IQGAP1 promotes tumor progression by stabilizing MCM3/Nrf2 in cholangiocarcinoma

Figure 1 IQGAP1 was upregulated in cholangiocarcinoma tissues. A: Venn diagram of differential genes shared by four public databases. The transcriptome data of cholangiocarcinoma (CCA) in this study were sourced from four publicly available authoritative databases: (1) The Cancer Genome Atlas (TCGA) database’s TCGA-CHOL cohort, which included RNA-seq data from 36 CCA tumor tissues and 9 paired normal bile duct tissues; (2) The Gene Expression Omnibus (GEO) database’s GSE76297 dataset, constructed based on the GPL17586 platform, which included chip data from 54 CCA tumor tissues and 53 paired non-tumor tissues; (3) The GEO database’s GSE26566 dataset, constructed based on the GPL6104 platform, which included chip data from 104 CCA tumor tissues and 65 control tissues; and (4) The GEO database’s GSE32879 dataset, constructed based on the GPL6244 platform, which included chip data from 16 introhepatic CCA tumor tissues and 7 normal liver tissues. All datasets were standardized and quality-controlled to ensure the comparability and reliability of the data; B: The intersection of common differential genes with the top ten genes in the GSE76296 database; C: The mRNA expression of IQGAP1 tissue in different types of cancer in Tumor Immune Estimation Resource 2.0; D-G: The mRNA expression of IQGAP1 in different databases. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP < 0.0001. TCGA: The Cancer Genome Atlas; TPM: Transcripts per million.

Figure 2 IQGAP1 promoted cholangiocarcinoma proliferation and migration in vitro. A and B: The mRNA and protein expression of five cell lines; C-G: Cell counting kit-8, 5-ethynyl-2’-deoxyuridine, plate cloning, Transwell, and wound healing experiments; H-K: Quantification of cell function experiments. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP < 0.0001. NC: Negative control; OE: Overexpression; OD: Optical density; si: Silence; EdU: 5-ethynyl-2’-deoxyuridine.

Figure 3 IQGAP1 recruited USP28 to stabilize MCM3 expression. A: Silver-stained plots of the IQGAP1 immunoprecipitated proteins, in which MCM3 interacted with IQGAP1; B: Co-IP consequence confirmed that IQGAP1 significantly precipitated MCM3 and USP28 in two cholangiocarcinoma (CCA) cell lines; C: After knocking down or overexpressing IQGAP1, MCM3 showed the same trend of change as IQGAP1, while USP28 protein levels remained unchanged; D: Overexpression of IQGAP1 led to an increase in MCM3 that interacted with USP28, while knockdown of IQGAP1 reduced the interaction between USP28 and MCM3; E: Immunofluorescence results showed that MCM3 and IQGAP1 were co-localized in CCA cells. IgG: Immunoglobulin G; NC: Negative control; OE: Overexpression; si: Silence; IP: Immunoprecipitation.

Figure 4 IQGAP1 stabilized MCM3 expression by recruiting USP28 for K48 ubiquitination modification. A: Molecular docking patterns of IQGAP1, MCM3, and USP28 (IQGAP1 in pink, MCM3 in green and USP28 in blue); B: ASP-709, LYS-1653, LYS-1571 of IQGAP1 can form 4 hydrogen bonds with ASP-706, LYS-165, GLU-714 respectively on MCM3. It is proved that there is interaction between them; C: LYS-283, GLU-285, ASP-255, GLU-250 of USP28 can form 4 hydrogen bonds with LYS-248, LYS-177, GLN-386, GLU-185 respectively on MCM3. It is proved that there is interaction between them; D: SER-1084, ASP-1081, GLU-1078, ASP-1090 of IQGAP1 can form 4 hydrogen bonds with SER-205, THR-207, ARG-247, LYS-262, LYS-99 respectively on USP28. It is proved that there is interaction between them; E: The altered protein amount of MCM3 regulated by USP28 could be terminated by the addition of the proteasome inhibitor MG132; F: The addition of actinotide inhibited intracellular protein synthesis and faster degradation of MCM3 was observed upon knockdown of USP28 and lower degradation of MCM3 was observed upon overexpression of USP28; G: The expression of USP28 was inversely proportional to the ubiquitination level of MCM3; H: USP28 prevented MCM3 degradation through K48 ubiquitin chain-dependent breakout. NC: Negative control; si: Silence; WT: Wild type; UB: Ubiquitination; IP: Immunoprecipitation.

Figure 5 IQGAP1 promoted the progression of cholangiocarcinoma by mediating the MCM3/Nrf2 axis. A: The altered protein amount of MCM3 regulated by IQGAP1 could be terminated by the addition of the proteasome inhibitor MG132; B: The addition of actinotide inhibited intracellular protein synthesis and faster degradation of MCM3 was observed upon knockdown of IQGAP1, and the addition of actinotide inhibited intracellular protein synthesis and lower degradation of MCM3 was observed upon overexpression of IQGAP1; C: IQGAP1 prevented MCM3 degradation through ubiquitin-dependent breakout; D: The deubiquitination of MCM3 mediated by IQGAP1 could be reversed by USP28 knockdown; E and F: MCM3 regulated by IQGAP1 competitively bound Nrf2 to KEAP1, resulting in a positive correlation between the expression of Nrf2 and the change of MCM3; G: Dihydroethidium staining of cells assessed oxidative stress levels; H: Flow cytometry was used to detect apoptosis. NC: Negative control; OE: Overexpression; si: Silence; UB: Ubiquitination; IP: Immunoprecipitation.

Figure 6 IQGAP1 promoted the progression of cholangiocarcinoma by regulating MCM3. A and B: TUNEL staining results and flow cytometry analysis showed that the cell apoptosis inhibited by overexpression (OE) of IQGAP1 could be restored by knocking down MCM3; C-G: HUCCT1 transfected with OE of IQGAP1 showed higher proliferative capacity compared with NC in cell counting kit-8, plate cloning, 5-ethynyl-2’-deoxyuridine, Transwell and wound healing tests which was restored when MCM3 was knocked down. ^aP < 0.001. NC: Negative control; OE: Overexpression; si: Silence; EdU: 5-ethynyl-2’-deoxyuridine; OD: Optical density.

Figure 7 IQGAP1 promoted the proliferation and migration of cholangiocarcinoma by regulating MCM3 in vivo. A-C: Subcutaneous xenografts from three groups of differently treated cells. Subcutaneous tumor volume and weight were largest in the presence of IQGAP1, and this was MCM3 dependent; D: Ki-67, IQGAP1, and MCM3 histochemical staining of xenografts. ^aP < 0.0001. NC: Negative control; HE: Hematoxylin and eosin.