Materials and methods

Patients

Tissue samples were obtained from 236 patients diagnosed with ESCC who underwent esophagectomy at our hospital between 2017 and 2019. The collected specimens included formalin-fixed, paraffin-embedded (FFPE) tumor tissues and matched adjacent non-tumorous tissues located more than 5 cm from the tumor margins. All samples were histopathologically confirmed by experienced pathologists. None of the patients received radiotherapy or chemotherapy prior to surgery. Tumor staging was conducted according to the American Joint Committee on Cancer (AJCC) eighth edition guidelines (Rice et al., 2017). Detailed clinical and pathological characteristics of the enrolled patients are presented in Table 1.

Clinical characteristics | Cases | Percent
Age (years)
≥64 | 148 | 62.7
<64 | 88 | 37.3
Gender
Male | 178 | 75.4
Female | 58 | 24.6
T stage
T1 | 23 | 9.7
T2 | 53 | 22.5
T3 | 147 | 62.3
T4 | 13 | 5.5
N stage
N0 | 153 | 64.8
N1 | 48 | 20.3
N2 | 27 | 11.4
N3 | 8 | 3.4
M stage
M0 | 225 | 95.3
M1 | 11 | 4.7
Clinical stage
Locoregional (I–II) | 47 | 19.9
Advanced (III–IV) | 189 | 80.1

Ethical approval for this study was granted by the Ethics Committee of the Fifth People’s Hospital of Wujiang District, and written informed consent was obtained from all participants. RNA sequencing (RNA-seq) of the ESCC tissue samples was performed by BGI Genomics (Shenzhen, China).

Transcriptome deep sequencing

Whole RNA-seq was performed on normal esophageal tissues and matched ESCC tumor tissues from three patients by BGI Genomics using the MGISEQ-2000 sequencing platform, as previously described (Lang et al., 2021). For visualization, a heatmap was generated using OmicShare tools, a freely accessible online data analysis platform (http://www.omicshare.com/tools). Gene Ontology (GO) analysis of the RNA-seq data was conducted using the R programming language.

qPCR

Quantitative polymerase chain reaction (qPCR) assays were conducted using the TaqMan method, following the manufacturer’s protocol. Primers and TaqMan Gene Expression Assay probes for the target genes were obtained from Thermo Fisher Scientific Inc. The assays were performed in duplicate, including a no-template negative control to ensure specificity. qPCR reactions were carried out on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, USA). The specific probes used in this study were c-Myc, Cyclin D1, MMP9, MEK, ERK, IQGAP2, and GAPDH, with GAPDH serving as the internal control. Relative gene expression levels were calculated using the 2−ΔΔCT method.

Cell culture

EC9706, a poorly differentiated ESCC cell line, was obtained from the Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific Inc., Waltham, USA). Cultures were maintained in a humidified incubator with 5% CO2 at 37 °C.

Plasmid construction and lentiviral transfection

The IQGAP2 overexpression construct (pCDH-MSCV-MCS-IQGAP2) and the short hairpin RNA (shRNA) construct targeting IQGAP2 (pHY-LV-KD-IQGAP2) were synthesized and obtained from Sangon Biotech (Shanghai, China). Lentiviral packaging and production were carried out by Genomeditech (Shanghai, China). EC9706 cells were transduced with either the IQGAP2 overexpression lentivirus (IQGAP2-OE) or the IQGAP2 knockdown lentivirus (shIQGAP2). Untreated parental cells were used as the control for IQGAP2 overexpression experiments and knockdown experiments. Following infection, cells were subjected to puromycin selection to generate stable 1QGAP2-overexpressing and IQGAP2-knockdown cell lines.

Western blot

EC9706 cells were lysed using RIPA buffer supplemented with a 1× protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, USA) and 1% phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, USA) to preserve protein integrity. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of total protein (20 μg per lane) were separated by SDS-polyacrylamide gel electrophoresis (Thermo Fisher Scientific) and subsequently transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, USA).

Membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with the following primary antibodies: anti-IQGAP2 (#MA5-44712, Thermo Fisher, 1:1,500), anti-MEK (#9122, CST, 1:1,000), anti-phospho-MEK (Ser217/221, #9154, CST, 1:1,000), anti-ERK (#4695, CST, 1:2000), anti-phospho-ERK (Thr202/Tyr204, #4370, CST, 1:1,000), and anti-GAPDH (#2118, CST, 1:2000). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, and bands were visualized using enhanced chemiluminescence (Sigma-Aldrich). Band intensity was quantified using ImageJ software.

CCK-8 assay

To assess the proliferative capacity of EC9706 cells with IQGAP2 overexpression or knockdown, cells were seeded into 96-well plates at a density of 1 × 103 cells per well. Cell viability was measured at 0, 24, 48, and 72 h using the Cell Counting Kit-8 (CCK-8; Beyotime). At each time point, 10 μL of CCK-8 reagent was added to each well and incubated for the recommended duration. Absorbance was then measured at 450 nm using a microplate reader, serving as a quantitative indicator of cell viability. The 0-h reading, obtained after cells adhered to the plate, served as the baseline. MEK-specific inhibitor rescue experiment was conducted to confirm pathway dependence. We performed a gold-standard rescue experiment using the MEK-specific inhibitor PD98059 (10 μM) in IQGAP2-knockdown EC9706 cells, with three groups set as follows: shControl + DMSO, shIQGAP2 + DMSO, shIQGAP2 + PD98059.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9.0 (https://www.graphpad.com/). Continuous variables were expressed as mean ± standard deviation (SD). Comparisons between groups were conducted using Student’s t-test (two-tailed, unpaired) or two-way ANOVA, as appropriate. P-values less than 0.05 were considered statistically significant. In this study, GO enrichment analysis was performed using the clusterProfiler R package (version 4.8.3), and the org. Hs.e.g.,.db R package (version 3.18.0) was used for human gene ID conversion.