SOX18 knockdown inhibits colorectal cancer metastasis and angiogenesis through regulation of SAPK/JNK and PI3K/AKT/mTOR signaling pathways

Introduction

Colorectal cancer (CRC) is a common malignancy that is the second biggest cause of cancer-related deaths globally due to its high mortality rate (1). In China, CRC mortality rates are the sixth highest among malignant tumors, constituting a severe health risk (2). Tumor recurrence and liver metastases are prevalent in patients with CRC, despite the current treatment procedures that include surgical resection, pre- and postoperative chemotherapy, and molecular-targeting therapeutic agents (3). Therefore, a deeper understanding of the molecular pathways underlying CRC initiation and progression is urgently needed to develop improved preventive, diagnostic, and prognostic strategies.

SOX18 belongs to the SOX protein family, which is involved in numerous biological activities (4). It belongs to the F subfamily, along with SOX7 and SOX17 (5). The SOX18 gene encodes a 384-amino-acid protein and is found on chromosome 20q13.3 (6). SOX18 is extensively expressed in a wide range of fetal and adult tissues, and is essential for cardiogenesis, vasculogenesis, and lymphangiogenesis, and its dysregulation has been linked to disease development (5,7,8). SOX18 has recently been identified as a crucial cancer-related protein in a number of malignancies (9,10). SOX18 expression in tumor tissues is aberrant, and it could be exploited as a diagnostic and prognostic biomarker (11-13). Additionally, SOX18 contributes to tumor progression and metastasis, making it a potential therapeutic target for cancer treatment (14,15).

Previous studies have shown that SOX18 expression is aberrant in CRC tissues, and patients with high SOX18 expression exhibit poorer survival (15). Functional studies have further demonstrated that SOX18 overexpression increases cell proliferation, promotes S-phase cell cycle progression, and reduces apoptosis in CRC cells (16). Additionally, the role of SOX18 in cancer metastasis and angiogenesis has been explored in other tumor types (11). However, despite these preliminary findings, the exact molecular mechanisms by which SOX18 drives CRC progression remain unclear.

SAPK/JNK is a member of the mitogen-activated protein kinase (MAPK) family, and the SAPK/JNK signaling pathway is involved in autophagy, necroptosis, and apoptotic cell death. The SAPK/JNK signaling pathway is activated in response to a variety of factors, including pro-inflammatory cytokines and oxidative stress (17). Several studies have revealed JNK’s crucial involvement in initiating apoptosis, and evidence has also shown JNK’s role in cancer development by boosting cell proliferation and survival (18,19).

PI3K/AKT is a core intracellular signaling pathway governing cell growth, proliferation, differentiation, and migration. AKT, a serine/threonine kinase downstream of PI3K, mediates PI3K‑driven tumor progression (20). In CRC, AKT phosphorylation is closely linked to cell growth and survival, and AKT further activates mTOR to promote metabolism, protein synthesis, and angiogenesis (21). The PI3K/AKT/mTOR pathway is frequently dysregulated in multiple malignancies, including CRC, and controls proliferation, metastasis, survival, and angiogenesis (22,23).

The hypoxia-inducible factor (HIF)-1 heterodimer, which is composed of the HIF-1α and HIF-1β subunits, is a transcription factor that plays a critical role in regulating cellular responses to hypoxia. HIF-1β is constitutively nuclear and oxygen‑insensitive, whereas HIF1α is tightly regulated by oxygen tension (24). When cells are hypoxic, HIF-1α is stabilized and translocated to the nucleus, where it upregulates VEGF and other target genes to promote angiogenesis (25). Importantly, the PI3K/AKT/mTOR pathway is well-established to promote HIF-1α protein synthesis and stability (26), while SAPK/JNK signaling modulates HIF-1α activity and degradation in various cancer contexts (27).

Despite the importance of the SAPK/JNK and PI3K/AKT/mTOR axis in CRC, little is known about the functional relationship between these signaling pathways and the SOX18 gene. Distinct from previous studies focusing on SOX18 in single tumors or single phenotypes, this study innovatively explores whether SOX18 coordinately regulates CRC metastasis and angiogenesis via SAPK/JNK and PI3K/AKT/mTOR dual pathways, aiming to define its pro-tumor mechanism and provide a novel therapeutic target for CRC. In light of the preceding findings indicating a potential stimulatory role for SAPK/JNK, PI3K/AKT/mTOR, and SOX18 in the mechanism of CRC, we investigated its functional role in CRC. We present this article in accordance with the MDAR reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0235/rc).

Methods

Cell lines and treatment

Human umbilical vein endothelial cells (HUVEC, Homo sapiens, CVCL_2959) were grown in RPMI-1640 medium (HyClone, USA). OUMS‑23 (CVCL_3088) and HCT116 (CVCL_0291) human colorectal carcinoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS, Gibco) at 37 ℃ with 5% CO₂ environment. All cell lines were obtained directly from Cellverse Co., Ltd. (Shanghai, China) in December 2024 and passaged for fewer than 4 months prior to experimentation. For treatment, cells were treated for 48 h with either 100 ng/mL anisomycin (MedChemExpress, USA) for JNK pathway rescue or 1 µM MK‑2206 (MedChemExpress) for AKT pathway validation. Cell line authentication was performed by the supplier using short tandem repeat (STR) profiling, and certificates confirming species identity and absence of interspecies contamination were provided with the cell stocks. No reauthentication was required, as cells were used within 6 months of receipt from a certified cell bank.

Cytotoxicity assay

The MTT assay kit (Beyotime, China) was used to assess cell viability. The cells were planted at a density of 1×10⁴ cells/well in a 96-well plate and incubated for 24 h. The cells were then transfected for 48 h with the relevant siRNA with or without a specific agonist. Each well received 10 µL MTT reagent after transfection. After a 4 h incubation period at 37 ℃, the culture medium was carefully aspirated, and 100 µL of dimethyl sulfoxide was added to each well. The plates were then shaken for 10 min to thoroughly dissolve the formazan crystals, and the absorbance was measured at 570 nm using a microplate reader (Molecular Devices SpectraMax iD5, USA). All experiments were performed in triplicate to ensure reproducibility.

RNA isolation and quantitative real-time PCR (qPCR)

Cells were seeded at a density of 5×10⁶ cells per well in a 6-well plate. According to the manufacturer’s instructions, total RNA was isolated using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, China). cDNA was synthesized from 500 ng RNA using PrimeScript™ RT Master Mix (Takara, Japan). Quantitative PCR was performed with Power SYBR™ Green PCR Master Mix (Applied Biosystems, USA) in triplicate on a QuantStudio™ 5 Real-Time PCR System (Thermo Fisher, USA). The GAPDH gene expression was used to standardize the relative gene expression. All experiments were performed in triplicate to ensure reproducibility.

Primer sequences: SOX18-forward: 5'-CGGACGCCTACCTCAACATC-3', SOX18-reverse: 5'-GGTACTTGTCGGGGGAGATG-3'. GAPDH-forward: 5'-GGAGCGAGATCCCTCCAAAAT-3', GAPDH-reverse: 5'-GGCTGTTGTCATACTTCTCATGG-3'.

Western blot assay

The cells were lysed for 30 min on ice in RIPA lysis buffer (Thermo Fisher) containing Protease & Phosphatase Inhibitor Cocktail (MedChemExpress), then centrifuged for 15 min at 4 ℃ at 13,000 rpm. Protein concentration was quantified with the BCA Protein Assay Kit (Thermo Fisher). The proteins were denatured in SDS loading buffer, separated on a 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and electroblotted onto 0.22-µm PVDF membranes. The membranes were then blocked for 1 h at room temperature in 5% (w/v) BSA in Tris-buffered saline with 0.1% (v/v) Tween-20 (TBST), followed by overnight incubation with the primary antibodies at 4 ℃. The membranes were rinsed with TBST and incubated for 1 h at room temperature with HRP-conjugated secondary antibodies: anti-rabbit IgG (CST, USA, 7074S) and anti-mouse IgG (CST, 7076S) diluted 1:5,000 in 5% BSA/TBST. After washing with TBST, Immobilon ECL Ultra western HRP Substrate was used to visualize the proteins recognized by the particular antibodies, and pictures were captured using a G-Box system (Syngene, UK). All experiments were performed in triplicate to ensure reproducibility.

Primary antibody information: SOX18 (Abcam, UK, ab284424, 1:1,000), p-JNK (CST, 4668, 1:1,000), JNK (Santa Cruz Biotechnology, USA, sc-7345, 1:2,000), cleaved Caspase 3 (CST, 9664, 1:1,000), Caspase 3 (Proteintech, USA, 19677-1-AP, 1:2,000), p-AKT (CST, 4060, 1:1,000), AKT (Proteintech, 60203-2-Ig, 1:3,000), p-mTOR (CST, 5536, 1:1,000), mTOR (CST, 2983, 1:2,000), HIF-1α (BD Biosciences, USA, 610959, 1:500), VEGF (Proteintech, 66828-1-Ig, 1:1,000), GAPDH (Proteintech, 60004-1-Ig, 1:5,000).

Plasmids and transfection

Lipofectamine 2000 reagent (Thermo Fisher) was used to transfect plasmids and control-siRNA or SOX18-siRNA into cells according to the manufacturer’s procedure. Transfected cells were collected after 48 h of incubation, and transfection efficiency was evaluated by qPCR and Western blot. All experiments were performed in triplicate to ensure reproducibility.

Transfection sequences: SOX18-siRNA: 5'-GGAUCUACCGUGGACAUCATT-3'; Control-siRNA: 5'-UUCUCCGAACGUGUCACGUTT-3'.

Flow cytometry for apoptosis detection

The Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) was used to identify early apoptosis in the cells. Cells and supernatants were collected after treatment and washed once with PBS before staining with 5 µL Annexin V-FITC and 5 µL PI. Cell death was quantified with a BD flow cytometer and analyzed by FlowJo software. All experiments were performed in triplicate to ensure reproducibility.

Cell migration and invasion assay

Transwell assays (8 µm pore size, 24 wells; Corning, USA) were used to assess the migration and invasion abilities of OUMS-23 and HCT116 cells. Cells were resuspended in serum-free media (5×10⁴/mL) and seeded into the upper chamber (100 µL/well) after being transfected for 48 h. Each hole in the bottom chambers held 500 µL of media containing 10% FBS as a chemoattractant. In the invasion tests, unlike the migratory experiments, the chambers were precoated with 60 µL of Matrigel matrix (dilution 1:6; BD Biosciences). After a 24 h incubation period, cells that had passed through the membrane were fixed for 15 min with 4% paraformaldehyde and stained for 10 min with crystal violet (0.2%). In each chamber, 5 fields were chosen at random for cell counting. All experiments were performed in triplicate to ensure reproducibility.

Endothelial tube-formation assays

HUVECs (1×10⁵ cells/well) were planted in 96-well plates that had been precoated with 200 µL Matrigel (1:1, BD Biosciences) and incubated for 4 h at 37 ℃. The angiogenesis ability was determined by counting the number of branches, loops, and overall length of the cell cords. All experiments were performed in triplicate to ensure reproducibility.

Bioinformatics analysis

TCGA‑CRC dataset was downloaded from The Cancer Genome Atlas (TCGA) database. Gene expression data (RNA‑Seq, FPKM) and corresponding clinical information were extracted. SOX18 expression was dichotomized into high‑ and low‑expression groups using the median value as the cutoff. Kaplan–Meier survival analysis and log‑rank test were performed to compare overall survival (OS) between the two groups. Multivariate Cox proportional hazards regression analysis was used to identify independent prognostic factors. The correlation between SOX18 expression and clinicopathological parameters (pathological stage, T stage) was analyzed using the chi‑square test. All bioinformatics analyses were conducted using R software (v4.2.1).

Statistical analysis

All experiments in this study were performed with three independent biological replicates (n=3). The data were expressed by mean ± standard deviation (SD). Comparison between groups was performed using the Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. P<0.05 was considered statistically significant.

Results

High SOX18 expression is associated with poor prognosis in CRC

To evaluate the clinical significance of SOX18 in CRC, we analyzed the correlation between SOX18 expression, patient prognosis, and clinicopathological characteristics using a clinical cohort (clinicopathological details are shown in Table S1) and the TCGA-CRC dataset. Kaplan-Meier survival analysis revealed that patients in the SOX18 high-expression group exhibited significantly shorter OS compared with those in the low-expression group (P=0.0013; Figure S1A). Multivariate Cox proportional hazards regression analysis confirmed that high SOX18 expression was an independent risk factor for poor prognosis in CRC patients [hazard ratio (HR) =1.63, 95% confidence interval (CI): 1.03–2.58, P=0.039; Figure S1B]. Furthermore, we examined the association between SOX18 expression and In the TCGA cohort, SOX18 expression showed no statistically significant difference across pathological stages (Stage I–IV) (P=0.082; Figure S1C), but was significantly associated with T category (T1–T4) (P=0.043; Figure S1C). These findings indicate that SOX18 expression is closely associated with patient survival, supporting its potential value as a prognostic indicator in CRC.

SOX18 knockdown inhibits proliferation, migration, invasion, and induces apoptosis in CRC cells

To investigate the functional role of SOX18 in CRC progression, we performed SOX18 knockdown in OUMS-23 and HCT116 cells using specific siRNA. qPCR and Western blot analyses confirmed significant downregulation of SOX18 mRNA and protein expression (Figure 1A,1B; Figure S2A,S2B). MTT assays showed that SOX18 knockdown markedly suppressed cell proliferation in a time-dependent manner (Figure 1C; Figure S2C). Transwell assays further demonstrated that silencing SOX18 significantly inhibited cell migration and invasion in both OUMS-23 and HCT116 cells (Figure 1D-1G; Figure S2D-S2G). Concurrently, SOX18 depletion significantly promoted apoptosis, evidenced by increased early apoptotic cell population in flow cytometry (Figure 2A,2B) and elevated cleaved Caspase-3 expression via Western blot, without altering total Caspase-3 levels (Figure 2C,2D). These findings collectively indicate that SOX18 drives CRC malignancy by coordinating the regulation of cell proliferation, migratory/invasive capacities, and apoptosis resistance.

Figure 1 SOX18 knockdown inhibits proliferation, migration, and invasion of OUMS-23 cells. (A) qPCR analysis of SOX18 mRNA expression after transfection with control-siRNA or SOX18-siRNA. (B) Cropped regions of the Western blot showing SOX18 protein levels following siRNA transfection. (C) Cell proliferation was assessed by MTT assay at 24, 48, and 72 h post-transfection. (D,E) Cell migration ability was evaluated by Transwell assay (crystal violet staining, Scale bar = 100 μm); representative images (D) and quantitative analysis (E). (F,G) Cell invasion ability was assessed by Matrigel Transwell assay; representative images (F) and quantitative analysis (G). Data are presented as mean ± SD (n=3). **P<0.01. mRNA, messenger RNA; OD, optical density; qPCR, quantitative real-time polymerase chain reaction; SD, standard deviation; siRNA, small interfering RNA.

Figure 2 SOX18 knockdown induces apoptosis in OUMS-23 cells. (A,B) Flow cytometry analysis of apoptosis using Annexin V-FITC/PI double staining. (A) Representative dot plots showing the distribution of viable (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), and necrotic (Annexin V⁻/PI⁺) cells. (B) Quantitative analysis of the total apoptosis rate (early + late apoptotic cells). (C,D) Cropped regions of the Western blot showing apoptosis-related proteins. (C) Representative images showing cleaved Caspase-3 and total Caspase-3 protein levels. (D) Quantitative analysis of the cleaved Caspase 3/Caspase 3 ratio. Data are presented as mean ± SD (n=3). **P<0.01. FITC, fluorescein isothiocyanate; PI, propidium iodide; SD, standard deviation; siRNA, small interfering RNA.

SOX18 knockdown suppresses angiogenic capacity and downregulates pro-angiogenic factors in endothelial cells

To investigate the role of SOX18 in tumor angiogenesis, functional and mechanistic analyses were performed using HUVECs. Tube formation assays demonstrated significantly impaired angiogenic capacity in SOX18 knockdown groups compared to controls (Figure 3A,3B), evidenced by sparse vascular networks and reduced branch points. Mechanistic investigations revealed that SOX18 knockdown substantially downregulated key pro-angiogenic regulators in HUVECs. Western blot analysis confirmed decreased HIF-1α protein expression with corresponding reduction in its downstream effector VEGF (Figure 3C). qPCR analysis further demonstrated downregulation of HIF-1α mRNA (Figure 3D) and VEGF mRNA (Figure 3E) in SOX18-knockdown HUVECs. These findings suggest SOX18 mediates tumor angiogenesis through regulation of the HIF-1α/VEGF signaling axis.

Figure 3 SOX18 knockdown impairs tube formation and downregulates HIF-1α/VEGF expression in endothelial cells. (A) Representative bright-field images of tube networks formed by HUVECs transfected with control, control-siRNA or SOX18-siRNA on Matrigel-coated plates (Scale bar = 100 μm). (B) Quantitative analysis of tube formation capacity. (C) Cropped regions of the Western blot showing pro-angiogenic factors in HUVEC endothelial cells. (D,E) qPCR analysis of HIF-1α (D) and VEGF (E) mRNA expression levels. Data are presented as mean ± SD (n=3). **P<0.01. HUVEC, human umbilical vein endothelial cells; qPCR, quantitative real-time polymerase chain reaction; SD, standard deviation; siRNA, small interfering RNA.

SOX18 knockdown disrupts oncogenic signaling by suppressing SAPK/JNK and PI3K/AKT/mTOR pathways

To elucidate the molecular mechanisms underlying SOX18 regulation in CRC progression, we examined activation states of key oncogenic signaling pathways. Western blot analysis revealed that SOX18 knockdown in OUMS-23 cells specifically suppressed phosphorylation of signaling molecules (Figure 4A). Compared with control groups, p-JNK expression was significantly reduced in SOX18-knockdown cells (Figure 4B). Concurrent impairment of phosphorylation was observed at critical nodes of the PI3K/AKT/mTOR pathway, manifested by decreased p-AKT and p-mTOR expression (Figure 4C,4D). qPCR analysis demonstrated stable mRNA expression of JNK, AKT, and mTOR across all groups (Figure 4E-4G). These results demonstrate that SOX18 drives malignant tumor progression by modulating phosphorylation-dependent cascades in both SAPK/JNK and PI3K/AKT/mTOR signaling pathways.

Figure 4 SOX18 knockdown suppresses key signaling pathways in cancer cells. (A-D) Cropped regions of the Western blot showing phosphorylation and total protein levels in the SAPK/JNK and PI3K/AKT/mTOR pathways in OUMS-23 cells. Representative images (A) and quantitative analysis of p-JNK/JNK (B), p-AKT/AKT (C), p-mTOR/mTOR (D) protein expression. (E-G) qPCR analysis of JNK (E), AKT (F), and mTOR (G) mRNA expression levels in OUMS-23 cells. Data are presented as mean ± SD (n=3). **P<0.01. mRNA, messenger RNA; qPCR, quantitative real-time polymerase chain reaction; SD, standard deviation; siRNA, small interfering RNA.

JNK pathway activation reverses the tumor-suppressive effects of SOX18 knockdown

To validate the critical role of the SAPK/JNK pathway within the SOX18 regulatory network, SOX18-knockdown OUMS-23 cells were treated with the JNK agonist anisomycin or the AKT inhibitor MK-2206. Western blot analysis showed that SOX18 knockdown significantly decreased the phosphorylation levels of JNK, AKT, and mTOR. Anisomycin treatment selectively restored p-JNK expression without affecting p-AKT or p-mTOR, whereas MK-2206 further suppressed p-AKT and p-mTOR without influencing p-JNK (Figure 5A,5B; Figure S3). Consistent with this post-translational regulation, qPCR analysis demonstrated unaltered JNK mRNA expression (Figure 5C). Functional rescue experiments demonstrated that JNK pathway activation counteracted the tumor-suppressive effects of SOX18 knockdown: MTT assays showed enhanced proliferative capacity (Figure 6A), Transwell migration and invasion assays revealed improved migratory and invasive abilities (Figure 6B-6E), while flow cytometry indicated suppressed apoptosis (Figure 6F,6G), accompanied by reduced cleaved Caspase-3 expression (Figure 6H,6I). In endothelial cell models, anisomycin treatment similarly restored angiogenic capacity (Figure 7A,7B). These results establish the SAPK/JNK pathway as the central mediator coordinating SOX18-regulated tumor malignancy and angiogenesis.

Figure 5 Verification of JNK pathway activation by anisomycin in SOX18-knockdown OUMS-23 cells. (A) Cropped regions of the Western blot showing p‑JNK and total JNK protein levels. (B) Quantification of the p‑JNK/JNK ratio. (C) qPCR analysis of JNK mRNA expression levels. Data are presented as mean ± SD (n=3). **P<0.01. mRNA, messenger RNA; qPCR, quantitative real-time polymerase chain reaction; SD, standard deviation; siRNA, small interfering RNA.

Figure 6 JNK activation reverses the inhibitory effects of SOX18 knockdown on proliferation, migration, invasion, and apoptosis in OUMS-23 cells. (A) Cell proliferation was assessed by MTT assay at 24, 48, and 72 h. (B,C) Cell migration ability was evaluated by Transwell assay (crystal violet staining, Scale bar = 100 µm); representative images (B) and quantitative analysis (C). (D,E) Cell invasion ability was assessed by Matrigel Transwell assay (crystal violet staining, Scale bar = 100 µm); representative images (D) and quantitative analysis (E). (F,G) Apoptosis was analyzed by flow cytometry with Annexin V-FITC/PI double staining; representative images (F) and quantitative analysis (G). (H,I) Cropped regions of the Western blot showing apoptosis-related protein expression; representative images (H) and quantitative analysis (I). Data are presented as mean ± SD (n=3). **P<0.01. FITC, fluorescein isothiocyanate; OD, optical density; PI, propidium iodide; SD, standard deviation; siRNA, small interfering RNA.

Figure 7 JNK activation reverses SOX18 knockdown-impaired tube formation in endothelial cells. (A) Representative bright-field images (Scale bar = 100 µm) of tube networks formed by HUVECs transfected with SOX18-siRNA or SOX18-siRNA combined with the JNK agonist anisomycin on Matrigel-coated plates. (B) Quantitative analysis of tube formation capacity. Data are presented as mean ± SD (n=3). **P<0.01. HUVEC, human umbilical vein endothelial cells; SD, standard deviation; siRNA, small interfering RNA.

Discussion

Metastasis and angiogenesis in CRC significantly contribute to poor prognosis (28). This study demonstrates that SOX18 drives CRC progression and angiogenesis by coordinately regulating both SAPK/JNK and PI3K/AKT/mTOR signaling pathways. Mechanistically, both pathways act as critical downstream effectors of SOX18 in mediating these oncogenic processes.

As a member of the SOXF transcription factor family (29), SOX18 has been implicated in various cancers, yet its regulatory role in CRC progression remained poorly defined. This study identifies a previously unreported dual mechanism through which SOX18 coordinately regulates autonomous tumor cell behaviors and non-autonomous microenvironment remodeling in CRC. Specifically, SOX18 knockdown significantly suppressed cell migration and invasion capabilities, a finding that resonates with the proposed SOX18-PD-L1/CXCL12 pro-metastatic axis previously observed in liver cancer (15). In the context of angiogenesis, our data reveal that SOX18 directly downregulates HIF-1α and VEGF expression within endothelial cells in the tumor microenvironment, leading to a substantial reduction in vascular branching. This contrasts with its well-established role in developmental angiogenesis reported by Hong et al. (30). Our results provide novel insights into tumor-endothelial crosstalk and expand the understanding of SOXF family proteins in tumor angiogenesis (31).

At the signaling level, SOX18 silencing inhibited phosphorylation of both SAPK/JNK and PI3K/AKT/mTOR. Activation of JNK by anisomycin reversed the anti-tumor and anti-angiogenic effects of SOX18 knockdown. Treatment with the AKT inhibitor MK-2206 further suppressed PI3K/AKT/mTOR activity without affecting JNK signaling. Together, these data confirm that both pathways are functionally regulated by SOX18 in CRC cells.

While this study provides evidence that SOX18 promotes CRC metastasis and angiogenesis through SAPK/JNK and PI3K/AKT/mTOR signaling, several limitations should be noted. First, the conclusions are primarily derived from cell-based models, and lack in vivo validation using patient-derived xenografts (PDX) or transgenic mouse models to definitively establish the physiological function of this axis. Second, although JNK rescue experiments confirmed its functional role, the regulatory contribution of the PI3K/AKT/mTOR pathway was only verified by Western blot, without functional rescue assays. Third, angiogenesis assays were performed in HUVEC monoculture, which directly demonstrates the cell‑autonomous pro‑angiogenic role of SOX18 in endothelial cells. Further tube formation assays under CRC cell–HUVEC co‑culture conditions, which better recapitulate the tumor microenvironment, were not performed in this study and will be addressed in future investigations. Finally, the clinical relevance and prognostic value of SOX18 expression levels and JNK activation status in human tumors require further investigation through large-scale cohort studies.

Conclusions

In summary, this study demonstrates that high SOX18 expression correlates with poor prognosis in CRC patients. Mechanistically, SOX18 drives CRC metastasis and angiogenesis by coordinately regulating the SAPK/JNK and PI3K/AKT/mTOR signaling pathways. These findings identify SOX18 and its downstream signaling axes as novel therapeutic targets for metastatic CRC. Targeting SOX18 may provide new opportunities for combination therapy with anti‑angiogenic agents in the clinical management of CRC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0235/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0235/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0235/prf

Funding: This work was financially supported by the Jiangxi Provincial Natural Science Foundation of China (Project No. 20202BABL206077).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0235/coif). The 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.

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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