VWA2 fuels colon cancer growth and immune evasion through Wnt/β-catenin signaling

Summary

Colon adenocarcinoma (COAD) progression involves complex interactions between tumor cell intrinsic pathways and immune evasion. Here, we show that VWA2 is markedly upregulated in COAD and correlates with poor prognosis across multiple cancer types. Using patient-derived data, cell-based assays, and in vivo models, we find that VWA2 binds β-catenin, promotes its nuclear translocation, and activates Wnt signaling. This axis drives transcription of CEMIP via TCF4, enhancing tumor cell proliferation and resistance to apoptosis, while simultaneously upregulating PD-L1 expression. The latter impairs CD8⁺ T cell function, revealing a dual pro-tumor role: direct growth promotion and immune suppression. These findings position VWA2 as a nodal regulator of Wnt-dependent malignant progression and immune evasion, suggesting that targeting this pathway may offer a therapeutic strategy to both curb tumor aggressiveness and restore anti-tumor immunity in COAD.

Graphical abstract

Highlights

• •VWA2 is upregulated in COAD and linked to poor prognosis across cancers
• •VWA2 promotes proliferation and apoptosis resistance in COAD cells
• •VWA2 binds β-catenin, activates Wnt signaling, and upregulates CEMIP via TCF4
• •VWA2 upregulates PD-L1 via Wnt/β-catenin to impair CD8⁺ T cell function

Molecular biology; Immunology

Introduction

Colorectal cancer (CRC) ranks among the most prevalent malignancies and is a leading cause of cancer-related mortality globally.^1,2 Among all types of CRC, colon adenocarcinoma (COAD) is the most common histological type, accounting for as high as 90% of the occurrences. Multiple factors contribute to an elevated risk of developing COAD, including aging, smoking, dietary habits, obesity, and physical inactivity.^3,4 Despite the rising incidence of early-onset COAD in individuals under 50 years of age worldwide, about 30% of patients are diagnosed at advanced stages due to the asymptomatic nature of the disease in its early phases.^5,6 Thus, improving patient survival requires a thorough understanding of how COAD cells grow and avoid apoptosis.

von Willebrand factor A domain containing 2 (VWA2), a member of the von Willebrand factor A-like domain protein superfamily, functions as a structural component in basement membranes or anchoring structures.^7,8,9 It has been suggested that the increased risk of Alzheimer’s disease in sporadic cases is associated with homozygous and complex heterozygous missense mutations in VWA2.¹⁰ In congenital anomalies of the kidney and urinary tract (CAKUT), a homozygous missense mutation in VWA2 was identified, which resulted in decreased translocation of monomeric VWA2 protein and increased translocation of aggregated VWA2 protein into the extracellular space.¹¹ VWA2 expression has been reported to be elevated in the majority of COAD cases, suggesting its potential as a specific biomarker.¹² In COAD cell lines, hypermethylation of VWA2 was observed and found to correlate with the expression of Myc and Myc-target genes.¹² The strong correlation with Myc is considered a crucial factor in VWA2’s role in promoting tumor malignancy. Nevertheless, the precise role and underlying mechanisms of VWA2 in enhancing the malignant phenotype of COAD require further investigation.

In our study, we found that VWA2 was upregulated in COAD cells and tissues, and this upregulation was linked to poor clinical prognosis in patients with COAD. We determined that VWA2 facilitates tumor proliferation and inhibits apoptosis by binding β-catenin and promoting β-catenin nuclear translocation. Activation of the Wnt/β-catenin signaling pathway subsequently increased CEMIP expression through TCF4 transcription. In addition, our study identified that VWA2 impacted the tumor microenvironment by inhibiting the function of CD8⁺ T cells through the upregulation of PD-L1 expression in tumor cells. These findings suggest that VWA2 may serve as a prognostic marker and a potential therapeutic target for COAD.

Results

VWA2 is upregulated in COAD and associated with poor prognosis

To explore the expression and prognostic value of VWA2 in COAD, we analyzed The Cancer Genome Atlas (TCGA) database. The result showed that the expression of VWA2 in tumors was significantly higher than that in normal tissues in colon adenocarcinoma (COAD), breast invasive carcinoma (BRCA), lung adenocarcinoma (LUAD), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), and rectum adenocarcinoma (READ) (Figure 1A). We also found that high expression of VWA2 portended a poor prognosis in COAD (Figure 1B). VWA2 was then detected in 60 colon cancer tissues and paired with normal tissues through immunohistochemical staining (IHC) using anti-VWA2 primary antibody. It showed that VWA2 expression was elevated in tumor tissues, and was higher in tumors with stages III and IV compared with stages I and II (Figure 1C). Kaplan-Meier survival analysis indicated that high VWA2 expression was associated with poor prognosis of patients with COAD(Figure 1D). Besides, high VWA2 expression was related to poor prognosis in liver, lung, and pancreatic cancers according to the Kaplan-Meier plotter database (Figure 1E). To sum up, these results suggest that VWA2 has important clinical value in colon cancer and a variety of tumors.

Figure 1.

VWA2 expression and prognosis in GC and other cancers

(A) VWA2 expression was upregulated in COAD, BRCA, LUAD, OV, PAAD, and READ according to TCGA data.

(B) Kaplan-Meier analyses of correlations between VWA2 expression and overall survival of CRC patients according to TCGA data.

(C) VWA2 IHC staining was increased in tumor tissues compared with normal tissues, according to the COAD cohort. Anti-VWA2 primary antibody (Proteintech, Cat# 8714-1-AP, diluted 1:100) was used.

(D) Kaplan-Meier analyses of correlations between VWA2 expression and overall survival of 60 patients with COAD, according to IHC staining.

(E) High expressed VWA2 was associated with poor prognosis in Liver cancer, Lung cancer and Pancreatic cancer.

Data are represented as mean ± SEM. ∗p < 0.05 by Student’s t test (A and C) or by one-way ANOVA (C).

VWA2 promotes the malignant phenotype of COAD in vivo and in vitro

We assessed VWA2 expression in NCM460 and various CRC cell lines. Based on these results, LoVo cells (with low endogenous VWA2 expression) and SW620 cells (with high endogenous VWA2 expression) were selected for subsequent in vitro and in vivo experiments (Figures 2A and S1A). Firstly, we overexpressed VWA2 in LoVo cells and knocked down VWA2 in SW620 cells (Figures 2B and 2C). We found that VWA2 overexpression was able to enhance the anti-apoptotic ability and proliferation ability of LoVo cells (Figures 2D, 2E, and S1B). In this experiment, the control cells exhibited a slightly higher baseline apoptosis rate than usual due to culture conditions; however, all groups were processed in parallel, making the relative intergroup comparisons reliable. Downregulation of VWA2 was capable of decreasing the anti-apoptotic ability and proliferation ability of SW620 cells (Figures 2D and 2E). We further established an orthotopic allograft model by inoculating CT26 cells (with or without VWA2 knockdown, Figure S1C) into the cecal wall of BALB/c mice. Both primary cecal tumor growth and distant metastasis were monitored by bioluminescence imaging. Results showed that VWA2 knockdown significantly inhibited the growth of orthotopic tumors and reduced the incidence and burden of metastases (Figures 2F and 2G). In addition, we inoculated SW620 cells with VWA2 knockdown into nude mice to establish subcutaneous xenograft models. Downregulation of VWA2 significantly inhibited tumor proliferation (Figure 2H). Ki67 and cleaved caspase-3 staining demonstrated that knockdown of VWA2 inhibited tumor proliferation and promoted tumor apoptosis (Figure 2I). These results suggest that VWA2 plays an important role in the proliferation and anti-apoptosis of COAD.

Figure 2.

The role of VWA2 in the malignant phenotype of COAD

(A) VWA2 expression in NCM460 and COAD cell lines.

(B and C) VWA2 mRNA and protein expression in LoVo and SW620 cells when VWA2 was upregulated or downregulated.

(D) Apoptosis analysis of LoVo or SW620 cells when VWA2 was upregulated or downregulated.

(E) Cell proliferation analysis of LoVo or SW620 cells when VWA2 was upregulated or downregulated.

(F) Representative bioluminescence images and quantitative analysis of orthotopic allograft tumors (CT26 cells) and their metastases.

(G) Representative image and statistics of orthotopic allograft tumors.

(H) Representative image and weight and volume of subcutaneous xenograft tumors when VWA2 was downregulated.

(I) Ki67 and cleaved caspase-3 staining of subcutaneous xenograft tumors.

n = 3 technical replicates from 3 biological replicates (A–E). n = 3 technical replicates from 5 biological replicates (F, G, H, and I). Data are represented as mean ± SD. p < 0.05 by Student’s t test (B, D, F, G, H, and I), one-way ANOVA (B and D), or two-way ANOVA (E and H).

VWA2 activates Wnt signaling pathway through binding β-catenin

To investigate the mechanism of VWA2 affecting tumor growth and apoptosis, we divided patients with COAD in TCGA into two groups according to high expression and low expression of VWA2 (Figure 3A). There were 2,436 genes increased and 819 genes decreased in the two groups (Figure 3A). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of different expressed genes revealed that the Wnt signaling pathway was the most significant enrichment pathway (Figure 3B). β-Catenin was the key regulator in the Wnt signaling pathway and was found to increase when VWA2 was overexpressed and decreased when VWA2 was knocked down (Figure 3C). We then overexpressed or knocked down β-catenin in LoVo or SW620 cells and found that β-catenin promoted anti-apoptosis and proliferation of COAD cells (Figures 3D and 3E). Furthermore, β-catenin overexpression reversed the decreased anti-apoptosis and proliferation induced by the knockdown of VWA2 (Figure 3F). Wnt3a was used to activate the Wnt signaling pathway, and it incited that the Wnt3a supplement could also reverse the decreased anti-apoptosis and proliferation induced by VWA2 knockdown (Figure 3G). Notably, co-immunoprecipitation (coIP) assay and GST-pull-down assay confirmed that VWA2 bound β-catenin (Figures 3H, S1D, and S1E). We also found VWA2 enhanced β-catenin nuclear translocation (Figure 3I). β-Catenin nuclear translocation is an important prerequisite for its function. Our findings demonstrated that VWA2 promoted the entry of β-catenin into the nucleus to induce tumor progression. Finally, we found that knocking down VWA2 decreased the expression of anti-apoptotic BCL2 and enhanced the expression of pro-apoptotic BAX, which could be reversed by overexpression of β-catenin or addition of Wnt3a (Figures 3J and 3K). These results indicate that VWA2 activates the Wnt signaling pathway by binding β-catenin to promote COAD malignancy.

Figure 3.

The signaling pathway activated by VWA2

(A) Volcano map of different expressed genes in VWA2 low and high expressed COAD patients.

(B) KEGG pathway enrichment of different expressed genes when VWA2 was low or high expressed in COAD patients.

(C) β-Catenin mRNA and protein level when VWA2 was upregulated or downregulated.

(D) Apoptosis analysis of LoVo and SW620 cells when VWA2 was upregulated or downregulated.

(E) Cell proliferation of LoVo and SW620 cells when VWA2 was upregulated or downregulated.

(F) Apoptosis and cell proliferation of SW620 cells when VWA2 was downregulated and β-catenin was upregulated.

(G) Apoptosis and cell proliferation of LoVo cells when VWA2 was upregulated and β-catenin was wnt3a supplement.

(H) coIP analysis of the binding of VWA2 and β-catenin.

(I) Subcellular distribution of β-catenin protein in LoVo cells after VWA2 overexpression. GAPDH and Histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively.

(J and K) BCL2 and BAX mRNA expression in SW620 cells when VWA2 was downregulated and β-catenin was upregulated (or wnt3a was supplemented).

n = 3 technical replicates from 3 biological replicates for all. Data are represented as mean ± SD. p < 0.05 by Student’s t test (C and D), one-way ANOVA (C, D, F, G, J, and K), or two-way ANOVA (E, F, and G).

VWA2 promotes CEMIP expression through Wnt/β-catenin/TCF4 pathway

To identify the genes regulated by VWA2, we searched the STRING database and found 10 genes associated with VWA2, among which CEMIP had a close correlation with VWA2 (Figure 4A). We verified that CEMIP expression increased when VWA2 was overexpressed in LoVo cells and decreased When VWA2 was knocked down in SW620 cells (Figure 4B). According to the GEPIA database, VWA2 expression was highly positively correlated with CEMIP expression (Figure 4C). CEMIP was observed to be highly expressed in COAD and associated with poor prognosis in patients with COAD (Figures 4D and 4E). We then overexpressed VWA2 in LoVo cells and silenced VWA2 in SW620 cells, and apoptosis assays showed that CEMIP promoted COAD anti-apoptosis (Figures 4F and 4G). Inhibition of CEMIP could diminish the increased apoptosis caused by VWA2 overexpression, and knockdown of CEMIP diminished decreased apoptosis caused by VWA2 underexpression (Figure 4H). Further, we found that VWA2 regulated CEMIP expression through β-catenin (Figure 4I). TCF4 is the key transcription factor downstream of the Wnt/β-catenin signaling pathway. It was found that there were two predicted binding sites in CEMIP promoter (Figure 4J), and VWA2 promoted TCF4 expression (Figure 4K). Dual-Luciferase reporter assay and binding site mutation proved that TCF4 could bind CEMIP promoter (Figure 4L). The apoptosis experiment indicated that CEMIP could diminish the apoptosis effect of β-catenin on COAD cells (Figure 4M). The aforementioned results suggest that VWA2 promotes CEMIP expression through the Wnt/β-catenin/TCF4 pathway, which promotes COAD cell anti-apoptosis.

Figure 4.

VWA2 regulates CEMIP expression by activating its transcription

(A) STRING database analysis of genes associated with VWA2.

(B) CEMIP mRNA and protein expression in LoVo or SW620 cells when VWA2 was upregulated or downregulated.

(C) Correlation of VWA2 and CEMIP expression in COAD.

(D) CEMIP expression in tumors and normal tissues of patients with COAD.

(E) Kaplan-Meier analyses of correlations between CEMIP expression and overall survival of patients with COAD.

(F) CEMIP mRNA and protein expression in LoVo or SW620 cells when CEMIP was upregulated or downregulated.

(G) Apoptosis analysis of LoVo or SW620 cells when CEMIP was upregulated or downregulated.

(H) Apoptosis analysis of LoVo and SW620 cells when VWA2 was upregulated or downregulated with CEMIP downregulated or upregulated.

(I) CEMIP mRNA expression of LoVo and SW620 cells when VWA2 was upregulated or downregulated with β-catenin downregulated or upregulated.

(J) The TCF4 motif and the predicted binding sites in CEMIP promoter.

(K) TCF4 mRNA and protein expression in LoVo cells when VWA2 was upregulated.

(L) Dual-luciferase reporter assay analysis of TCF4 binding CEMIP promoter.

(M) Apoptosis analysis of LoVo and SW620 cells when β-catenin was upregulated or downregulated with CEMIP downregulated or upregulated.

n = 3 technical replicates from 3 biological replicates for all. Data are represented as mean ± SD. ∗p < 0.05 by Student’s t test (B, D, F, G, K, and L) or one-way ANOVA (B, G, H, I, and M).

VWA2 inhibits CD8⁺ T cells function through overexpressing PD-L1

Cancer immunotherapy has been validated to be an important approach for cancer treatment. To explore the effect of VWA2 on the tumor microenvironment, we analyzed immune cell infiltration in COAD with high or low VWA2 expression. The results showed significant changes in infiltration of CD8⁺ T cell, B cell, natural killer (NK) cell, and monocyte (Figure 5A). Since CD8⁺ T cells are the most important cells for tumor killing and were negatively correlated with VWA2 in COAD (Figure 5B), we chose CD8⁺ T cells for further study. Firstly, we found that high expression of β-catenin could also decrease CD8⁺ T cell infiltration (Figure 5C). Tumor cells could highly express immune checkpoints, such as PD-L1, causing T cells to lose the activity of killing tumor cells so as to achieve immune escape. We detected PD-L1 expression and found that VWA2 promoted PD-L1 expression, which was mediated by β-catenin (Figures 5D and 5E). We then co-cultured CD8⁺ T cells with colon cancer cells (Figures S1F and S1G) and found that overexpressing VWA2 in LoVo cells inhibited CD8⁺ T cells function, and knockdown of VWA2 in SW620 cells enhanced CD8⁺ T cells function (Figure 5F). We also observed that PD-1 expression on CD8⁺ T cells was similarly regulated by VWA2 expression in the tumor cells (Figure 5G). Furthermore, the VWA2-mediated alterations in CD8⁺ T cell function were dependent on β-catenin (Figure 5H). Consistently, in mouse tumor tissues, we also observed that VWA2 expression affected the infiltration of CD8⁺ T cells (Figure 5I). IHC staining showed that CD8⁺ T cells infiltration was negatively correlated with VWA2 expression in COAD (Figure 5J). Prognosis analysis showed that patients with low CD8⁺ T cells infiltration and high VWA2 expression had worse prognosis (Figure 5J). In conclusion, we confirm that VWA2 inhibits CD8⁺ T cell infiltration and function, therefore leading to adverse outcomes for patients with COAD.

Figure 5.

VWA2 inhibits CD8⁺ T cells infiltration in COAD

(A) Infiltration of immune cells in VWA2 low and high expressed COAD according to TCGA data.

(B) Correlation of VWA2 and CD8⁺ T cells infiltration in patients with COAD in Timer 2.0 database.

(C) Infiltration of CD8⁺ T cells in β-catenin low and high expressed according to TCGA data.

(D) PD-L1 mRNA and protein expression of LoVo and SW620 cells when VWA2 was upregulated or downregulated.

(E) PD-L1 mRNA and protein expression of LoVo and SW620 cells when VWA2 was upregulated or downregulated with β-catenin downregulated or upregulated.

(F) Flow cytometry analysis of cytokine secretion of CD8⁺ T cells co-cultured with LoVo or SW620 cells.

(G) Flow cytometry analysis of PD-1 expression of CD8⁺ T cells co-cultured with LoVo or SW620 cells.

(H) Flow cytometry analysis of cytokine secretion of CD8⁺ T cells co-cultured with LoVo or SW620 cells when VWA2 was upregulated or downregulated with β-catenin downregulated or upregulated.

(I) CD8⁺ T cells infiltration in orthotopic allograft tumors (CT26 cells).

(J) Correlation of VWA2 and CD8⁺ T cells infiltration in the COAD cohort and prognosis analysis of VWA2 expression with CD8⁺ T cells infiltration. Scale bars, 100 μm.

n = 3 technical replicates from 3 biological replicates for all. Data are represented as mean ± SD. p < 0.05 by Student’s t test (A, C, D, F, and I) or one-way ANOVA (D, E, F, G, and H).

Discussion

COAD is one of the most common malignant tumors of the digestive tract, which seriously threatens human health and life.¹³ Although surgery and medication have improved the survival of patients with COAD, there are still a considerable number of patients who progress to the advanced stage of the tumor.^14,15,16 Once advanced, the survival rate of patients will sharply decline. It was reported that the 5-year survival rate of COAD in the early stage is about 90%, and in patients with local metastasis, the 5-year survival rate is about 70%.^17,18,19 If it progresses to an advanced stage, the 5-year survival rate is only about 15%.²⁰ Therefore, it is particularly necessary to explore the molecular mechanism underlying the malignant phenotype of COAD and to screen therapeutic targets for improving prognosis. In our study, we found that VWA2 is highly expressed in COAD and is highly associated with poor prognosis in patients with COAD, liver cancer, lung cancer, and pancreatic cancer.

VWA2 is a member of the von Willebrand factor A-like domain protein superfamily. It was found that epigenetic and transcriptional dysregulation of VWA2 was associated with a myc-driven oncogenic program in CRC,¹² which provided an explanation that the role of VWA2 in COAD may be related to myc. We found that VWA2 promoted COAD cells to escape apoptosis by inducing activation of the Wnt/β-catenin signaling pathway. Although VWA2 is a secreted extracellular matrix (ECM) protein, its ability to activate intracellular β-catenin signaling may be mediated through integrin-dependent outside-in signaling as well as through modulation of ECM stiffness and Wnt receptor trafficking. These processes collectively facilitate β-catenin nuclear translocation and subsequent Wnt pathway activation. The Wnt/β-catenin signaling pathway is a complex network of protein interactions that are most common in embryonic development and cancer.^21,22,23 The Wnt/β-catenin signaling pathway was reported to participate in cancer cell apoptosis, metabolism, drug resistance, and immunity.^24,25 In COAD, drugs or molecular inhibitors targeting the Wnt/β-catenin signaling pathway is a promising strategy for treating cancer clinically.²⁶ Our study deepens the understanding that the Wnt/β-catenin signaling pathway contributes to the malignant phenotype of COAD.

Immune cells are an important part of the tumor microenvironment, including innate immune cells (macrophages, neutrophils, dendritic cells, innate lymphocytes, myeloid suppressor cells, and NK cells) as well as adaptive immune cells (T cells and B cells).^27,28 There is growing evidence that the interaction between tumor cells and immune cells shapes the inhibitory microenvironment that drives tumor growth, invasion, metastasis, and treatment failure.^28,29 For example, tumor-associated macrophages are the largest number of bone marrow-derived cells in the tumor microenvironment and play an important role in angiogenesis, extracellular matrix remodeling, tumor cell metastasis, and immune regulation.³⁰ CD8⁺ T cells are central antitumor effector cells in the tumor microenvironment, and their number and functional status largely determine the antitumor effect.^31,32 The advent of novel cancer treatments, such as immune checkpoint blockers, neoantigen vaccines, and chimeric antigen receptor T (CAR-T) cell therapy, points to the fact that CD8⁺ T cell-based immunotherapy has gained dramatic breakthroughs.^33,34In our study, we found that VWA2 inhibited tumor infiltration of multiple immune cells, including CD8⁺ T, B, and NK cells. VWA2 overexpression in tumors increased PD-L1 expression through the Wnt/β-catenin signaling pathway, which severely impaired T cell function. Our study revealed the important relationship between VWA2 and the tumor microenvironment, providing new theoretical and experimental evidence for the immunotherapy of COAD.

We would like to note a limitation in our apoptosis assays: the control group exhibited a somewhat elevated baseline apoptosis rate, which may be attributed to suboptimal culture conditions at that time. Nonetheless, all experimental groups were processed in parallel, and the relative differences remained statistically significant. Future studies with further optimized culture conditions would help to strengthen these observations. We also acknowledge that the functional assays in this study are primarily limited to CCK-8 proliferation assays, apoptosis detection, and subcutaneous xenograft models. While these experiments are fundamental for assessing tumor growth and anti-apoptotic capacity, they do not fully capture other critical malignant phenotypes of colorectal cancer, such as cell migration, invasion, epithelial-mesenchymal transition, or resistance to chemotherapy. Moreover, the subcutaneous xenograft model lacks the native tumor microenvironment of colon cancer, which may influence the observed phenotypes. Therefore, additional in vitro assays (e.g., Transwell migration/invasion, wound healing, and sphere formation) and orthotopic or patient-derived xenograft models are warranted in future studies to comprehensively evaluate the role of VWA2 in colon cancer progression.

Overall, the conclusions of this study indicate that VWA2 activated the Wnt/β-catenin signaling pathway, subsequently promoting the expression of CEMIP and PD-L1. This dual mechanism contributed to increased proliferation and anti-apoptotic properties of COAD cells, while simultaneously inhibiting the function of CD8⁺ T cells. It is conceivable that VWA2 could be developed into a potent target for anti-COAD in clinical practice.

Limitations of the study

Several limitations should be noted. First, the control groups in our apoptosis assays showed a somewhat elevated baseline apoptotic rate due to suboptimal culture conditions, although the relative differences remained significant. Second, our functional experiments mainly focused on proliferation and apoptosis resistance, without addressing other malignant traits such as migration, invasion, or chemoresistance. Third, we did not perform in vivo rescue experiments (e.g., PD-L1 blockade or β-catenin inhibition) to definitively confirm that VWA2-driven immune suppression is mediated exclusively through this axis. These issues warrant further investigation using additional models and intervention strategies.

Resource availability

Lead contact

Further information and requests for resources should be directed to the lead contact, Tao Yang (joyceqin@d.sxmu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •Data reported in this paper will be shared by the lead contact upon request.
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper are available from the lead contact upon request.

Acknowledgments

The financial support of this study is received from the NATCMs Project of High-level Construction of Key TCM Disciplines (grant no. zyyzdxk-2023188) and Guizhou Science and Technology Project (grant nos. Basic-ZK[2023] General 437 and Basic-ZK[2024] General 400).

Author contributions

W.X. and T.Y., funding acquisition, methodology, project administration, supervision, validation, visualization, writing – original draft, and writing – review and editing; Y.H., J.C., and H.D., methodology, software, visualization, writing – original draft, and writing – review and editing; X.R. and T.T., formal analysis, methodology, software, and visualization.

Declaration of interests

All authors of this manuscript state that they do not have any conflict of interest, and there is nothing to disclose.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal anti-VWA2	Proteintech	Cat# 18714-1-AP
Rabbit polyclonal anti-CEMIP	Proteintech	Cat# 21129-1-AP
Rabbit polyclonal anti-β-catenin	Proteintech	Cat# 51067-2-AP
Rabbit monoclonal anti-GAPDH	Cell Signaling Technology	Cat# 2118
Rabbit polyclonal anti-TCF4	Proteintech	Cat# 22337-1-AP
Biological samples
Colon adenocarcinoma tissues	This study	IACUC-2020-008
Chemicals, peptides, and recombinant proteins
RPMI 1640	Gibco	Cat#12633020
DMEM	Gibco	Cat#11965118
Penstrep	Gibco	Cat#15140163
Amphotericin B	Gibco	Cat#15290026
Trypsin-0.05% EDTA	Gibco	Cat#25300120
Fetal Bovine Serum	Gibco	Cat#A5256701
DMSO	Solarbio	Cat#D8371
Triton X-100	Solarbio	Cat#T8200
NC membrane	BIO-RAD	Cat#1620112
Wnt3a protein	SinoBiological	Cat#WNT007-02H
recombinant IL-2	SinoBiological	Cat#GMP-11848-HNAE
Critical commercial assays
EasySep™ Human CD8+ T Cell Isolation Kit	Stemcell	Cat# 17953
Deposited data
RNA-seq	Gene expression	TCGA: https://www.cancer.gov/ccg/research/genome-sequencing/tcga; Kaplan-Meier plotter: https://kmplot.com/analysis/; GEPIA: http://gepia.cancer-pku.cn/
STRING	Protein-protein interaction	STRING: https://string-db.org/
Timer 2.0	Immune cell infiltration	Timer 2.0: https://compbio.cn/timer2/
Experimental models: Cell lines
NCM460	Procell	CL-0749
LoVo	ATCC	CCL-229
SW620	ATCC	CCL-227
CT26	ATCC	CRL-2638
HEK293T	ATCC	CRL-3216
DiFi	COBIOER	CBP61017
Caco-2	ATCC	HTB-37
KM12	Procell	CL-1131
HCT8	ATCC	CCL-244
DLD-1	ATCC	CCL-221
SW480	ATCC	CCL-228
Experimental models: Organisms/strains
BALB/c-nu/nu nude	Beijing Vital River Laboratory Animal Technology	N/A
BALB/c	Beijing Vital River Laboratory Animal Technology	N/A
Oligonucleotides
qPCR primers for VWA2	This paper	N/A
qPCR primers for CEMIP	This paper	N/A
qPCR primers for β-catenin	This paper	N/A
qPCR primers for BAX	This paper	N/A
qPCR primers for VWA2	This paper	N/A
qPCR primers for BCL-2	This paper	N/A
qPCR primers for PD-L1	This paper	N/A
Software and algorithms
GraphPad Prism 9.0	GraphPad Software	https://www.graphpad.com/
R software (version 4.1.0)	R Foundation	https://www.r-project.org/
BIO-RAD CFX Manager	Bio-Rad	Not applicable
Living Image software (for IVIS bioluminescence)	PerkinElmer	Not applicable
ImageLAb	Bio-Rad	https://www.bio-rad.com/
FlowJo	BD Biosciences	https://www.flowjo.com/
GraphPad Prism	GraphPad	https://www.graphpad.com/

Experimental model and study participant details

Mouse models and clinical samples

This study was conducted strictly following the Guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. All breeding and experiments were undertaken with review and approval from the Animal Ethical and Experimental Committee of the Guizhou University of Traditional Chinese Medicine (IACUC-2020-008). Human primary colon adenocarcinoma tissues and paired adjacent normal colon tissues were obtained from 60 patients who underwent surgical resection at the First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. All patients provided written informed consent for the use of their specimens. The study was approved by the Ethics Committee of the First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine (KS2024284).

Cell lines

NCM460 and colorectal cells were stored in the National Key Laboratory of Integrated Prevention and Treatment of Digestive System Tumors. The frozen cells were removed from liquid nitrogen and quickly thawed in a water bath. Cells were then transferred to a medium containing fetal bovine serum and penicillin-streptomycin, and cultured in a CO₂ cell incubator at 37°C. Trypsin-EDTA was used to digest the cells, and after centrifugation, washing and re-suspension, the cells were inoculated into culture bottles for further culture. Cell lines were authenticated by the respective providers. All Cell lines were tested for mycoplasma contamination and the results were negative.

Method details

RNA extraction

RNA extraction was performed according to the instruction (AXYGEN, USA). First, the medium in the 6-well plate was discarded, and Trizol was added to lyse cells. Second, chloroform was added, followed by vigorous shaking, and the samples were centrifuged at 4°C for 15 min. Third, the samples were divided into yellow organic phase, middle layer and upper colorless water phase, and the water phase was added with isopropyl alcohol for 10 min and then centrifuged. Finally, the samples were washed and precipitated with 75% ethanol and were dissolved in RNase-free water.

Real-time PCR

cDNA was prepared according to reverse transcription kit (TAKARA, Japan). 12 μL Nuclease-Free Water, 4 μL SweScript RT I Enzyme Mix, and 4 μL Total mRNA were prepared. The mRNA was placed in a PCR apparatus for reverse transcription at 37°C for 15 min, 85°C 5 s, and 4°C 30 min. Real-time PCR was performed according to manufacturer’s recommendation (TAKARA, Japan). 20 μL system was prepared (Forward Primer 1 μL, Reverse Primer 1 μL, SYBR Premix Ex Taq II 10 μL, cDNA 2 μL and Nuclease-Free Water 6 μL). Samples suffered predenaturation at 95°C 30 s, denaturation at 95°C 15 s, and annealing extension at 60°C 30 s. The primer sequences used were as follows: for VWA2, forward 5′-ATGGGTCTAACAGCGTC.

GG-3′ and reverse 5′-AGGAGTGGAACTGAACTGGAA-3′; for CEMIP, forward 5′-CACGGTCTATTCCATCCACATC-3′ and reverse 5′-GGTTCGCAAAACAATC.

GGCT-3′; for β-catenin, forward 5′-CCCACTAATGTCCAGCGTTT-3′ and reverse 5′-AACGCATGATAGCGTGTCTG-3′; for BAX, forward 5′-AGCTGCAGAGGAT.

GATTGC-3′ and reverse 5′-GTTGAAGTTGCCGTCAGAAA-3′; for BCL-2, forward 5′-TGCCTTTGTGGAACTGTACG-3′ and reverse 5′-GGCCAAACTGAGC.

AGAGTC-3′; for PD-L1, forward 5′-GACGTGAAGCCTAGACAGC-3′ and reverse 5′-CAAAGAATTTTGGTTGTGGA-3′. The experimental data were collected and analyzed by BIO-RAD CFX Manager and GraphPad Prism software.

Protein extraction

When the cell density increased to 80%, after washing with PBS, cells were added to cell lysis solution (Thermo Fisher, USA) for 30 min. Cells were scraped by cell scraper and transferred to 1.5 mL; centrifuge tube. Samples were centrifuged at 12000 rpm at 4°C for 1 5min. Samples were transferred to a new tube, followed by protein concentration detection. Protein loading buffer was added proportionally, denatured and stored at −80°C.

Western Blot

Protein Ladder and protein samples were added in the gel electrophoresis. The constant pressure is set at 200 V-300 V, and the electrophoresis time is 15-30 min. The protein was then transferred into NC nitrocellulose through Bio-Rad Trans-Blot SD. The parameter is set to a constant pressure of 25 V and 14 min. The sealing buffer was prepared according to instructions (Beyotime, China), and the protein was incubated in a shaker at room temperature for 1 h. The primary antibodies were diluted and added at 4°C overnight, including anti-VWA2 (Proteintech, cat# 18714-1-AP), anti-CEMIP (Proteintech, cat# 21129-1-AP), anti-β-catenin (Proteintech, cat# 51067-2-AP), anti-GAPDH (CST, cat# 2118), anti-Histone H3 (CST, cat# 9715), and anti-TCF4 (Proteintech, cat# 22337-1-AP). The antibody was diluted and added at 4°C overnight. After TBST washing, the protein was incubated with a secondary antibody. The Bio-Rad imager was used for protein banding imaging.

Co-culture of CD8⁺ T cells and tumor cells

For co-culture assays, CD8⁺ T lymphocytes were first enriched from the peripheral blood mononuclear fraction of healthy volunteers using a negative selection strategy (EasySep Human CD8⁺ T Cell Isolation Kit, Stemcell Technologies). Prior to co-incubation with cancer cells, the T cells were stimulated using CD3/CD28-conjugated beads (Thermo Fisher Scientific) and maintained in OpTmizer medium containing recombinant IL-2 (10 ng/mL). The co-culture setup involved a 6-well plate, where 1×10⁵ adherent tumor cells were seeded in the plate and 0.5×10⁶ stimulated CD8⁺ T cells were added. Following a 48-hour incubation period, the CD8⁺ T cells were retrieved from the plate and processed for flow cytometric assessment.

Immunoprecipitation (Co-IP)

Co-immunoprecipitation was performed to validate the interaction between VWA2 and β-catenin in LoVo and SW620 cells. Cells were lysed in IP lysis buffer (Beyotime, China) containing protease and phosphatase inhibitors (Thermo Fisher, USA) on ice for 30 minutes. After centrifugation at 12,000 × g for 15 minutes at 4°C, the supernatant was collected and protein concentration was quantified using a BCA kit. For each reaction, 500 μg of total protein lysate was incubated with 2 μg of anti-VWA2 antibody or anti-β-Catenin antibody (Proteintech, USA) or an equal amount of normal rabbit IgG (Cell Signaling Technology, USA) as a negative control, with gentle rotation overnight at 4°C. Subsequently, 30 μL of Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, USA) was added and incubated for another 4 hours at 4°C. The beads were then washed three times with IP lysis buffer, and bound proteins were eluted by boiling in SDS loading buffer for 5 minutes. The immunoprecipitates were subjected to Western blotting.

Dual-Luciferase reporter assay

Putative TCF4 binding sites in the CEMIP promoter were predicted using the JASPAR database. Wild-type (WT) and mutant (MT) promoter fragments were cloned into the pGL3-Basic vector (Promega). 293T cells were co-transfected with the reporter construct, pRL-TK Renilla vector, and pcDNA3.1-TCF4 or empty vector using Lipofectamine 3000 (Thermo Fisher). After 48 h, firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). Relative luciferase activity was calculated as firefly/Renilla ratio. Each experiment was performed in triplicate.

Immunohistochemistry (IHC)

Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections derived from 60 primary colon adenocarcinoma (COAD) tissues and their paired adjacent normal colon tissues collected from surgical specimens. Sample slices of patients were placed in the oven at 60-65°C for 2 hours, followed by being immersed in xylene for 10 min. Sample slices were then sequentially placed in 100%, 95%, 80% and 70% alcohol for 5 min. The specimens were treated with Citrate Antigen Retrieval Solution for antigen retrieval. 3% H₂O₂ aqueous solution was prepared, and the slide was soaked in this solution for 15 min to remove endogenous peroxidase. After blocking, the primary antibody was added and incubated at 4°C overnight. After PBS washing, the remaining liquid was removed, and the slides were incubated with secondary antibody for 10 min. Horseradish peroxidase (HRP)-conjugated secondary antibody was then applied, and the chromogenic reaction was developed using 3,3′-diaminobenzidine (DAB) substrate solution (Zhongshan Golden Bridge, Beijing, China) for 1-3 minutes at room temperature, followed by rinsing with distilled water to stop the reaction. Subsequently, sections were counterstained with hematoxylin for 2 minute and washed under running tap water for 3 minutes. All stained slides were scanned using a digital slide scanner. VWA2 expression was evaluated using the H-score method. Staining intensity was graded as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The H-score was calculated as: [1 × (% of weakly stained tumor cells) + 2 × (% of moderately stained tumor cells) + 3 × (% of strongly stained tumor cells)], yielding a total range of 0–300.

Tumor-bearing experiment

For subcutaneous xenograft models, SW620 cells (5 × 10⁶) were injected subcutaneously into the right thigh of 4–6-week-old male nude mice (BALB/c-nu/nu). For orthotopic allograft models, luciferase-labeled CT26 cells (5 × 10⁵) were injected into the cecal wall of 4–6-week-old male BALB/c mice. Specifically, logarithmic cells were resuspended in PBS at 1×10⁷ cells/mL, mixed 1:1 with Matrigel, and kept on ice. Mice were anesthetized, and 30 μL of the mixture was injected into the cecal wall. The incision was closed, and mice were monitored postoperatively. In the orthotopic allograft model, tumor metastasis to distant organ was monitored by bioluminescence imaging and confirmed at necropsy. For bioluminescence imaging, mice were intraperitoneally injected with D-luciferin (150 mg/kg) under isoflurane anesthesia. Images were acquired 15 minutes later using an IVIS Spectrum system, and total photon flux was quantified with Living Image software. Mice were sacrificed when subcutaneous tumor volume reached 1500–2000 mm³ or at three weeks post-orthotopic inoculation.

Flow cytometry

Single-cell suspension was prepared. PBS buffer solution was used for washing and resuspension. Dilute Zombie NIR dye at a ratio of 1:100∼1000, and add it to samples at room temperature for 15∼30 min. Bio Legend Cell Staining Buffer was used. Goat serum was added to the cell precipitation and incubated at 4°C in the dark for 10 min. After blocking, the antibody was added to the system and incubated at 4°C in the dark for 30 min. After incubation, 1 mL flow washing solution was added into the system and centrifuged at 1000 rpm at 4°C for 5 min. Finally, samples were suspended using a flow washing solution and analyzed by flow cytometer.

Bioinformatic analyses and database usage

The following public databases were used in this study. TCGA (The Cancer Genome Atlas, https://portal.gdc.cancer.gov/) provided RNA-seq expression profiles and corresponding clinical data for COAD, BRCA, LUAD, OV, PAAD, and READ. Kaplan-Meier plotter (http://kmplot.com/analysis/) was used to evaluate the prognostic value of VWA2 in liver, lung, and pancreatic cancers. GEPIA (http://gepia.cancer-pku.cn/) was applied to analyze the correlation between VWA2 and CEMIP expression. STRING (https://string-db.org/) predicted potential VWA2-interacting proteins, and Timer 2.0 (http://timer.cistrome.org/) was used to assess immune cell infiltration, particularly CD8⁺ T cells, in relation to VWA2 expression.

Quantification and statistical analysis

All statistical analyses were performed using GraphPad Prism 9.0 and R software (version 4.1.0). Data are presented as mean ± standard deviation (SD) from at least three independent experiments. For comparisons between two groups, a two-tailed unpaired Student’s t-test was applied. For comparisons involving more than two groups, one-way or two-way ANOVA was used, followed by Tukey’s post hoc test. Survival curves were constructed using the Kaplan-Meier method and compared by the log-rank test. Correlation analyses between VWA2 expression and CEMIP or CD8⁺ T cell infiltration were performed using Pearson’s correlation coefficient. A p value of less than 0.05 was considered statistically significant. Statistical significance thresholds were set at ∗p < 0.05, ∗∗p < 0.01; ns, not significant. Statistical methods used for each analysis are described in the corresponding figure legends.