brms1la Antibody

What is BRMS1L and what are the typical applications for BRMS1L antibodies in cancer research?

BRMS1L (Breast Cancer Metastasis Suppressor 1-Like) is a protein that functions as a transcriptional repressor and is a component of the mSin3a/HDAC1 repressive machinery . BRMS1L antibodies are widely used in multiple research applications:

BRMS1L antibodies are particularly valuable for studying tumor suppression mechanisms as BRMS1L has been shown to inhibit proliferation and metastasis in several cancer types, including breast cancer, lung cancer, and gliomas .

What are the key considerations for validating a BRMS1L antibody before experimental use?

Proper validation of BRMS1L antibodies is critical for experimental reliability:

• Specificity testing: Verify using positive controls such as MCF-7 and HeLa cells, which are known to express BRMS1L .

• Molecular weight confirmation: BRMS1L has a calculated molecular weight of 38 kDa but is typically observed at approximately 40 kDa in Western blots .

• Multiple detection techniques: Cross-validate using different methods (e.g., Western blot and IHC).

• Knockdown/overexpression validation: Use BRMS1L-knockdown or overexpressing cell models to confirm antibody specificity.

• Sample preparation optimization: For Western blotting, researchers should note that BRMS1L can appear as two bands at approximately 40 kDa in various cell types .

What sample preparation methods are optimal for BRMS1L antibody applications?

For optimal results with BRMS1L antibodies across different applications:

Western Blotting:

• Lyse cells in RIPA buffer with protease inhibitors on ice for 30 minutes

• Separate proteins by 10% SDS-PAGE

• Transfer to PVDF membrane

• Block with 5-10% normal goat serum or BSA

Immunohistochemistry:

• For paraffin-embedded tissues: Cut 3μm thick slices

• Dewax twice in xylol and rehydrate in graded ethanol series

• Perform antigen retrieval by boiling in 20mM citrate buffer (pH 6.0) for 10 minutes

• Treat with 0.7% hydrogen peroxide and 10% normal goat serum before antibody incubation

Immunofluorescence:

• Fix cells in 4% paraformaldehyde

• Permeabilize with 0.1-0.5% Triton X-100

• Block with 1-5% BSA before antibody incubation

How do BRMS1L expression patterns differ between normal and cancerous tissues?

Research has revealed distinct expression patterns of BRMS1L in normal versus cancerous tissues:

Intriguingly, multiple studies have observed discrepancies between mRNA and protein expression. In gliomas, despite BRMS1L mRNA overexpression, protein levels appear significantly decreased compared to normal brain tissue . Similarly, in breast cancer, mRNA levels can be significantly higher than in normal epithelial cells, yet protein expression is reduced . This suggests post-transcriptional regulation mechanisms affecting BRMS1L protein levels .

What explains the discrepancy between BRMS1L mRNA and protein expression observed in cancer?

The paradoxical finding of high BRMS1L mRNA but low protein levels in various cancers appears to involve multiple post-transcriptional regulatory mechanisms:

• MicroRNA regulation: Several microRNAs, including miR-423, miR-125a-5p, and miR-3200-5p, have been found to lower BRMS1L protein levels by binding to the 3'UTR region of BRMS1L mRNA .

• Protein degradation pathways: Casein kinase 2α can trigger degradation of BRMS1L protein through phosphorylation of serine 30, resulting in cytoplasmic localization and poly-ubiquitination .

• Compensatory feedback: The elevation in mRNA might represent a cellular attempt to compensate for reduced protein levels, suggesting a feedback mechanism .

• Tissue-specific regulation: The Human Protein Atlas data indicates different regulation patterns across cell types, with neurons strongly expressing BRMS1L protein while astrocytes and oligodendrocytes show high mRNA but low protein expression .

This discrepancy highlights the importance of analyzing both mRNA and protein expression when studying BRMS1L's role in cancer.

What is the relationship between BRMS1L expression and clinical outcomes in different cancer types?

The relationship between BRMS1L expression and clinical outcomes varies across cancer types:

Gliomas:

• Surprisingly, survival-time analysis revealed no significant difference between high/low BRMS1L expression groups

• Gliomas grade 2/3, which have better prognosis than glioblastoma, displayed stronger BRMS1L mRNA and protein expression

These findings suggest BRMS1L may have tissue-specific roles in cancer progression, with particularly strong evidence for its prognostic value in NSCLC and breast cancer.

How can BRMS1L antibodies be optimized for Chromatin Immunoprecipitation (ChIP) assays?

Optimizing ChIP assays with BRMS1L antibodies requires careful consideration:

• Antibody selection: Choose ChIP-validated antibodies targeting BRMS1L. Monoclonal antibodies like those from Abcam (ab134968) have been successfully used in IP applications and may be suitable for ChIP .

• Cross-linking optimization: For transcription factors like BRMS1L that are part of larger complexes (mSin3a/HDAC1), use 1% formaldehyde for 10 minutes at room temperature.

• Sonication parameters: Optimize sonication to achieve chromatin fragments of 200-500bp, which is critical for resolution in identifying BRMS1L binding sites.

• Controls: Include:

  • Input chromatin control

  • IgG negative control

  • Positive control targeting known BRMS1L-binding regions

• Target validation: Based on published research, focus on promoter regions of BRMS1L target genes such as FZD10 or regions containing candidate p53-binding sites .

• Data validation: Confirm ChIP results using methods such as reporter assays. For example, luciferase assays have been used to validate BRMS1L promoter binding and transcriptional effects .

In one study, researchers successfully performed ChIP assays to demonstrate direct binding of p53 family proteins to BRMS1L regulatory elements, identifying two binding sites (RE1-BRMS1L and RE2-BRMS1L) located 3241bp upstream and 469bp downstream of the first exon, respectively .

What molecular mechanisms explain BRMS1L's role in suppressing cancer metastasis?

BRMS1L inhibits cancer metastasis through several interconnected molecular mechanisms:

• Transcriptional repression: As a component of the mSin3A/HDAC1 complex, BRMS1L functions as a transcriptional repressor . It can recruit histone deacetylases to specific promoter regions, leading to epigenetic silencing of target genes.

• Wnt signaling inhibition: In breast cancer, BRMS1L induces epigenetic silencing of FZD10, a Wnt receptor, thereby downregulating Wnt signaling which is critical for EMT and metastasis .

• Modulation of oxidative stress response: In NSCLC, BRMS1L transcriptionally inhibits GPX2-mediated oxidative stress repair. Overexpression of BRMS1L downregulates glutathione peroxidase 2 (GPX2), causing abnormal glutathione metabolism and increased ROS levels, inducing oxidative stress injury and apoptosis .

• Regulation of cell adhesion and migration: BRMS1L affects the invasion and migration capabilities of cancer cells by regulating cell adhesion molecules. In gliomas, BRMS1L has been shown to suppress invasion, migration, and adhesion .

• p53 pathway interaction: BRMS1L appears to be regulated by p53 family proteins and may function as a mediator of the p53 pathway in suppressing metastasis .

This multifaceted action allows BRMS1L to exert context-dependent tumor suppressive effects across different cancer types.

How can researchers investigate the interplay between BRMS1L and the p53 family proteins?

Investigating the BRMS1L-p53 relationship requires several specialized approaches:

• Expression correlation studies:

  • Analyze BRMS1L mRNA and protein expression after modulating p53 family proteins (p53, p73β, p63γ)

  • Perform immunoblotting and real-time RT-PCR to quantify changes

  • Use immunofluorescence staining to observe endogenous BRMS1L expression in p53-transfected versus non-transfected cells

• Promoter binding analysis:

  • Perform in silico analysis to identify putative p53-binding sites in the BRMS1L gene

  • Validate with ChIP assays using p53 family protein antibodies

  • Use known p53-binding sites (e.g., in p21 gene) as positive controls

• Functional studies:

  • Perform knockdown/overexpression of p53 family members and assess BRMS1L expression

  • Use p53-null and p53-wildtype cells to compare basal and induced BRMS1L expression

  • Implement luciferase reporter assays with BRMS1L promoter constructs

• Clinical relevance:

  • Analyze BRMS1L expression in relation to p53 mutation status in cancer tissues

  • Use databases like Oncomine to look for correlations between p53 status and BRMS1L expression

What are the most common technical challenges when using BRMS1L antibodies and how can they be addressed?

Researchers working with BRMS1L antibodies commonly encounter several technical challenges:

For BRMS1L in particular, be aware that expression differences between mRNA and protein levels are commonly observed in research , representing a biological phenomenon rather than a technical artifact.

How should researchers design experiments to investigate BRMS1L's role in specific cancer types?

A comprehensive experimental design for studying BRMS1L in cancer should include:

• Expression profiling:

  • Analyze BRMS1L mRNA using qRT-PCR with primers targeting the 40-120 amino acid region

  • Detect protein using validated BRMS1L antibodies in Western blot (1:500-1:2000 dilution)

  • Perform IHC on tissue microarrays comparing normal vs. cancerous tissues (1:30-1:150 dilution)

• Functional studies:

  • Generate stable cell lines with BRMS1L overexpression or knockdown using retroviral/lentiviral vectors

  • Verify successful modification by qRT-PCR and Western blotting

  • Assess effects on:

    • Proliferation (colony formation assays, proliferation assays)

    • Migration (wound healing assays)

    • Invasion (transwell invasion assays)

    • Apoptosis (flow cytometry)

• Mechanistic investigations:

  • Identify BRMS1L target genes using RNA-seq after BRMS1L modulation

  • Perform ChIP followed by qPCR to identify direct binding targets

  • Use luciferase reporter assays to confirm transcriptional regulation

  • Investigate protein interactions with co-immunoprecipitation

• Clinical correlations:

  • Analyze BRMS1L expression in relation to clinical parameters (stage, grade, survival)

  • Use public databases (TCGA, Oncomine, Kaplan-Meier Plotter) to validate findings

  • Consider correlation with therapy response

• In vivo validation:

  • Establish xenograft models with BRMS1L-modified cells

  • Assess tumor growth, metastasis, and response to therapy

This comprehensive approach allows for thorough characterization of BRMS1L's role in cancer progression and its potential as a biomarker or therapeutic target.

What are the considerations for developing therapeutic approaches targeting the BRMS1L pathway?

Developing therapeutics targeting the BRMS1L pathway requires consideration of several key factors:

• Target validation:

  • Establish clear causal relationship between BRMS1L dysfunction and disease progression

  • Determine whether increasing or decreasing BRMS1L activity would be beneficial

  • Identify patient subpopulations likely to benefit based on expression patterns

• Mechanism selection:

  • For cancers with low BRMS1L protein expression: Develop approaches to increase BRMS1L levels

    • Inhibitors of specific microRNAs (miR-423, miR-125a-5p) that target BRMS1L

    • Inhibitors of pathways promoting BRMS1L protein degradation (casein kinase 2α)

  • For targeting BRMS1L's downstream effects: Focus on epigenetic modifiers

    • HDAC inhibitors may affect BRMS1L-containing complexes

    • Wnt pathway inhibitors could synergize with BRMS1L functions

• Exploiting synthetic lethality:

  • In NSCLC with low BRMS1L expression, cells maintain redox balance through alternative mechanisms

  • These cells show increased sensitivity to ROS inducers like piperlongumine

  • BRMS1L expression could potentially serve as a biomarker for predicting response to oxidative stress-targeting therapies

• Delivery approaches:

  • For protein replacement: Consider nanoparticle-based delivery systems for recombinant BRMS1L

  • For genetic approaches: Viral vectors or lipid nanoparticles for BRMS1L expression constructs

  • For mRNA stabilization: Antisense oligonucleotides targeting microRNAs that regulate BRMS1L

• Biomarker development:

  • Develop robust IHC protocols for assessing BRMS1L protein in clinical samples

  • Consider both mRNA and protein detection due to observed discrepancies

  • Create companion diagnostics to identify likely responders to BRMS1L-targeting therapies

This strategic approach acknowledges the complex biology of BRMS1L while identifying practical routes toward therapeutic development.

How can single B cell antibody discovery platforms be leveraged to develop novel BRMS1L antibodies?

Advanced single B cell platforms offer promising approaches for next-generation BRMS1L antibody development:

• Function-first screening approaches:

  • The Opto B Discovery Application on Beacon® platforms allows for function-first, high-throughput single B cell screening

  • This enables screening tens of thousands of B cells for BRMS1L-specific antibodies in a single run

  • Multiple functional assays can be conducted simultaneously, including antigen specificity, affinity, and cross-reactivity

• Species diversity advantages:

  • These platforms can screen antibodies across diverse species (human, mouse, rabbit, alpaca)

  • This diversity increases the likelihood of generating unique epitope coverage across the BRMS1L protein

  • For challenging epitopes in highly conserved regions of BRMS1L, non-traditional host species may produce more effective antibodies

• Experimental design considerations:

  • Immunize hosts with full-length recombinant BRMS1L protein

  • Additionally, use peptide immunogens from specific regions (N-terminal, C-terminal, internal domains)

  • Screen resulting antibodies against both native and denatured forms of BRMS1L

• Validation methodologies:

  • Implement multi-parameter screening assays

  • Test cross-reactivity with related proteins (BRMS1, other HDAC complex components)

  • Verify function in multiple applications (WB, IP, ChIP, IHC)

  • Confirm specificity using BRMS1L-knockout cell lines

• Applications in BRMS1L research:

  • Generate antibodies that can distinguish between post-translationally modified forms

  • Develop antibodies specific to different BRMS1L isoforms

  • Create antibodies with enhanced sensitivity for detecting low levels of BRMS1L in cancer samples

These advanced platforms can address current limitations in BRMS1L detection and potentially reveal new insights into BRMS1L biology through more precise and sensitive detection methods.

What is the potential for BRMS1L as a biomarker for cancer prognosis and therapeutic response?

BRMS1L shows considerable promise as both a prognostic biomarker and predictor of therapeutic response:

The dual assessment of BRMS1L mRNA and protein levels might provide more comprehensive prognostic information given the documented discrepancies between them in various cancer types .

How does BRMS1L interact with other epigenetic regulators in the context of cancer progression?

BRMS1L functions within a complex network of epigenetic regulators that collectively influence cancer progression:

• mSin3A/HDAC complex interactions:

  • BRMS1L is a component of the mSin3A family of histone deacetylase complexes (HDAC)

  • It promotes HDAC1 binding to promoter regions, facilitating histone deacetylation and gene silencing

  • This interaction is critical for BRMS1L's function as a transcriptional repressor

• NF-κB pathway modulation:

  • BRMS1L can downregulate transcription activation by NF-κB

  • It promotes deacetylation of RELA at 'Lys-310'

  • This results in reduced expression of anti-apoptotic genes controlled by NF-κB

  • In gliomas, BRMS1 can bind to the NFκB region of the urokinase plasminogen-activator (uPA) promoter

• p53 family regulatory network:

  • p53 family proteins (p53, p73β, p63γ) can upregulate BRMS1L expression

  • ChIP assays identified direct binding of p53 family proteins to BRMS1L regulatory elements

  • This positions BRMS1L as a potential effector in p53-mediated tumor suppression

• Wnt signaling suppression:

  • In breast cancer, BRMS1L induces epigenetic silencing of FZD10, a Wnt receptor

  • This leads to downregulation of Wnt signaling, which is critical for EMT and metastasis

  • The interaction involves recruitment of the HDAC complex to the FZD10 promoter

• Regulation of oxidative stress response:

  • In NSCLC, BRMS1L transcriptionally inhibits GPX2 expression

  • This causes abnormal glutathione metabolism and increased ROS levels

  • The mechanism links BRMS1L to metabolic reprogramming in cancer cells