CCDC25 Human

What is CCDC25 and what is its basic function in human cells?

CCDC25 (Coiled-Coil Domain Containing 25) is a protein-coding gene that functions as a transmembrane receptor capable of DNA binding activity. It is primarily located in the endomembrane system and serves as an integral component of the plasma membrane . CCDC25's fundamental function involves sensing neutrophil extracellular traps (NETs), which are DNA fibers released by neutrophils during inflammation. Upon binding to NETs, particularly 8-OHdG-enriched DNA, CCDC25 triggers the ILK-PARVB pathway, initiating cytoskeleton rearrangement and promoting directional cell migration . This mechanism plays a crucial role in positive regulation of cell motility under both normal and pathological conditions.

Where is CCDC25 expressed in human tissues and at what levels?

CCDC25 shows variable expression across human tissues, with abnormally high expression observed in various malignancies compared to corresponding normal tissues . In particular, CCDC25 is overexpressed in human clear cell renal cell carcinoma (ccRCC) tissues and cell lines compared to normal kidney tissues and cell lines . Similar overexpression patterns have been documented in hepatocellular carcinoma . For experimental investigation of CCDC25 expression, quantitative PCR (qPCR) and Western blotting are commonly employed methodologies, allowing researchers to compare expression levels between normal and diseased tissues.

What are the known molecular interactions of CCDC25?

CCDC25 has been identified to interact with several key molecular partners:

• DNA Binding: CCDC25 specifically binds to NETs through its extracellular region, with particular affinity for 8-OHdG-enriched DNA present in NETs .

• ILK Interaction: CCDC25 interacts with integrin-linked kinase (ILK), coordinating the activation of downstream signaling pathways . This interaction appears to be critical for its cellular functions.

• Signaling Pathway Activation: Upon binding to NETs and recruiting ILK, CCDC25 initiates the ILK-PARVB cascade and activates the NF-κB signaling pathway .

To investigate these interactions, co-immunoprecipitation assays, protein-protein interaction studies, and signaling pathway analyses using Western blotting for phosphorylated pathway components are recommended methodological approaches.

How does CCDC25 contribute to cancer progression?

CCDC25 contributes to cancer progression through multiple mechanisms:

• Enhanced Cell Proliferation: Overexpression of CCDC25 promotes cancer cell proliferation, as demonstrated in ccRCC cell lines using CCK8 assays .

• Increased Migration and Invasion: CCDC25 significantly enhances the migratory and invasive capabilities of cancer cells, shown through wound healing and transwell invasion assays .

• Metastasis Promotion: CCDC25 senses NETs and promotes directional migration of tumor cells toward these structures, facilitating metastatic spread .

• Signaling Pathway Activation: CCDC25 activates the ILK-NF-κB signaling pathway, which regulates various oncogenic processes including proliferation, survival, and invasion .

For investigating these mechanisms, researchers commonly employ knockdown or overexpression of CCDC25 in cancer cell lines, followed by functional assays including proliferation assays, migration assays, invasion assays, and in vivo tumor formation experiments in nude mice .

What is the prognostic significance of CCDC25 expression in different cancers?

CCDC25 expression has emerging prognostic significance in several cancer types:

• Hepatocellular Carcinoma (HCC): CCDC25 may serve as a potential diagnostic and prognostic marker for HCC, with higher expression potentially correlating with worse outcomes .

• Clear Cell Renal Cell Carcinoma (ccRCC): Overexpression of CCDC25 in ccRCC tissues suggests it could be a prognostic indicator, though comprehensive survival analyses are still being developed .

To assess the prognostic value of CCDC25, researchers should conduct Kaplan-Meier survival analyses correlating CCDC25 expression levels with patient outcomes, multivariate Cox regression analyses to determine independent prognostic value, and meta-analyses of multiple datasets to establish robust prognostic significance.

How does CCDC25 interact with neutrophil extracellular traps in cancer metastasis?

CCDC25 functions as a specialized receptor for neutrophil extracellular traps (NETs) in the context of cancer metastasis:

• Specific NET Recognition: CCDC25's extracellular domain specifically recognizes and binds to NET-DNA, particularly 8-OHdG-enriched DNA regions .

• Directional Migration: Upon binding to NETs, CCDC25 activates the ILK-PARVB cascade, inducing cytoskeletal rearrangements that facilitate directional migration of cancer cells toward NETs .

• Metastatic Niche Formation: NETs can act as a chemotactic factor for cancer cells expressing CCDC25, potentially guiding them to pre-metastatic niches .

Methodologically, researchers can investigate this interaction using fluorescently labeled NET-DNA binding assays, competitive binding experiments with oligopeptides, cell migration assays toward purified NETs, and in vivo metastasis models with NET inhibition or CCDC25 knockdown .

What are effective methods for modulating CCDC25 expression in experimental settings?

Several approaches can be employed to modulate CCDC25 expression:

• RNA Interference (RNAi): siRNA or shRNA targeting CCDC25 can effectively knockdown its expression. This approach has been successfully used in ccRCC cell lines to demonstrate the functional role of CCDC25 .

• CRISPR-Cas9 Gene Editing: For permanent knockout or modification of CCDC25, CRISPR-Cas9 technology offers precise genome editing capability.

• Overexpression Systems: Plasmid-based overexpression of wild-type or mutant CCDC25 can be achieved using appropriate expression vectors with strong promoters .

• Small Molecule Inhibitors: While specific inhibitors of CCDC25 are still under development, oligopeptides that block CCDC25 interaction with DNA have shown promise in inhibiting its function .

When designing experiments, researchers should include appropriate controls, validate the modulation efficiency by qPCR and Western blotting, and consider potential off-target effects, especially with RNAi approaches.

What assays are optimal for assessing CCDC25 function in vitro?

Several assays are particularly useful for investigating CCDC25 function:

• Proliferation Assays: CCK8 assay can effectively measure the impact of CCDC25 modulation on cell proliferation rates .

• Migration Assays: Wound healing (scratch) assays allow for assessment of directional migration capacity influenced by CCDC25 expression .

• Invasion Assays: Transwell invasion assays with Matrigel coating provide insights into invasive capacity regulated by CCDC25 .

• Protein-Protein Interaction Assays: Co-immunoprecipitation and proximity ligation assays can identify CCDC25 interaction partners, particularly with ILK .

• DNA Binding Assays: Fluorescence anisotropy can quantitatively determine binding kinetics between CCDC25 and various DNA structures, especially NET-DNA .

• Signaling Pathway Activation: Western blotting for phosphorylated components of the ILK-NF-κB pathway can reveal CCDC25's impact on downstream signaling .

When implementing these assays, researchers should optimize conditions for their specific cell lines, include appropriate positive and negative controls, and consider complementary approaches to validate findings.

What in vivo models are most appropriate for studying CCDC25 function?

Several in vivo models have proven valuable for investigating CCDC25 function:

• Xenograft Tumor Models: Subcutaneous implantation of CCDC25-modulated cancer cells in nude mice provides insights into its role in tumor growth .

• Metastasis Models: Tail vein injection or orthotopic implantation followed by spontaneous metastasis assessment can evaluate CCDC25's role in metastatic spread .

• Genetic Mouse Models: While not yet widely reported, conditional knockout or knockin mice for CCDC25 could provide valuable insights into its physiological function.

• NET-Induced Metastasis Models: Models that incorporate NET formation (e.g., through LPS injection) combined with CCDC25 modulation can specifically assess its role in NET-mediated metastasis .

Methodological considerations include proper sample size calculation, blinded assessment of outcomes, detailed monitoring of tumor growth and metastatic burden, and comprehensive histological and molecular analyses of tissue specimens.

What approaches are being developed to target CCDC25 therapeutically?

Several therapeutic approaches targeting CCDC25 are under investigation:

• Oligopeptide Inhibitors: Precise oligopeptides have been designed to block CCDC25 interaction with DNA in NETs, showing promise in inhibiting tumor metastasis . These peptides compete with CCDC25 for binding to NET-DNA.

• RNA Interference-Based Therapeutics: Though still in preclinical stages, siRNA or shRNA targeting CCDC25 has shown effectiveness in reducing tumor growth and metastasis in experimental models .

• Small Molecule Inhibitors: While specific small molecule inhibitors of CCDC25 are still being developed, compounds that disrupt protein-protein interactions between CCDC25 and ILK could have therapeutic potential.

For researchers developing CCDC25-targeted therapeutics, methodological considerations include optimizing drug delivery systems, conducting comprehensive pharmacokinetic and pharmacodynamic studies, and designing appropriate combination therapy approaches.

How can CCDC25 expression be utilized as a biomarker in cancer diagnosis or prognosis?

CCDC25 shows promise as a biomarker in several contexts:

• Diagnostic Biomarker: Elevated CCDC25 expression has been observed in multiple cancer types compared to normal tissues, suggesting potential utility as a diagnostic marker, particularly for hepatocellular carcinoma and clear cell renal cell carcinoma .

• Prognostic Biomarker: Preliminary evidence suggests CCDC25 expression levels may correlate with disease progression and patient outcomes, making it a candidate prognostic marker .

• Predictive Biomarker: As therapies targeting CCDC25 or its pathways develop, expression levels might predict treatment response.

Methodologically, researchers should develop standardized immunohistochemistry protocols, establish appropriate cutoff values for high versus low expression, validate findings in independent cohorts, and integrate CCDC25 status with other established biomarkers for optimal clinical utility.

How does CCDC25 regulate the ILK-NF-κB signaling pathway at the molecular level?

This advanced question addresses the detailed molecular mechanisms of CCDC25 signaling:

• Structural Biology: Determining the crystal structure of CCDC25-ILK complexes to identify interaction domains.

• Mutational Analysis: Generating CCDC25 mutants with alterations in potential ILK-binding domains to identify critical residues.

• Proximity Labeling: Techniques such as BioID or APEX2 could map the proximal proteome of CCDC25 in different cellular compartments.

• Phosphoproteomics: Mass spectrometry-based approaches to identify phosphorylation cascades triggered by CCDC25 activation.

• Live-Cell Imaging: FRET or BRET approaches to visualize real-time interactions between CCDC25, ILK, and downstream components.

Researchers should consider the dynamic nature of these interactions under different cellular conditions and the potential for cell-type specific regulation.

What is the evolutionary conservation of CCDC25 function across species and what does this reveal about its fundamental roles?

This evolutionary biology question explores CCDC25's conservation:

Investigating the evolutionary conservation of CCDC25 across species can provide insights into its fundamental biological roles. Methodological approaches include:

• Comparative Genomics: Analyzing CCDC25 sequences across species ranging from lower eukaryotes to mammals using phylogenetic analysis.

• Functional Complementation: Testing whether CCDC25 from different species can rescue phenotypes in human cell models with CCDC25 knockout.

• Domain Conservation Analysis: Identifying which domains are most highly conserved, suggesting functional importance.

• Cross-Species Expression Studies: Examining expression patterns of CCDC25 orthologs in model organisms to identify conserved regulatory mechanisms.

• Synteny Analysis: Evaluating the genomic context of CCDC25 across species to identify potentially co-evolved gene clusters.

Understanding evolutionary conservation can guide researchers to the most fundamental functions of CCDC25 that have been maintained through natural selection.

How does CCDC25 contribute to the formation of pre-metastatic niches in cancer progression?

This advanced cancer biology question explores CCDC25's role in metastasis:

The contribution of CCDC25 to pre-metastatic niche formation represents a cutting-edge area of research with significant therapeutic implications. Methodological approaches include:

• Single-Cell Analysis: Using single-cell RNA sequencing to characterize CCDC25-expressing cells within the pre-metastatic niche.

• Spatial Transcriptomics: Mapping the spatial distribution of CCDC25 expression in relation to other pre-metastatic niche markers.

• In Vivo Lineage Tracing: Following the fate of CCDC25-expressing cells during metastatic colonization.

• NET Formation Models: Developing models that specifically examine how CCDC25-NET interactions influence recruitment of immune cells and cancer cells to pre-metastatic sites.

• Secretome Analysis: Investigating how CCDC25-expressing cells might alter the local microenvironment through secreted factors.

This research direction may lead to novel approaches for interrupting the metastatic cascade by targeting CCDC25-mediated processes in pre-metastatic niche formation.

What inconsistencies exist in the literature regarding CCDC25 function, and how might these be resolved?

Several areas of controversy or inconsistency exist in CCDC25 research:

• Tissue-Specific Effects: CCDC25 may have different or even opposing effects in different cancer types or tissues. Methodologically, comprehensive pan-cancer analyses using consistent experimental approaches could help resolve these discrepancies.

• Upstream Regulators: The mechanisms controlling CCDC25 expression remain poorly characterized, with potentially conflicting reports about its regulation. Systematic promoter analyses, epigenetic profiling, and transcription factor binding studies across multiple cell types would provide clarity.

• Role in Normal Physiology: While CCDC25's pathological roles are increasingly documented, its normal physiological functions remain underexplored. Conditional knockout mouse models in specific tissues could help elucidate these functions.

Researchers should explicitly address these inconsistencies by designing experiments that directly test competing hypotheses and by replicating key findings across multiple experimental systems.

What are the most significant unanswered questions in CCDC25 research?

The field of CCDC25 research contains several important knowledge gaps:

• Structure-Function Relationship: The three-dimensional structure of CCDC25 remains unsolved, limiting our understanding of how it recognizes and binds to NETs and interacts with ILK. X-ray crystallography or cryo-EM studies are needed to address this gap.

• Regulation of Expression: The transcriptional, post-transcriptional, and post-translational regulation of CCDC25 remains poorly characterized. Comprehensive analyses of promoter elements, miRNA targeting, and protein modifications are warranted.

• Non-Cancer Functions: The role of CCDC25 in non-malignant conditions, including inflammatory and autoimmune diseases, remains largely unexplored. Studies in relevant disease models could reveal new functions.

• Therapeutic Targeting Challenges: The feasibility and potential side effects of CCDC25 inhibition as a therapeutic strategy require further investigation. Development of conditional knockout models and careful toxicology studies of CCDC25 inhibitors would address this gap.

Addressing these questions will require multidisciplinary approaches combining structural biology, biochemistry, cell biology, and in vivo disease models.

What are the technical challenges in developing specific antibodies for CCDC25 detection?

Developing specific antibodies for CCDC25 presents several technical challenges:

• Membrane Protein Nature: As an integral membrane protein, CCDC25 presents difficulties in maintaining native conformation during immunization and antibody production.

• Cross-Reactivity: Ensuring specificity against related coiled-coil domain-containing proteins requires careful epitope selection and extensive validation.

• Isoform Specificity: Antibodies may need to distinguish between potential CCDC25 isoforms for accurate detection.

Methodological approaches to address these challenges include:

• Developing monoclonal antibodies against carefully selected unique epitopes

• Comprehensive validation using knockout/knockdown controls

• Testing across multiple applications (IHC, IF, WB, IP) to ensure consistent performance

• Employing synthetic peptide competition assays to confirm specificity

How can researchers best standardize CCDC25 expression analysis for clinical applications?

Standardization of CCDC25 expression analysis is crucial for clinical applications:

• Reference Standards: Establish calibrated reference materials with known CCDC25 expression levels for normalization across laboratories.

• Protocol Standardization: Develop consensus protocols for tissue processing, staining conditions, and scoring systems for immunohistochemistry.

• Quantification Methods: Implement digital pathology and automated scoring to reduce inter-observer variability.

• Multi-Center Validation: Conduct ring trials across multiple laboratories to ensure reproducibility of CCDC25 detection methods.

• Combined Biomarker Approaches: Develop standardized panels that include CCDC25 alongside other established biomarkers for improved clinical utility.