Work overview

Section 03 of 05

Results

Reduced IQGAP2 promotes esophageal squamous cell carcinoma by regulating MEK/ERK MAPK pathway

Liangcheng Zhao · 2026

Contents

Section 03 of 05

  1. 01Introduction
  2. 02Materials and methods
  3. 03Results
  4. 04Discussion
  5. 05Conclusion
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Work overview

Section 3 of 5

Results

Liangcheng Zhao · about 6 minutes

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The expression of IQGAP2 in ESCC samples of patients

A total of 236 patients diagnosed with ESCC were enrolled in this study, and the clinical characteristics of the cohort are summarized in Table 1. RNA-seq analysis revealed a set of differentially expressed genes between tumor and adjacent normal tissues. Among them, IQGAP2 was notably downregulated in ESCC tumor tissues, as illustrated by the heatmap in Figure 1A. To validate these findings, qPCR was performed on all 236 paired tissue samples. Consistently, qPCR results confirmed a significant decrease in IQGAP2 expression in tumor tissues compared to adjacent non-tumorous tissues (Figure 1B).

FIGURE 1: Panel A displays a heatmap of gene expression with hierarchical clustering, comparing normal samples and ESCC samples; IQGAP2 is indicated with a red arrow. Panel B presents a bar graph showing significantly lower IGQAP2 mRNA expression in ESCC tissues compared to normal tissues, marked with double asterisks indicating statistical significance.

FIGURE 1: Expressions of IQGAP2 in ESCC samples and normal tissue. (A) Heatmaps. Red represents high levels of expression and purple represents low levels of expression. N = 3 patients. (B) The expression levels of IQGAP2 in the ESCC samples and normal tissue were measured by qPCR. Data are represented as mean ± SD. N = 236 patients. T-test was performed; **p < 0.01.

To analyze the correlation between IQGAP2 expression and clinicopathological parameters, we divided the 236 patients into IQGAP2 high-expression and low-expression groups based on the median IQGAP2 expression level, and performed Pearson’s chi-square test to analyze the correlation between IQGAP2 expression and clinicopathological parameters (Supplementary Table S1). The results showed that low IQGAP2 expression was significantly associated with higher T stage (p = 0.002), lymph node metastasis (N+, p < 0.001), and advanced clinical stage (stage III-IV, P < 0.001), while no significant correlation was found with age or gender. These results directly demonstrate that downregulation of IQGAP2 is closely related to the malignant progression of ESCC, supporting its role as a biomarker for the malignant phenotype of ESCC. According to the Kaplan-Meier analysis, the ESCC patients who presented with low IQGAP2 expression had a shorter survival time than the patients with high IQGAP2 expression (Supplementary Figure S1). Furthermore, we investigated the functional pathways of IQGAP2 through GSEA. The enrichment outcomes revealed a significant accumulation of IQGAP2 in the MAPK signaling pathway (Supplementary Figure S2). The results showed that low expression of IQGAP2 was accompanied by exceptional activation of several important cancer-related pathways, of which the MAPK signaling pathway was the most significant.

Effects of IQGAP2 on ESCC cell proliferation in vitro

To explore the role of IQGAP2 in ESCC progression, EC9706 cell lines with stable 1QGAP2 overexpression (IQGAP2-OE) and knockdown (siIQGAP2) were established. Both mRNA and protein levels of IQGAP2 were significantly increased in EC9706 cells transfected with the IQGAP2-OE plasmid, as demonstrated by qPCR and Western blot analyses (Figures 2A–C). Conversely, treatment with siIQGAP2 resulted in a marked reduction of IQGAP2 expression at both the mRNA and protein levels (Figures 2D–F).

FIGURE 2: Panel A shows a bar graph comparing relative mRNA of IQGAP2 between Vector and IQGAP2-OE, with a significant increase in IQGAP2-OE. Panel B contains two western blots, one for IQGAP2 and one for GAPDH, with higher IQGAP2 band in IQGAP2-OE. Panel C shows a bar graph of relative intensity, significantly greater in IQGAP2-OE than Vector. Panel D displays relative mRNA levels of IQGAP2 in shControl versus shIQGAP2, showing a significant decrease in shIQGAP2. Panel E contains two western blots, IQGAP2 and GAPDH, with reduced IQGAP2 band in shIQGAP2. Panel F displays a bar graph of relative intensity, with a significant decrease in shIQGAP2 compared to shControl. Statistical significance is indicated by asterisks.

FIGURE 2: Overexpression or knockdown of IQGAP2 in EC9706 cells. The expression levels of IQGAP2 in the EC9706 were measured by qPCR (A,D) and Western blotting (B,E). The quantified relative band intensity was determined by ImageJ (C,F). Overexpression: IQGAP2-OE, knockdown: shIQGAP2. Data are represented as mean ± SD. N = 3. T-test was performed; *p < 0.05. **p < 0.01. ***p < 0.001.

Functionally, overexpression of IQGAP2 significantly inhibited the proliferative capacity of EC9706 cells compared to controls (Figure 3A). In contrast, knockdown of IQGAP2 notably enhanced EC9706 cell proliferation (Figure 3B).

FIGURE 3: Panel A line graph compares absorbance at four time points for Vector (red circles) and IQGAP2-OE (blue squares), showing higher absorbance in Vector at 48 and 72 hours with significant differences. Panel B line graph shows shControl (red circles) and shIQGAP2 (blue squares), with shIQGAP2 displaying significantly higher absorbance at all later time points. Error bars represent standard deviation. Asterisks indicate statistical significance.

FIGURE 3: IQGAP2 inhibits ESCC cell proliferation in vitro. CCK-8 assays comparing cell growth of IQGAP2-OE (A) or shIQGAP2 (B) cells as compared with their controls. Data are represented as mean ± SD. N = 3. T-test was performed; *p < 0.05. **p < 0.01.

IQGAP2 knockdown promotes ESCC cell proliferation through regulation of MEK/ERK MAPK pathway

The MEK/ERK MAPK pathway plays a critical role in the survival and development of various tumor cells (Guo et al., 2020). To investigate whether IQGAP2 modulates this pathway in ESCC, changes in MEK/ERK signaling were analyzed in EC9706 cells following IQGAP2 overexpression or knockdown (Figures 4A,B). Western blot analysis demonstrated that overexpression of IQGAP2 did not alter the total protein levels of MEK and ERK but significantly reduced the phosphorylation of both p-MEK and p-ERK (Figures 4A,C–F). In contrast, knockdown of IQGAP2 led to a marked increase in MEK and ERK phosphorylation, indicating enhanced activation of this signaling cascade (Figures 4A,C–F).

FIGURE 4: Western blot images and bar graphs display the expression levels of IQGAP2, p-MEK, MEK, p-ERK, ERK, and GAPDH in control, shIQGAP2, and IQGAP2-OE samples. Quantification graphs show statistical significance with asterisks for IQGAP2, p-MEK, and p-ERK expression changes, with no significant differences noted for MEK and ERK.

FIGURE 4: Reduced IQGAP2 promotes esophageal squamous cell carcinoma cell proliferation through regulation of MEK/ERK MAPK pathway. (A) Western blot showing the expression of phospho-MEK and phospho-ERK in EC9706 with IQGAP2 overexpression (IQGAP2-OE) or knockdown (shIQGAP2). GAPDH was used as a loading control. The quantified relative band intensity was determined by ImageJ (B–F). Data are represented as mean ± SD. N = 3. ANOVA was performed; **p < 0.01; ***p < 0.001.

We performed additional qPCR assays to detect the mRNA expression of classic downstream proliferation-related target genes in the MEK/ERK MAPK pathway, namely, c-Myc, Cyclin D1 and MMP9, in IQGAP2-OE, shIQGAP2 and control EC9706 cells (Supplementary Figures S3A-C). The results showed that IQGAP2 overexpression markedly reduced the mRNA levels of c-Myc, Cyclin D1 and MMP9 (all p < 0.01), consistent with decreased MEK and ERK phosphorylation. Accordingly, IQGAP2 knockdown significantly elevated the expression of these downstream molecules (all p < 0.001), which was consistent with hyperactivation of the MEK/ERK signaling cascade.

A rescue experiment using the MEK-specific inhibitor PD98059 was further performed. CCK-8 proliferation assay results showed that PD98059 treatment significantly abolished the promotive effect of IQGAP2 knockdown on ESCC cell proliferation (Supplementary Figure S4). These results clearly demonstrate that the pro-proliferative effect of reduced IQGAP2 in ESCC is dependent on the activation of the MEK/ERK MAPK pathway.

Moreover, we performed all core functional validation experiments in KYSE-150, a widely used moderately differentiated ESCC cell line. We generated IQGAP2-OE and shIQGAP2 KYSE-150 cells and verified transfection efficiency by qPCR (Supplementary Figure S5A). CCK-8 proliferation assay showed completely consistent results with EC9706 cells: overexpression of IQGAP2 significantly inhibited the proliferation of KYSE-150 cells, while knockdown of IQGAP2 markedly promoted cell proliferation (Supplementary Figure S3B). It was also shown that overexpression of IQGAP2 and knockdown of IQGAP2 significantly changes in the target genes of the MEK/ERK MAPK pathway that are directly associated with cell proliferation, including c-Myc, Cyclin D1 and MMP9 (Supplementary Figures S5C-E).

These results demonstrate that our findings are not cell line-specific, and have good generalizability in ESCC cell lines with different differentiation degrees.