CPNE7 Antibody

What is CPNE7 and why is it significant in cancer research?

CPNE7 (Copine-7) is a calcium-dependent phospholipid-binding protein that plays critical roles in calcium-mediated intracellular processes. It belongs to the copine family, which includes other members such as CPNE1, CPNE3, and CPNE6 . The significance of CPNE7 in cancer research stems from its differential expression and functional impact across multiple cancer types. In colorectal cancer (CRC), CPNE7 is significantly upregulated in tumor tissues compared to normal tissues, with expression patterns correlating with disease progression and patient survival outcomes . While initially characterized as a potential tumor suppressor gene in some cancers like breast cancer, recent evidence demonstrates its oncogenic role in colorectal cancer and oral squamous cell carcinoma, highlighting its context-dependent functionality in different cancer types . This dual nature makes CPNE7 an intriguing target for cancer biology investigations.

How does CPNE7 expression vary across colorectal cancer cell lines compared to normal cells?

CPNE7 expression demonstrates significant variation across colorectal cancer cell lines compared to normal colon cells. Quantitative RT-PCR analysis reveals that CPNE7 expression is consistently higher in colorectal cancer cell lines (SW480, SW620, HCT116, HT29) compared to the normal colon fibroblast cell line CCD-18-co . Among the cancer cell lines, SW480 and HCT116 exhibit particularly elevated expression levels. Western blotting confirms these findings at the protein level, with all tested CRC cell lines showing high CPNE7 protein expression . This differential expression pattern provides a molecular basis for studying CPNE7's role in colorectal carcinogenesis and validates the relevance of these cell line models for CPNE7-focused research. These expression patterns also suggest that colorectal cancer cells may depend on elevated CPNE7 levels for maintaining their malignant phenotype.

What subcellular localization patterns does CPNE7 exhibit?

CPNE7 exhibits complex subcellular localization patterns that vary by cell type and experimental conditions. While traditionally classified as a membrane protein due to its phospholipid-binding properties, immunohistochemical staining of colorectal cancer patient tissues reveals CPNE7 presence in the cytoplasm and nucleus as well as the cell membrane . Western blot analysis using cellular fractionation confirms this diverse localization pattern, with CPNE7 detected in both nuclear and cytoplasmic fractions of HeLa cells . This multifaceted distribution pattern suggests CPNE7 may perform distinct functions in different cellular compartments, potentially interacting with varied binding partners depending on its localization. When designing immunofluorescence experiments with CPNE7 antibodies, researchers should anticipate detecting signals across multiple cellular compartments and consider using co-localization studies with compartment-specific markers to fully characterize CPNE7's distribution patterns in their specific experimental system.

How does CPNE7 influence epithelial-mesenchymal transition (EMT) in colorectal cancer?

CPNE7 functions as a positive regulator of epithelial-mesenchymal transition (EMT) in colorectal cancer, as evidenced by gene expression changes following CPNE7 knockdown. When CPNE7 is suppressed in CRC cell lines, significant alterations occur in key EMT markers: epithelial markers E-cadherin and Collagen A1 increase dramatically (by approximately 600% and 1800%, respectively), while mesenchymal markers generally decrease . Specifically, EMT-associated transcription factors ZEB1 and ZEB2 decrease by approximately 1100% and 250% respectively, while MMP2, MMP3, and SNAIL show approximately 40% reduction in expression . These changes indicate that CPNE7 normally promotes the mesenchymal phenotype in CRC cells. The mechanism likely involves CPNE7's participation in signaling pathways that regulate these EMT genes, though the precise molecular interactions require further elucidation. Researchers investigating EMT mechanisms should consider CPNE7 as a potential upstream regulator and may benefit from examining its interaction with known EMT pathway components such as Wnt/β-catenin or TGF-β signaling pathways.

What experimental approaches have proven effective for CPNE7 knockdown studies?

Multiple experimental approaches have demonstrated efficacy for CPNE7 knockdown studies, with both transient and stable suppression systems yielding consistent functional effects. Research shows successful CPNE7 suppression using:

• siRNA-mediated transient knockdown: Achieved greater than 80% reduction in CPNE7 expression across multiple colorectal cancer cell lines, with SW480 showing the most significant mRNA reduction and SW620 exhibiting the most pronounced protein level reduction .

• shRNA-mediated stable knockdown: Generated through viral infection and selection, providing sustained CPNE7 suppression for long-term studies including in vivo experiments .

How does CPNE7 expression correlate with patient survival in colorectal cancer?

CPNE7 expression demonstrates significant negative correlation with patient survival in colorectal cancer, as evidenced by comprehensive Kaplan-Meier survival analysis. Data from approximately 250 CRC patients shows that those with high CPNE7 expression exhibit markedly worse survival outcomes compared to those with low expression . At the 40-month follow-up point, approximately 58% of patients with high CPNE7 expression had died, compared to only 27% in the low-expression group . The survival disparity becomes even more pronounced at longer follow-up periods, with 120-month data showing virtually complete mortality in the high-expression group (127 deaths out of 128 patients) versus significantly better survival in the low-expression cohort (3 deaths out of 123 patients) . Statistical analysis confirms this correlation is significant (p=0.048 for univariate and p=0.032 for multivariate analysis) . Importantly, CPNE7 expression also correlates with lymphatic invasion (p=0.003), suggesting a potential biological mechanism for its impact on survival through promotion of metastatic spread . This survival correlation indicates CPNE7 may serve as a valuable prognostic biomarker in CRC and justifies further investigation into therapeutic strategies targeting CPNE7 or its downstream pathways.

What are the optimal antibody validation methods for CPNE7 detection in cancer tissues?

Comprehensive validation of CPNE7 antibodies requires a multi-platform approach to ensure specificity and reliability across different experimental applications. Based on current research protocols, the following validation workflow is recommended:

• Western blot validation:

  • Test the antibody against both recombinant CPNE7 protein and endogenous CPNE7 from cell lysates

  • Include positive controls (cells known to express CPNE7, such as SW480 or HCT116)

  • Include negative controls (CPNE7 knockdown cells or non-expressing cells like CCD-18-co)

  • Verify the detected band matches the predicted molecular weight (approximately 70 kDa)

• Immunohistochemistry validation:

  • Test on FFPE tissue sections with known CPNE7 expression status

  • Include isotype controls to assess non-specific binding

  • Validate subcellular localization patterns (membrane, cytoplasmic, and nuclear staining)

  • Compare staining patterns between normal and tumor tissues from the same patient

• Specificity validation:

  • Pre-absorption with recombinant CPNE7 antigen should eliminate specific staining

  • Cross-reactivity testing against other copine family members (CPNE1, CPNE3, CPNE6) is essential due to high sequence homology

  • Genetic validation using CPNE7 knockout or knockdown systems

• Application-specific optimization:

  • Determine optimal antibody concentration for each application (IHC: typically 1-10 μg/mL; WB: approximately 1 μg/mL)

  • Optimize antigen retrieval methods for fixed tissues (heat-induced vs. enzymatic retrieval)

  • Test different blocking reagents to minimize background

These rigorous validation steps ensure reliable antibody performance across experimental platforms and strengthen the validity of subsequent research findings.

What technical considerations are important when designing CPNE7 functional studies?

Designing robust CPNE7 functional studies requires careful consideration of several technical factors to ensure reliable and reproducible results. Key considerations include:

• Cell model selection:

  • Choose cell lines with varying baseline CPNE7 expression levels (e.g., SW480 and HCT116 for high expression; CCD-18-co for low expression)

  • Consider patient-derived primary cells to complement established cell lines

  • For in vivo studies, both knockout mouse models and xenograft models have proven effective

• Knockdown/overexpression strategies:

  • For transient effects, siRNA approaches achieve >80% knockdown efficiency

  • For stable manipulation, shRNA or CRISPR-Cas9 systems provide sustained alteration

  • Rescue experiments with wild-type CPNE7 are essential to confirm specificity of observed phenotypes

  • Inducible expression systems help study dose-dependent effects

• Functional assay selection:

  • Proliferation: Multiple timepoints (24h, 48h, 72h) are necessary to capture growth kinetics

  • Migration/invasion: Both Transwell and wound-healing assays provide complementary data

  • Colony formation: Semi-solid agar assays assess anchorage-independent growth

  • EMT assessment: Combine morphological analysis with molecular marker quantification (E-cadherin, N-cadherin, etc.)

• Controls and normalization:

  • Include both positive controls (known EMT inducers) and negative controls (non-targeting siRNA)

  • Normalize cell functional data to baseline proliferation rates

  • Control for transfection/transduction efficiency in all experiments

  • Account for cell-cycle effects when interpreting proliferation data

• Downstream molecular analysis:

  • Examine both mRNA (qRT-PCR) and protein (Western blot) levels of CPNE7 and target genes

  • Consider pathway analysis to identify signaling networks affected by CPNE7 manipulation

  • Evaluate calcium-dependent effects given CPNE7's calcium-binding properties

Addressing these technical considerations ensures robust experimental design and facilitates meaningful interpretation of CPNE7's functional impact.

How can researchers differentiate between CPNE7's direct effects and secondary consequences in signaling pathways?

Differentiating between CPNE7's direct molecular interactions and its secondary effects on signaling pathways requires sophisticated experimental approaches that establish causality and temporal relationships. Researchers should implement the following strategies:

• Temporal analysis of signaling events:

  • Conduct time-course experiments following CPNE7 manipulation to establish the sequence of molecular changes

  • Early events (0-6 hours) after CPNE7 knockdown or overexpression are more likely to represent direct effects

  • Late events (24+ hours) may reflect secondary or compensatory mechanisms

• Protein-protein interaction studies:

  • Conduct co-immunoprecipitation (Co-IP) assays to identify direct binding partners of CPNE7

  • Perform proximity ligation assays (PLA) to visualize in situ protein interactions

  • Use mass spectrometry-based interactome analysis to comprehensively identify the CPNE7 protein complex

• Domain-specific functional analysis:

  • Generate CPNE7 mutants lacking specific functional domains (calcium-binding C2 domains or von Willebrand A-like domain)

  • Test these mutants in rescue experiments to determine which domains are essential for specific functions

  • Identify critical residues through site-directed mutagenesis

• Pathway inhibitor studies:

  • Use specific inhibitors of EMT-related pathways (e.g., TGF-β, Wnt/β-catenin, NF-κB) in combination with CPNE7 manipulation

  • Determine if pathway inhibition prevents CPNE7-induced phenotypic changes

  • This approach helps position CPNE7 within established signaling hierarchies

• Direct target validation:

  • Perform chromatin immunoprecipitation (ChIP) if nuclear localization suggests transcriptional regulatory roles

  • Use reporter assays with promoter regions of putative target genes

  • Employ EMSA (Electrophoretic Mobility Shift Assay) to detect direct DNA-protein interactions

By integrating these approaches, researchers can construct a mechanistic model that distinguishes CPNE7's direct molecular actions from downstream consequences, providing clearer insights into its role in complex cellular processes like EMT and cancer progression.

How reliable is CPNE7 as a prognostic biomarker in colorectal cancer patient stratification?

CPNE7 demonstrates significant potential as a prognostic biomarker in colorectal cancer, supported by both univariate and multivariate analyses of patient outcomes. Statistical evidence from a cohort of approximately 250 CRC patients reveals that CPNE7 expression is an independent predictor of patient survival with hazard ratios of 1.54 (p=0.048) in univariate analysis and 1.58 (p=0.032) in multivariate analysis . The table below summarizes key prognostic associations of CPNE7 expression:

What are the challenges in developing therapeutic strategies targeting CPNE7?

Developing therapeutic strategies targeting CPNE7 presents several significant challenges that span from basic molecular understanding to practical drug development considerations:

• Target specificity challenges:

  • CPNE7 shares high homology with other copine family members (CPNE1, CPNE3, CPNE6)

  • Designing inhibitors with specificity for CPNE7 over other copines requires detailed structural knowledge

  • Potential for off-target effects on other calcium-binding proteins with similar domains

• Functional complexity:

  • CPNE7 demonstrates context-dependent functions (oncogenic in colorectal cancer but potentially tumor-suppressive in other cancers)

  • Therapeutic inhibition might have unpredictable effects in different tissues

  • CPNE7 knockout mice show developmental defects in dental tissues, suggesting potential for systemic toxicity

• Molecular targeting approaches:

  • As a protein without enzymatic activity, CPNE7 lacks obvious catalytic sites for small molecule inhibition

  • Protein-protein interaction disruption requires identification of critical binding interfaces

  • Calcium-binding disruption may affect numerous physiological processes beyond CPNE7

• Delivery challenges:

  • Nuclear and cytoplasmic localization of CPNE7 necessitates delivery systems capable of accessing these compartments

  • Tissue-specific delivery would be required to minimize effects on normal CPNE7 functions

  • Potential for compensatory upregulation of other copine family members

• Patient selection considerations:

  • Need for predictive biomarkers to identify patients most likely to benefit from CPNE7 targeting

  • Development of companion diagnostics to quantify CPNE7 expression levels

  • Understanding resistance mechanisms to CPNE7-targeted therapies

• Validation requirements:

  • Demonstration of target engagement in vivo

  • Correlation between CPNE7 inhibition and therapeutic efficacy

  • Development of pharmacodynamic markers for clinical trials

Addressing these challenges requires collaborative efforts between structural biologists, medicinal chemists, and cancer biologists to develop targeted approaches that can effectively modulate CPNE7 function while minimizing off-target effects and toxicity.

What emerging technologies might enhance our understanding of CPNE7's role in cancer biology?

Several cutting-edge technologies hold promise for elucidating CPNE7's complex functions in cancer biology, enabling researchers to address current knowledge gaps with unprecedented precision:

• Single-cell multi-omics approaches:

  • Single-cell RNA sequencing can reveal heterogeneity in CPNE7 expression within tumors

  • Integration with spatial transcriptomics would map CPNE7 expression patterns within the tumor microenvironment

  • Single-cell proteomics could identify cell populations where CPNE7 exerts its strongest effects

  • These approaches would clarify whether CPNE7's oncogenic effects are universal or cell-type specific

• Advanced protein structural analysis:

  • Cryo-electron microscopy could resolve CPNE7's structure in different calcium-binding states

  • Hydrogen-deuterium exchange mass spectrometry would identify dynamic regions involved in protein interactions

  • AlphaFold2 and other AI-based structure prediction tools could model CPNE7 complexes with potential partners

  • These structural insights would facilitate rational design of CPNE7 inhibitors

• Genome and epigenome editing technologies:

  • CRISPR-Cas9 screening approaches could identify synthetic lethal interactions with CPNE7

  • Base editing technologies allow introduction of specific CPNE7 mutations to assess their functional impact

  • Epigenome editing could investigate regulatory mechanisms controlling CPNE7 expression

  • These genetic tools would establish causal relationships between CPNE7 and downstream effects

• Advanced in vivo models:

  • Patient-derived organoids expressing variable CPNE7 levels would provide physiologically relevant models

  • Humanized mouse models could better recapitulate CPNE7's effects in human cancer

  • CRISPR-engineered animal models with tissue-specific CPNE7 manipulation would clarify its context-dependent roles

  • These models would bridge the gap between in vitro findings and clinical observations

• Calcium signaling visualization techniques:

  • Genetically encoded calcium indicators combined with CPNE7 fluorescent tagging would enable real-time visualization of CPNE7 dynamics during calcium fluctuations

  • Optogenetic tools could trigger calcium release to study immediate CPNE7 responses

  • These approaches would illuminate CPNE7's calcium-dependent functions in living cells

Integrating these advanced technologies would provide unprecedented insights into CPNE7's multifaceted roles in cancer biology and potentially reveal new therapeutic vulnerabilities in CPNE7-expressing tumors.

How might CPNE7 research findings in colorectal cancer translate to other cancer types?

The translation of CPNE7 research findings from colorectal cancer to other cancer types requires systematic comparative analysis and consideration of tissue-specific factors. Several approaches and considerations are essential for this translation:

• Cross-cancer expression analysis:

  • Comprehensive profiling of CPNE7 expression across cancer types using multi-omics databases (TCGA, ICGC)

  • Correlation of expression patterns with clinical outcomes in each cancer type

  • Identification of cancer types with similar CPNE7 expression patterns to CRC

• Functional conservation assessment:

  • Validation of CPNE7's oncogenic functions in cell line models representing diverse cancer types

  • Comparison of phenotypic changes following CPNE7 manipulation across cancer types

  • Evaluation of whether CPNE7-regulated EMT processes are consistent across different tissues of origin

• Mechanistic pathway comparison:

  • Analysis of whether CPNE7 interacts with the same downstream effectors across cancer types

  • Identification of tissue-specific binding partners through comparative interactome studies

  • Determination if calcium-dependent functions of CPNE7 are universally conserved

• Context-dependent effects:

  • Investigation of potential tumor-suppressive roles in certain cancer types versus oncogenic roles in others

  • Analysis of how tissue-specific microenvironments might influence CPNE7 function

  • Consideration of how different genetic landscapes might modify CPNE7's impact

• Therapeutic implications:

  • Assessment of whether CPNE7-targeting strategies developed for CRC could apply to other cancers

  • Identification of cancer types most likely to benefit from CPNE7 modulation

  • Development of biomarkers to predict responsiveness to CPNE7-targeted therapies across cancer types

This translational approach would determine whether CPNE7 represents a broad oncogenic driver across multiple cancers or if its functions are context-dependent, guiding the development of targeted therapeutic strategies with appropriate patient selection criteria.

What are the best practices for incorporating CPNE7 analysis in cancer research studies?

Incorporating CPNE7 analysis in cancer research requires methodological rigor and attention to technical details. Based on current evidence, the following best practices are recommended:

• Experimental design considerations:

  • Include paired tumor and normal tissues when analyzing CPNE7 expression in patient samples

  • Utilize multiple cancer and normal cell lines to account for biological variability

  • Design time-course experiments to capture both immediate and delayed effects of CPNE7 manipulation

  • Implement both gain-of-function and loss-of-function approaches for comprehensive functional assessment

• Technical methodology recommendations:

  • For protein detection: Use validated antibodies with demonstrated specificity against CPNE7 (e.g., mouse monoclonal antibodies at 1 μg/mL concentration for Western blotting)

  • For gene expression analysis: Design primers spanning exon junctions to prevent genomic DNA amplification

  • For knockdown studies: Compare siRNA and shRNA approaches, validating knockdown efficiency at both mRNA and protein levels

  • For functional assays: Implement multiple complementary assays (proliferation, migration, invasion, colony formation) to comprehensively assess phenotypic changes

• Data analysis and interpretation:

  • Stratify patient cohorts by CPNE7 expression levels using clearly defined thresholds

  • Perform both univariate and multivariate analyses when correlating CPNE7 with clinical outcomes

  • Consider calcium signaling context when interpreting CPNE7 functional data

  • Analyze EMT markers comprehensively, including both epithelial (E-cadherin, Collagen A1) and mesenchymal (N-cadherin, ZEB1/2, SNAIL) markers

• Reporting standards:

  • Clearly document antibody validation procedures, including specificity controls

  • Report full experimental conditions including cell density, passage number, and culture conditions

  • Include comprehensive methods descriptions enabling reproducibility

  • Document statistical approaches and significance thresholds