E2F1 transactivates IQGAP3 and promotes proliferation of hepatocellular carcinoma cells through IQGAP3-mediated PKC-alpha activation

Abstract

For decades, E2F1 has been recognized as a retinoblastoma protein (RB) binding transcription factor that regulates the cell cycle. E2F1 binds preferentially to RB and accelerates the cell cycle in most cancer cells. However, it is thought that E2F1 modulates cell proliferation in other ways as well. Herein, it has been discovered that in pathological tissues derived from hepatocellular carcinoma (HCC) patients, E2F1 correlates positively with IQGAP3 and that both of these factors are highly expressed (N = 164, R = 0.6716). In addition, a high level of E2F1 or IQGAP3 predicted poor survival in HCC patients. Further study determined that E2F1 transactivates IQGAP3, the GTPase binding protein in MHCC-97H cells. Co-immunoprecipitation analysis indicated that IQGAP3 interacts with PKCδ and competitively inhibits the interaction between PKCδ and PKCα, resulting in phosphorylation of PKCα activation and promotion of cell proliferation. This study reveals that highly expressed E2F1 not only transactivates cell-cycle-related factors but also promotes HCC proliferation by activating the phosphorylation of PKCα.

Keywords: E2F1; IQGAP3; PKCα activation; cell proliferation; hepatocellular carcinoma.

Figure 2

E2F1 transactivates IQGAP3 in HCC cells. (A) Total proteins were extracted from L02, HepG2 and MHCC-97H cells. The level of E2F1 and IQGAP3 was determined by immunoblot. (B) HepG2 cells were transfected with 2 μg pCMV-E2F1 or pCMV vectors. MHCC-97H cells were transfected with E2F1-specific siRNAs or control siRNAs. Samples were harvested 48 hours after transfection. The level of E2F1 and IQGAP3 was determined by immunoblot. (C) Schematic representation of the promoter region of human IQGAP3. The predicted E2F1 binding sites are indicated. (D) Products of ChIP-PCR in the input, IgG and ChIP groups were analyzed using agarose gel electrophoresis. It was demonstrated that E2F1 interacted with the promoter of IQGAP3 at P1. (E) Quantitative PCR analysis of samples in the input, IgG and ChIP groups using P1-specific primers. (F) Luciferase activity was measured after MHCC-97H cells were transfected with pGP4.19-IQGAP3 promoter/pGP4.19-IQGAP3 promoter P1-mutant, pRL-TK and vector/c-Myc. Data in (D and E) are mean ± SEM. Two-tailed Student’s t test was used for statistical calculation.

Figure 5

Upregulation of IQGAP3 promotes cell proliferation in vitro. (A) HepG2 cells were transfected with 0.5 μg pCMV-E2F1, pCMV-IQGAP3 or pCMV vectors. MHCC-97H cells were transfected with E2F1, IQGAP3-specific siRNAs, or control siRNAs. Cell viability was measured using MTS assay 0, 24, 48 and 72 hours after transfection. (B) Cells were treated with vectors and siRNAs as described in (A). Representative immunofluorescence images from a BrdU study of HepG2 and MHCC-97H cells. Total DNA (blue) and DNA under replication (red) are shown (scale bar: 50 μm). (C) Percentage of cells with DNA under replication in (B) was measured using image-J. Data in (A and C) are mean ± SEM. Two-tailed Student’s t test was used for statistical calculation.

Figure 1

E2F1 is positively correlated with IQGAP3 in pathological tissues derived from HCC patients. (A) The lead gene, IQGAP3, was significantly reduced in MHCC-97H^siE2F1 compared with MHCC-97H^siNC. (B) The correlation between E2F1 and IQGAPs in HCC pathological tissues (based on the TCGA database, N = 423) is represented using linear correlativity. (C) E2F1 and IQGAPs differentially expressed in pathological HCC tissues vs adjacent liver tissues (N = 50). Red color represents increased expression and green indicates reduced expression. (D) Expression of E2F1 and IQGAP3 in HCC based on individual cancer stage (based on TCGA database, N = 390). (E) Overall survival of probability of HCC patients with high/low E2F1 (high: n = 92; low: n = 273) and IQGAP3 (high: n = 91; low: n = 274) expression (based on TCGA database). (F) Immunohistochemical analysis of E2F1 and IQGAP3 in pathological HCC (scale bar: 200 μm). (G) The correlation among concurrent immunostaining scores of E2F1 and IQGAP3 in HCC tissues. (H) The overall survival of HCC patients with low and high expression of E2F1 or IQGAP3 is shown. Data in (D) are mean ± SEM. Two-tailed Student’s t test was used for statistical calculation.

Figure 3

IQGAP3 interacts with PKCδ and competitively inhibits the interaction between PKCδ and PKCα, resulting in phosphorylation and activation of PKCα. (A) HepG2 cells were transfected with 2 μg pCMV-E2F1, pCMV-IQGAP3 or pCMV vectors. MHCC-97H cells were transfected with E2F1, IQGAP3-specific siRNAs or control siRNAs. Samples were harvested 48 hours after transfection. The level of PKCδ was determined by immunoblot. (B) Co-immunoprecipitation and immunoblot analysis of IQGAP3 and PKCδ in MHCC-97H cells transfected for 48 hours with pCMV-IQGAP3 or pCMV vectors. The proteins were immunoprecipitated using Protein A/G beads conjugated with anti-IQGAP3 or anti-PKCδ antibodies. (C) Co-immunoprecipitation and immunoblot analysis of PKCα and PKCδ in MHCC-97H cells transfected for 48 hours with pCMV-IQGAP3 or pCMV vectors. The proteins were immunoprecipitated using Protein A/G beads conjugated with anti-PKCα or anti-PKCδ antibodies. (D) Co-immunoprecipitation and immunoblot analysis of PKCα and PKCδ in MHCC-97H cells transfected for 48 hours with IQGAP3-siRNA or control-siRNA. The proteins were immunoprecipitated using Protein A/G beads conjugated with anti-PKCα antibody. (E) Schematic representation of IQGAP3- and PKCα-PKCδ interaction. IQGAP3 interacted with PKCδ and competitively inhibited the interaction between PKCδ and PKCα, resulting in PKCα activation in HCC cells. Inhibition of IQGAP3 promoted PKCα-PKCδ interaction and activated PKCα. Data in (D) are mean ± SEM. Two-tailed Student’s t test was used for statistical calculation.

Figure 4

Upregulation of IQGAP3 facilitates phosphorylation of PKCα and activates the AKT signaling pathway. (A) HepG2 cells were transfected with 2 μg pCMV-E2F1, pCMV-IQGAP3 or pCMV vectors. MHCC-97H cells were transfected with E2F1, IQGAP3-specific siRNAs or control siRNAs. Samples were harvested 48 hours after transfection. The levels of E2F1, IQGAP3 and factors in the AKT pathway were determined by immunoblot. (B) Relative expression levels of the proteins indicated in (A). Data are mean ± SEM. Two-tailed Student’s t test was used for statistical calculation.

Figure 6

Upregulation of IQGAP3 promotes cell proliferation in vivo. (A) HepG2^vector, HepG2^IQGAP3, MHCC-97H^shV and MHCC-97H^shIQGAP3 cells were injected subcutaneously into nude mice to generate xenograft models. Three weeks after injection, the animals were sacrificed and xenografts were obtained. (B) Tumor weights of xenografts. (C) Immunohistochemical analysis of E2F1 and IQGAP3 in xenografts (scale bar: 200 μm). Data in (B) are mean ± SEM. Two-tailed Student’s t test was used for statistical calculation.

Figure 7

Overview of IQGAP3-mediated signaling pathways in HCC cells. E2F1 transactivates IQGAP3, and IQGAP3 competitively inhibits the interaction between PKCδ and PKCα, resulting in phosphorylation and activation of PKCα and promotion of cell proliferation in HCC cells.