Phospho-ESR1 (S167) Antibody

What is Phospho-ESR1 (S167) Antibody and what is its significance in breast cancer research?

Phospho-ESR1 (S167) Antibody is a specialized antibody that specifically recognizes the estrogen receptor alpha (ESR1) protein when it is phosphorylated at serine residue 167. This antibody is crucial for studying post-translational modifications of ESR1, which play significant roles in breast cancer biology.

The significance of this antibody in breast cancer research stems from multiple clinical studies demonstrating that phosphorylation of ESR1 at Ser167 is predictive of response to endocrine therapy in hormone receptor-positive breast cancer patients . Unlike phosphorylation at other sites such as Ser118, Ser167 phosphorylation has been specifically associated with better clinical outcomes, including longer survival after relapse and increased likelihood of response to endocrine therapy .

Mechanistically, ESR1 can be phosphorylated at Ser167 by various kinases such as S6K1, RSK, and Aurora A, promoting ESR1-dependent transcription and cellular proliferation . This modification has been linked to endocrine therapy response mechanisms, making it a valuable biomarker in breast cancer management.

What are the recommended applications and protocols for Phospho-ESR1 (S167) Antibody use?

Phospho-ESR1 (S167) Antibody can be used in multiple experimental applications, with specific protocols optimized for each technique:

Western Blotting (WB):

• Recommended dilutions range from 1:500-1:2000, with 1:1000 being commonly used

• Use standard SDS-PAGE and transfer protocols

• Expected molecular weight: approximately 66-67 kDa

• Include appropriate positive controls (such as EGF-stimulated cell lysates)

Immunohistochemistry (IHC):

• Recommended dilution: 1:200

• Heat-induced antigen retrieval using citrate buffer is recommended

• Score nuclear staining according to both intensity (scale 0-3) and percentage of positive cells (0-100%)

• IHC-scores are calculated by multiplying intensity by percentage, generating a 0-300 scale

Enzyme-Linked Immunosorbent Assay (ELISA):

• Recommended concentration: 0.01-0.1 μg/mL

• Optimal dilutions should be determined by the end user based on specific assay requirements

Immunofluorescence (IF):

• Recommended dilution: 1:100-1:800

• Paraformaldehyde fixation is suitable for this application

How is the specificity of Phospho-ESR1 (S167) Antibody validated?

Validating the specificity of phospho-specific antibodies is critical to ensure accurate experimental results. For Phospho-ESR1 (S167) antibody, multiple validation approaches have been documented:

• A non-phosphorylated ERα peptide of the same sequence

• A peptide phosphorylated at a different site (e.g., T311)

Immunoblotting with Controlled Stimulation:

• In studies with transfected COS-7 cells, P-S167-ERα becomes inducibly phosphorylated in response to EGF but not E2 treatment

• This differential response can be used to validate antibody specificity

Cross-Reactivity Testing:
The antibody should specifically recognize ESR1 phosphorylated at Ser167 and not cross-react with:

• Non-phosphorylated peptide

• ESR1 phosphorylated at other sites (such as Ser106 or Ser118)

These validation methods ensure that the antibody provides specific detection of the phosphorylated form of ESR1 at serine 167.

What is the relationship between ESR1 Ser167 phosphorylation and clinical outcomes in breast cancer?

Multiple clinical studies have established significant correlations between ESR1 Ser167 phosphorylation and breast cancer outcomes:

Association with Favorable Prognostic Factors:
In ER-positive breast cancer patients, Ser167 phosphorylation has been associated with:

Endocrine Therapy Response:

• Phosphorylation of ER-α Ser167 in primary breast tumors is predictive of response to endocrine therapy after relapse

• Patients with high phosphorylation of ER-α Ser167 had significantly longer survival after relapse compared to those with low phosphorylation

Multivariate Analysis:
Studies involving 290 primary breast cancer biopsies demonstrated that Ser167 phosphorylation predicts likelihood of response to endocrine therapies in ER-positive breast cancer patients, making it a valuable prognostic marker .

These findings collectively suggest that assessment of Ser167 phosphorylation status could help in selecting patients who may benefit from endocrine therapy and serve as a prognostic marker in metastatic breast cancer.

How does ESR1 Ser167 phosphorylation differ from other phosphorylation sites in terms of function and clinical significance?

ESR1 can be phosphorylated at multiple sites, each with distinct functional consequences and clinical implications:

Comparison of Ser167 vs. Ser118 Phosphorylation:

Additionally, while seven phosphorylated ERα forms have been detected in breast cancer (P-S104/106-ERα, P-S118-ERα, P-S167-ERα, P-S282-ERα, P-S294-ERα, P-T311-ERα, and P-S559-ERα) , the phosphorylation of Ser167 has emerged as particularly significant for predicting endocrine therapy response.

What kinases are responsible for ESR1 Ser167 phosphorylation and what signaling pathways are involved?

Multiple kinases have been identified as responsible for phosphorylating ESR1 at Ser167, connecting this modification to several important signaling pathways:

Primary Kinases Phosphorylating Ser167:

• S6K1 (p70 ribosomal S6 kinase 1)

• RSK (p90 ribosomal S6 kinase)

• Aurora A kinase

Signaling Pathways:

• The MAPK pathway: Phosphorylated MAPK (p44/p42) has been strongly associated with Ser167 phosphorylation (P < 0.0005)

• The PI3K/AKT/mTOR pathway: S6K1 is downstream of this pathway

• Growth factor signaling: EGF stimulation induces Ser167 phosphorylation

Cellular and Molecular Consequences:

• Phosphorylation on Ser167 promotes ERα-dependent transcription

• Enhances cellular proliferation

• May contribute to tamoxifen resistance in some contexts

Interestingly, while HER2 positivity is associated with phosphorylation of Ser118, it is not associated with Ser167 phosphorylation . This suggests that other cell surface receptors may be important in regulating the activities of MAPK and RSK that lead to Ser167 phosphorylation in breast cancer.

What are the best practices for quantifying Phospho-ESR1 (S167) expression in immunohistochemistry studies?

Accurate quantification of Phospho-ESR1 (S167) in immunohistochemistry studies is critical for generating reliable and reproducible results:

Scoring Methodology:

• Use semi-quantitative IHC-scores derived from:

  • Staining intensity (scale 0–3)

  • Percentage of positive cells (0–100%)

  • Multiply these values to generate a final score (0–300)

• Focus only on nuclear staining when evaluating ESR1 phosphorylation

Cut-off Selection:

• No universally accepted clinical cut-off points exist for phosphorylated ESR1 sites

• Many studies define positivity based on the 25th percentile of IHC-scores (often equating to scores >0)

• Independent evaluation by multiple investigators is recommended, with re-evaluation of discordant cases to reach consensus

Validation Practices:

• Include appropriate positive and negative controls

• Perform parallel staining with immunoabsorbed antibodies to demonstrate specificity

• Compare with total ESR1 expression in sequential sections

Technical Considerations:

• Use automated tissue immunostainers when possible to enhance reproducibility

• Perform heat-induced antigen retrieval in citrate buffer (CC1)

• Evaluate TMA (tissue microarray) cores in duplicate or triplicate to account for tumor heterogeneity

How do different growth factors and hormones affect ESR1 Ser167 phosphorylation?

The phosphorylation status of ESR1 at Ser167 is dynamically regulated by various growth factors and hormones, with distinct patterns of response:

Epidermal Growth Factor (EGF):

• EGF treatment induces phosphorylation of ESR1 at Ser167

• This has been demonstrated in transfected COS-7 cells using immunoblotting with phospho-specific antibodies

• EGF activates the MAPK pathway, which in turn activates RSK, leading to Ser167 phosphorylation

Estradiol (E2):

• In contrast to EGF, treatment with E2 alone does not significantly affect Ser167 phosphorylation in experimental systems

• When ERα becomes phosphorylated at Ser167 in response to EGF, this phosphorylation status is not further affected by E2 treatment

Combined Growth Factor and Hormone Effects:

• Co-treatment with EGF and E2 maintains EGF-induced phosphorylation of Ser167

• This suggests that growth factor signaling may operate independently of classical estrogen signaling for this particular phosphorylation site

Understanding these differential effects is important when designing experiments to study the dynamics of ESR1 phosphorylation and when interpreting results from different experimental conditions.

What experimental challenges exist in detecting Phospho-ESR1 (S167) in different research contexts?

Researchers face several challenges when attempting to detect and quantify Phospho-ESR1 (S167) in various experimental settings:

Tissue Preservation and Processing Effects:

• Phosphorylation status can be affected by ischemic time before fixation

• Formalin fixation and paraffin embedding can reduce phospho-epitope detection

• Proper tissue handling and processing protocols are critical to preserve phosphorylation status

Antibody Specificity Concerns:

• Cross-reactivity with similar phosphorylation sites must be rigorously controlled

• Batch-to-batch variation in antibody production may affect consistency

• Validation using peptide competition assays is essential

Transient Nature of Phosphorylation:

• Phosphorylation is a dynamic process that can change rapidly

• Experimental timing is critical when studying phosphorylation events

• Phosphatase activity during sample preparation can reduce signal

Quantification Challenges:

• Establishing appropriate cut-off values for positivity remains subjective

• Comparison between different studies is complicated by varied scoring systems

• Inter-observer variability must be addressed through consensus scoring

Technical Recommendations:

• Use freshly prepared or properly stored antibodies

• Include appropriate positive and negative controls

• Consider using recombinant antibodies for superior lot-to-lot consistency

• For long-term storage, store at -20°C or lower and aliquot to avoid repeated freezing and thawing

How can Phospho-ESR1 (S167) status be integrated with other biomarkers for improved breast cancer prognosis?

Integration of Phospho-ESR1 (S167) status with other biomarkers provides a more comprehensive approach to breast cancer prognosis and treatment selection:

Complementary Biomarkers for Integrated Assessment:

Multivariate Analysis Approach:
Studies have demonstrated that phosphorylation of ER-α Ser167 remains an independent prognostic factor even after adjusting for other clinicopathological variables, suggesting its unique contribution to outcome prediction .

Practical Implementation:

• Developing multiplex assays that simultaneously detect multiple phosphorylation sites

• Creating integrated scoring systems that weight different biomarkers appropriately

• Incorporating phosphorylation status into existing prognostic models