MIB1 upregulates IQGAP1 and promotes pancreatic cancer progression by inducing ST7 degradation

Abstract

Despite recent progress in cancer treatment, the prognosis of patients with pancreatic cancer still remains poor. Pancreatic tumors are reported to display high molecular heterogeneity. Elucidating the molecular mechanisms underlying pancreatic cancer progression is essential for improving patient treatment and survival. The overexpression of E3 ubiquitin ligase mind bomb 1 (MIB1) was previously described in pancreatic cancer cells, where it enhanced tumor cell proliferation. However, the role of MIB1 in pancreatic cancer progression remains elusive. In the present study, we confirmed that MIB1 expression is elevated in pancreatic cancer tissues and that high levels of MIB associate with unfavorable prognosis. Overexpression of MIB1 enhanced proliferation and invasion of pancreatic cancer cells both in vitro and in vivo. We further investigated the molecular mechanisms downstream of MIB1 and observed for the first time that MIB1 targets suppressor of tumorigenicity 7 protein (ST7), previously described as suppressor of tumorigenicity, for proteasomal degradation. Furthermore, we found that ST7 suppressed tumor growth by downregulating IQ motif containing GTPase activating protein 1 (IQGAP1) in pancreatic tumor cells. Thus, these data show that MIB1 promotes pancreatic cancer progression by inducing ST7 degradation followed by downregulation of IQGAP1 in pancreatic cancer cells. In conclusion, our research shows that the MIB1/ST7/IQGAP1 axis is essential for pancreatic cancer progression, and MIB1 inhibition may serve as a novel therapeutic strategy in patients with pancreatic cancer.

Keywords: IQGAP1; MIB1; ST7; pancreatic cancer.

Fig. 1

Aberrantly overexpressed MIB1 is associated with poor prognosis in pancreatic cancer. (A) The mRNA expression level of MIB1 in the TCGA dataset (http://www.cbioportal.org/). (B) Analysis of the mRNA expression level of MIB1 by using the GEPIA web tool (http://gepia.cancer‐pku.cn/). Wilcoxon signed‐rank test was used to determine the statistical significance. The error bar indicates SD. *, P < 0.01. (C) Representative IHC images stained with MIB1 in the tissue microarray. The scale bar as indicated in the figure. (D) The protein levels of MIB1 from the tissue microarray were determined by IHC analysis, Wilcoxon signed‐rank test was used to determine the statistical significance. P value as indicated in the figure. The error bar indicates SD. (E, F) The protein level of MIB1 from pancreatic cancer tissues (n = 12) and adjacent normal pancreatic tissues (n = 12) was detected by western blotting analysis, P = 0.0005. Wilcoxon signed‐rank test was used to determine the statistical significance. P value as indicated in the figure. (G, H) HPDE6‐C7, PANC‐1, BxPC‐3, SW1990, AsPC‐1, and MIA PaCa‐2 were harvested for western blotting analysis (G), RT‐qPCR analysis (H). For panel H, data presented as mean ± SD with three replicates (n = 3). Statistical significance was determined by one‐way ANOVA. *, P < 0.05; ***, P < 0.001. (I) The overall survival rate in high/low MIB1 group was analyzed by the human protein atlas (http://www.proteinatlas.org/), Log‐rank test was used to determine the statistical significance. P < 0.05.

Fig. 2

MIB1 is contributed to promoting pancreatic cancer cells progression. (A‐F), BxPC‐3 and SW1990 cells were infected with indicated shRNAs. 72 h post‐infection, cells were harvested for western blotting analysis (A), RT‐qPCR analysis (B), MTS assay (C), colony formation assay (D), and transwell assay (E). For panel B‐E, data presented as mean ± SD with three replicates. Statistical significance was determined by one‐way ANOVA. ***, P < 0.001. For panel E, the size of the scale bar on microscopy images was 100 μm. (G‐I) BxPC‐3 cells were infected with indicated shRNAs. After 72 h puromycin selection, cells were harvested and subcutaneously injected into nude mice for xenografts assay. The image of tumor was shown in panel G. The tumor mass was demonstrated in panel H. The tumor growth curve was indicated in panel I. Data presented as mean ± SD with five replicates. Student's t test was used to determine the statistical significance. ***, P < 0.001. (J‐M) BxPC‐3 and SW1990 cells were transfected indicated constructs. After 48 h, cells were harvested for western blotting analysis (J), RT‐qPCR analysis (K), MTS assay (L), and transwell assay (M). For panel K‐M, data presented as mean ± SD with three replicates. Student's t test was used to determine the statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001. For panel M, the size of the scale bar on microscopy images was 100 μm.

Fig. 3

MIB1 interacts with ST7 in pancreatic cancer cells. (A, B) western blotting analysis of the WCL of BxPC‐3 and SW1990 cells. (C, D), the PLA by using the indicated antibodies (panel C) to verify the interaction between MIB1 and ST7 in BxPC‐3 cells. The size of the scale bar on microscopy images was 20 μm. (E) A schematic diagram depicting the domain of MIB1. (F) Flag‐MIB1‐C and Flag‐MIB1‐N were translated in vitro; the immunoprecipitation was employed to examine the region of MIB1 binding with ST7.

Fig. 4

ST7 is a bona fide substrate of MIB1 in pancreatic cancer. (A and B) The pancreatic cancer cell lines (BxPC‐3, SW1990, AsPC‐1) were infected with indicated shRNAs. 72 h post‐infection, cells were harvested for western blotting analysis (A) and RT‐qPCR analysis (B). Data presented as mean ± SD with three replicates. Statistical significance was determined by one‐way ANOVA. ns, not significant. (C and D) The pancreatic cancer cell lines (BxPC‐3, SW1990, AsPC‐1) were transfected with indicated constructs. After 48 h, cells were harvested for western blotting analysis (C) and RT‐qPCR analysis (D). Data presented as mean ± SD with three replicates. ns, not significant. (E) BxPC‐3 cells were transfected with indicated constructs. After 48 h, the WCL of BxPC‐3 was subjected to western blotting analysis. Cells were treated with or without 20 µm of MG132 for 8 h before harvested. (F) BxPC‐3 cells were transfected with indicated constructs. After 48 h, the WCL of BxPC‐3 was subjected to western blotting analysis. (G) BxPC‐3 cells were infected with indicated shRNAs. After 72 h, cells were treated with cycloheximide (CHX) and cells were collected for western blotting analysis at different time points. (H) BxPC‐3 cells were transfected with indicated plasmids. After 48 h, cells were treated with cycloheximide (CHX) and cells were collected for western blotting analysis at different time points. The imagej software Laboratory for Optical and Computational Instrumentation (LOCI) of the University of Wisconsin‐Madison, Madison, WI, USA was used to quantified the protein level of ST7 and GAPDH. The relative protein level of ST7 to GAPDH was shown. (I) BxPC‐3 cells were infected with indicated plasmids. Seventy‐two hours postinfection, cells were collected for western blotting analysis after treated with MG132 for 8 h. The imagej software was used to quantified the protein level of ST7 and GAPDH. The relative protein level of ST7 to GAPDH was shown. (J) BxPC‐3 cells were transfected with indicated plasmids. After 48 h, cells were collected for western blotting analysis after treated with MG132 for 8 h. (K and L) The tissue microarray of pancreatic cancer was stained with MIB1 and ST7, respectively (n = 35). The typical IHC images stained with MIB1 and ST7 were shown in panel K. The size of the scale bar on microscopy images as indicated in the figure. The correlation of these two proteins was shown in panel L. Pearson correlation was used to determine statistical significance; the P value was indicated in the figure.

Fig. 5

ST7 is the key mediator for MIB1‐induced pancreatic cancer cells progression. (A‐E) BxPC‐3 and SW1990 cells were infected with indicated shRNAs. Seventy‐two hours post‐infection, cells were harvested for western blotting analysis (A), RT‐qPCR analysis (B), MTS assay (C), colony formation assay (D), and transwell assay (E). For panel B‐E, data presented as mean ± SD with three replicates. Statistical significance was determined by one‐way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001. For panel E, the size of the scale bar on microscopy images was 100 μm. (F and K), BxPC‐3 were infected with indicated shRNAs. Seventy‐two hours post‐infection and puromycin selection, cells were harvested for western blotting analysis (F), RT‐qPCR analysis (G), MTS assay (H), and xenograft assay (I‐K). For panel G and H, data presented as mean ± SD with three replicates (n = 3). Statistical significance was determined by one‐way ANOVA. ns, not significant; **, P < 0.01; ***, P < 0.001. The image of tumor was shown in panel I. The tumor mass was demonstrated in panel J. The tumor growth curve was indicated in panel K. Data presented as mean ± SD with five replicates (n = 5). Statistical significance was determined by one‐way ANOVA. ns, not significant; **P < 0.01;***P < 0.001.

Fig. 6

The MIB1/ST7/IQGAP1 signaling axis increases pancreatic cancer proliferation. (A) BxPC‐3 cells were transfected with indicated constructs for 48 h. Cells were subjected to RNA‐seq analysis and subsequent KEGG pathway enrichment. (B and C) BxPC‐3 and SW1990 cells were transfected with indicated constructs. After 48 h, cells were harvested for western blotting analysis (B) and RT‐qPCR analysis (C). Statistical significance was determined by one‐way ANOVA. Data presented as mean ± SD with three replicates (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) BxPC‐3 and SW1990 cells were infected with indicated shRNAs. Seventy‐two hours postinfection, cells were harvested for MTS assay. Statistical significance was determined by one‐way ANOVA on the fifth day. Data presented as mean ± SD with three replicates (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E and F) BxPC‐3 cells were infected with indicated shRNAs. Seventy‐two hours postinfection, cells were harvested for western blotting analysis (E) and RT‐qPCR analysis (F). Data presented as mean ± SD with three replicates. ns, not significant; *, P < 0.05; ***, P < 0.001. (G and H) BxPC‐3 cells were infected with indicated shRNAs. After 48 h, cells were transfected with indicated plasmids for other 24 h. Cells were harvested for western blotting analysis (G) and RT‐qPCR analysis (H). Data presented as mean ± SD with three replicates. ns, not significant; ***, P < 0.001. (I) a hypothesis model depicting that abnormally upregulated MIB1 degrades ST7 to increase IQGAP1 expression and promotes pancreatic cancer progression.