CCDC170 Antibody

What is CCDC170 and why is it significant in breast cancer research?

CCDC170 is a protein that has gained considerable attention due to its involvement in breast cancer pathogenesis. Its significance stems from the discovery of recurrent rearrangements between the estrogen receptor gene (ESR1) and CCDC170, which have been identified in 6-8% of luminal B breast cancers . These ESR1-CCDC170 gene fusions are associated with more aggressive forms of estrogen receptor-positive (ER+) breast cancer and potentially reduced endocrine therapy responsiveness . Additionally, CCDC170 affects breast cancer apoptosis through modulation of the IRE1 pathway, suggesting its importance in cancer cell survival mechanisms .

How does CCDC170 expression vary across breast cancer subtypes?

CCDC170 expression shows significant variation across breast cancer molecular subtypes. Analysis of data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases reveals that CCDC170 mRNA levels are higher in luminal A and luminal B subtypes, while they are considerably lower in HER2-positive and basal-like breast cancer subtypes . This subtype-specific expression pattern suggests that CCDC170 may play different roles depending on the molecular context and could potentially serve as a biomarker for certain breast cancer subtypes.

What is known about the subcellular localization of CCDC170?

The subcellular localization of CCDC170 has been investigated using electron microscopy in HeLa cells transiently expressing GFP-CCDC170. Researchers have used immunogold labeling with anti-GFP antibodies to track the protein's location within cellular compartments . Understanding the subcellular distribution pattern is crucial for interpreting immunofluorescence results and provides insights into potential protein functions. The protein's localization pattern suggests possible associations with specific cellular structures, which may inform hypotheses about its biological role.

What are the validated applications for CCDC170 antibodies in research?

CCDC170 antibodies have been validated for several research applications:

• Western blotting: For detecting both wild-type CCDC170 and truncated variants resulting from ESR1-CCDC170 gene fusions

• Immunohistochemistry (IHC): For analyzing CCDC170 expression in patient tissue samples and xenograft tumors

• Immunofluorescence: For examining subcellular localization and co-localization with other proteins

• Immunoprecipitation: For protein interaction studies and complex isolation

• Proximity labeling experiments: When combined with techniques like BioID for identifying protein interaction networks

Each application requires specific optimization and validation strategies to ensure reliable results.

How can CCDC170 antibodies be used to detect ESR1-CCDC170 fusion proteins?

ESR1-CCDC170 fusion proteins (ΔCCDC170) can be detected using antibodies targeting the C-terminal region of CCDC170, as this portion is retained in the fusion products. Western blot analysis using commercial polyclonal antibodies against the C-terminus of CCDC170 has successfully detected various fusion variants with predicted molecular weights of 41kDa (E2-E6), 39kDa (E2-E7), 30kDa (E2-E8), and 14kDa (E2-E10) in cells ectopically expressing these variants . When designing experiments to detect fusion proteins, researchers should:

• Select antibodies that recognize epitopes present in the truncated fusion products

• Include appropriate positive controls (cells expressing known fusion variants)

• Use negative controls (fusion-negative cell lines)

• Optimize protein extraction methods, particularly if the proteins associate with cellular structures

What validation strategies are essential when using CCDC170 antibodies?

Comprehensive validation of CCDC170 antibodies is crucial for generating reliable research data. Key validation strategies include:

How should researchers design experiments to study ESR1-CCDC170 fusion variants?

When investigating ESR1-CCDC170 fusion variants, researchers should consider this comprehensive experimental approach:

• Detection and characterization:

  • RT-PCR with primers spanning the fusion junction

  • Western blotting with C-terminal CCDC170 antibodies to identify fusion proteins

  • DNA sequencing to confirm fusion breakpoints

• Functional analysis:

  • Ectopic expression of fusion variants in appropriate breast cancer cell models (e.g., T47D, MCF7)

  • Comparison of different fusion variants (E2-E6, E2-E7, E2-E8, E2-E10) to determine variant-specific effects

  • Assessment of cellular phenotypes: proliferation, apoptosis, migration, invasion

• In vivo studies:

  • Xenograft models with estradiol supplementation

  • Comparison of tumor growth between fusion-expressing and control tumors

  • Evaluation of endocrine therapy response using tamoxifen treatment (25 μg/kg body weight)

• Mechanistic investigations:

  • Analysis of downstream signaling pathways (SRC/HER2/HER3 complex)

  • Examination of effects on estrogen receptor signaling

  • Protein interaction studies to identify fusion-specific binding partners

What are the optimal methods for manipulating CCDC170 expression in experimental models?

Researchers have successfully employed multiple strategies to manipulate CCDC170 expression:

• CRISPR/Cas9-mediated knockout:

  • Guide RNA design targeting CCDC170 exons

  • Cloning into appropriate vectors (e.g., px459)

  • Single-cell cloning to generate knockout lines

  • Validation by Sanger sequencing and Western blot

• Overexpression systems:

  • Cloning CCDC170 ORF into expression vectors

  • Transient transfection for short-term studies

  • Stable integration for long-term experiments

  • GFP-fusion constructs for localization studies

• RNA interference:

  • siRNA-mediated knockdown for transient depletion

  • shRNA for stable knockdown

  • Validation of knockdown efficiency by qPCR and Western blot

• Fusion protein expression:

  • Construction of ESR1-CCDC170 fusion variants (E2-E6, E2-E7, E2-E8, E2-E10)

  • Expression in appropriate cell models (T47D, MCF7)

  • Validation of expression by Western blot

How can researchers investigate the relationship between CCDC170 and the IRE1 pathway?

To investigate the relationship between CCDC170 and the IRE1 pathway, researchers should consider these experimental approaches:

• Expression correlation analysis:

  • Examining CCDC170 and IRE1 co-expression in breast cancer databases (TCGA, GEO)

  • Analyzing correlation with XBP1 and ESR1 expression

• Protein expression manipulation:

  • Measuring IRE1α, XBP1s, and ERα levels after CCDC170 overexpression or knockdown

  • Western blot analysis at multiple time points (24h, 48h) to capture temporal dynamics

  • Immunofluorescence to visualize changes in protein expression and localization

• Functional assays:

  • Assessing the impact of CCDC170 manipulation on cell survival under ER stress conditions

  • Measuring XBP1 splicing as a readout of IRE1 activity

  • Evaluating apoptosis rates in relation to CCDC170 and IRE1 expression

• Mechanistic studies:

  • Investigating direct or indirect interactions between CCDC170 and IRE1

  • Examining effects on downstream IRE1 signaling components

  • Exploring potential regulatory mechanisms

How should researchers interpret conflicting CCDC170 expression data?

When encountering conflicting CCDC170 expression data across studies, researchers should consider several factors:

• Methodological differences:

  • Antibody epitopes and specificity

  • Detection techniques (Western blot, IHC, qPCR)

  • Sample preparation and fixation methods

• Biological variables:

  • Breast cancer subtype heterogeneity

  • Presence of ESR1-CCDC170 fusions affecting antibody recognition

  • Patient treatment history

• Quantification approaches:

  • Threshold definitions for "high" versus "low" expression

  • Normalization methods

  • Statistical analyses and significance thresholds

• Validation strategies:

  • Correlation between protein and mRNA data

  • Use of multiple antibodies targeting different epitopes

  • Inclusion of appropriate positive and negative controls

What are common technical challenges with CCDC170 antibodies and their solutions?

How do ESR1-CCDC170 fusions affect endocrine therapy response?

ESR1-CCDC170 fusions appear to significantly impact endocrine therapy response in breast cancer:

• Clinical observations:

  • ESR1-CCDC170 fusions are associated with the luminal B subtype, which typically shows reduced endocrine sensitivity

  • Fusion-positive cases demonstrate higher Ki67 scores, indicating increased proliferation

• Experimental evidence:

  • T47D xenograft tumors expressing ESR1-CCDC170 fusion variants (E2-E7, E2-E10) show significantly reduced sensitivity to tamoxifen treatment

  • Kaplan-Meier analysis reveals significantly worse regression-free survival for both E2-E7 (P < 0.01) and E2-E10 (P < 0.001) fusion-expressing tumors treated with tamoxifen

  • The E2-E10 variant confers a more pronounced growth advantage (P = 0.000002) compared to the E2-E7 variant

• Potential mechanisms:

  • Activation of the SRC/HER2/HER3 signaling complex, which may bypass estrogen dependence

  • Induction of ligand-independent growth factor signaling

  • Modulation of cell survival pathways

• Therapeutic implications:

  • Combination therapy with SRC/HER2/HER3 inhibitors and tamoxifen has been proposed as a potential strategy for fusion-positive tumors

  • ESR1-CCDC170 status could potentially serve as a biomarker for predicting endocrine therapy response

What is the prognostic significance of CCDC170 expression in breast cancer?

The prognostic significance of CCDC170 expression shows some data source-dependent variation:

This complex picture suggests that CCDC170's impact on patient outcomes is context-dependent and warrants careful interpretation in relation to other clinicopathological factors.

How can researchers distinguish between wild-type CCDC170 and fusion variants in patient samples?

Distinguishing between wild-type CCDC170 and fusion variants in patient samples requires a multi-modal approach:

• Nucleic acid-based detection:

  • RT-PCR with primers spanning potential fusion junctions

  • RNA sequencing to identify fusion transcripts

  • DASE (Differential Allele-Specific Expression) analysis at the CCDC170-ESR1 locus

• Protein-based detection:

  • Western blotting using antibodies against the C-terminus of CCDC170

  • Identification of truncated protein products (41kDa, 39kDa, 30kDa, or 14kDa) corresponding to fusion variants

  • Comparison with wild-type CCDC170 (approximately 55kDa)

• Tissue analysis:

  • IHC to assess CCDC170 expression patterns

  • Correlation with Ki67 index (fusion-positive cases typically have higher scores)

  • Co-expression analysis with ERα and potential downstream targets

• Validation approaches:

  • Sequencing confirmation of fusion breakpoints

  • Correlation of protein expression with transcript data

  • Functional assays to assess endocrine sensitivity

What therapeutic strategies might target CCDC170 or its fusion variants?

Based on current research, several therapeutic strategies could potentially target CCDC170 or its fusion variants:

• Targeting downstream signaling pathways:

  • Combination of SRC/HER2/HER3 inhibitors with endocrine therapy for ESR1-CCDC170-positive tumors

  • IRE1 pathway inhibitors, given the relationship between CCDC170 and IRE1α

• Fusion-specific approaches:

  • Development of small molecules targeting the unique protein interfaces created by fusion events

  • Peptide-based inhibitors designed to disrupt fusion protein interactions

• Gene therapy approaches:

  • siRNA or antisense oligonucleotides targeting fusion-specific junctions

  • CRISPR/Cas9-based strategies to disrupt fusion genes

• Biomarker-guided therapy:

  • Using ESR1-CCDC170 status to guide treatment decisions

  • Monitoring fusion status during treatment to detect resistance mechanisms

• Combination strategies:

  • Integrating endocrine therapy with targeted agents based on fusion status

  • Personalizing treatment regimens according to molecular profiles

While these approaches show promise in experimental settings, clinical translation requires further validation in appropriate models and eventually in clinical trials.