WFDC2 Human, His

What is WFDC2/HE4 and what are its basic molecular characteristics?

WFDC2, also known as HE4 (Human Epididymis Protein 4), is a small secretory protein that belongs to the WAP (whey acidic protein) four-disulfide core domain family. It functions as a biomarker in various cancers, particularly ovarian cancer, where it promotes proliferation, metastasis, and chemoresistance while suppressing cytotoxic lymphocytes . The protein contains conserved cysteine residues that form disulfide bonds, contributing to its structural stability. WFDC2/HE4 is encoded by a gene located on chromosome 20q13.12 and undergoes post-translational modifications, including glycosylation, before secretion into biological fluids where it can be detected and measured for diagnostic purposes .

How does WFDC2/HE4 expression vary across different human tissues?

WFDC2/HE4 shows differential expression across human tissues. While initially identified in the epididymis (hence its name), it is now known to be expressed in multiple epithelial tissues including respiratory epithelium, female reproductive tract tissues, and kidney. In pathological conditions, particularly in ovarian cancer, WFDC2/HE4 expression is significantly elevated . Interestingly, in prostate cancer, WFDC2 expression appears to be negatively correlated with Gleason score and metastasis, suggesting a potential tumor suppressor role in this specific context . This tissue-specific expression pattern makes WFDC2/HE4 a valuable biomarker for differential diagnosis in clinical settings.

What is the current consensus on the physiological function of WFDC2/HE4?

The physiological function of WFDC2/HE4 remains an active area of investigation with multiple proposed roles. Research indicates that WFDC2/HE4 may function as an immunomodulator, affecting immune cell function and cytokine expression in peripheral blood mononuclear cells . It has been shown to promote angiogenesis through upregulation of factors like CXCL8 (IL8) and HIF1A . Additionally, WFDC2 appears to interact with cell signaling pathways, particularly EGFR signaling, which may explain its differential effects in various cancer types . Some studies suggest protease inhibitory functions similar to other WAP domain-containing proteins, though the specific proteases it inhibits remain under investigation.

What are the optimal methods for measuring WFDC2/HE4 expression and activity in experimental settings?

For measuring WFDC2/HE4 expression and activity, researchers employ multiple complementary techniques. Quantitative PCR (qPCR) using validated primers is effective for measuring mRNA expression levels, as demonstrated in studies examining WFDC2/HE4 regulation of immune-related genes . For protein detection, western blotting using specific antibodies (such as Origene TA307787, dilution 1:2000) provides reliable results . Immunofluorescence and immunohistochemistry are valuable for tissue localization studies, while ELISA assays (such as those using Quantikine® kits) enable precise quantification in biological fluids . For functional studies, recombinant WFDC2/HE4 protein (typically used at 1-20 nM concentrations) can be employed to treat cells, followed by downstream assays to assess effects on proliferation, angiogenesis, or immune function .

What experimental models are most suitable for studying WFDC2/HE4 functions?

Multiple experimental models have proven effective for studying WFDC2/HE4 functions. Cell culture systems using cancer cell lines (such as SKOV3 for ovarian cancer) allow for manipulation of WFDC2/HE4 expression through overexpression or knockdown approaches . Primary cells, like human peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVECs), have been successfully used to study WFDC2/HE4's effects on immune function and angiogenesis, respectively . In vivo models, particularly mouse xenografts, provide systems for studying WFDC2/HE4's role in tumor growth and metastasis. For mechanistic studies, co-immunoprecipitation assays have been employed to identify protein-protein interactions, such as WFDC2 binding to the extracellular domain of EGFR .

What are the key considerations when designing experiments to assess WFDC2/HE4 interactions with signaling pathways?

When designing experiments to assess WFDC2/HE4 interactions with signaling pathways, researchers should consider several critical factors. First, concentration-dependent effects are important; studies typically use recombinant HE4 at concentrations ranging from 1-20 nM, with 20 nM being common for in vitro studies . Temporal dynamics must be considered, as pathway activation may be rapid (minutes to hours) or delayed (requiring longer incubation periods). Control experiments should include pathway-specific inhibitors (such as STAT3 inhibitor VIII used at 5 μM) to confirm specificity of observations. To comprehensively assess pathway activation, both phosphorylation status of key intermediates (using phospho-specific antibodies) and downstream gene expression changes should be measured. For mechanistic insights, rescue experiments (restoring pathway activity after WFDC2/HE4 manipulation) provide strong evidence for causality.

How does WFDC2/HE4 contribute to ovarian cancer progression and metastasis?

WFDC2/HE4 contributes to ovarian cancer progression and metastasis through multiple mechanisms. It promotes proliferation and has been shown to induce a pro-angiogenic tumor microenvironment by upregulating factors like CXCL8 (IL8) and HIF1A . A significant positive correlation exists between HE4 and IL8 intensity levels in serous adenocarcinoma tissue (Pearson r = 0.4423, p = 0.002) . WFDC2/HE4 also appears to modulate immune responses, potentially creating an immunosuppressive environment that facilitates tumor growth. This is supported by the observed inverse relationship between CD8+ T cell counts and HE4 serum levels (r = -0.3694; p = 0.032) . Additionally, WFDC2/HE4 promotes microvascular density, as evidenced by the positive correlation between serum HE4 and CD34+ area (r = 0.5680; p = 0.017) . These multiple effects collectively contribute to enhanced tumor growth and metastatic potential.

What is the evidence for WFDC2/HE4 as a tumor suppressor in prostate cancer?

In contrast to its oncogenic role in ovarian cancer, evidence suggests WFDC2/HE4 functions as a tumor suppressor in prostate cancer. Bioinformatic analyses have shown that WFDC2 expression is negatively correlated with Gleason score and metastasis in prostate cancer patients . Mechanistically, WFDC2 has been demonstrated to suppress prostate cancer metastasis by binding to the extracellular domain of the epidermal growth factor receptor (EGFR) . This binding leads to inactivation of the EGFR/AKT/GSK3β/Snail signaling pathway, which in turn restrains the progression of epithelial-mesenchymal transition (EMT), a critical process for cancer cell invasion and metastasis . Experimental evidence from both in vitro and in vivo studies supports that overexpression of WFDC2 or addition of recombinant HE4 protein significantly inhibits prostate cancer metastasis, providing functional validation of its tumor suppressor role in this context .

How do the effects of WFDC2/HE4 differ between cancer types?

The effects of WFDC2/HE4 appear to be cancer type-specific, with contradictory roles observed in different malignancies. In ovarian cancer, WFDC2/HE4 functions as an oncogenic factor promoting proliferation, metastasis, chemoresistance, and creating an immunosuppressive microenvironment . Conversely, in prostate cancer, WFDC2/HE4 acts as a tumor suppressor, with expression negatively correlating with disease progression and metastasis . This dichotomy may be explained by tissue-specific molecular contexts, particularly the differential regulation and expression of receptor systems with which WFDC2/HE4 interacts. In prostate cancer, WFDC2/HE4 inhibits metastasis by binding to EGFR and inactivating downstream signaling , while in ovarian cancer, it appears to activate STAT3 signaling and promote angiogenesis . These findings highlight the importance of cancer-specific studies when investigating WFDC2/HE4 as a potential therapeutic target.

How does WFDC2/HE4 modulate immune cell function?

WFDC2/HE4 exerts significant modulatory effects on immune cell function through direct interactions with peripheral blood mononuclear cells (PBMCs). Research utilizing qPCR arrays has revealed that treatment of normal human PBMCs with 20 nM recombinant HE4 substantially alters the expression of multiple immune-related genes . Most notably, WFDC2/HE4 strongly upregulates CSF3 (colony stimulating factor 3), which plays a critical role in granulocyte production and function . Additionally, WFDC2/HE4 increases expression of CXCL8 (IL8) and HIF1A, which contribute to immune modulation and angiogenesis . The immunosuppressive effects of WFDC2/HE4 are further evidenced by the inverse correlation observed between HE4 serum levels and CD8+ T cell counts in epithelial ovarian cancer tissue (r = -0.3694; p = 0.032) . These findings suggest that WFDC2/HE4 may promote tumor progression in part by creating an immunosuppressive microenvironment that inhibits cytotoxic T cell function.

What are the key genes and pathways regulated by WFDC2/HE4 in immune cells?

WFDC2/HE4 regulates several key genes and pathways in immune cells, with significant implications for immunity and cancer progression. Treatment of peripheral blood mononuclear cells (PBMCs) with recombinant HE4 (20 nM for 6 hours) has been shown to upregulate multiple genes . The most dramatically upregulated gene is CSF3 (colony stimulating factor 3), which plays a crucial role in granulocyte development and function . Other significantly upregulated genes include CXCL8 (IL8) and HIF1A, both of which are STAT3-regulated and involved in promoting angiogenesis . The consistent upregulation pattern observed across experimental replicates (Pearson r = 0.8884, p < 0.0001 between array sets) suggests these are robust effects . Pathway analysis indicates STAT3 activation may be a key mediator of WFDC2/HE4 effects, as many of the upregulated genes are STAT3 targets . These findings suggest that WFDC2/HE4 modulates immune function through coordinated regulation of genes involved in inflammation, angiogenesis, and cellular migration.

What is the relationship between WFDC2/HE4 and angiogenesis in the tumor microenvironment?

WFDC2/HE4 promotes angiogenesis in the tumor microenvironment through multiple mechanisms. Research demonstrates that WFDC2/HE4 upregulates pro-angiogenic factors, particularly CXCL8 (IL8) and HIF1A, in both immune cells and cancer cells . In vitro studies using human umbilical vein endothelial cells (HUVECs) have shown that recombinant HE4 (1 nM) enhances tube formation, a critical process in angiogenesis . This effect appears to be mediated through STAT3 activation, as STAT3 inhibitor VIII (5 μM) blocks the pro-angiogenic effects of HE4 . The clinical relevance of these findings is supported by immunohistochemistry studies in epithelial ovarian cancer tissue, which revealed a significant positive correlation between serum HE4 levels and CD34+ area (r = 0.5680; p = 0.017), indicating enhanced microvascular density in tumors with high HE4 expression . Additionally, HE4 and IL8 levels show a positive correlation in cancer tissue (Pearson r = 0.4423, p = 0.002), further supporting the role of WFDC2/HE4 in promoting a pro-angiogenic tumor microenvironment .

How does WFDC2/HE4 interact with the EGFR signaling pathway?

WFDC2/HE4 directly interacts with the epidermal growth factor receptor (EGFR) signaling pathway, though with differential effects across cancer types. In prostate cancer, evidence indicates that WFDC2 binds to the extracellular domain of EGFR, as demonstrated through co-immunoprecipitation assays . This binding leads to inactivation of the EGFR/AKT/GSK3β/Snail signaling cascade, thereby restraining epithelial-mesenchymal transition (EMT) progression and suppressing metastasis . The inhibitory effect on this pathway appears to contribute to WFDC2's tumor suppressor function in prostate cancer . Conversely, in ovarian cancer, WFDC2/HE4 may interact differently with receptor-mediated signaling pathways, potentially activating rather than inhibiting certain pathways. This context-dependent interaction with EGFR signaling may explain the opposing roles of WFDC2/HE4 observed in different cancer types and highlights the importance of tissue-specific studies when investigating its molecular mechanisms.

What is known about the relationship between WFDC2/HE4 and STAT3 signaling?

The relationship between WFDC2/HE4 and STAT3 signaling appears to be significant in mediating WFDC2/HE4's biological effects. Research indicates that WFDC2/HE4 promotes STAT3 activation, which may be responsible for the upregulation of STAT3-regulated genes like CXCL8 (IL8) and HIF1A . These genes are involved in promoting angiogenesis and modulating immune responses . The functional significance of this relationship is supported by experiments using STAT3 inhibitor VIII (5 μM), which blocks the pro-angiogenic effects of recombinant HE4 on human umbilical vein endothelial cells (HUVECs) . This suggests that STAT3 activation is necessary for WFDC2/HE4-mediated angiogenesis. The exact mechanism by which WFDC2/HE4 activates STAT3 remains under investigation, though it likely involves interaction with upstream receptors or signaling components that converge on STAT3 phosphorylation. Understanding this relationship is particularly important given STAT3's established role in promoting cancer progression through effects on cell survival, proliferation, angiogenesis, and immune evasion.

How do post-translational modifications affect WFDC2/HE4 function?

Post-translational modifications (PTMs) play crucial roles in determining WFDC2/HE4 functionality, though this area requires further investigation. WFDC2/HE4 undergoes glycosylation, which affects its stability, secretion, and potentially its binding interactions with receptors like EGFR . The glycosylation pattern may vary across different tissues and pathological conditions, potentially contributing to the context-specific effects of WFDC2/HE4 observed in different cancer types. Other potential PTMs, such as phosphorylation, acetylation, or ubiquitination, may also regulate WFDC2/HE4 function, though these modifications are less well-characterized. Research using recombinant HE4 protein has provided valuable insights, but differences between recombinant and endogenous WFDC2/HE4 in terms of PTMs should be considered when interpreting experimental results . Studies utilizing mass spectrometry and other proteomic approaches would be valuable for comprehensively characterizing the PTM landscape of WFDC2/HE4 and understanding how these modifications impact its biological functions in normal and pathological conditions.

What are the key variables to control when studying WFDC2/HE4 in cell culture systems?

When designing experiments to study WFDC2/HE4 in cell culture systems, researchers must carefully control several key variables. First, the concentration of recombinant HE4 is critical; studies typically use 1-20 nM, with 20 nM being common for immune cell experiments . Exposure time varies by experimental endpoint, ranging from 6 hours for gene expression studies to 24 hours or longer for functional assays like angiogenesis . Cell density must be standardized (e.g., approximately 5,000 cells/well for viability assays, 10,000 HUVECs/well for tube formation) . The specific cell type dramatically influences results, with different responses observed in cancer cells, immune cells, and endothelial cells . Serum concentration in culture media should be controlled as serum components may interact with WFDC2/HE4. For knockdown or overexpression studies, verification of manipulation efficiency through qPCR and western blot is essential. Finally, appropriate controls must be included: vehicle controls for recombinant protein experiments, scrambled/non-targeting controls for knockdown studies, and empty vector controls for overexpression experiments.

What methods are recommended for accurate quantification of WFDC2/HE4 in clinical samples?

For accurate quantification of WFDC2/HE4 in clinical samples, a combination of methods is recommended depending on the sample type and research question. For protein quantification in serum or plasma, enzyme-linked immunosorbent assay (ELISA) using validated kits such as the Quantikine Immunoassay Control Set for Human HE4/WFDC2 provides reliable results . These assays should include appropriate standards and quality controls to ensure accuracy across different batches . For tissue samples, immunohistochemistry or immunofluorescence using specific antibodies (such as Origene TA307787, 1:2000 dilution) allows visualization and semi-quantitative assessment of WFDC2/HE4 expression patterns. Digital image analysis using calibrated systems and intensity thresholding enables objective quantification from immunostained samples . For mRNA quantification, quantitative PCR with validated primers after RNA extraction is effective, though researchers should be aware that mRNA and protein levels may not always correlate. Regardless of the method, standardized sample collection, processing, and storage protocols are critical to minimize pre-analytical variables that could affect measurements.

How can researchers reconcile the opposing roles of WFDC2/HE4 in ovarian versus prostate cancer?

Researchers can reconcile the opposing roles of WFDC2/HE4 in different cancers through several analytical approaches. First, context-dependent protein interactions should be investigated; WFDC2/HE4 may interact with different binding partners across tissue types, explaining its divergent effects . The receptor landscape varies between ovarian and prostate tissues, potentially causing WFDC2/HE4 to activate different signaling cascades. In prostate cancer, WFDC2 inhibits metastasis by binding to EGFR and inactivating the EGFR/AKT/GSK3β/Snail pathway , while in ovarian cancer, it may interact with different receptors leading to STAT3 activation . Additionally, alternative splicing or post-translational modifications might produce tissue-specific WFDC2/HE4 variants with distinct functions. Concentration-dependent effects should also be considered; WFDC2/HE4 might have biphasic effects depending on its local concentration. Finally, the tumor microenvironment differs substantially between cancer types, potentially modifying WFDC2/HE4's effects through interactions with tissue-specific factors. Comprehensive mechanistic studies addressing these possibilities would help resolve the apparent contradictions.

What techniques can help determine whether discrepancies in WFDC2/HE4 research findings are methodological or biological?

To determine whether discrepancies in WFDC2/HE4 research findings stem from methodological or biological factors, researchers should employ several analytical techniques. Standardization studies using identical protocols across different laboratories can identify method-dependent variations. Meta-analyses of published data, stratifying results by experimental approach, can reveal systematic biases associated with specific methodologies. Antibody validation studies are crucial, as different antibodies may recognize distinct epitopes or isoforms of WFDC2/HE4, leading to discrepant results . Cross-platform validation, using multiple techniques (e.g., ELISA, western blot, immunohistochemistry) to measure WFDC2/HE4 in the same samples, can identify method-specific artifacts. Reproducibility studies with larger sample sizes increase statistical power to detect true biological effects. Isoform-specific analyses can determine whether different WFDC2/HE4 variants explain divergent findings . Finally, comprehensive reporting of experimental conditions, including cell densities, protein concentrations, incubation times, and passage numbers, enables more effective comparison across studies and identification of variables that might explain discrepancies.

How do genetic variations in the WFDC2 gene impact experimental findings and clinical correlations?

Genetic variations in the WFDC2 gene can significantly impact experimental findings and clinical correlations through multiple mechanisms. Single nucleotide polymorphisms (SNPs) in the WFDC2 gene may affect protein expression levels, potentially explaining variability in serum HE4 levels across different populations. Promoter region variants can alter transcriptional regulation, affecting how WFDC2/HE4 responds to various stimuli or treatment conditions. Coding region variations may modify protein structure or function, potentially altering WFDC2/HE4's interaction with binding partners like EGFR or its effects on downstream pathways like STAT3 signaling . Alternative splicing variations can generate different WFDC2/HE4 isoforms with distinct functional properties. These genetic factors may contribute to the heterogeneity observed in clinical correlations and experimental outcomes. Researchers should consider genotyping study populations and experimental models for known WFDC2 variants and controlling for these variables in analyses. Additionally, studying the functional consequences of specific genetic variations through targeted mutagenesis approaches can provide insights into structure-function relationships of WFDC2/HE4.

What are the most promising therapeutic strategies targeting WFDC2/HE4 in cancer?

The development of therapeutic strategies targeting WFDC2/HE4 must account for its context-dependent roles in different cancers. For ovarian cancer, where WFDC2/HE4 promotes tumor progression, several approaches show promise: neutralizing antibodies that bind and inactivate circulating WFDC2/HE4; small molecule inhibitors that disrupt WFDC2/HE4 interactions with key binding partners; antisense oligonucleotides or siRNA technologies to downregulate WFDC2/HE4 expression; and inhibitors of downstream pathways activated by WFDC2/HE4, particularly STAT3 inhibitors . Conversely, for prostate cancer, where WFDC2/HE4 functions as a tumor suppressor, therapeutic strategies might include recombinant WFDC2/HE4 administration; gene therapy approaches to increase WFDC2/HE4 expression; or peptide mimetics that replicate WFDC2/HE4's inhibitory effect on EGFR signaling . For both cancer types, combination therapies targeting WFDC2/HE4 alongside other established treatments may enhance efficacy. Precision medicine approaches that stratify patients based on WFDC2/HE4 expression patterns and genetic background will be essential for optimizing therapeutic outcomes.

What novel experimental models could advance our understanding of WFDC2/HE4 biology?

Advanced experimental models could significantly enhance our understanding of WFDC2/HE4 biology. Three-dimensional organoid cultures derived from patient tissues would provide more physiologically relevant systems than traditional cell lines, preserving tissue architecture and cellular heterogeneity . Genetically engineered mouse models with tissue-specific, inducible WFDC2/HE4 expression or deletion would enable in vivo studies of WFDC2/HE4 function in both normal development and cancer progression. Patient-derived xenografts maintain tumor heterogeneity and microenvironment interactions, allowing for more translational studies of WFDC2/HE4's role in cancer. CRISPR-Cas9 engineered cell lines with specific WFDC2 mutations or domain deletions would facilitate structure-function analyses. Co-culture systems combining cancer cells with immune cells and endothelial cells would better recapitulate the complex interactions in the tumor microenvironment where WFDC2/HE4 operates . Microfluidic "organ-on-a-chip" platforms could model dynamic processes like angiogenesis and metastasis under controlled conditions. Finally, computational models integrating multi-omics data could predict WFDC2/HE4 interactions and effects across different cellular contexts.

What are the critical unanswered questions in WFDC2/HE4 research?

Several critical questions remain unanswered in WFDC2/HE4 research. The molecular basis for WFDC2/HE4's opposing roles in different cancers requires elucidation—what specific interactions or modifications determine whether it functions as an oncogene or tumor suppressor? The comprehensive interactome of WFDC2/HE4 across different tissues remains undefined; identifying all binding partners would provide insights into its diverse functions. The precise mechanism by which WFDC2/HE4 activates STAT3 signaling in some contexts while inhibiting EGFR signaling in others needs clarification . The physiological role of WFDC2/HE4 in normal tissues, particularly in immune regulation and tissue homeostasis, remains poorly understood. The potential role of WFDC2/HE4 in resistance to various cancer therapies requires investigation, as does its utility as a predictive biomarker for treatment response. The relationship between different WFDC2/HE4 isoforms and their specific functions has not been fully characterized. Finally, the evolutionary conservation and divergence of WFDC2/HE4 across species could provide insights into its fundamental biological importance and specialized functions that have developed in humans.