WFDC3: Novel Prognostic Biomarker in Pancreatic Cancer

Introduction: The Challenge of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PAAD) remains one of the deadliest cancers, with a five-year survival rate near 10%. Its silent onset, rapid spread, and resistance to current treatments make early detection and targeted therapy especially difficult. Immunotherapies that have revolutionized treatment in other cancers show limited benefit in PAAD, largely due to its immunosuppressive tumor microenvironment. Identifying reliable biomarkers for prognosis and new therapeutic targets is therefore a critical priority.

Whey-Acidic-Protein Four-Disulfide Core (WFDC) Family: A Brief Background

The WFDC family comprises 18 small, secreted proteins defined by conserved “WFDC” domains stabilized by four disulfide bonds. These proteins, broadly expressed in tissues like lung and reproductive organs, modulate protease activity, host defense, and immune responses. Dysregulated WFDC proteins have been linked to cancer progression—for example, WFDC2 (HE4) in ovarian and lung cancer—yet the role of WFDC3 in PAAD was previously unexplored.

Study Overview

Liu et al. performed a comprehensive multi-omic investigation of WFDC3 in PAAD, integrating:

1. Pan-cancer bioinformatics (TCGA & GTEx) to chart WFDC expression and survival associations.

2. Single-cell RNA-seq analysis (GSE154778) to localize WFDC3 in PAAD cell types.

3. Functional assays in PAAD cell lines to test effects on migration, invasion, and immune interactions.

4. Prognostic modelling using LASSO regression to build a WFDC-based risk score.

Key Findings

1. WFDC3 Is Overexpressed and Prognostic in PAAD

• Tumor vs. normal: WFDC3 mRNA is significantly higher in PAAD tissues (TCGA-GTEx data).

• Survival impact: High WFDC3 correlates with poorer overall, disease-specific, and progression-free survival (e.g., OS HR 1.89, P = 0.003).

2. Malignant Epithelial Specificity Revealed by Single-Cell Analysis

• Cell type mapping: In GSE154778, WFDC3 expression localizes almost exclusively to epithelial clusters, not stromal or immune cells (UMAP plots on p. 6).

• Malignant vs. normal: InferCNV distinguishes malignant ductal cells with high chromosomal instability, which show markedly higher WFDC3 than normal epithelium (P < 1e-12).

3. WFDC3 Drives Migration, Invasion, and EMT

• In vitro assays: Overexpressing WFDC3 in PANC-1 cells increases migration and invasion; knockdown in MIA PaCa-2 cells has the opposite effect (Transwell; Figure 7A–B).

• EMT markers: WFDC3 upregulation elevates mesenchymal markers (N-cadherin, Vimentin, Snail) and reduces epithelial markers (E-cadherin, ZO-1); knockdown reverses these changes (Figures 7C–F).

4. WFDC3 Suppresses T-Cell Cytotoxicity

• Immune profiling: High WFDC3 tumors have fewer CD8⁺ and cytotoxic T cells, more M0 macrophages and Tregs (CIBERSORT; Figure 6A–C).

• Functional assays:

  • Recombinant WFDC3 or WFDC3-rich conditioned media reduce Granzyme B expression in Jurkat T cells (flow cytometry; P < 0.01).

  • T cells co-cultured with WFDC3-overexpressing tumor cells show lowered IFNG, IL2, Perforin, GZMB, and Granulysin transcripts (qRT-PCR; P < 0.05).

  • WFDC3 knockdown enhances T cell–mediated tumor killing (CCK-8 and LDH assays).

5. A Four-Gene WFDC Signature Stratifies Patient Risk

• Model construction: LASSO regression of 18 WFDC genes identifies WFDC3, WFIKKN1, PI3, and SLPI as prognostic.

• Risk formula:

  Risk Score = 0.068 × WFDC3 – 0.540 × WFIKKN1 + 0.041 × PI3 + 0.074 × SLPI

• Performance: High- vs. low-risk groups show clear survival separation (KM P = 0.0014); 5-year AUC 0.84; validated in two GEO cohorts (GSE62452 & GSE78229).

Practical Implications and Next Steps

• Prognostic tool: The WFDC3-based signature offers a potential assay for patient stratification and monitoring.

• Therapeutic targeting: As a secreted protein, WFDC3 could be targeted by neutralizing antibodies or small molecules to inhibit metastasis and relieve immunosuppression.

• Mechanistic studies: Future work should identify WFDC3’s binding partners, downstream signaling (e.g., STAT, NF-κB), and test in in vivo PAAD models.

• Clinical translation: Measuring circulating WFDC3 may aid early detection and assess response to immunotherapies in PAAD.

Conclusion

This study uncovers WFDC3 as a dual-role driver of pancreatic cancer progression—promoting epithelial–mesenchymal transition and suppressing T cell–mediated immunity—while providing a robust four-gene prognostic signature. Targeting WFDC3 holds promise for improving outcomes in this hard-to-treat malignancy.

 

The translation of the preceding English text in Chinese:

 

介绍：胰腺癌的挑战

胰腺导管腺癌（PAAD）仍然是最致命的癌症之一，五年生存率仅约 10%。其发病隐匿、进展迅速且对现有治疗耐药，使得早期检测和靶向治疗尤其困难。在其他癌症中已带来革命性变化的免疫疗法在PAAD中受益有限，这主要归因于其免疫抑制性肿瘤微环境。因此，识别可靠的预后生物标志物和新的治疗靶点成为迫切要务。

乳清酸性蛋白四二硫键核心（WFDC）家族：简要背景

WFDC 家族由 18 种小型分泌蛋白组成，其特征为高度保守的“WFDC”结构域，并由四个二硫键稳定。这些蛋白广泛分布于肺部、生殖器官等组织中，参与蛋白酶活性调节、宿主防御和免疫反应。WFDC 蛋白的失调已被证明与癌症进展有关，例如在卵巢癌和肺癌中高表达的 WFDC2（HE4）。然而，WFDC3 在PAAD中的作用此前尚未受到研究。

研究概述

Liu 等人对 WFDC3 在 PAAD 中的作用进行了全面的多组学研究，整合了以下内容：

• 全癌种生物信息学分析（TCGA & GTEx）以绘制 WFDC 表达谱及其与生存的关联；

• 单细胞 RNA 测序分析（GSE154778）以定位 WFDC3 在 PAAD 细胞类型中的分布；

• PAAD 细胞系功能实验以测试其对细胞迁移、侵袭及免疫相互作用的影响；

• 基于 LASSO 回归构建 WFDC 基因风险评分的预后模型。

主要发现

1. WFDC3 在 PAAD 中高表达且具有预后意义

   • 肿瘤 vs. 正常：TCGA-GTEx 数据显示，PAAD 组织中 WFDC3 mRNA 水平显著升高。

   • 生存影响：高表达组的总体生存（OS）、疾病特异生存（DSS）和进展无事件生存（PFS）均较差（例如，OS 风险比 HR=1.89，P=0.003）。

2. 单细胞分析揭示其在恶性上皮细胞中的特异性

   • 细胞类型定位：在 GSE154778 数据集中，WFDC3 几乎仅在上皮细胞亚群中表达，而在基质或免疫细胞中不表达。

   • 恶性 vs. 正常：InferCNV 分析显示，具有高染色体不稳定性的恶性导管细胞中 WFDC3 显著高于正常上皮细胞（P<1e-12）。

3. WFDC3 促进细胞迁移、侵袭和上皮-间质转化（EMT）

   • 体外实验：在 PANC-1 细胞中过表达 WFDC3 可增强迁移和侵袭能力；在 MIA PaCa-2 细胞中敲低则相反（Transwell 实验）。

   • EMT 标志物：WFDC3 上调可提高间质标志物（N-钙黏蛋白、Vimentin、Snail）表达，降低上皮标志物（E-钙黏蛋白、ZO-1）表达；敲低可逆转该变化。

4. WFDC3 抑制 T 细胞细胞毒性

   • 免疫浸润分析：高 WFDC3 肿瘤中 CD8⁺ 及细胞毒性 T 细胞减少，M0 巨噬细胞和调节性 T 细胞（Treg）增多。

   • 功能实验：重组 WFDC3 蛋白或含高 WFDC3 的条件培养基可降低 Jurkat 细胞中 Granzyme B 表达（流式细胞术，P<0.01）；与 WFDC3 过表达肿瘤细胞共培养的 T 细胞中 IFNG、IL2、Perforin、GZMB 和 Granulysin 转录水平显著下降（qRT-PCR，P<0.05）；WFDC3 敲低可增强 T 细胞介导的肿瘤细胞杀伤（CCK-8 和 LDH 实验）。

5. 基于四基因的 WFDC 风险评分可对患者进行分层

   • 模型构建：对 18 个 WFDC 基因进行 LASSO 回归筛选，确定 WFDC3、WFIKKN1、PI3 和 SLPI 具有预后价值。

   • 风险公式：
     Risk Score = 0.068 × WFDC3 – 0.540 × WFIKKN1 + 0.041 × PI3 + 0.074 × SLPI

   • 性能评估：高低风险组生存明显分离（KM P=0.0014）；五年 AUC 为 0.84；并在两个 GEO 队列（GSE62452 & GSE78229）中得到验证。

实践意义与下一步

• 预后工具：该基于 WFDC3 的四基因签名可作为患者分层和监测的潜在检测手段。

• 治疗靶向：作为分泌蛋白，WFDC3 可通过中和抗体或小分子进行靶向，以抑制转移并缓解免疫抑制。

• 机制研究：后续工作应聚焦于识别 WFDC3 的结合伙伴、下游信号通路（如 STAT、NF-κB）及在体内 PAAD 模型中的功能验证。

• 临床转化：测定循环中的 WFDC3 水平或有助于早期检测及评估免疫疗法反应。

结论

本研究首次揭示了 WFDC3 在胰腺癌进展中的双重驱动作用——促进上皮-间质转化并抑制 T 细胞介导的免疫杀伤，同时提出了具有良好预后价值的四基因签名。针对 WFDC3 的干预策略或可改善这一难治性恶性肿瘤的治疗效果。

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Reference:

Bohan Liu, Xuqing Shi, Tianqi Liu, Huanwen Wu, Zhiyong Liang

WFDC3 identified as a prognostic and immune biomarker in pancreatic cancer.

Biomol Biomed [Internet]. 2025 May 8 [cited 2025 Jul. 29];

Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/12444

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