BCAR1 Antibody

What is BCAR1 and what are its primary functions in cell biology?

BCAR1, also known as p130Cas, is a docking protein that plays a central coordinating role in tyrosine kinase-based signaling related to cell adhesion. BCAR1 functions as a human homologue of the adapter protein p130Cas and is implicated in:

• Induction of cell migration and cell branching

• Cancer cell invasion and metastasis

• Resistance to anoikis (a form of programmed cell death)

• Epithelial-to-mesenchymal transition (EMT)

• Antiestrogen resistance in breast cancer cells

Research has demonstrated that BCAR1 is overexpressed in various malignancies, including cancers of the breast, lung, liver, and brain, where it promotes invasion and metastasis .

What is the molecular structure and genetic location of BCAR1?

BCAR1 consists of 870 amino acids with a calculated molecular weight of 93 kDa, though it often appears as 116 kDa in observed molecular weight due to post-translational modifications. Genomic analysis has revealed that BCAR1 consists of seven exons and is located at chromosome 16q23.1 . The protein contains multiple functional domains that facilitate its role as a scaffold protein in signaling pathways.

What are the primary applications for BCAR1 antibodies in cancer research?

BCAR1 antibodies are used across multiple research applications that investigate its role in cancer progression:

These applications allow researchers to investigate BCAR1 expression patterns, protein-protein interactions, and its role in signaling pathways in cancer cells .

How can BCAR1 antibodies be used to detect circulating tumor cells (CTCs)?

BCAR1 antibodies can be used to detect CTCs through specialized methods such as:

• The CanPatrol method: This technique combines BCAR1 antibody staining with detection of epithelial and mesenchymal markers to identify different CTC populations. BCAR1-positive CTCs can be classified according to the extent of BCAR1 expression as BCAR1(+) CTCs (containing one signal point) or BCAR1(++) CTCs (containing 2 or more signal points) .

• The CytoploRare method: This approach involves collecting blood samples, removing erythrocytes using red blood cell lysis, depleting CD45+ leukocytes with magnetic beads, and then staining the remaining cells with BCAR1 antibodies to identify CTCs .

These methods have revealed that BCAR1-positive CTCs often co-express both epithelial and mesenchymal markers, suggesting a "limited" EMT state that may enhance their metastatic potential .

What optimization steps are necessary when using BCAR1 antibodies for immunohistochemistry?

For optimal BCAR1 detection in tissue samples through immunohistochemistry:

• Antigen retrieval: Use TE buffer at pH 9.0 for optimal results. Alternatively, citrate buffer at pH 6.0 can be used but may yield different staining patterns.

• Antibody dilution: Start with dilutions between 1:250-1:1000 and optimize based on your specific tissue and fixation methods.

• Detection system: Use a polymer-based detection system compatible with the host species of your primary antibody.

• Positive controls: Include known BCAR1-positive tissues such as breast cancer or colon cancer samples.

• Counterstaining: Use hematoxylin for nuclear counterstaining to facilitate interpretation of BCAR1 localization .

It is recommended to titrate the antibody in each testing system to obtain optimal results, as sample-dependent factors can influence staining patterns .

How can researchers validate the specificity of BCAR1 antibodies?

To ensure specificity of BCAR1 antibodies:

• Western blot validation: Compare observed molecular weight (approximately 116 kDa) with the expected weight of 93 kDa. The difference is due to post-translational modifications.

• Knockout/knockdown controls: Use BCAR1 knockout cells (via CRISPR-Cas9) as negative controls to confirm antibody specificity.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to block specific binding sites.

• Cross-species reactivity testing: Validate antibody performance across different species if working with animal models.

• Multiple antibody comparison: Use antibodies targeting different epitopes of BCAR1 to confirm consistent detection patterns .

How does BCAR1 expression in CTCs correlate with cancer prognosis?

Research has shown significant correlations between BCAR1 expression in CTCs and cancer prognosis:

• In lung adenocarcinoma (LUAD): Patients with BCAR1(++) CTCs in peripheral blood before surgery were more prone to recurrence or metastasis after 2 years. Cox analysis showed that patients with higher abundance of BCAR1(++) CTCs had a poorer prognosis (hazard ratio [HR] = 1.712, 95% confidence interval [CI] = 1.077–2.272, p = 0.023) .

• EMT marker correlation: BCAR1-positive CTCs more commonly co-expressed both epithelial and mesenchymal markers, suggesting a "dual impact" of BCAR1 on EMT markers. BCAR1 in tumor tissues was significantly positively correlated with the expression of both epithelial markers (e.g., CK8/18/19) and mesenchymal markers (e.g., vimentin and twist) .

• Predictive value: High BCAR1 expression in tumor tissues was predictive of poor prognosis (HR = 2.654, 95% CI = 1.239–5.686, p = 0.012), as validated by The Cancer Genome Atlas (TCGA) database (HR = 2.217, 95% CI = 1.069–4.595, p = 0.032) .

These findings suggest that BCAR1 expression in CTCs can serve as a valuable biomarker for cancer progression and patient outcomes.

What are the molecular mechanisms through which BCAR1 promotes cancer progression?

BCAR1 promotes cancer progression through multiple molecular mechanisms:

• EMT regulation: BCAR1 can promote EMT by inhibiting transforming growth factor β1 (TGF-β1), activating Smad3, and enhancing the coupling of TGF-β1 with mitogen-activated protein kinases (p38) .

• Cell proliferation: In lung adenocarcinoma, BCAR1 promotes proliferation and cell growth via upregulation of RNA polymerase II subunit A (POLR2A) and subsequent enhancement of catalytic and transferase activities .

• Anoikis resistance: BCAR1 enhances resistance to anoikis, a form of programmed cell death that occurs when cells detach from the extracellular matrix, thereby promoting cancer cell survival during metastasis .

• Immunoevasion: BCAR1 up-regulates CD274 (PD-L1) expression probably by shuttling the short isoform of BRD4 (BRD4-S) into the nucleus, potentially contributing to cancer immune evasion .

• RAC1 interaction: RAC1 functions with BCAR1 to induce EMT and to enhance cell proliferation, colony formation, cell invasion and migration in cancer cells .

What methods are available for quantifying BCAR1 protein in clinical samples?

Several methods have been developed for quantifying BCAR1 protein in clinical samples:

• ELISA: A specific ELISA has been developed for the quantitative measurement of BCAR1 in human breast cancer tissue extracts. This method involves recombinant fragment production, antibody generation in chickens and rabbits, affinity purification, and ELISA construction .

• Western blotting: Western blot analysis using specific BCAR1 antibodies can provide semi-quantitative measurement of BCAR1 protein levels. High concentrations of BCAR1 measured by Western blotting in primary breast tumor cytosols have been associated with early disease progression and failure of tamoxifen therapy .

• Immunohistochemistry scoring: Semi-quantitative scoring of BCAR1 expression in tissue sections can be performed using specific antibodies and appropriate scoring systems that consider both staining intensity and percentage of positive cells .

• Proteomics analysis: Mass spectrometry-based approaches can be used to quantify BCAR1 protein levels and identify post-translational modifications in complex samples .

How can researchers effectively study BCAR1 function through genetic manipulation?

To study BCAR1 function through genetic manipulation, researchers can employ these approaches:

• CRISPR-Cas9 knockout: BCAR1 can be knocked out in cell lines using CRISPR-Cas9 technology to study its functional effects. For example, cell proliferation of H1975 and H1299 lung adenocarcinoma cells was significantly inhibited following BCAR1 knockout, demonstrating its role in cancer cell growth .

• Overexpression systems: Transfection of BCAR1 cDNA into cells (e.g., ZR-75-1 breast cancer cells) can result in sustained cell proliferation in the presence of antiestrogens, confirming BCAR1's role in antiestrogen resistance .

• Protein interaction studies: Co-immunoprecipitation (Co-IP) can be used to study the interaction between BCAR1 and other proteins, such as RAC1 and BRD4, helping to elucidate the signaling pathways involved .

• RNA interference: siRNA-mediated knockdown of BCAR1 can be used to study its role in various cellular processes. Research has shown that RNA interference of BCAR1 in A549 cells significantly reduced phosphorylation of p38, inhibiting the EMT process .

• Bioinformatics analysis: Utilizing online databases like STRING and Cytoscape to analyze potential interactions between BCAR1 and other proteins can guide experimental designs for functional studies .

How is BCAR1 being investigated as a potential therapeutic target in cancer?

Current research on BCAR1 as a therapeutic target focuses on several approaches:

• Targeting BCAR1-mediated signaling pathways: Inhibition of the interaction between BCAR1 and RAC1 could potentially disrupt cancer progression, as RAC1 functions with BCAR1 to induce EMT and enhance cancer cell proliferation and invasion .

• Combination with immunotherapy: Given BCAR1's role in regulating CD274 (PD-L1) expression, combining BCAR1 inhibition with immune checkpoint inhibitors might enhance anti-tumor immune responses .

• Targeting BCAR1 in CTCs: As BCAR1-positive CTCs show enhanced survival and metastatic potential, developing strategies to target these cells could potentially prevent metastasis formation .

• Addressing antiestrogen resistance: Since BCAR1 overexpression confers antiestrogen resistance in breast cancer cells, targeting BCAR1 might restore sensitivity to endocrine therapies like tamoxifen .

• Exploiting synthetic lethality: Identifying genes that, when inhibited in combination with BCAR1, lead to selective cancer cell death could provide novel therapeutic approaches.

What are the current challenges in studying BCAR1 in different cancer types?

Researchers face several challenges when studying BCAR1 across cancer types:

• "Dual impact" phenomenon: BCAR1 can have different effects on EMT markers in tumor tissues versus CTCs due to micro-environmental disparities, making it challenging to develop unified models of BCAR1 function .

• Isoform complexity: Multiple BCAR1 isoforms have been reported (at least 7 isoforms), complicating the interpretation of experimental results and potentially requiring isoform-specific approaches .

• Context-dependent functions: BCAR1's functions may vary depending on cancer type, stage, and microenvironment, necessitating careful experimental design and interpretation.

• Technical limitations in CTC research: The rarity of CTCs in peripheral blood and technical challenges in their isolation and characterization limit comprehensive studies of BCAR1 in these cells .

• Translation to clinical applications: Despite established correlations between BCAR1 expression and poor prognosis, translating these findings into clinically useful biomarkers or therapeutic targets remains challenging.