FOXF2 may inhibit esophageal squamous cell carcinoma growth and metastasis by regulating the EZR-ERBB2 axis

Introduction

Esophageal cancer (EC) is the seventh most common cancer worldwide, with more than 50% of patients being of Chinese ethnicity (1). In China, more than 90% of the EC cases are esophageal squamous cell carcinoma (ESCC) (2). In addition, patients with ESCC are usually diagnosed at a late stage and do not benefit from targeted therapies due to the lack of biomarkers, which facilitate early diagnosis and precise treatment, leading to a poor prognosis. Despite significant advances in surgical technique, ESCC has a poor outlook, and the 5-year survival rate is less than 30% (3). Therefore, there is an urgent need to develop novel prognostic biomarkers and therapeutic targets for ESCC.

FOXF2 is a member of the transcription factor FOX family proteins. In recent years, several studies have indicated that FOXF2 may play a critical role in tumorigenesis, progression, and therapeutic resistance. For example, in breast cancer, a deficiency in FOXF2 activates the VEGF-C/VEGFR3 signaling pathway and facilitates lymphangiogenesis and metastasis (4). Higashimori et al. found that FOXF2 expression is lower in gastric cancer than in normal mucosa and that the knockdown of FOXF2 expression promotes proliferation and migration ability of gastric cancer cell line (5). Moreover, FOXF2 may be critical for Hedgehog/Wnt feedback signaling and for maintaining intestinal homeostasis, with suppressed function of FOXF2 being associated with the tumorigenesis of colorectal cancer (6). Similar results have been reported for other human cancers (7,8). While the FOXF2 gene has been implicated in various cancer types, the precise mechanisms underlying its action remain to be fully elucidated. In the context of breast cancer, its role appears to be contingent upon the specific tumor subtype (9). Current research predominantly concentrates on the gene’s biological function as a transcription factor. Nevertheless, the intricate mechanisms by which FOXF2 exerts its tumor-inhibitory effects, particularly within the diverse tumor microenvironments, warrant further in-depth investigation. In our previous study, we observed that FOXF2 had a lower expression in ESCC. In addition, patients with ESCC with lower FOXF2 expression were associated with a more advanced stage of disease and shorter survival (10).

We subsequently conducted this study, in which knockdown of FOXF2 expression significantly enhanced the growth, invasion, and metastasis of ESCC cells both in vitro and in vivo. We further identified EZR as a downstream gene of FOXF2. FOXF2 may bind to the promoter of EZR and inhibit its transcriptional expression, resulting in the suppressed ERBB2 signal function and ultimately halting ESCC growth and metastasis. Our research addresses a significant gap by providing an experimental exploration of the FOXF2 gene’s biological function in the context of ESCC. Notably, our study introduces a novel finding that the EZR gene potentially participates in the modulation of the ERBB2 signaling cascade, a role previously attributed to the FOXF2 gene. This involvement suggests a critical contribution to the pathogenesis and progression of ESCC. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2365/rc).

Methods

Data collection

A range of open-source, publicly accessible databases were utilized in our research. For instance, we accessed RNA-seq data related to esophageal cancer patients from The Cancer Genome Atlas (TCGA) and the University of California Santa Cruz (UCSC) databases. Additionally, we employed the R packages “TFBSTools”, “JASPAR”, and “PROMO” (R version 4.4.0) for our analysis. To further analyze protein localization, subcellular localization, function, and interactions, we utilized the GoMiner (11), GeneCards (https://www.genecards.org/), Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, https://string-db.org/), and COMPARTMENTS subcellular localization databases (https://compartments.jensenlab.org). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Cell lines

Ten different ESCC cell lines, including KYSE30, KYSE140, KYSE410, KYSE510, HK, TE1, KYSE180, EC18, EC109, and CE81t, were obtained from Sun Yat-sen University Cancer Center, Guangdong Esophageal Cancer Institute, Guangzhou, China. All cell lines were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco) and 100 IU/mL of penicillin and 100 mg/mL of streptomycin (Gibco) and incubated at 37 ℃ in a humidified incubator containing 5% CO₂.

RNA isolation and real-time quantitative reverse-transcription polymerase chain reaction

Total RNA was isolated using Revert Aid First Strand cDNA Synthesis (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed using a Power SYBR Green PCR Master Mix Kit (Applied Biosystems Inc., Thermo Fisher Scientific, Waltham, MA, USA) on a real-time PCR system. The relative expression of messenger RNA (mRNA) in EC cells (fold) was calculated using the 2^-ΔCt method and normalized to GAPDH.

Western blot analysis

The cells were subjected to lysis using a lysis buffer and were followed by centrifugation at 4 ℃ to isolate and retrieve the proteins present in the supernatant fraction. Protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit (Pierce, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. For Western blot analysis, equal amounts of protein extract were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Pall Corp., Port Washington, NY, USA). The PVDF membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween20 for 1 hour and incubated overnight with primary antibodies according to the manufacturer’s instructions. Antibodies of anti-EZR (1:1,000; CST, 3145), antibodies of anti-FOXF2 (1:1,000; Abcam, ab198283) were purchased and used.

Plasmid transfection

The FOXF2 expression of each ESCC cell line was analyzed by qRT-PCR and Western blot analysis (Figure 1A). The ESCC cells with higher FOXF2 expression (CE81t) were transfected with lentivirus carrying FOXF2 short hairpin RNA (shRNA) and FOXF2 control (CON) vectors (GeneChem Co., Shanghai, China) to knockdown the expression of FOXF2; ESCC cells with lower FOXF2 expression (KYSE30) were transfected with lentivirus carrying FOXF2-ORF (open reading frame) and FOXF2-CON vectors (GeneChem Co.) to overexpress FOXF2. Stably transfected cell lines, namely KYSE30-ORF, KYSE30-CON, CE81t-SH, and CE81t-CON, were established via the addition of puromycin.

Figure 1 FOXF2 inhibited ESCC cell proliferation, invasion, and metastasis. (A) FOXF2 expression in different EC cell lines. (B) KYSE30 was transfected with FOXF2-ORF or control vector, CE81t was transfected with FOXF2-SH or control vector, and the expression of FOXF2 was detected by qRT-PCR and Western blotting. (C-F) Cells with lower FOXF2 expression demonstrated enhanced cell growth, invasion, and metastasis. (D) Clone formation and (F) wound-healing assay. *, P<0.05; **, P<0.01; ***, P<0.001. ORF, open reading frame; CON, control; SH, short hairpin RNA; ESCC, esophageal squamous cell carcinoma; EC, esophageal cancer; qRT-PCR, real-time quantitative reverse-transcription polymerase chain reaction.

To evaluate the interaction between FOXF2 and EZR, CE81t-FOXF2SH was transfected with a lentivirus carrying EZR-shRNA and EZR-CON vector (GeneChem Co.) to knockdown the expression of FOXF2. Stably transfected cell lines, namely CE81t-FOXF2SH-EZRCON and CE81t-FOXF2SH-EZRSH, were established via the addition of chloramphenicol.

Cell Counting Kit-8 analysis

The ESCC cells were seeded onto 96-well plates and incubated for 48 hours. Subsequently, Cell Counting Kit-8 (10 µL) was added into the culture medium for 4 hours, and the optical density (OD) value at 450 nm was detected using a microplate reader (Elx808; BioTek Instruments, Winooski, VT, USA).

Colony formation assay

A total of 2,000 viable ESCC cells were seeded into six-well plates and incubated for 10 days to allow for growth. After incubation, the colonies were fixed with phosphate-buffered saline (PBS) and stained with 1% crystal violet for 30 minutes.

Scratch assay

A scratch assay was performed to study cell migration changes. Trypsinized cells were seeded into six-well plates and grown until full confluence. A straight scratch was created with a sterile pipette tip. Subsequently, the cells were incubated with fresh complete medium. The area of the wound closed was calculated at indicated time points.

Migration and invasion assay

For the migration or invasion assay, 2,000 cells were incubated in the upper chamber with solid growth factor-reduced Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), with the lower chamber filled with complete medium. Cells were incubated for 12 hours at 37 ℃ to allow for migration. Next, the cells that had migrated through the filter pores from the underside of the membrane were fixed and stained.

Luciferase assay

Cells were seeded into six-well plates, and then FOXF2 overexpression plasmid and EZR promoter-driven luciferase plasmids were simultaneously cotransfected into the cells via Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA). After 48 hours of transfection, a luciferase assay kit was applied to examine the luciferase expression in the cells according to the manufacturer’s instructions of Lipofectamine 2000.

Functional enrichment analysis

Metascape (http://metascape.org) is a free, efficient, effective, user-friendly gene-list analysis tool for gene annotation and analysis (12) and based on enriched biological pathways and protein complexes contained within the OMICs datasets. Metascape integrates functional enrichment, interactome analysis, and gene annotation, serving as a comprehensive tool to elucidate shared and distinct pathways across various independent target-discovery studies. In this study, Metascape was used to conduct pathway and process enrichment analysis of the top 100 genes positively correlated with EZR in multiple types of cancer. Gene Ontology (GO) terms for biological processes, cellular component, and molecular function categories, as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, were enriched using the Metascape online tool.

Xenograft mouse model

Four-week-old male C57BL/6 nude mice were purchased for this experiment (Cyagen, Guangzhou, China). In the subcutaneous xenograft model, 0.5×10⁷ stable ESCC cells were subcutaneously injected into the abdomen of nude mice. All nude mice were harvested on the 21st day after tumor inoculation and euthanized on the 28th day. A protocol was prepared before the study without registration. Animal experiments were performed under a project license (No. L2021ZSLYEC-106) granted by the Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University, in compliance with the national guidelines for the care and use of animals.

Statistical analysis

Experiments were repeated three times, and data are presented as the mean ± standard deviation. Data were analyzed using SPSS version 19.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 8.0 (GraphPad Software, Boston, MA, USA). One-way analysis of variance followed by the Tukey post hoc test was used to analyze differences between multiple groups, while the Student t-test was applied to assess differences between two groups. P<0.05 indicated a statistically significant difference.

Results

FOXF2 promoted ESCC growth, metastasis, and drug resistance

The FOXF2 expression of each ESCC cell line was analyzed via qRT-PCR and Western blot analysis (Figure 1A). CE81t cells exhibited the highest expression level of FOXF2 among the ESCC cell lines, while KYSE30 exhibited the slowest expression level. We knocked down the FOXF2 expression in CE81t (CE81t-SH and CE81t-CON) cells and overexpressed FOXF2 in KYSE30 (KYSE30-ORF and KYSE30-CON) cells. The efficiency of FOXF2 transfection was assessed via qRT-PCR and Western blot analysis (Figure 1B). In the cell viability assay, ESCC cells with a relative low level of FOXF2 expression (KYSE30-CON and CE81t-SH) had a greater proliferative (Figure 1C), clone formation (Figure 1D), migration and invasion ability (Figure 1E), and wound-healing ability (Figure 1F).

EZR as a potential downstream gene of FOXF2

From TCGA database, we extracted the genome and transcriptome information of 184 cases of ESCC and analyzed the potential factor coexpression with FOXF2. A positive association with FOXF2 expression was observed in 1,518 genes, and a negative association with FOXF2 expression was observed in 1,270 genes (Figure 2A). Through the GoMiner database and GeneCard database, we identified the localization and function of the protein products of these candidate genes. Using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, we evaluated the gene-protein interaction relationship and selected EZR as the key downstream gene of FOXF2 (Figure 2B,2C).

Figure 2 FOXF2 gene may be involved in the occurrence and development of tumors through various regulatory modes. (A) The color represents the Z-score, with blue indicating low expression and red indicating high expression, which provides a comparison of how the expression of each gene changes under different conditions. (B) Gene classifications into categories of biological processes (colored red), cellular components (colored blue), and molecular functions (colored green) are presented. In these classifications, the height of each bar corresponds to the number of genes involved. (C) Scatter plot of FOXF2 association results with Pearson correlation coefficient on the horizontal axis and −log₁₀(P value) on the vertical axis. The red dots denote statistically significant positive correlations with the FOXF2 gene, whereas the green dots indicate statistically significant negative correlations.

FOXF2 could inhibit the transcription through biding the promoter of EZR

Based on the COMPARTMENTS subcellular localization database (13), we predicted the subcellular locations of FOXF2 and EZR. In Figure 3A, the confidence of the evidence is color coded, ranging from light green [1] for low confidence to dark green [5] for high confidence, while white [0] indicates an absence of localization evidence. The FOXF2 protein was primarily located in the nucleus, and the EZR protein was enriched in the cytosol, plasma membrane, and nucleus.

Figure 3 FOXF2 inhibited transcription through biding the promoter of EZR. (A) Subcellular locations of the FOXF2 and EZR. (B) FOXF2 could bind to the promoter region of EZR. The x-axis represents the sequence positions from the 5' to 3' end on the DNA. (C) The luciferase relative activity of firefly/Renilla indicated that the FOXF2 protein was a negative regulator of the EZR promoter, which verified the direct transcriptional inhibition of EZR by FOXF2. (D,E) According to the Western blotting and qRT-PCR analysis, FOXF2 expression was negatively correlated with EZR. **, P<0.01; ***, P<0.001. ORF, open reading frame; CON, control; SH, short hairpin RNA; qRT-PCR, real-time quantitative reverse-transcription polymerase chain reaction.

The sequence of the EZR promoter region was evaluated by a review of the PubMed database and verified in the UCSC database. Prediction of transcription factors binding sites in the EZR promoter region was examined with the “TFBSTools”, “JASPAR”, and “PROMO” packages. As shown in Figure 3B, FOXF2 can bind to the promoter region of EZR.

To further determine whether FOXF2 negatively regulates the EZR gene, an FOXF2 overexpression plasmid was transfected into HEK293T cells. We performed a dual-luciferase reporter (DLR) assay to determine whether FOXF2 could regulate the promoter of the EZR gene. The luciferase relative activity of firefly/Renilla indicated that the FoxF2 protein was a negative regulator of the EZR promoter, confirming that FOXF2 inhibits the transcription of EZR by anchoring at its promoter (Figure 3C). Western blot and qRT-PCR analysis indicated that FOXF2 expression was negatively correlated with EZR, which is accordance with the aforementioned relationship (Figure 3D,3E).

ERBB2 maybe the downstream signaling pathway of EZR

We further analyzed the downstream signaling pathway of EZR through the database and found that there was a significant correlation between the ERBB signaling pathway and EZR (Figure 4A). Further analysis of the mRNA levels in the EC database in TCGA showed that this relationship was particularly evident in ERBB2; that is, EZR was negatively correlated with FOXF2 and positively correlated with ERBB2 (Figure 4B). Western Blot analysis based on the constructed EZR stable knockdown cell line and its control group (CE81t-FOXF2SH-EZRSH vs. CE81t-FOXF2SH-EZRCON) showed that the cell line with the higher EZR expression level had a higher HER2 expression level (Figure 4C). Moreover, the phosphorylation levels of PI3K and Akt protein were high, suggesting that EZR may exert its effect by activating HER2 and its downstream PI3K/Akt signaling pathway.

Figure 4 EZR upregulation activated the ERBB2 signaling pathway. (A) There was a significant correlation between the ERBB signaling pathway and the EZR expression matrix in TCGA database. (B) EZR was negatively correlated with FOXF2 but positively correlated with ERBB2 in the esophageal cancer database of TCGA. (C) Western blot analysis was performed on the stable EZR-knockdown cell line and its control group. FDR, false discovery rate; TPM, transcripts per kilobase of exon model per million mapped reads; CON, control; SH, short hairpin RNA; TCGA, The Cancer Genome Atlas database.

Xenograft mouse model confirmed the impact of the FOXF2/EZR signal pathway on tumor progression

To determine the effect of FOXF2-EZR on tumorigenicity of ESCC in vivo, an EC xenograft model was constructed (Figure 5A). The tumors formed by the FOXF2-knockdown EC cell line (CE81t-FOXF2SH) were much larger and heavier than were those in the control group (CE81t-FOXF2CON) (Figure 5B,5C). Inhibition of EZR protein expression reversed the trend of tumorigenesis in the low FOXF2 protein expression group. The tumors formed by the EZR-knockdown EC cell line (CE81t-FOXF2SH-EZRSH) were much smaller and lighter than were those with normal EZR expression (CE81t-FOXF2SH) (Figure 5B,5C).

Figure 5 The xenograft mouse model confirmed the effect of the FOXF2/EZR signaling pathway on tumor progression. (A) Establishment of the xenograft mouse model with different expressions of FOXF2-EZR. (B,C) The accelerated tumor progression caused by the decrease in FOXF2 expression was inhibited by decreased EZR expression.

Discussion

In this study, we found that FOXF2 would suppress the growth of ESCC by inhibiting EZR expression and by downregulating the related HER2 pathways.

FOXF2 has been demonstrated to be a tumor-suppressor gene in various human cancers. For example, Fang et al. found that suppressed FOXF2 expression is often observed in thyroid cancer and is associated with epithelial-mesenchymal transition (EMT) and metastasis (14). Jia et al. showed that augmenting lung stromal Foxf2 mediates an immunosuppressive milieu and inhibits lung colonization of prostate cancer (15). The diminished expression of FOXF2 protein presents novel opportunities for tumor development. However, the precise factors contributing to this reduction remain to be comprehensively identified. Extensive research has demonstrated that the methylation of gene promoters can lead to the suppression of gene expression. The relationship between the downregulation of the FOXF2 gene in ESCC and its promoter methylation status is a subject of ongoing debate. Some researchers have suggested that FOXF2, classified as a tumor suppressor gene, when hypermethylated at the promoter level, may contribute to oncogenesis and tumor progression. For instance, Tian et al. revealed that methylation within the CpG islands of the FOXF2 proximal promoter region plays a role in the regulation of FOXF2 isoform-specific expression in breast cancer cells (16). Chen et al. examined the methylation status of the FOXF2 gene promoter in tumor tissue from 135 patients with esophageal squamous carcinoma and found that higher levels of methylation showed a poor prognosis (17). These studies underscore the significance of the FOXF2 gene in tumorigenesis and development. Nevertheless, the intricate mechanisms by which FOXF2 is involved in the initiation, progression, and metastasis of ESCC are yet to be fully elucidated. In our previous study, we performed qRT-PCR analysis in 188 resected ESCC samples, including 33 pairs of tumor and non-tumor tissues (10). We found that the FOXF2 mRNA was downregulated in tumor tissues compared to paired non-tumor tissues. Moreover, a lower level FOXF2 mRNA expression was an independent predictor of lymph node metastasis and shorter survival, even after adjustments were made for other confounders. These results were in high accordance with the present study, as ESCC with suppressed FOXF2 expression was associated with enhanced cell proliferation, colony formation, migration, and invasion. Our studies suggest that further research to clarify the role of FOXF2 is warranted.

EZR is a cytoskeletal protein belonging to ezrin-radixin-moesin (ERM) family and contains three major domains: the amino-terminal FERM domain, the α-helical domain, and the carboxy-terminal ERM association domain (C-ERMAD) (18,19). In an inactive closed status, EZR is localized in the cytoplasm where the FERM and C-ERMAD domains bind to each other (20). In an active open status, EZR is recruited to the plasma membrane where the C-ERMAD domain binds to filamentous actin (F-actin) and the FERM domain binds with cell membrane lipids, transmembrane proteins, and other membrane-associated proteins (21). Therefore, EZR is prime bridging protein, regulating various cytoskeleton-membrane interactions and modulating a diverse range of physiological processes (22,23). Overexpression of EZR is frequently observed in various human cancers and is associated with cancer progression and metastasis. Using an in vitro experiment, Zhu et al. found that the overexpression of EZR led to the formation of more metastatic foci in nude mice with liver metastasis (24). Moreover, Xie et al. demonstrated that DOCK1 overexpression can lead to greater aggression in endometrial cancer, with the underlying mechanism potentially involving the upregulation of EZR expression (25). The pivotal role of EZR in modulating cancer progression and metastasis in ESCC has also been indicated in a series of studies. For example, Deng et al. found that although PALM2 overexpression could enhance cell migration in ESCC, knockout of EZR expression could reduce invasiveness (26). In addition, L1CAM was found to upregulate the expression of EZR to enhance the cell growth, migration, and invasiveness of ESCC (27). In this study, we found that FOXF2 could influence the invasiveness of ESCC by regulating the transcriptional expression of EZR. Overall, these findings suggest that EZR might be a potential and promising therapeutic target for ESCC.

ERBB2 is overexpressed and/or amplified in almost 20% of patients with EC (28,29). Anti-ERBB2 therapeutic antibody has long been used in combination with cytotoxic therapy as the standard therapeutic approach for ERBB2-positive gastroesophageal cancer but is associated with poor prognosis in patients with ESCC (30,31). On this basis, future studies should focus on two key areas. The first is clarifying the interaction mechanism between EZR and ERBB2. A recent study has indicated that EZR might interact with ERBB2 through some intermediate element (32). The other is to evaluate the efficacy of the combined application of anti-EZR and anti-ERBB2 therapy for ESCC through in vivo and in vitro experiments.

Conclusions

In conclusion, suppression of FOXF2 expression is associated with enhanced cell growth, migration, and invasion in ESCC.

Acknowledgments

Funding: This study was supported by the National Natural Science Foundation of China (No. 82102955 to Y.Z.Z.).

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2365/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2365/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2365/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2365/coif). Y.Z.Z. reports funding support from the National Natural Science Foundation of China (No. 82102955). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Animal experiments were performed under a project license (No. L2021ZSLYEC-106) granted by the Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University, in compliance with the national guidelines for the care and use of animals

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Gray)