AKR1C3 Antibody

What is AKR1C3 and why is it important in research?

AKR1C3 (Aldo-Keto Reductase Family 1, Member C3), also known as 3-alpha hydroxysteroid dehydrogenase type II or 17-beta hydroxysteroid dehydrogenase type 5, is an enzyme involved in the biosynthesis of steroid hormones and prostaglandins. It catalyzes the reduction of Δ4-androstene-3,17-dione to yield testosterone, the reduction of 5α-dihydrotestosterone to yield 3α- and 3β-androstanediol, and the reduction of estrone to yield 17β-estradiol . The enzyme plays crucial roles in hormone-dependent cancers, particularly prostate and breast cancers, where it regulates ligand access to androgen and estrogen receptors . AKR1C3 is also implicated in leukemia, showing particularly high expression in T acute lymphoblastic leukemia/lymphoma (T-ALL) . Its significance extends to its dual function as both an enzyme and an androgen receptor coactivator, making it a valuable target for cancer research and therapeutic development .

How do I choose between monoclonal and polyclonal AKR1C3 antibodies for my research?

Selection between monoclonal and polyclonal AKR1C3 antibodies should be based on your specific research application, required specificity, and experimental design:

Monoclonal Antibodies:

• Offer higher specificity with less cross-reactivity, particularly important given AKR1C3's >86% sequence identity with related human aldo-keto reductases (AKR1C1, AKR1C2, and AKR1C4)

• Mouse monoclonal clone NP6.G6.A6 (Sigma/Millipore) has demonstrated superior specificity compared to rabbit polyclonal antibodies in immunohistochemistry evaluation

• Provide consistent results between batches, beneficial for longitudinal studies

• Ideal for specific epitope targeting, particularly when discriminating between highly similar proteins

Polyclonal Antibodies:

• Recognize multiple epitopes, potentially increasing detection sensitivity

• May provide stronger signals in applications where the target protein is expressed at low levels

• Could offer greater tolerance to protein denaturation in certain applications

• Often more economical for preliminary studies

In comparative evaluations, mouse monoclonal antibody clone NP6.G6.A6 demonstrated higher specificity than rabbit polyclonal antibodies (e.g., Thermo Fisher Scientific Clone#PA5-23667) when tested against cell line controls including HCT116 (negative control) and HCT116 with AKR1C3 overexpression . For applications requiring high discrimination between AKR1C family members, the characterized high-titer isoform-specific monoclonal antibody described by Guise et al. offers confirmed specificity without cross-reactivity with human AKR1C1, AKR1C2, AKR1C4, AKR1A1, or rat AKR1C9 .

What are the key differences among commercially available AKR1C3 antibodies?

Commercial AKR1C3 antibodies differ in several important characteristics that influence their performance in various applications:

When selecting an antibody, researchers should consider:

• Target species and cross-reactivity requirements

• Specific application needs (WB, IHC, IF, IP, etc.)

• Target region of interest (N-terminal, C-terminal, or specific domains)

• Validation status in relevant models or tissues

• Knockout (KO) validation, which provides strong evidence for specificity

For detecting AKR1C3 in T-ALL samples, the Sigma/Millipore Anti-AKR1C3 antibody (mouse monoclonal, clone NP6.G6.A6) has been specifically recommended based on comprehensive validation studies comparing multiple antibodies .

What are the optimal protocols for AKR1C3 immunohistochemistry staining?

For optimal AKR1C3 immunohistochemical (IHC) staining, the following validated protocol has demonstrated reliable results in clinical research settings:

Sample Preparation:

• Cut tissue sections at 4 μm thickness and mount on positively charged slides (e.g., Fisher Superfrost Plus)

• Bake and deparaffinize slides according to standard protocols

• Perform antigen retrieval with 1× Tris-EDTA retrieval buffer for 15 minutes at 110°C

Staining Procedure:

• Use an automated slide stainer (e.g., Biocare IntelliPath IHC) for consistent results

• Dilute concentrated AKR1C3 antibody (mouse monoclonal clone NP6.G6.A6 recommended) to 2 μg/mL in appropriate diluent (e.g., Biocare Devinci)

• Apply at least 300 μL of diluted antibody per slide

• Include quality control tissue containing both positive and negative elements for AKR1C3 staining in every run

Evaluation:

• Define AKR1C3 protein positivity as nuclear and/or cytoplasmic staining

• Grade expression levels as dim (1+), moderate (2+), or strong (3+)

• Calculate H-score to quantify percent of nuclear immunoreactivity

For research on T-ALL and B-ALL samples, this protocol has successfully differentiated between expression levels, with T-ALL samples typically showing higher H-scores (172-190) compared to B-ALL cases (H-score 30-160) . When comparing antibodies, the mouse monoclonal NP6.G6.A6 (Sigma/Millipore) demonstrated superior specificity compared to rabbit polyclonal antibodies in controlled evaluations .

For prostate and breast tissue, AKR1C3 antibodies have revealed distinct localization patterns: in normal prostate, immunoreactivity is primarily limited to stromal cells with only faint staining in epithelial cells, while adenocarcinoma shows elevated staining in both endothelial and carcinoma cells .

How can I validate the specificity of my AKR1C3 antibody?

Validating AKR1C3 antibody specificity is crucial given the high sequence homology (>86%) with related isoforms AKR1C1, AKR1C2, and AKR1C4 . A comprehensive validation approach should include:

1. Cell Line Controls:

• Negative control: HCT116 cell line (lacking AKR1C3 expression)

• Positive control: Genetically modified HCT116 with AKR1C3 overexpression

• Additional controls: Nalm and TF1 cell lines for comparative expression

2. Western Blot Analysis:

• Test for cross-reactivity with purified recombinant proteins:

  • AKR1C1 (20α-hydroxysteroid dehydrogenase)

  • AKR1C2 (type 3 3α-hydroxysteroid dehydrogenase)

  • AKR1C3 (type 2 3α-hydroxysteroid dehydrogenase)

  • AKR1C4 (type 1 3α-hydroxysteroid dehydrogenase)

  • AKR1A1 (human aldehyde reductase)

  • AKR1C9 (rat 3α-hydroxysteroid dehydrogenase)

• Verify a single band at approximately 36 kDa for specific AKR1C3 detection

3. Serial Antibody Titration:

• Determine optimal antibody concentration (e.g., 0.2 μg/mL for Protein Wes)

• Test against varying protein extract concentrations (e.g., 5, 20, and 100 μg/mL)

4. Knockout/Knockdown Validation:

• Use CRISPR/Cas9 knockout or siRNA knockdown of AKR1C3

• Compare antibody reactivity in wildtype versus knockout/knockdown samples

• Choose KO-validated antibodies when available

5. Multi-method Concordance:

• Verify concordance between antibody-based detection (IHC, Western blot, Protein Wes) and mRNA expression (RT-qPCR)

• Compare antibody performance across different detection platforms to ensure consistent results

For example, in T-ALL research, AKR1C3 expression measured by Protein Wes using the mouse monoclonal NP6.G6.A6 antibody showed concordance with RNA expression by RT-PCR in relapsed/refractory and minimal residual disease cases, providing stronger validation of antibody specificity and performance .

What are the recommended protocols for Western blot detection of AKR1C3?

For optimal Western blot detection of AKR1C3, the following validated protocol has provided consistent and specific results:

Sample Preparation:

• Measure protein concentration using a standard assay (e.g., BioRad Protein Assay)

• Normalize input volumes to ensure equal loading

• Use appropriate lysis buffers that preserve AKR1C3 structure while ensuring complete extraction

Western Blot Protocol:

• Gel Preparation and Electrophoresis:

  • Use reducing conditions with appropriate gel percentage (typically 10-12% for AKR1C3)

  • Load 20-30 μg of total protein per lane

• Transfer:

  • Transfer to PVDF membrane (recommended over nitrocellulose for AKR1C3)

  • Use Immunoblot Buffer Group 1 for optimal results

• Antibody Incubation:

  • Primary antibody: Mouse Anti-Human AKR1C3 Monoclonal Antibody at 0.5-1.0 μg/mL

  • Secondary antibody: HRP-conjugated Anti-Mouse IgG

• Detection:

  • Use enhanced chemiluminescence (ECL) system

  • Expected band size: approximately 36 kDa

Controls and Validation:

• Positive Controls:

  • A549 human lung carcinoma cell line

  • HepG2 human hepatocellular carcinoma cell line

  • LNCaP prostate cancer cells stimulated with recombinant Human/Mouse/Rat ActivinA (50 ng/mL for 24 hours)

• Alternative Methods:

  • For precise quantification, consider using Protein Wes (automated capillary Western system):

    • Optimal antibody concentration: 0.2 μg/mL

    • Optimal protein extract concentration: 20 μg/mL

In validation studies using the mouse monoclonal NP6.G6.A6 antibody, Western blot consistently showed a specific band at approximately 36 kDa in positive control cell lines, with no cross-reactivity with other AKR family members , making this approach reliable for AKR1C3 detection and quantification in research settings.

How is AKR1C3 expression implicated in different cancer types?

AKR1C3 exhibits diverse expression patterns and functional roles across various cancer types, with significant implications for disease progression, therapeutic resistance, and prognosis:

Research has revealed that AKR1C3 functions beyond its enzymatic role in cancer. In prostate cancer, AKR1C3 acts as an AR-selective coactivator that promotes the growth of both androgen-dependent prostate cancer and CRPC . The combined effects of its enzymatic activity (converting androgen precursors to testosterone) and coactivator function make it a particularly important player in castration-resistant disease .

In breast cancer, AKR1C3's role in modulating the estradiol:progesterone ratio potentially increases ERα signaling while decreasing PR signaling, though this hypothesis requires further investigation . The enzyme's involvement in prostaglandin metabolism may also generate hormone-independent proliferative signals .

The distinct expression patterns in different cancer types suggest potential utility as a diagnostic biomarker, particularly in distinguishing NSCLC from SCLC and in characterizing acute lymphoblastic leukemia subtypes .

What is the significance of AKR1C3 in therapeutic resistance mechanisms?

AKR1C3 contributes to therapeutic resistance through multiple mechanisms, presenting both challenges and opportunities for targeted intervention:

1. Androgen-Dependent Cancers (Prostate Cancer):

• AKR1C3 facilitates intratumoral androgen synthesis, converting weak androgen precursors to potent androgens like testosterone

• Functions as an AR-selective coactivator, enhancing AR signaling even in low androgen environments

• Promotes castration resistance by maintaining AR signaling despite androgen deprivation therapy

• Confers resistance to AR-targeted therapies through persistent AR activation

• Inhibiting AKR1C3 with selective inhibitors can reverse resistance to AR-targeted therapies

2. Leukemia:

• In chronic myeloid leukemia (CML), high AKR1C3 expression correlates with resistance to imatinib

• Operates through a novel miR-379-5p/AKR1C3/ERK signaling axis

• Combination therapy with imatinib and indomethacin (an AKR1C3 inhibitor) significantly prolongs survival in mouse models

• In T-ALL, higher expression correlates with relapsed/refractory disease

3. Oropharyngeal Squamous Cell Carcinoma:

• AKR1C3 inhibition potentially enhances effectiveness of cisplatin therapy

• Highest expression observed in HPV-negative tumors, which typically have worse prognosis

• Positive correlation between AKR1C3 expression and poorer survival outcomes in both HPV-positive and the entire cohort of OPSCC cases

4. Molecular Mechanisms of Resistance:

• Modulates MAPK/ERK signaling pathways

• Activates NF-κB by inducing autoubiquitination of TRAF6

• Enhances STAT3 phosphorylation through release of proinflammatory factors

• Establishes a positive regulatory feedback loop through direct binding of STAT3 to the AKR1C3 promoter

• Promotes epithelial-mesenchymal transition (EMT), enhancing invasive and metastatic potential

The multifaceted role of AKR1C3 in therapeutic resistance underscores its potential as a druggable target. Combining AKR1C3 inhibitors with existing therapies represents a promising strategy to overcome resistance and improve treatment outcomes across multiple cancer types .

How can AKR1C3 be targeted therapeutically, and what role do antibodies play in developing these approaches?

AKR1C3 presents a unique therapeutic opportunity as both an enzyme and coactivator, with antibodies playing crucial roles in target validation, therapeutic development, and companion diagnostics:

1. Therapeutic Targeting Strategies:

2. Role of Antibodies in AKR1C3-Targeted Therapeutic Development:

Target Validation:

• Highly specific antibodies confirm AKR1C3 expression in potential target tissues

• Monoclonal antibodies like NP6.G6.A6 enable precise qualification of AKR1C3 in patient samples

• Immunohistochemistry with validated antibodies establishes correlation between AKR1C3 expression and disease progression/prognosis

Mechanism Elucidation:

• Co-immunoprecipitation studies using AKR1C3 antibodies revealed its interaction with AR in prostate cancer cells, xenografts, and human CRPC samples

• Chromatin immunoprecipitation with AKR1C3 antibodies demonstrated recruitment to androgen-responsive gene promoters

• These studies established AKR1C3's novel function as an AR coactivator, expanding therapeutic strategies beyond enzyme inhibition

Companion Diagnostics:

• AKR1C3 antibodies enable patient stratification for clinical trials of AKR1C3 inhibitors

• IHC with validated antibodies identifies patients likely to benefit from AKR1C3-targeted therapies

• H-scoring systems using specific antibodies provide quantitative assessment of expression levels

Therapeutic Monitoring:

• Sequential tissue sampling with antibody-based detection can monitor treatment effects on AKR1C3 expression

• Concordance between protein detection (antibody-based) and mRNA expression confirms true biological changes during treatment

The unique status of AKR1C3 as the first of more than 200 known nuclear hormone receptor coactivators that can be pharmacologically targeted makes it a particularly exciting therapeutic opportunity . The development of AKR1C3-selective competitive inhibitors has already shown promise in inhibiting both the coactivator and growth-promoting functions of AKR1C3 in preclinical models , highlighting the importance of continued antibody-based research to advance these therapeutic approaches.

How do different AKR1C3 antibodies perform in detecting specific AKR1C3 functions in cancer research?

Different AKR1C3 antibodies exhibit variable performance in detecting the enzyme's dual functions as both a steroidogenic enzyme and nuclear receptor coactivator:

1. Detection of Enzymatic Function vs. Coactivator Function:

2. Localization Studies:

• Nuclear vs. cytoplasmic localization of AKR1C3 correlates with its different functions

• In confocal microscopy studies, AKR1C3 antibodies have revealed:

  • Primarily cytoplasmic localization in normal tissues (consistent with enzymatic function)

  • Increased nuclear localization in certain cancer types (consistent with coactivator function)

  • Both nuclear and cytoplasmic staining in T-ALL samples

• The specificity of antibodies is critical for accurate subcellular localization studies

3. Protein-Protein Interaction Studies:

• Co-immunoprecipitation studies using AKR1C3 antibodies have demonstrated:

  • Direct interaction between AKR1C3 and AR in prostate cancer cells

  • Presence of this interaction in xenografts and human CRPC samples

• Mouse monoclonal antibodies have shown superior performance in pulling down specific complexes without cross-reactivity with other AKR1C family members

4. Comparative Performance in Research Applications:

• In comparative studies evaluating antibody performance in detecting specific AKR1C3 functions:

  • Mouse monoclonal NP6.G6.A6 demonstrated superior specificity and consistent performance across multiple applications

  • Rabbit monoclonal antibodies showed intermediate performance

  • Some rabbit polyclonal antibodies exhibited nonspecific reactivity

The selection of an appropriate antibody should be guided by the specific research question regarding AKR1C3 function. For studies investigating AKR1C3's coactivator function, antibodies validated in chromatin immunoprecipitation and co-immunoprecipitation studies are preferred . For enzymatic function studies, antibodies that specifically detect AKR1C3 without cross-reactivity to other family members are essential .

What are common troubleshooting challenges with AKR1C3 antibodies and how can they be addressed?

Researchers working with AKR1C3 antibodies encounter several common challenges that can impact experimental outcomes. Here are key troubleshooting approaches:

1. Cross-Reactivity with Other AKR1C Family Members:

2. Inconsistent Immunohistochemistry Results:

3. Discordance Between Protein and mRNA Expression:

4. Technical Challenges in Western Blot:

5. Validation in Complex Samples:

Research has shown that comprehensive validation using multiple approaches provides the most reliable results. In studies of T-ALL, concordance between AKR1C3 expression measured by Protein Wes (using NP6.G6.A6 antibody) and RT-qPCR confirmed true biological signal in relapsed/refractory and minimal residual disease cases , demonstrating the value of multi-method validation.

How can researchers integrate AKR1C3 antibodies with emerging technologies for advanced cancer research?

Integrating AKR1C3 antibodies with cutting-edge technologies opens new avenues for investigating this multifunctional protein in cancer research:

1. Single-Cell Analysis Technologies:

2. Spatial Biology Approaches:

3. Functional Genomics Integration:

4. Liquid Biopsy Applications:

5. Drug Development Applications:

Research integrating AKR1C3 antibodies with these advanced technologies has already yielded important insights. For example, studies in prostate cancer have used proximity ligation assays with AKR1C3 antibodies to visualize its interaction with AR in tissue specimens, confirming its coactivator function in the native tumor environment . Similarly, combining Protein Wes with RT-qPCR has established concordance between protein and mRNA expression in leukemia, validating AKR1C3 as a biomarker for minimal residual disease .

As these technologies continue to evolve, their integration with well-validated AKR1C3 antibodies will enable increasingly sophisticated investigations into this protein's multifaceted roles in cancer biology and therapeutic resistance.