activates β-catenin transcription by a luciferase promoter analysis. (E) The overexpression of IQGAP1 induced β-catenin translocation from cytoplasm to nucleus. LMNB1 was a nuclear protein control. GAPDH was a cytoplasmic protein control. ** Student’s t test p<0.01, *** Student’s t test p<0.001.

Fig 4. IQGAP1 regulates cell proliferation and migration ability.

Overexpression of IQGAP1 in HepG2 cells enhanced cell proliferation (A) and cell migration (C, D). Knockdown of IQGAP1 decreased cell growth (B) and migration (E, F). Control: HepG2 cells transfected with empty pCMV6 plasmids; flag-IQ:HepG2 cells transfected with pFlag-IQGAP1 plasmids. siNC: HepG2 cells transfected with control siRNA; si-IQ: HepG2 cells transfected with IQGAP1 siRNA. Scale bar represents 100 μm (original magnification×200).

Fig 5. β-catenin regulates cell growth and migration.

(A) β-catenin mRNA exhibited a recovery increase in IQGAP1-knockdown HepG2 cells re-transfected β-catenin plasmids. The upregulation of β-catenin induced the mRNA expression of c-myc, cyclin D1 and Axin2 (B), and enhanced cell proliferation (E) and migration (C, D). Control: HepG2 cells transfected with empty vectors. flag-β-cat: HepG2 cells transfected with pFlag-β-catenin plasmids. si-IQ: HepG2 cells transfected with IQGAP1 siRNA; flag-β-cat+si-IQ: HepG2 cells transfected with pFlag-β-catenin plasmids on the condition of IQGAP1 knockdown. ** Student’s t test p<0.01, *** Student’s t test p<0.001. Scale bar: 100 μm (original magnification×200).

Fig 6. The interacting proteins with IQGAP1 and β-catenin analyzed by a bioinformatics software STRING.

The functional partners were predicted by different methods, which were shown in different line colors. black (\, coexpression), means genes co-expressed in same or in other species; purple (\, experiments), shows a significant protein interaction from literatures; light blue (\, database), shows a significant protein interaction group gathered from curated databases; yellow (\, textmining), shows protein interaction groups extracted from scientific literatures; grey blue (\, homology), shows a protein interaction group gathered from homology. The predicted functional partners include as follows. APC, adenomatous polyposis coli; CDC42, cell division cycle 42; CDH5, cadherin 5; CDH2, cadherin 2, type 1, N-cadherin; CDH1, cadherin 1, type 1, E-cadherin; AXIN1, axin 1; GSK3B, glycogen synthase kinase 3 beta; LEF1, lymphoid enhancer-binding factor 1; PSEN1, presenilin 1; TCF7L2, transcription factor 7-like 2.