VOPP1 Antibody

What is VOPP1 and why is it significant in cancer research?

VOPP1 (Vesicular, Overexpressed in Cancer, Prosurvival Protein 1) is a protein that has emerged as a significant player in cancer biology, particularly in breast tumorigenesis. Research has identified VOPP1 as a novel oncogene that promotes carcinogenesis by inhibiting tumor suppressor functions. VOPP1 is overexpressed in approximately 25% of breast tumors compared to normal breast tissue, with particularly elevated levels observed in ERBB2 breast cancer subtypes . The protein contains a transmembrane domain and signal peptide, along with endosome/lysosome targeting sequences (YXXΦ motif and dileucine sequence), suggesting its function in protein sorting to endosomes, Golgi apparatus, and lysosomes . Its clinical significance is underscored by findings that high VOPP1 expression is associated with reduced metastasis-free survival in breast cancer patients, particularly in those with WWOX-positive tumors .

What epitopes of VOPP1 are typically targeted by research antibodies?

Commercial antibodies against VOPP1 target various regions of the protein, with different epitopes offering distinct advantages depending on the research application. Common epitopes include the central region (amino acids 65-95) and a broader segment (amino acids 23-172) . For polyclonal antibody production in research settings, immunization strategies have utilized dual peptide approaches targeting amino acids 101-116 (TRQPPNPGPGTQQPGP) and 137-151 (AFQVPPNSPQGSVAC) . When selecting a VOPP1 antibody, researchers should consider whether the epitope is likely to be accessible in their experimental conditions, particularly when working with fixed or denatured samples versus native conformations.

What applications are VOPP1 antibodies validated for?

VOPP1 antibodies have been validated for multiple applications in cancer research, with performance varying by host species, clonality, and targeted epitope. The most common applications include:

When using these antibodies, researchers should implement appropriate controls to ensure specificity, particularly when comparing expression levels across different experimental conditions.

How should VOPP1 expression be assessed in clinical breast cancer samples?

When analyzing VOPP1 expression in clinical breast cancer samples, multiple complementary approaches should be employed. Quantitative RT-PCR has been successfully used to identify VOPP1 overexpression (defined as ≥2.5-fold increase compared to normal breast tissue) in 25% of breast tumors . This molecular approach should be supplemented with immunohistochemical staining to assess protein levels and localization patterns. In breast tumors, VOPP1 typically displays a characteristic granular cytoplasmic distribution reflecting its vesicular localization . For accurate interpretation, researchers should consider molecular subtyping of breast tumors (luminal A, luminal B, ERBB2, triple-negative) as VOPP1 expression patterns may vary across these subtypes, with ERBB2 tumors showing slightly higher VOPP1 levels . For validation purposes, publicly available datasets such as Oncomine can be consulted to confirm expression patterns across larger patient cohorts.

What methodologies can verify VOPP1-WWOX protein interaction?

The interaction between VOPP1 and WWOX tumor suppressor represents a significant mechanism in breast cancer biology that can be investigated through multiple complementary approaches. Initial identification of this interaction employed yeast two-hybrid screening, which revealed VOPP1 as a binding partner of both full-length WWOX and its shorter isoform (WWOX v2) . To validate these findings in mammalian systems, co-immunoprecipitation assays can be performed in both overexpression systems (using tagged constructs in HEK-293T cells) and with endogenous proteins in breast cancer cell lines such as MDA-MB-468 . For mechanistic studies, researchers should generate mutant constructs targeting the key interaction domains – specifically, the WW1 domain of WWOX (using the Y33R mutant) and the PPPY165 motif in VOPP1's proline-rich C-terminal region . These mutational analyses are essential for establishing the specificity and structural requirements of the interaction. Complementary approaches like proximity ligation assays or FRET-based methods can provide additional spatial information about where in the cell these interactions occur.

How can researchers assess the functional consequences of VOPP1 overexpression?

To evaluate the functional impact of VOPP1 overexpression, researchers should implement a multi-faceted experimental approach. In vitro transformation assays using non-cancerous cell lines (e.g., NIH3T3) represent a foundational method, revealing morphological changes characterized by the appearance of small, refractile, spindle-shaped cells in VOPP1-overexpressing clones . Soft agar colony formation assays provide a critical assessment of anchorage-independent growth capabilities, a hallmark of transformation, with both colony number and size serving as quantitative metrics . For in vivo validation, xenograft studies in immunocompromised mice can demonstrate the tumorigenic potential of VOPP1-overexpressing cells, with tumor growth rate, final tumor volume, and immunohistological analyses of proliferation markers (Ki67) serving as essential endpoints . To link VOPP1 overexpression to specific molecular pathways, researchers should assess its impact on WWOX-dependent apoptosis through Annexin V/PI staining coupled with flow cytometry, and analysis of apoptotic markers like cleaved PARP . These functional studies should be complemented with VOPP1 silencing experiments to establish necessity in addition to sufficiency for the observed phenotypes.

What experimental approaches best demonstrate VOPP1's inhibition of WWOX-mediated apoptosis?

To establish VOPP1's role in suppressing WWOX-mediated apoptosis, a comprehensive experimental strategy is required. RNA interference approaches targeting VOPP1 in breast cancer cell lines with high endogenous expression (e.g., MDA-MB-468) provide a direct assessment of how VOPP1 depletion affects cell viability and apoptotic markers . Critical readouts include cell viability assays, expression of cleaved PARP (using Western blotting), and Annexin V/PI staining coupled with flow cytometry to quantify apoptotic populations . To establish specificity for WWOX-dependent apoptosis, comparative experiments should be performed in cells with and without WWOX depletion, demonstrating that VOPP1 suppression induces apoptosis only in WWOX-expressing cells . Complementary gain-of-function experiments in appropriate cellular models (e.g., A549 cancer cells) can demonstrate that ectopic VOPP1 expression overcomes WWOX-mediated cell death . For mechanistic insight, subcellular localization studies using confocal microscopy with appropriate markers should demonstrate VOPP1-mediated sequestration of WWOX in lysosomes, preventing its association with pro-apoptotic factors like p73α . Mutation studies targeting the VOPP1-WWOX interaction domains provide additional confirmation of the specific molecular requirements for this functional relationship.

How should researchers evaluate VOPP1 antibody specificity in experimental systems?

Ensuring antibody specificity is critical for reliable VOPP1 research. A comprehensive validation strategy should include multiple complementary approaches. First, researchers should perform Western blotting with positive controls (cells known to express VOPP1, such as MDA-MB-468) and negative controls (either VOPP1-knockout cells or cells treated with effective VOPP1 siRNA that demonstrates at least 70% knockdown efficiency) . For immunohistochemistry applications, researchers should compare staining patterns in normal breast tissue versus breast tumors with known VOPP1 overexpression . The characteristic granular cytoplasmic staining pattern reflecting VOPP1's vesicular localization should be consistent across samples and detection methods . When performing interaction studies, specificity controls should include competition with the immunizing peptide and evaluation of non-specific binding to relevant protein partners. For antibodies targeting specific domains, researchers should test recognition of VOPP1 mutant constructs with alterations in the target epitope. Cross-reactivity testing against related proteins should be performed when possible, particularly when working in systems where homologous proteins may be present.

What are the most effective experimental designs to study VOPP1's role in different cancer types?

When investigating VOPP1's role across different cancer types, researchers should implement a systematic experimental framework that allows for meaningful cross-cancer comparisons. Begin with expression profiling of VOPP1 at both mRNA and protein levels across a panel of cancer cell lines representing diverse tissue origins, similar to the approach used in breast cancer studies examining ten distinct cell lines . For each cancer type, establish the correlation between VOPP1 expression and WWOX status, as VOPP1's oncogenic function appears most relevant in WWOX-positive contexts . Functional studies should employ both gain-of-function approaches (overexpression in low-VOPP1 expressing cells) and loss-of-function approaches (siRNA or CRISPR-based knockout in high-VOPP1 expressing cells) across multiple cancer types. Key phenotypic assays should include proliferation, apoptosis resistance, migration, invasion, and in vivo tumorigenicity. Molecular mechanism studies should determine whether the VOPP1-WWOX interaction is universally conserved across cancer types or if tissue-specific co-factors modify this relationship. For clinical relevance, analyze VOPP1 expression in patient cohorts representing different cancer types, with careful stratification by molecular subtypes and correlation with clinical outcomes such as metastasis-free survival, as has been done for breast cancer .

How should researchers optimize immunohistochemical detection of VOPP1 in tissue samples?

Optimizing immunohistochemical detection of VOPP1 requires careful attention to several technical parameters. For formalin-fixed, paraffin-embedded (FFPE) samples, antigen retrieval conditions are critical – researchers should evaluate both heat-induced epitope retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0) to determine optimal conditions for their specific antibody . When selecting antibodies, those targeting the central region (amino acids 65-95) or broader segments (amino acids 23-172) have demonstrated efficacy in IHC applications . For visualization, both unconjugated primary antibodies with secondary detection systems and directly conjugated antibodies (HRP, FITC, or biotin-conjugated) are available and should be selected based on the specific application requirements . Researchers should establish appropriate positive controls (breast cancer tissues with known VOPP1 overexpression) and negative controls (normal breast tissue or VOPP1-depleted samples) . For scoring and interpretation, both staining intensity and the characteristic granular cytoplasmic distribution pattern should be evaluated, as this vesicular pattern is consistent with VOPP1's subcellular localization and function . Multiplexed immunofluorescence approaches combining VOPP1 with markers of subcellular compartments (e.g., lysosomal markers) can provide additional insights into its functional localization in tissue contexts.

What are the critical controls when using VOPP1 antibodies for co-immunoprecipitation studies?

When designing co-immunoprecipitation (co-IP) experiments to study VOPP1 interactions, implementing rigorous controls is essential for reliable results. Primary antibody specificity controls should include performing parallel IPs with isotype-matched control antibodies and pre-immune serum to identify non-specific binding . Input controls (typically 5-10% of the lysate used for IP) must be run alongside IP samples to verify the presence of target proteins in the starting material . For studying the VOPP1-WWOX interaction specifically, reciprocal co-IPs are essential – precipitating with anti-VOPP1 and blotting for WWOX, then precipitating with anti-WWOX and blotting for VOPP1 . Competition controls using excess immunizing peptide can confirm binding specificity. To establish the structural requirements for interaction, researchers should include mutant constructs targeting key domains (WWOX Y33R mutant affecting the WW1 domain and VOPP1 PPPY165 mutant) . Additional controls should address potential confounding variables such as DNA-mediated interactions (by including DNase treatment) and detergent effects (by testing multiple lysis conditions to ensure the interaction is not an artifact of specific detergent usage). For endogenous interaction studies in cell lines like MDA-MB-468, verification of antibody performance in both overexpression systems and with endogenous proteins provides additional confidence in co-IP results .

What methodological approaches can resolve contradictory findings about VOPP1 function?

When researchers encounter contradictory findings regarding VOPP1 function, a systematic troubleshooting approach can help resolve discrepancies. First, carefully evaluate cell type-specific effects – VOPP1's impact appears to depend on WWOX status, so contradictory results might stem from variations in endogenous WWOX expression across experimental models . Antibody-related artifacts should be addressed by using multiple antibodies targeting different epitopes and comparing their performance in the same experimental system . For functional studies, comprehensive loss-of-function approaches combining transient (siRNA) and stable (shRNA or CRISPR) knockdown strategies can distinguish between acute and adaptive responses to VOPP1 depletion . When contradictions arise regarding VOPP1's subcellular localization, co-localization studies with markers for specific cellular compartments (endosomes, lysosomes, Golgi) can provide clarification, as VOPP1 has been shown to sequester WWOX specifically in lysosomes . For interaction studies producing inconsistent results, consider the impact of experimental conditions including cell confluence, serum levels, and stress conditions that might affect VOPP1-WWOX dynamics. Meta-analysis approaches comparing VOPP1 expression and clinical outcomes across multiple independent patient cohorts can help resolve contradictions in prognostic significance . Finally, genetic rescue experiments reintroducing wild-type or mutant VOPP1 into knockout backgrounds provide definitive evidence of specific functional requirements.

How should researchers interpret VOPP1 expression patterns in relation to breast cancer prognosis?

Interpreting VOPP1 expression in the context of breast cancer prognosis requires careful consideration of multiple variables. Researchers should first establish a clear threshold for VOPP1 overexpression, such as the ≥2.5-fold increase compared to normal breast tissue used in previous studies . When analyzing survival outcomes, stratification by breast cancer molecular subtypes (luminal A, luminal B, ERBB2, triple-negative) is essential, as VOPP1's impact may vary across these distinct biological entities . Most critically, VOPP1's prognostic significance appears intimately linked to WWOX status – high VOPP1 expression is associated with reduced metastasis-free survival specifically in patients with WWOX-positive tumors, but not in those with WWOX-negative tumors . This interaction highlights the mechanistic basis of VOPP1's role in sequestering and inhibiting WWOX tumor suppressor function rather than functioning as an independent prognostic marker . For comprehensive analysis, researchers should employ multivariate models that account for established prognostic factors including tumor size, grade, lymph node status, and treatment regimens. Validation across independent cohorts is essential, utilizing tools like Kaplan-Meier survival analysis with appropriate statistical testing (log-rank test) to establish significance . Time-dependent analyses may also reveal whether VOPP1's prognostic impact varies across different post-diagnosis intervals.

What experimental design best evaluates VOPP1 as a potential therapeutic target?

Evaluating VOPP1 as a potential therapeutic target requires a comprehensive experimental framework spanning in vitro, in vivo, and ex vivo approaches. Initial target validation should establish both necessity (through loss-of-function studies in high-VOPP1 expressing cells) and sufficiency (through gain-of-function studies in model systems) for oncogenic phenotypes . Critical phenotypes to assess include cell viability, apoptosis resistance (measuring cleaved PARP and Annexin V/PI staining), and tumorigenic potential in vivo . For therapeutic development, researchers should design approaches that specifically disrupt the VOPP1-WWOX interaction, such as peptide or small molecule inhibitors targeting the WW1 domain of WWOX or the PPPY165 motif of VOPP1 . High-throughput screening assays measuring WWOX release from lysosomes or restoration of WWOX-p73α interaction could identify potential therapeutic candidates . Patient-derived xenograft models stratified by VOPP1 and WWOX expression can provide clinically relevant systems for testing candidate therapies. Combination approaches exploring potential synergy between VOPP1 inhibition and standard-of-care treatments for breast cancer should be systematically evaluated. For biomarker development, researchers should establish whether VOPP1 expression or VOPP1-WWOX interaction status can predict response to specific therapeutic approaches, potentially through immunohistochemical or molecular analyses of patient samples prior to treatment.

How can researchers establish the functional significance of VOPP1 across diverse cancer types?

To establish VOPP1's functional significance across cancer types, researchers should implement a systematic comparative oncology approach. Begin with comprehensive expression profiling of VOPP1 at both mRNA and protein levels across tumor types, similar to analyses that have identified VOPP1 overexpression in breast cancer and other tumor types referenced in the literature . For each cancer type, determine the correlation between VOPP1 expression and WWOX status, as this relationship appears critical for interpreting VOPP1's functional impact . Employing tissue microarrays representing multiple cancer types with matched normal tissues can efficiently establish expression patterns while controlling for technical variables. Functional studies should utilize cell line panels representing diverse cancer types with genetic manipulation of VOPP1 (overexpression and knockdown) followed by standardized assays measuring proliferation, apoptosis, migration, invasion, and drug sensitivity. Xenograft studies comparing VOPP1-manipulated cells across cancer types can reveal tissue-specific differences in in vivo tumorigenic potential . Mechanistic investigations should determine whether the lysosomal sequestration of WWOX by VOPP1 is a universal mechanism across cancer types or if tissue-specific pathways exist . Mining published multi-cancer genomic datasets for VOPP1 alterations (mutations, amplifications, deletions) can identify cancer types where genetic alterations rather than expression changes might drive VOPP1-related pathology.

What strategies can resolve inconsistent VOPP1 antibody performance across applications?

When facing inconsistent VOPP1 antibody performance across different applications, researchers should implement a systematic troubleshooting approach. First, analyze epitope accessibility issues – antibodies targeting amino acids 65-95 or 23-172 regions may perform differently in applications where protein conformation varies (native vs. denatured conditions) . For western blotting applications with suboptimal results, optimization of protein extraction methods is critical, with evaluation of different lysis buffers that may better preserve VOPP1's membrane-associated properties . Sample preparation conditions including heating temperature, reduction status, and detergent concentration should be systematically tested. For immunohistochemistry applications, comprehensive antigen retrieval optimization (testing multiple pH conditions and retrieval methods) may resolve inconsistent staining . When antibodies perform well in overexpression systems but poorly with endogenous proteins, sensitivity can be enhanced through signal amplification systems (tyramide signal amplification for IHC/IF or enhanced chemiluminescence for Western blotting) . Cross-validation with multiple antibodies targeting distinct epitopes can differentiate between technical issues and true biological variation . For co-immunoprecipitation applications specifically, optimizing immunoprecipitation conditions (antibody concentration, incubation time, wash stringency) and using protein A vs. protein G beads based on antibody isotype can improve performance . Finally, verify antibody lot-to-lot consistency through standardized control samples, as manufacturing variations can significantly impact performance.

How can researchers distinguish between specific and non-specific staining patterns in VOPP1 immunohistochemistry?

Distinguishing specific from non-specific staining in VOPP1 immunohistochemistry requires implementation of multiple validation approaches. Researchers should first establish the expected subcellular localization pattern – VOPP1 typically displays a characteristic granular cytoplasmic distribution reflecting its vesicular nature . Peptide competition controls, where the immunizing peptide is pre-incubated with the primary antibody before staining, can identify specific staining that disappears upon competition . Cell line control slides containing VOPP1-positive (e.g., MDA-MB-468) and VOPP1-knockdown cells prepared under identical fixation conditions provide critical positive and negative controls . Correlation between IHC staining intensity and quantitative methods (qRT-PCR, Western blotting) across the same samples adds confidence in staining specificity . Multiple antibody validation comparing staining patterns from antibodies targeting different VOPP1 epitopes can confirm true signal versus artifact . When evaluating breast tumor samples specifically, comparison with normal breast tissue serves as an important reference point, as VOPP1 shows significantly higher expression in tumors . For challenging cases, dual-labeling immunofluorescence with lysosomal markers can confirm the expected subcellular localization of VOPP1 . Finally, automated image analysis tools can help quantify staining patterns objectively and distinguish between specific signal and background across sample sets.