ANKDD1A Antibody

What is ANKDD1A and why is it important for cancer research?

ANKDD1A is a protein containing nine ankyrin repeats and one death domain, located at chromosome 15q22.31. It has been identified as a potential tumor suppressor in several cancers, including breast cancer and glioblastoma multiforme. The importance of ANKDD1A in cancer research stems from its decreased expression in malignant tissues compared to normal tissues, and its correlation with better prognosis in patients with higher expression levels . The ankyrin repeat domain enables protein-protein interactions, which is critical for ANKDD1A's biological functions, particularly in immune processes and hypoxia response pathways .

What sample types can be analyzed using ANKDD1A antibodies?

ANKDD1A antibodies can be used to analyze various sample types including:

• Formalin-fixed paraffin-embedded (FFPE) tissue sections

• Frozen tissue sections

• Cell lines (both primary cultures and established lines)

• Protein lysates for western blotting

Research indicates that ANKDD1A expression can be successfully detected in both normal brain tissues and breast tissues, as well as in corresponding cancer samples. Expression analysis has been performed in both para-cancerous breast tissues and breast cancer tissues, as well as in glioma samples versus normal brain tissues .

What are the recommended applications for ANKDD1A antibodies in cancer research?

Based on the current literature, ANKDD1A antibodies are valuable for multiple research applications:

• Immunohistochemistry (IHC) to assess protein expression patterns in tissue samples and correlate with clinical parameters

• Western blotting to quantify protein levels and analyze post-translational modifications

• Immunoprecipitation to study protein-protein interactions, particularly with the HIF1α pathway components

• Immunofluorescence to examine subcellular localization, especially under hypoxic conditions

• Flow cytometry to analyze ANKDD1A expression in different cell populations, particularly in immune cell subsets

For breast cancer research, ANKDD1A antibodies have been used to correlate expression with different molecular subtypes, ER status, and immune cell infiltration . In glioma research, these antibodies have helped establish ANKDD1A's role in hypoxia response pathways through its interaction with FIH1 .

How should researchers interpret variations in ANKDD1A expression across different cancer types?

When interpreting ANKDD1A expression data, researchers should consider several key factors:

In breast cancer, ANKDD1A expression varies by molecular subtype, with the highest expression in normal-like subtype, followed by basal, luminal A, and luminal B, with the lowest expression in HER2-enriched type . Additionally, ANKDD1A is upregulated in ER-negative breast cancers compared to ER-positive cases, and shows higher expression in infiltrating lobular carcinoma compared to infiltrating ductal carcinoma .

In glioblastoma, most tumor samples exhibit low ANKDD1A expression, while normal brain tissues generally show medium to high expression levels . These expression patterns correlate with patient survival outcomes, as higher ANKDD1A expression predicts better prognosis in both breast cancer and glioma patients .

The interpretation of expression variations should consider the specific cellular context, hypoxic conditions, and the immune microenvironment, as ANKDD1A appears to play different roles depending on these factors.

How does DNA methylation affect ANKDD1A expression, and what methodologies can detect this epigenetic regulation?

ANKDD1A expression is significantly influenced by DNA methylation, particularly in glioblastoma. The ANKDD1A promoter contains a CpG island extending from approximately -400 bp to +400 bp from the transcription start site, making it susceptible to epigenetic silencing through hypermethylation . This hypermethylation appears to be a key mechanism for the decreased expression of ANKDD1A observed in glioma tissues.

To investigate this epigenetic regulation, researchers can employ several methodologies:

• Methylation-specific PCR (MSP) to detect methylation status of specific CpG sites

• Bisulfite sequencing for comprehensive analysis of all CpG sites within the promoter region

• Methylation arrays for genome-wide methylation profiling

• Pyrosequencing for quantitative methylation analysis

• Treatment of cells with demethylating agents (e.g., 5-aza-2'-deoxycytidine) followed by expression analysis using ANKDD1A antibodies

When designing experiments to study ANKDD1A methylation, researchers should consider analyzing both the CpG island in the promoter region and potential enhancer regions that might influence gene expression .

What are the optimal conditions for investigating ANKDD1A's interaction with FIH1 and its role in hypoxia response?

To effectively study ANKDD1A's interaction with FIH1 (Factor Inhibiting HIF1, also known as HIF1AN) and its role in hypoxia response, researchers should consider the following experimental conditions:

• Hypoxia induction: Culture cells at 1-2% O₂ for 24-48 hours to properly activate hypoxia response pathways

• Co-immunoprecipitation assays: Use ANKDD1A antibodies to pull down protein complexes and probe for FIH1, or vice versa

• Domain mapping: Generate constructs expressing specific domains of ANKDD1A (particularly the ankyrin repeat domain) to determine the precise interaction interface with FIH1's N-terminal domain

• Functional assays: Measure HIF1α hydroxylation status and transcriptional activity using reporter assays under both normoxic and hypoxic conditions

• Live-cell imaging: Use fluorescently tagged proteins to track the dynamics of ANKDD1A-FIH1 interaction in real-time during hypoxia

Research has confirmed that the ankyrin repeat domain of ANKDD1A directly binds to the N-terminal domain of FIH1, and this interaction appears to regulate HIF1α stability and activity . Under hypoxic conditions, ANKDD1A expression leads to decreased HIF1α levels by promoting its ubiquitination and degradation through upregulation of FIH1 and PHD2 .

How can researchers effectively analyze the correlation between ANKDD1A expression and immune cell infiltration in tumor microenvironments?

To analyze the correlation between ANKDD1A expression and immune cell infiltration in tumor microenvironments, researchers should employ a multi-faceted approach:

• Multiplex immunohistochemistry or immunofluorescence:

  • Use ANKDD1A antibody in combination with immune cell markers

  • Analyze spatial relationships between ANKDD1A-expressing cells and different immune cell populations

• Flow cytometry and cell sorting:

  • Isolate tumor-infiltrating lymphocytes and analyze ANKDD1A expression in different immune cell subsets

  • Correlate with functional markers of T cell activation and exhaustion

• Transcriptomic analysis:

  • Examine correlation between ANKDD1A and immune cell gene signatures

  • Focus particularly on T cell markers that have shown strong correlation with ANKDD1A, such as:

    • TBX21 (R=0.422)

    • STAT4 (R=0.418)

    • CD3D (R=0.417)

    • PDCD1 (R=0.400)

    • ITGAX (R=0.400)

• Computational deconvolution of bulk RNA-seq data:

  • Use algorithms like CIBERSORT or xCell to estimate immune cell proportions

  • Correlate with ANKDD1A expression levels

Studies have shown that ANKDD1A is highly correlated with CD4+ T cells (R=0.455), dendritic cells (R=0.381), and neutrophils (R=0.339) . This suggests ANKDD1A may play a role in regulating T cell function, particularly Th1 cells, which has implications for anti-tumor immunity.

What experimental approaches can resolve conflicting data regarding ANKDD1A's effects in different cancer types?

Resolving conflicting data regarding ANKDD1A's effects across different cancer types requires systematic experimental approaches:

• Context-dependent molecular characterization:

  • Perform comprehensive proteomic analysis to identify cancer-specific ANKDD1A binding partners

  • Use proximity labeling techniques (BioID or APEX) to map the ANKDD1A interactome in different cancer types

  • Analyze post-translational modifications of ANKDD1A that might alter its function

• Isogenic cell line panels:

  • Generate ANKDD1A knockout and overexpression models in multiple cancer cell lines

  • Perform parallel phenotypic assays under standardized conditions

  • Compare effects on proliferation, invasion, and response to hypoxia

• Patient-derived models:

  • Establish patient-derived xenografts (PDXs) or organoids with varying ANKDD1A expression levels

  • Characterize growth patterns and molecular profiles

  • Test response to therapy in relation to ANKDD1A status

• Conditional expression systems:

  • Develop inducible ANKDD1A expression systems to study temporal effects

  • Monitor changes in signaling pathways upon ANKDD1A induction or repression

Research has shown that while ANKDD1A acts as a tumor suppressor in both breast cancer and glioblastoma, its mechanisms differ: in breast cancer, it appears to function primarily through immune-related pathways , whereas in glioblastoma, it regulates hypoxia response through interaction with FIH1 . These context-dependent functions may explain seemingly conflicting observations across cancer types.

What methodological considerations should be taken when analyzing ANKDD1A's role in Th1/Th2 cell differentiation and immune response?

When investigating ANKDD1A's role in Th1/Th2 cell differentiation and immune response, researchers should consider these methodological approaches:

• T cell isolation and differentiation assays:

  • Isolate naïve CD4+ T cells and culture under Th1 or Th2 polarizing conditions

  • Manipulate ANKDD1A expression using siRNA, CRISPR, or overexpression systems

  • Assess impact on differentiation markers and cytokine production

• ChIP-seq analysis:

  • Map binding of transcription factors associated with Th1/Th2 differentiation (such as T-bet, GATA3) at the ANKDD1A locus

  • Identify potential enhancers or repressors that regulate ANKDD1A in different T cell subsets

• Single-cell analysis:

  • Perform single-cell RNA-seq of tumor-infiltrating lymphocytes

  • Correlate ANKDD1A expression with T cell differentiation states and functional markers

  • Create trajectory analyses to understand ANKDD1A's role in T cell differentiation

• Functional immune assays:

  • Measure cytokine production (IFN-γ, IL-2, IL-4, IL-13) in T cells with altered ANKDD1A expression

  • Assess T cell proliferation, cytotoxicity, and exhaustion markers

  • Evaluate the impact on antigen presentation and T cell activation

Functional enrichment analysis has revealed that ANKDD1A co-expressed genes are involved in multiple immune processes, including regulation of leukocyte activation, T cell activation, lymphocyte activation, and leukocyte differentiation . ANKDD1A is strongly associated with Th1 cell markers (TBX21, STAT4) and T cell exhaustion markers (PDCD1), suggesting its involvement in T cell function regulation . The ratio of Th1/Th2 cells in breast cancer tissues is considered an optimal immune prognostic factor, with increased Th1 proportion indicating favorable prognosis .

What are the most common technical challenges when using ANKDD1A antibodies, and how can they be addressed?

Several technical challenges may arise when working with ANKDD1A antibodies:

• Antibody specificity:

  • Validate antibody specificity using positive and negative controls

  • Confirm results with multiple antibodies targeting different epitopes

  • Use ANKDD1A-knockout or knockdown cells as negative controls

• Low endogenous expression:

  • Optimize signal amplification methods for IHC/IF

  • Use sensitive detection systems like tyramide signal amplification

  • Consider concentrating proteins for western blotting when working with samples having low expression

• Background signal:

  • Optimize blocking conditions (consider using 5% BSA or commercial blocking reagents)

  • Increase washing steps and duration

  • Titrate primary antibody concentration carefully

• Cross-reactivity:

  • Perform peptide competition assays to confirm specificity

  • Use monoclonal antibodies when possible for increased specificity

  • Pre-absorb antibodies with potential cross-reacting proteins

When interpreting results, it's important to remember that ANKDD1A expression varies significantly between cancer types and even within cancer subtypes. For instance, ANKDD1A shows differential expression across breast cancer molecular subtypes and varies between infiltrating lobular carcinoma and infiltrating ductal carcinoma .

How should researchers design experiments to study the relationship between ANKDD1A methylation status and protein expression?

To effectively study the relationship between ANKDD1A methylation status and protein expression, researchers should design experiments that incorporate these methodological considerations:

• Integrated analysis approach:

  • Analyze DNA methylation and protein expression in matched samples

  • Use bisulfite sequencing or methylation-specific PCR to characterize the ANKDD1A promoter

  • Correlate methylation patterns with protein expression using ANKDD1A antibodies

• Experimental manipulation:

  • Treat cells with demethylating agents (5-aza-2'-deoxycytidine)

  • Monitor changes in ANKDD1A expression using western blotting and qRT-PCR

  • Perform time-course experiments to track the dynamics of demethylation and re-expression

• Targeted methylation modification:

  • Use CRISPR-dCas9 systems with DNA methyltransferases (DNMT3A) or TET enzymes

  • Target specific CpG sites in the ANKDD1A promoter

  • Assess the impact on protein expression

• Clinical correlation:

  • Analyze methylation status in patient samples using methylation arrays

  • Perform IHC with ANKDD1A antibodies on matched samples

  • Correlate findings with clinical parameters and survival data

Research has shown that the ANKDD1A promoter contains a CpG island region extending from -400 bp to +400 bp from the transcription start site . Hypermethylation of this region appears to be a key mechanism for the decreased expression of ANKDD1A observed in glioma tissues, suggesting epigenetic silencing plays an important role in regulating this gene .

What emerging technologies could enhance our understanding of ANKDD1A's function in cancer biology?

Several cutting-edge technologies hold promise for advancing our understanding of ANKDD1A's function in cancer:

• Spatial transcriptomics and proteomics:

  • Map ANKDD1A expression within the tumor microenvironment with spatial resolution

  • Correlate with immune cell infiltration patterns and hypoxic regions

  • Identify microenvironmental factors that regulate ANKDD1A expression

• CRISPR-based functional genomics:

  • Perform genome-wide CRISPR screens to identify synthetic lethal interactions with ANKDD1A

  • Use CRISPR activation/inhibition to modulate ANKDD1A expression

  • Employ base editors to introduce specific mutations in ANKDD1A regulatory regions

• Protein structure determination:

  • Resolve the 3D structure of ANKDD1A, particularly the ankyrin repeat domain

  • Characterize the structural basis of the ANKDD1A-FIH1 interaction

  • Use this information to develop small molecules that mimic ANKDD1A function

• Single-cell multi-omics:

  • Integrate single-cell transcriptomics, proteomics, and epigenomics

  • Track ANKDD1A expression and function at single-cell resolution

  • Identify cell-specific roles across diverse tumor microenvironments

These technologies could help resolve the context-dependent functions of ANKDD1A observed in different cancer types. For instance, understanding why ANKDD1A functions primarily through immune-related pathways in breast cancer but regulates hypoxia response in glioblastoma would provide valuable insights into its broader role in cancer biology.

How might ANKDD1A function as a biomarker for patient stratification and therapeutic response prediction?

ANKDD1A shows significant potential as a biomarker for patient stratification and treatment response prediction:

• Prognostic applications:

  • Develop standardized IHC protocols using validated ANKDD1A antibodies

  • Establish scoring systems for ANKDD1A expression in different cancer types

  • Integrate with existing prognostic markers to improve risk stratification

• Predictive applications:

  • Correlate ANKDD1A expression with response to immunotherapies

  • Evaluate its predictive value for treatments targeting hypoxia-related pathways

  • Assess its utility in predicting response to epigenetic therapies

• Multi-marker panels:

  • Combine ANKDD1A with immune cell markers (particularly T cell markers)

  • Develop integrated scores incorporating ANKDD1A methylation and expression

  • Create cancer-specific panels based on ANKDD1A's context-dependent functions

• Liquid biopsy approaches:

  • Investigate ANKDD1A methylation in circulating tumor DNA

  • Develop sensitive detection methods for monitoring treatment response

  • Correlate with tissue-based ANKDD1A expression

Research indicates that patients with higher ANKDD1A expression have more favorable prognosis in both breast cancer and glioblastoma . In breast cancer, ANKDD1A's strong association with T cell markers suggests it may help identify patients likely to respond to immunotherapies . In glioblastoma, its role in hypoxia response pathways indicates potential utility in predicting response to therapies targeting these pathways .

How does ANKDD1A compare with other ankyrin repeat domain-containing proteins in experimental applications?

ANKDD1A belongs to the ankyrin repeat domain-containing protein family, which includes numerous members with diverse functions. When comparing ANKDD1A with other family members in experimental applications:

Key experimental differences to consider:

• Antibody cross-reactivity:

  • Due to the conserved nature of ankyrin repeats, validate antibody specificity against other family members

  • Confirm specificity using knockout controls or peptide competition assays

• Functional redundancy:

  • Design experiments to account for potential compensation by other family members

  • Consider combinatorial knockdown/knockout approaches

• Interaction partners:

  • ANKDD1A uniquely interacts with FIH1 through its ankyrin repeat domain

  • This interaction should be considered when designing co-immunoprecipitation experiments

• Tissue expression patterns:

  • ANKDD1A shows more restricted expression patterns than some widely expressed family members

  • This can affect choice of experimental models and controls

Unlike many ankyrin repeat proteins that function in cytoskeletal organization, ANKDD1A appears specialized for tumor suppression through immune regulation and hypoxia response pathways , making it uniquely valuable for cancer research applications.