FAM53A Antibody

What is FAM53A and why is it significant in cancer research?

FAM53A, also known as dorsal neural-tube nuclear protein (DNTNP), is an uncharacterized protein that has attracted attention for its potential role in tumorigenesis. Originally identified for its importance in neurodevelopment, particularly in specifying the fate of dorsal cells within the neural tube, FAM53A has now been implicated in cancer biology . Recent research indicates that FAM53A may function as a tumor suppressor in p53-positive breast cancer while potentially promoting oncogenic activity in p53-negative breast cancer contexts. This dual role makes it particularly interesting for researchers studying the molecular mechanisms of cancer progression and developing targeted therapies . The protein's interaction with the MEK-ERK pathway and its relationship with p53 status highlights its significance in understanding the complex signaling networks that govern cancer cell behavior.

What are the key specifications of commercially available FAM53A antibodies?

The typical FAM53A antibody used in research is a rabbit polyclonal primary antibody that targets the Family With Sequence Similarity 53, Member A protein. These antibodies are generally unconjugated and belong to the IgG isotype class . Most commercially available anti-FAM53A antibodies are reactive with human samples and have been validated for Western blot applications. The antibodies are typically raised against synthetic peptides corresponding to the C-terminal region of FAM53A. A common immunogen peptide sequence used is RGLRTSPVHPNLWASRESVTSDGSRRSSGDPRDGDSVGEEGVFPRARWEL . These antibodies undergo immunogen affinity purification to ensure specificity and are usually supplied in PBS buffer with 2% sucrose at a volume of 100 μl .

How is FAM53A localized in breast cancer cells?

Immunofluorescence studies have revealed that FAM53A is present in both the cytoplasm and nucleus of breast cancer cells. This dual localization has been demonstrated in multiple breast cancer cell lines including MCF-7, T47D, MDA-MB-231, and BT-549, as well as in the non-malignant human mammary epithelial cell line MCF-10A . The subcellular distribution pattern may have functional implications, particularly regarding its role in signaling pathway regulation and transcriptional activities. Understanding the localization patterns of FAM53A in different cell types and under various conditions can provide insights into its functional roles in normal and pathological states.

What is the recommended protocol for immunohistochemical detection of FAM53A in tissue samples?

For immunohistochemical detection of FAM53A in tissue samples, researchers should follow this validated protocol:

• Fixation and Processing: Surgically removed tumor specimens should be fixed in 10% neutral formalin, embedded in paraffin, and cut into 4-μm thick sections .

• Deparaffinization and Antigen Retrieval: Bake sections at 70°C for 2 hours, then dewax using xylene followed by rehydration through graded alcohols (absolute ethanol to distilled water). Perform antigen retrieval by boiling the sections in 0.01 M citrate buffer (pH 6.0) at high temperature and pressure for 2 minutes .

• Blocking: Block endogenous peroxidase activity with 0.3% hydrogen peroxide, then incubate with 5% normal goat serum for 30 minutes at 20°C to reduce non-specific binding .

• Primary Antibody Incubation: Incubate tissue sections with FAM53A antibody at a 1:100 dilution overnight at 4°C .

• Detection System: Visualize the reaction using an HRP-based immunohistochemistry detection kit and 3,3′-diaminobenzidine (DAB) .

• Counterstaining: Counterstain nuclei with hematoxylin .

• Evaluation: Assess FAM53A expression using both percentage of positive cells (PP) and staining intensity (SI) to calculate an immune response score (IRS) where IRS = PP × SI .

This protocol has been successfully used to analyze FAM53A expression in breast cancer tissues and correlate its levels with clinicopathological features.

How should FAM53A expression levels be quantified in immunohistochemical studies?

For quantification of FAM53A expression in immunohistochemical studies, a semi-quantitative scoring system based on the immune response score (IRS) is recommended. This system evaluates expression based on two parameters:

• Percentage of Positive Cells (PP):

  • 0: no dye

  • 1: 1–25% positive cells

  • 2: 26–50% positive cells

  • 3: 51–75% positive cells

  • 4: 76–100% positive cells

• Staining Intensity (SI):

  • 0: no staining

  • 1: weak staining

  • 2: medium staining

  • 3: strong staining

The final IRS is calculated by multiplying these two scores (PP × SI), resulting in a value ranging from 0 to 12. For analytical purposes, samples are typically categorized as having "high FAM53A expression" (scores >3) or "low FAM53A expression" (scores ≤3) . This standardized evaluation method enables consistent assessment across different samples and studies, facilitating reliable comparison of results between research groups.

What transfection protocols are effective for modulating FAM53A expression in breast cancer cell lines?

Two effective transfection protocols have been validated for modulating FAM53A expression in breast cancer cell lines:

For FAM53A Overexpression:

• Cell Preparation: Plate cells in 2 mL of complete growth medium, aiming for 50–70% confluency at transfection time .

• DNA-Polymer Complex Formation: Add 5 μg of plasmid DNA (pCMV6-ddk-myc-FAM53A) to Xfect Reaction Buffer to a final volume of 100 μl, then add 1.5 μL Xfect Polymer and incubate for 10 minutes at room temperature (15–25°C) .

• Transfection: Add the solution dropwise to the cell culture medium and incubate cells at 37°C for 4 hours .

• Medium Replacement: Replace with 2 mL of fresh complete growth medium and incubate at 37°C for 48 hours before analysis .

For FAM53A Knockdown:

• Cell Preparation: Plate cells in 2 mL of complete growth medium, aiming for 30–50% confluence at transfection time .

• siRNA Complex Formation: Add 37.5 ng of FAM53A siRNA to 100 μL of serum-free culture medium, add 3 μL of HiPerFect Transfection Reagent, vortex, and incubate for 5–10 minutes at room temperature .

• Transfection: Add complexes to the cells and incubate for 48 hours prior to analysis .

These protocols have been successfully used to manipulate FAM53A expression levels in MCF-7 and MDA-MB-231 breast cancer cell lines to study its functional effects.

How does FAM53A expression correlate with p53 status in breast cancer, and what are the functional implications?

Analysis of 199 breast cancer tissue specimens has revealed a significant negative correlation between FAM53A expression levels and wild-type p53 status (P < 0.001) . This relationship appears to be functionally relevant, as demonstrated by the contrasting effects of FAM53A in cell lines with different p53 status.

In the p53-wild-type breast cancer cell line MCF-7, FAM53A overexpression inhibits cell migration, invasion, and proliferation. It downregulates the expression of several proteins involved in cell cycle progression and epithelial-mesenchymal transition, including Snail, cyclin D1, RhoA, RhoC, and MMP9. It also decreases MEK and ERK phosphorylation while upregulating E-cadherin and p21 expression levels .

Conversely, in the p53-null breast cancer cell line MDA-MB-231, opposite trends were observed, suggesting that FAM53A functions as a tumor suppressor in p53-positive breast cancer by modulating the MEK-ERK pathway, while potentially promoting tumor progression in p53-negative contexts . These findings have significant implications for understanding the context-dependent roles of FAM53A in breast cancer biology and for developing targeted therapeutic approaches based on p53 status.

What signaling pathways are modulated by FAM53A in breast cancer cells?

FAM53A primarily exerts its effects in breast cancer cells through modulation of the MEK-ERK signaling pathway, with p53 status determining the directionality of this modulation . In p53-wild-type breast cancer cells, FAM53A overexpression decreases MEK and ERK phosphorylation, suggesting an inhibitory effect on this pathway. Conversely, in p53-null cells, FAM53A appears to promote MEK-ERK signaling .

The functional relevance of this pathway interaction was demonstrated using the MEK inhibitor PD98059. Treatment with PD98059 (10 μM for 1 hour in MCF-7 cells and 25 μM for 2 hours in MDA-MB-231 cells) reduced the biological effects of FAM53A knockdown in MCF-7 cells and FAM53A overexpression in MDA-MB-231 cells . This provides strong evidence that FAM53A affects breast cancer progression primarily through the MEK-ERK pathway.

Additionally, FAM53A influences multiple downstream targets involved in epithelial-mesenchymal transition (EMT) and cell cycle regulation, including Snail, E-cadherin, cyclin D1, p21, RhoA, RhoC, and MMP9 . The differential regulation of these targets depending on p53 status suggests complex crosstalk between FAM53A, p53, and the MEK-ERK pathway in determining breast cancer cell behavior.

How does FAM53A expression affect chemotherapy response in different breast cancer subtypes?

Expression quantitative trait loci variants of FAM53A have been identified in TP53-based interaction analysis and are associated with therapeutic responses to doxorubicin in breast cancer . The response patterns vary significantly based on p53 status and breast cancer subtype:

• In triple-negative TP53-missense mutant breast cancer cells (MDA-MB-231), downregulation of FAM53A increases doxorubicin resistance .

• In luminal B p53-truncated mutant cells (MDA-MB-361), downregulation of FAM53A results in increased sensitivity to doxorubicin .

• In luminal A p53-wild-type cells (MCF7), downregulation of FAM53A also leads to increased sensitivity to doxorubicin .

These contrasting effects highlight the context-dependent role of FAM53A in modulating chemotherapy responses. Understanding these differential effects can potentially help in developing personalized treatment strategies for breast cancer patients based on tumor subtype and p53 status. The mechanisms underlying these varied responses likely involve the interaction between FAM53A, p53 signaling, and the MEK-ERK pathway, though further research is needed to fully elucidate these relationships.

What are the optimal storage conditions for FAM53A antibodies to maintain reactivity?

To maintain optimal reactivity, FAM53A antibodies should be stored at -20°C and protected from repeated freeze-thaw cycles . Most commercial FAM53A antibodies are supplied in PBS buffer containing 2% sucrose as a cryoprotectant to help maintain antibody integrity during freezing . When working with the antibody, it's advisable to aliquot the stock solution into smaller volumes upon first thawing to minimize the number of freeze-thaw cycles any portion of the antibody experiences. For short-term storage during experimental procedures, antibodies can be kept at 4°C, but should be returned to -20°C for long-term storage. Always follow manufacturer-specific recommendations, as buffer compositions may vary slightly between suppliers, potentially affecting optimal storage conditions.

How should researchers validate FAM53A antibody specificity for their experimental systems?

To validate FAM53A antibody specificity for experimental systems, researchers should implement a multi-step validation approach:

• Positive and Negative Controls: Use cell lines or tissues with known FAM53A expression levels. Based on published data, MDA-MB-231 cells show high FAM53A expression and can serve as a positive control, while MCF-7 cells have relatively lower expression .

• Knockdown/Overexpression Validation: Perform siRNA knockdown or plasmid-based overexpression of FAM53A following the protocols described earlier, then confirm the corresponding decrease or increase in signal intensity using the antibody .

• Western Blot Analysis: Validate the antibody detects a band of the expected molecular weight for FAM53A. Look for a clean, single band rather than multiple non-specific bands.

• Cross-reactivity Testing: If working with non-human samples, test the antibody against the species of interest, noting that most commercial FAM53A antibodies are validated for human reactivity only .

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide (if available) before application to samples. This should abolish specific staining if the antibody is indeed specific.

These validation steps are crucial for ensuring experimental results are truly reflective of FAM53A biology rather than artifacts of non-specific antibody binding.

What factors should be considered when designing experiments to study FAM53A's role in different breast cancer subtypes?

When designing experiments to study FAM53A's role across breast cancer subtypes, researchers should consider:

• p53 Status: Given the strong negative correlation between FAM53A expression and p53 status, it's essential to characterize and consider the p53 status of any cell lines or tissue samples used . Experiments should include both p53-wild-type (e.g., MCF-7) and p53-mutant/null (e.g., MDA-MB-231) models to capture the context-dependent effects.

• Molecular Subtype Representation: Include cell lines representative of different breast cancer molecular subtypes (luminal A, luminal B, HER2-enriched, and triple-negative) as FAM53A may have distinct functions in each.

• MEK-ERK Pathway Assessment: Include assays to measure MEK and ERK phosphorylation status, as this pathway appears central to FAM53A's mechanism of action . Consider incorporation of pathway inhibitors like PD98059 as experimental controls.

• Comprehensive Phenotypic Analysis: Assess multiple cancer-related phenotypes including proliferation, migration, invasion, and apoptosis, as FAM53A appears to influence several aspects of cancer cell behavior simultaneously .

• Translation to Clinical Relevance: Correlate in vitro findings with patient data, particularly examining associations between FAM53A expression, p53 status, and clinical outcomes or treatment responses .

• Consideration of Other FAM53 Family Members: FAM53B and FAM53C may have overlapping or compensatory functions, so consider their expression and potential functional redundancy in experimental design and interpretation .

By systematically addressing these factors, researchers can develop more comprehensive and clinically relevant models of FAM53A function in breast cancer.