hoxb13a Antibody

What is HOXB13 and why is it important in research?

HOXB13 is a key lineage homeobox transcription factor that plays a critical role in the differentiation of the prostate gland. It has gained significant research interest because of its involvement in prostate cancer development and progression. HOXB13 expression increases during later stages of prostate development, and its expression pattern across different stages of cancer provides valuable insights into disease progression. Unlike other prostatic lineage markers (e.g., PSA, NKX3.1), HOXB13 expression is largely independent of androgen receptor (AR) signaling, making it a particularly valuable biomarker in advanced castration-resistant prostate cancers where AR signaling may be compromised .

How do I select the appropriate HOXB13 antibody for my experiments?

Selection of the appropriate HOXB13 antibody requires consideration of several factors:

• Antibody validation: Choose antibodies validated with genetic controls. For example, the rabbit monoclonal antibody (clone D7N8O, Cell Signaling Technologies) has been extensively validated using western blot analysis on cell lines with known HOXB13 expression and gain-of-function models .

• Application compatibility: Verify that the antibody is suitable for your specific application (WB, IHC, IF/ICC). For example, the 26384-1-AP antibody has been tested for WB (1:500-1:1000 dilution), IHC (1:50-1:500 dilution), and IF/ICC (1:50-1:500 dilution) .

• Species reactivity: Confirm the antibody's reactivity with your species of interest. Some antibodies are specific to human HOXB13, while others may cross-react with murine or other species .

• Clone type: Consider whether a monoclonal or polyclonal antibody is more suitable for your application. Polyclonal antibodies often have lower working dilutions than monoclonal antibodies .

What are the recommended protocols for optimizing HOXB13 antibody use in IHC?

Optimization of HOXB13 antibody for IHC requires:

• Antigen retrieval: For HOXB13 IHC, suggested antigen retrieval methods include TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 .

• Dilution optimization: Test a range of dilutions to determine the optimal concentration. For example, the 26384-1-AP antibody has a recommended dilution range of 1:50-1:500 for IHC .

• Positive and negative controls: Include tissue samples with known HOXB13 expression patterns. For positive controls, prostate cancer tissues are recommended. For negative controls, non-prostatic tissues can be used as HOXB13 shows high specificity (99%) for prostatic origin .

• Background minimization: All steps of IHC experiments must be optimized to visualize specific staining and minimize non-specific background signals .

• Signal detection system: Choose an appropriate detection system based on your tissue type and expected expression level.

How can I validate the specificity of a HOXB13 antibody for research applications?

Rigorous validation of HOXB13 antibody specificity requires multiple complementary approaches:

A comprehensive validation ensures that observed staining patterns accurately reflect HOXB13 distribution rather than non-specific binding .

What are the advantages and limitations of different types of HOXB13 antibodies for detecting treatment-resistant prostate cancer?

Different antibody types offer distinct advantages and limitations for detecting treatment-resistant prostate cancer:

Monoclonal Antibodies (e.g., clone D7N8O):

• Advantages: High specificity, consistent lot-to-lot performance, excellent for diagnostic applications with 97% sensitivity and 99% specificity for prostatic origin

• Limitation: May recognize a single epitope that could be masked in some contexts

Polyclonal Antibodies (e.g., 26384-1-AP):

• Advantages: Recognize multiple epitopes, potentially higher sensitivity for detecting HOXB13 in varied conformational states

• Limitations: Batch variation, potential for higher background, may require more extensive validation

For detecting HOXB13 in treatment-resistant prostate cancer specifically, key considerations include:

• AR independence: HOXB13 expression is largely AR-independent, making it valuable for detecting castration-resistant prostate cancers that have lost AR expression (~30% of cases)

• Sensitivity in advanced disease: HOXB13 shows greater sensitivity in detecting advanced metastatic prostate cancers compared to NKX3.1

• Epitope accessibility: Some antibodies may have reduced sensitivity in decalcified bone tissue, though clone D7N8O appears less vulnerable to pre-analytic differences

• Detection in lineage-plastic tumors: 84% of androgen receptor-negative castration-resistant prostate cancers and neuroendocrine prostate cancers (NEPC) retain detectable levels of HOXB13

How can I develop effective immunohistochemical panels incorporating HOXB13 for diagnosing challenging prostate cancer cases?

Developing effective IHC panels incorporating HOXB13 requires strategic selection of complementary markers:

Important considerations:

• HOXB13 has shown 97% sensitivity and 99% specificity for prostatic origin in a cohort of 837 patients (383 prostatic and 454 non-prostatic tumors)

• HOXB13 expression is maintained in most advanced prostate cancers even after treatment

• Include appropriate controls and standardize scoring criteria across pathologists

• Be aware that some tumors (4% of colorectal cancers, cauda equina neuroendocrine tumors, Ewing's sarcoma, and embryonal rhabdomyosarcoma) may express HOXB13

How does HOXB13 expression change throughout prostate development and cancer progression?

HOXB13 expression follows a dynamic pattern throughout development and cancer progression:

During Development:

• HOXB13 expression increases during later stages of murine prostate development

• In mouse embryos at E17.5, HOXB13 expression is restricted to the terminal segments of the tail, the distal rectum, and the urogenital sinus

• In the urogenital sinus, HOXB13 is expressed exclusively in epithelial cells, while in the tail it is predominantly expressed by stromal cells

In Localized Prostate Cancer:

• All localized prostate cancers show HOXB13 protein expression

• Lower HOXB13 expression levels are observed in higher-grade tumors (Gleason Score ≥9/Grade Group 5)

• African American men show significantly lower HOXB13 levels (mean difference −14, 95% CI −27 to −0.81, P=0.04)

In Advanced Metastatic Prostate Cancer:

• HOXB13 expression is retained in the majority of tumors

• Lower levels of HOXB13 protein and mRNA are observed in tumors with evidence of lineage plasticity

• 84% of androgen receptor-negative castration-resistant prostate cancers and neuroendocrine prostate cancers retain detectable levels of HOXB13

• Reduced expression in neuroendocrine prostate cancer is associated with a gain of HOXB13 gene body CpG methylation

These expression patterns make HOXB13 a valuable diagnostic biomarker across the disease spectrum of prostate cancer.

What is the current understanding of HOXB13's role in DNA damage response and therapy resistance?

Recent research has revealed HOXB13's critical role in DNA damage response and therapy resistance:

• DNA Damage Response:

  • HOXB13 assembles at DNA damage sites and colocalizes with γH2AX at double strand breaks despite Androgen Receptor antagonism

  • HOXB13 lysine 13 acetylation (K13-acetylated HOXB13) is rapidly induced in response to DNA damage caused by irradiation

  • HOXB13 is required for effective DNA replication following DNA damage and for the formation of nuclear puncta

• Radioresistance Mechanisms:

  • HOXB13-K13 acetylation promotes radioresistance of prostate cancer cells

  • HOXB13-K13A mutants (unable to be acetylated at lysine 13) show:

    • Increased accumulation of γH2AX-positive double strand breaks

    • Shorter DNA replication track lengths

    • Reduced G2/M arrest following DNA damage

    • Higher sensitivity to radiation treatment

• Therapy Resistance:

  • HOXB13 contributes to resistance to anti-androgen treatment

  • Ablation of HOXB13 sensitizes prostate cancer cells to:

    • Radiotherapy

    • Anti-androgen Enzalutamide

    • Combination therapies

  • HOXB13 mutants resistant to acetylation (K13A) are highly sensitive to combination therapy with irradiation and Enzalutamide but resistant to PARP inhibitor Olaparib

This mechanistic understanding suggests new therapeutic strategies targeting HOXB13 acetylation with CBP/p300 inhibitors in combination with DNA-damaging therapy to overcome anti-androgen resistance in prostate cancers .

How does HOXB13 regulate androgen receptor signaling in prostate cancer?

HOXB13 exhibits a complex regulatory relationship with androgen receptor (AR) signaling in prostate cancer:

Direct Interaction with AR:

• HOXB13 interacts with the DNA binding domain of AR

• This interaction occurs in a ligand-stimulated manner but also shows significant interaction with the apo (unliganded) receptor

• The interaction has been confirmed using multiple methodologies:

  • Mammalian two-hybrid assay

  • Bioluminescence resonance energy transfer (BRET) assay

  • Coimmunoprecipitation of endogenous complexes

Transcriptional Regulation:

• HOXB13 functions as both a positive and negative regulator of AR target gene transcription, creating distinct phenotypic clusters:

  • HOXB13 inhibits transcription of genes containing canonical androgen response elements (AREs)

  • HOXB13 confers androgen responsiveness to promoters containing specific HOXB13-response elements

  • HOXB13 and AR synergize to enhance transcription of genes containing a HOX element juxtaposed to an ARE

Functional Consequences:

• HOXB13 has profound effects on androgen-regulated:

  • Proliferation

  • Migration

  • Lipogenesis

• HOXB13 expression is mostly AR-independent, unlike other prostatic lineage markers (PSA, NKX3.1)

• Upregulation of HOXB13 is associated with an additive growth advantage of prostate cancer cells in the absence of or low androgen levels

This multifaceted relationship with AR makes HOXB13 a potentially valuable therapeutic target in both AR-dependent and AR-independent prostate cancer.

Can HOXB13 antibodies be reliably used for detecting and studying other cancer types?

While HOXB13 is primarily associated with prostate cancer, emerging research indicates applications for HOXB13 antibodies in other cancer types:

Hepatocellular Carcinoma (HCC):

• HOXB13 expression is significantly increased in HCC tissues compared to adjacent tissues

• HOXB13 expression positively correlates with tumor stage and survival of HCC patients

• High-level expression of HOXB13 is closely associated with tumor angiogenesis and poor prognosis in HCC

• HOXB13 can facilitate HCC progression by activation of the AKT/mTOR signaling pathway

Colorectal Cancer:

• A small subset (4%) of colorectal cancers show HOXB13 expression

• HOXB13 expression in colon is restricted to epithelial cells, making antibody staining patterns distinct from prostate tissue

Other Rare Tumors:

• Neuroendocrine tumors of the cauda equina may express HOXB13

• Pediatric tumors including Ewing's sarcoma and embryonal rhabdomyosarcoma have been suggested to express HOXB13

When using HOXB13 antibodies for non-prostate cancers, researchers should:

• Validate antibody specificity in the tissue of interest

• Include appropriate positive and negative controls

• Be aware that expression patterns and subcellular localization may differ from prostate tissue

• Consider using HOXB13 as part of a panel rather than a standalone marker

What are the key methodological considerations when studying HOXB13 knockout or overexpression models?

When developing and analyzing HOXB13 knockout or overexpression models, researchers should consider:

For Knockout Models:

• Method selection: CRISPR-Cas9 knockout has shown effective HOXB13 depletion in prostate cancer cell lines. Multiple guide RNAs should be tested to identify the most effective targeting strategy .

• Off-target effects: Always validate that observed phenotypes are due to HOXB13 knockout rather than guide RNA off-target effects. This can be done by:

  • Using multiple guide RNAs targeting different regions of HOXB13

  • Complementary approaches like shRNA (4 different sequences have been validated)

  • Rescue experiments by re-introducing HOXB13

• Cell type considerations: HOXB13 dependency varies across cell lines. DepMap 22Q1 data shows that HOXB13 knockout is highly selective to prostate cancer cell lines and doesn't significantly impact the proliferation of almost all non-prostatic cell lines (1059/1061) .

• In vivo validation: HOXB13 knockout causes significant reduction of prostate cancer engraftment in both AR-positive (LNCaP) and AR-negative (PC3) xenograft models .

For Overexpression Models:

• Inducible systems: Constitutive expression of HOXB13 can drive PCa cells to cell death. Use inducible systems (e.g., Tet-On) to avoid unwanted xenotoxic effects .

• Dosage control: HOXB13 should be expressed in a dose-responsive manner (e.g., using different Dox concentrations: 50, 100, 200 nM) .

• Tagging strategies: FLAG-tagged HOXB13 has been successfully used to distinguish exogenous from endogenous HOXB13 .

• Mutant variants: When studying specific functions, consider using mutant variants such as:

  • HOXB13-3A: DNA-binding defective mutant

  • HOXB13-K13A: Acetylation-defective mutant

These methodological considerations ensure robust and reproducible results when studying HOXB13 function in cancer models.

What are the challenges and solutions for producing recombinant HOXB13 antibodies with improved specificity?

Producing recombinant HOXB13 antibodies with improved specificity presents several challenges and potential solutions:

Challenges:

• Variable antibody performance: Many commercial HOXB13 antibodies are polyclonal and subject to batch variations, making them unsuitable for widespread application .

• Validation for FFPE tissues: The majority of previously published antibodies have not been formally validated for use in formalin-fixed paraffin-embedded tissues .

• Inconsistent reporting: The rate of reported HOXB13 expression in prostate cancer varies greatly across studies due to differences in antibodies and staining protocols .

• Cross-reactivity: Some HOXB13 antibodies show non-specific reactivity to proteins of different molecular weights (e.g., around 50kDa) .

• Pre-analytic sensitivity: Some HOXB13 antibodies show reduced sensitivity in decalcified bone tissue .

Solutions:

• Recombinant antibody production:

  • Obtain protein sequence using whole transcriptome shotgun sequencing or mass spectrometry

  • Design and order gene fragments based on variable and constant regions

  • Clone gene fragments into parent plasmids

  • Transfect human HEK293 suspension cells for production

• Improved validation protocols:

  • Use genetic controls (HOXB13 knockout and overexpression models)

  • Validate across multiple applications (WB, IHC, IF)

  • Test in cell lines with known HOXB13 expression patterns

  • Validate in developmental tissues with established expression patterns

• Monoclonal development: Develop rabbit monoclonal antibodies (like clone D7N8O) which show higher consistency between lots compared to polyclonal alternatives .

• Epitope optimization: Focus on epitopes that:

  • Are unique to HOXB13 (vs. HOXA13, HOXD13)

  • Remain accessible in fixed and processed tissues

  • Are not affected by post-translational modifications that may vary in disease states

• Standard protocols: Establish standardized staining protocols with optimized antigen retrieval methods (e.g., TE buffer pH 9.0 for HOXB13) .

These approaches can help produce recombinant HOXB13 antibodies with improved specificity and consistency for research and diagnostic applications.

What are the emerging applications of HOXB13 antibodies in precision medicine for prostate cancer?

Emerging applications of HOXB13 antibodies in precision medicine for prostate cancer include:

• Treatment stratification biomarker:

  • HOXB13 acetylation status (detected by acetyl-specific antibodies) could identify patients likely to benefit from combination therapy with CBP/p300 inhibitors and DNA-damaging agents

  • HOXB13 expression patterns may predict response to anti-androgen therapies, as HOXB13 contributes to resistance

• Detection of minimal residual disease:

  • High specificity (99%) and sensitivity (97%) for prostatic origin make HOXB13 antibodies valuable for detecting circulating tumor cells or micrometastases

  • HOXB13 expression is maintained in advanced disease where other markers may be lost

• Lineage plasticity assessment:

  • HOXB13 antibodies can help identify treatment-emergent neuroendocrine prostate cancer (t-NEPC) and AR-negative phenotypes

  • 84% of AR-negative and neuroendocrine prostate cancers retain HOXB13 expression

• Liquid biopsy development:

  • Development of ultrasensitive detection methods for HOXB13 protein in circulation

  • Correlation of HOXB13 protein levels with treatment response and disease progression

• Multi-marker panels:

  • Integration of HOXB13 antibodies into multiplexed immunohistochemistry panels

  • Combination with genomic markers (like HOXB13 G84E germline mutation status) for comprehensive risk assessment

How might new technological advances improve HOXB13 antibody sensitivity and specificity?

New technological advances that could improve HOXB13 antibody sensitivity and specificity include:

• Single-domain antibodies (nanobodies):

  • Smaller size allows better tissue penetration and epitope access

  • Higher stability and potentially improved specificity

  • Potential for detecting HOXB13 in complex tissue environments or decalcified specimens

• Bi-specific antibodies:

  • Design of antibodies that recognize both HOXB13 and another prostate-specific marker

  • Dramatically increased specificity through dual-epitope recognition

  • Reduced false positives in challenging diagnostic cases

• Machine learning-guided antibody engineering:

  • Computational prediction of optimal epitopes for specificity

  • Design of synthetic antibodies with improved binding characteristics

  • Systematic testing of variant antibodies against diverse tissue arrays

• Post-translational modification-specific antibodies:

  • Development of antibodies specific to acetylated HOXB13 (K13)

  • Phosphorylation-specific HOXB13 antibodies to detect activated forms

  • Correlation of specific modifications with disease progression and treatment response

• Proximity ligation assay (PLA) technology:

  • Detection of HOXB13 protein-protein interactions (e.g., with AR) in situ

  • Higher specificity through dual binding requirement

  • Visualization of functional HOXB13 complexes rather than just protein expression

• Quantum dot labeling:

  • Higher sensitivity and photostability compared to conventional fluorophores

  • Multiplexing capability for simultaneous detection of multiple markers

  • Potential for quantitative assessment of HOXB13 expression levels

These technological advances could substantially improve the utility of HOXB13 antibodies in both research and clinical applications.

What research gaps remain in understanding HOXB13 function that could be addressed using optimized antibodies?

Several critical research gaps in HOXB13 biology could be addressed using optimized antibodies:

• Molecular mechanisms of HOXB13 in therapy resistance:

  • Map the dynamic changes in HOXB13 localization during DNA damage response using super-resolution microscopy

  • Identify HOXB13 interaction partners at DNA damage sites using proximity labeling approaches

  • Characterize post-translational modifications beyond K13 acetylation that regulate HOXB13 function

• Cell-type specific HOXB13 interactome:

  • Define differences in HOXB13 protein-protein interactions between AR-positive and AR-negative prostate cancer models

  • High-confidence HOXB13-interacting proteins differ between cell lines (73 in LNCaP, 123 in 22Rv1, 153 in PC3 cells)

  • Map interaction domains to identify potential therapeutic targeting strategies

• Relationship between HOXB13 expression and tumor microenvironment:

  • Examine HOXB13 expression in relation to immune cell infiltration using multiplex immunohistochemistry

  • Explore potential roles in immune evasion mechanisms

  • Correlate with response to immunotherapy

• Developmental to pathological transition:

  • Map changes in HOXB13 chromatin occupancy from normal prostate development through cancer progression

  • Identify target genes differentially regulated by HOXB13 in normal versus cancer states

  • Determine epigenetic changes associated with altered HOXB13 function

• Non-nuclear functions of HOXB13:

  • Investigate potential cytoplasmic roles of HOXB13 using fractionation studies

  • Explore mitochondrial or other organelle-associated functions

  • Characterize HOXB13 nuclear puncta formation and its functional significance

• Lineage plasticity mechanisms:

  • Determine how HOXB13 gene body CpG methylation regulates its expression in neuroendocrine prostate cancer

  • Explore the relationship between HOXB13 and neuroendocrine differentiation markers

  • Identify factors that regulate HOXB13 in lineage plasticity transitions