TACC2 Antibody

What is TACC2 and what are its primary cellular functions?

TACC2 is a member of the transforming acidic coiled-coil-containing protein family associated with the centrosome-spindle apparatus during cell cycling. It plays a crucial role in the microtubule-dependent coupling of the nucleus and the centrosome . TACC2 is involved in regulating centrosome-mediated interkinetic nuclear migration (INM) of neural progenitors and may play a significant role in organizing centrosomal microtubules . Functionally, TACC2 works closely with Aurora A to promote correct formation of the mitotic apparatus, ensuring successful chromosomal alignment and segregation during cell division . Research suggests TACC2 may act as either a tumor suppressor protein or a tumor progression marker depending on the cancer type and context .

What are the known isoforms of TACC2 and how do they differ?

TACC2 exists in multiple isoforms, with research identifying at least two main variants:

The short isoform is predominantly expressed in LNCaP cells as confirmed by PCR primer design and protein expression studies . Androgen stimulation primarily upregulates the short isoform in prostate cancer cells, indicating potential functional differences between isoforms . The isoforms differ in their N-terminal regions, while all contain the characteristic C-terminal coiled-coil domain that defines the TACC family .

How is TACC2 expression regulated in normal and cancer tissues?

TACC2 expression exhibits tissue-specific patterns and is regulated through multiple mechanisms:

• Tissue distribution: TACC2 is predominantly expressed in postmitotic tissues, including heart, muscle, kidney, and brain .

• Hormonal regulation: In prostate cancer cells, TACC2 expression is androgen-responsive. Treatment with R1881 or dihydrotestosterone (DHT) induces TACC2 mRNA expression in a time-dependent manner, with approximately 2-fold elevation detected 24 hours after treatment . This induction can be significantly inhibited by antiandrogens such as bicalutamide .

• Dysregulation in cancer: TACC2 expression is frequently altered in cancer tissues compared to normal counterparts. Quantitative analysis of TACC2 transcript revealed higher levels of expression in breast tumors compared to normal tissues . TACC2 expression significantly increases in higher-grade breast tumors (grade 3 vs. grade 1, p=0.046) .

• Androgen receptor (AR) influence: In long-term androgen-deprived (LTAD) prostate cancer cells, TACC2 is upregulated compared to parental cells under hormone-depleted conditions. AR knockdown reduces TACC2 mRNA levels and androgen-mediated upregulation, suggesting AR overexpression contributes to increased TACC2 expression .

What is the prognostic significance of TACC2 expression in different cancer types?

TACC2 expression has emerged as a significant prognostic indicator in multiple cancer types, with particularly robust data in breast and prostate cancers:

Breast Cancer:

• TACC2 transcript levels are significantly higher in tumors from patients with moderate prognosis compared to those with good prognosis according to the Nottingham Prognostic Index (NPI) (p=0.045) .

• Expression is elevated in samples from patients with poor clinical outcomes (metastasis, recurrence, and breast cancer-related death) compared to those who remained disease-free .

• Disease-free survival is shorter for patients with high TACC2 expression (107 months, 95% CI: 91-122.8) compared to those with low TACC2 expression (137 months, 95% CI: 125-150.6, p=0.019) .

Prostate Cancer:

• TACC2 functions as a prognostic factor for prostate cancer progression and represents a potential therapeutic target for castration-resistant prostate cancer (CRPC) .

• Androgen-responsive TACC2 expression is significantly upregulated in long-term androgen-deprived (LTAD) prostate cancer cells compared to hormone-sensitive parental cells .

• TACC2 knockdown inhibits cell proliferation in both AR-positive hormone-sensitive and AR-negative hormone-refractory prostate cancer cell lines, suggesting its broader role in prostate cancer progression .

These findings collectively suggest that TACC2 overexpression correlates with aggressive disease features and poorer outcomes across multiple cancer types, highlighting its potential utility as a prognostic biomarker.

How does TACC2 modulate cell cycle progression and mitotic spindle formation?

TACC2 plays critical roles in cell cycle regulation and mitotic spindle formation through several mechanisms:

• Cell Cycle Progression: TACC2 knockdown studies in LTAD prostate cancer cells demonstrated significant G2/M accumulation and decreased proportion of cells in S-phase 24 hours after release from G0/G1 synchronization, indicating cell cycle inhibition at G2/M phase . Similar effects were observed following release from G2/M synchronization, with robust decrease in S-phase cells following TACC2 knockdown .

• Centrosome Function: Immunofluorescence analysis of LTAD cells treated with siTACC2 showed reduced γ-tubulin staining of centrosomes in most cells, suggesting TACC2's role in maintaining centrosome integrity .

• Mitotic Spindle Formation: TACC2 knockdown results in abnormal mitotic spindles with more than two centrosomes or single centrosomes, whereas control cells typically display two clear centrosomes during mitosis .

• Chromosomal Stability: Extended TACC2 knockdown (>5 days) in LTAD cells leads to chromatin instability during mitosis, highlighting TACC2's role in maintaining genomic integrity during cell division .

• Aurora A Interaction: TACC2 works in concert with Aurora A kinase to promote correct formation of the mitotic apparatus, ensuring successful chromosomal alignment and segregation during cell division .

These findings collectively establish TACC2 as a key regulator of multiple aspects of mitosis and cell cycle progression, explaining its impact on cancer cell proliferation and potential as a therapeutic target.

What are the molecular mechanisms by which TACC2 contributes to tumorigenesis or tumor suppression?

TACC2's role in tumorigenesis appears context-dependent, with evidence supporting both oncogenic and tumor-suppressive functions through several molecular mechanisms:

Potential Oncogenic Mechanisms:

• Cell Cycle Promotion: TACC2 facilitates cell cycle progression, particularly through the G2/M phase, supporting cancer cell proliferation .

• Androgen Signaling: In prostate cancer, TACC2 functions as an androgen-responsive gene that contributes to tumor growth through AR signaling pathways .

• Centrosomal Functions: TACC2 maintains centrosome integrity and proper mitotic spindle formation, potentially allowing cancer cells to continue dividing despite genomic instability .

• Histone Acetyltransferase Interaction: TACC2 may contribute to tumorigenesis through interactions with nuclear histone acetyltransferases, potentially altering gene expression patterns that favor cancer progression .

Potential Tumor-Suppressive Mechanisms:

• Genomic Stability: TACC2 promotes proper chromosomal alignment and segregation during mitosis, potentially preventing the genomic instability characteristic of many cancers .

• Differentiation Regulation: TACC2 may regulate cellular differentiation pathways in certain tissues, with its loss potentially contributing to dedifferentiation in some cancer types .

The seemingly contradictory roles of TACC2 in cancer progression may reflect tissue-specific functions, interaction with different molecular partners, or expression of different isoforms. This highlights the importance of context-specific analysis when evaluating TACC2 as a biomarker or therapeutic target.

What is the optimal protocol for detecting TACC2 in human tissue samples using antibody-based techniques?

Based on published research protocols, the following optimized methodologies are recommended for detecting TACC2 in human tissue samples:

Immunohistochemistry (IHC-P) Protocol:

• Sample Preparation: Use formalin-fixed paraffin-embedded (FFPE) sections of human tissue.

• Antigen Retrieval: Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol .

• Antibody Selection: Use a validated anti-TACC2 antibody (such as ab204891) at a 1/200 dilution .

• Detection System: Employ a sensitive detection system compatible with rabbit primary antibodies.

• Controls: Include both high-expression (e.g., human skeletal muscle) and low-expression (e.g., lymph node) tissues as controls .

Western Blotting (WB) Protocol:

• Sample Preparation: Prepare lysates from fresh or frozen tissue samples in a suitable lysis buffer containing protease inhibitors.

• Protein Quantification: Normalize protein loading (20-50 μg per lane).

• SDS-PAGE: Separate proteins on a 10% SDS-PAGE gel.

• Transfer: Transfer to PVDF or nitrocellulose membrane.

• Blocking: Block with 5% non-fat milk or BSA in TBST.

• Antibody Incubation: Incubate with anti-TACC2 antibody at 1/500 - 1/2000 dilution overnight at 4°C .

• Detection: Use HRP-conjugated secondary antibody and ECL detection reagents.

• Expected Bands: Look for a band at approximately 73 kDa, which corresponds to the short isoform of TACC2 .

Immunofluorescence/Immunocytochemistry (IF/ICC) Protocol:

• Cell Preparation: Culture cells on coverslips or use cytospin preparations.

• Fixation: Fix with 4% paraformaldehyde for 15 minutes.

• Permeabilization: Permeabilize with 0.1% Triton X-100 for 10 minutes.

• Blocking: Block with 5% normal serum in PBS for 1 hour.

• Antibody Incubation: Incubate with anti-TACC2 antibody at 1/20 - 1/200 dilution .

• Co-staining: Consider co-staining with centrosomal markers like γ-tubulin to visualize TACC2's centrosomal localization .

• Counterstaining: Counterstain with DAPI to visualize nuclei.

How can researchers effectively validate the specificity of TACC2 antibodies for experimental applications?

Thorough validation of TACC2 antibodies is essential for generating reliable experimental data. The following comprehensive validation approach is recommended:

1. Positive and Negative Controls:

• Use tissues or cell lines with known high (skeletal muscle) and low (lymph node) TACC2 expression as controls .

• Include TACC2 knockout or knockdown samples as negative controls when possible.

2. Multiple Detection Methods:

• Cross-validate findings using at least two independent detection methods (e.g., WB, IHC, and IF/ICC).

• Compare results from different antibody clones targeting distinct epitopes of TACC2.

3. Epitope Mapping:

• Confirm the specific TACC2 domain targeted by the antibody (e.g., antibodies targeting amino acids 200-350 versus 1250-1400) .

• Verify if the antibody recognizes all TACC2 isoforms or is isoform-specific.

4. Knockdown/Knockout Validation:

• Perform siRNA-mediated knockdown of TACC2 and confirm signal reduction in your experimental system .

• If using a cell line amenable to genetic manipulation, CRISPR/Cas9 knockout can provide stringent validation.

5. Peptide Competition Assay:

• Pre-incubate the antibody with the immunizing peptide/protein fragment.

• Verify signal elimination when the antibody is neutralized by its specific antigen.

6. Mass Spectrometry Correlation:

• For definitive validation, perform immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein.

7. Application-Specific Optimization:

• Optimize antibody concentration for each specific application (recommended dilutions: WB: 1/500-1/2000, IHC: 1/20-1/200, IF/ICC: 1/20-1/200) .

• Determine optimal incubation conditions (time, temperature) for each application.

What experimental approaches can be used to study the interaction between TACC2 and Aurora A in mitotic spindle formation?

To investigate the functional interaction between TACC2 and Aurora A in mitotic spindle formation, several complementary experimental approaches can be employed:

1. Co-immunoprecipitation (Co-IP) Assays:

• Immunoprecipitate endogenous TACC2 using validated antibodies and probe for Aurora A or vice versa.

• Perform under both native conditions and with cell cycle synchronization to examine cell cycle-dependent interactions.

• Include phosphatase inhibitors to preserve potential phosphorylation-dependent interactions.

2. Proximity Ligation Assay (PLA):

• Utilize PLA to visualize and quantify TACC2-Aurora A interactions in situ.

• Combine with cell cycle markers to map interaction dynamics throughout mitosis.

• Compare interactions in normal versus cancer cells to identify pathological alterations.

3. FRET/BRET Analysis:

• Generate fluorescent or bioluminescent protein fusions of TACC2 and Aurora A.

• Measure energy transfer to assess protein proximity and interaction dynamics in living cells.

• Track interactions throughout the cell cycle using time-lapse microscopy.

4. Pharmacological Inhibition:

• Treat cells with Aurora A kinase inhibitors (e.g., alisertib) and assess effects on TACC2 localization, phosphorylation, and function.

• Examine consequent changes in mitotic spindle formation and chromosome segregation.

5. Phosphorylation Site Mapping:

• Identify Aurora A-dependent phosphorylation sites on TACC2 using phospho-specific antibodies or mass spectrometry.

• Generate phospho-mimetic and phospho-deficient TACC2 mutants to assess functional consequences.

6. Live Cell Imaging:

• Utilize fluorescently tagged TACC2 and Aurora A to track their dynamic localization during mitosis.

• Employ high-resolution microscopy techniques (SIM, STORM) to visualize centrosomal co-localization at sub-diffraction resolution.

7. Functional Rescue Experiments:

• Deplete endogenous TACC2 using siRNA and express siRNA-resistant wild-type or mutant TACC2 lacking Aurora A interaction domains.

• Assess the ability of these constructs to rescue normal spindle formation and mitotic progression.

8. In Vitro Reconstitution:

• Purify recombinant TACC2 and Aurora A proteins.

• Perform in vitro kinase assays to confirm direct phosphorylation.

• Assess effects on microtubule dynamics using purified tubulin and total internal reflection fluorescence (TIRF) microscopy.

How can TACC2 expression analysis be incorporated into cancer prognostic assessments?

Based on emerging research, TACC2 expression analysis offers significant potential for enhancing cancer prognostic assessments, particularly in breast and prostate cancers:

Implementation Strategies:

• Quantitative Assessment Methods:

  • Quantitative real-time PCR to determine TACC2 transcript levels in fresh or frozen tumor tissues .

  • Immunohistochemistry with standardized scoring systems to evaluate TACC2 protein expression in FFPE samples .

  • Digital pathology algorithms for automated, objective quantification of TACC2 staining intensity and distribution.

• Integration with Established Prognostic Tools:

  • In breast cancer, combine TACC2 expression data with the Nottingham Prognostic Index (NPI) to refine risk stratification .

  • For prostate cancer, assess TACC2 expression alongside Gleason score and PSA levels to identify patients at higher risk of progression to castration resistance .

• Multi-marker Panels:

  • Develop prognostic panels that include TACC2 alongside other cell cycle and centrosomal markers.

  • Stratification algorithms that consider both TACC2 expression and androgen receptor status in hormone-dependent cancers .

Clinical Validation Data:

Implementation Considerations:

• Standardize tissue collection, processing, and staining protocols to ensure reproducible TACC2 assessment across institutions.

• Determine optimal cut-off values for categorizing "high" versus "low" TACC2 expression through large-scale validation studies.

• Consider tumor heterogeneity by analyzing multiple tumor regions when feasible.

• Validate prognostic value in diverse patient populations and treatment contexts.

What methodological approaches can detect alterations in TACC2 function in patient-derived cancer samples?

To comprehensively characterize TACC2 functional alterations in patient-derived cancer samples, researchers should employ multiple complementary methodologies:

1. Genomic Analysis:

• Next-generation sequencing (NGS) to identify mutations, copy number variations, and structural alterations affecting the TACC2 gene.

• Methylation analysis to assess epigenetic regulation of TACC2 expression.

• RNA-seq to detect splice variants and fusion transcripts involving TACC2.

2. Protein Expression and Modification Assessment:

• Multiplex immunohistochemistry (mIHC) to simultaneously visualize TACC2 with interaction partners like Aurora A and centrosomal markers.

• Reverse Phase Protein Array (RPPA) for high-throughput quantification of TACC2 expression and phosphorylation status across many samples.

• Mass spectrometry-based proteomics to identify post-translational modifications and altered protein-protein interactions.

3. Functional Assessment in Patient-Derived Models:

• Patient-derived organoids (PDOs) to study TACC2's role in 3D tumor architecture and cell division.

• Patient-derived xenografts (PDXs) for in vivo assessment of TACC2 function and therapeutic targeting.

• Primary cell cultures with live-cell imaging to visualize mitotic spindle formation and chromosome segregation.

4. Single-Cell Analysis:

• Single-cell RNA-sequencing to identify tumor subpopulations with altered TACC2 expression.

• CyTOF/mass cytometry to simultaneously measure TACC2 and other centrosomal proteins at the single-cell level.

• Single-cell Western blot for protein-level validation in rare tumor cell populations.

5. Spatial Analysis:

• Digital spatial profiling to map TACC2 expression within the tumor microenvironment.

• Multiplexed ion beam imaging (MIBI) or CODEX for high-parameter spatial analysis of TACC2 and related proteins.

6. Functional Genomic Screening:

• CRISPR-Cas9 screens in patient-derived cells to identify synthetic lethal interactions with TACC2 alterations.

• Drug sensitivity profiling to correlate TACC2 status with response to mitotic inhibitors.

By combining these methodologies, researchers can generate comprehensive functional profiles of TACC2 in patient samples, potentially identifying actionable alterations and therapeutic vulnerabilities.

What strategies could target TACC2 or its signaling pathways for cancer therapy?

Based on the emerging understanding of TACC2 biology, several therapeutic strategies show promise for targeting TACC2 or its associated pathways in cancer:

1. Direct TACC2 Targeting Approaches:

• RNA interference (RNAi): siRNA or shRNA targeting TACC2 has demonstrated efficacy in preclinical models, inhibiting cell proliferation in both AR-positive hormone-sensitive and AR-negative hormone-refractory prostate cancer cell lines .

• Antisense oligonucleotides (ASOs): Designed to specifically target TACC2 mRNA, potentially offering greater in vivo stability than siRNA.

• Proteolysis targeting chimeras (PROTACs): Bifunctional molecules that could target TACC2 protein for ubiquitin-mediated degradation.

2. Targeting TACC2-Protein Interactions:

• Aurora A kinase inhibitors: Given TACC2's functional relationship with Aurora A in mitotic spindle formation, Aurora A inhibitors (e.g., alisertib) could disrupt this critical interaction .

• Centrosome-targeting agents: Compounds that disrupt centrosome integrity could synergize with TACC2's role in centrosomal functions.

• Peptide-based inhibitors: Designed to interfere with specific protein-protein interactions involving TACC2.

3. Exploiting TACC2-Dependent Vulnerabilities:

• Mitotic checkpoint inhibitors: Cancer cells with altered TACC2 expression may be particularly vulnerable to agents targeting the spindle assembly checkpoint.

• Synthetic lethality approaches: Identifying genes that, when inhibited, are selectively lethal to cells with altered TACC2 expression.

• Cell cycle checkpoint inhibitors: Combining with DNA damage response inhibitors to exploit genomic instability following TACC2 disruption.

4. Targeting TACC2 Regulation:

• Hormonal therapies: For hormone-responsive cancers, modulating androgen signaling could affect TACC2 expression .

• Epigenetic modulators: Targeting epigenetic regulators of TACC2 expression.

• Transcriptional inhibitors: Compounds disrupting transcription factors driving TACC2 expression.

5. Combination Approaches:

• TACC2 inhibition + conventional chemotherapy: Potentially sensitizing cells to DNA-damaging agents by disrupting mitotic fidelity.

• TACC2 inhibition + radiotherapy: Exploiting the role of TACC2 in radiation response and DNA damage repair.

• TACC2 inhibition + immunotherapy: Exploring potential synergies with immune checkpoint inhibitors.

The optimal therapeutic strategy will likely depend on cancer type, molecular context, and whether TACC2 functions primarily as an oncogene or tumor suppressor in the specific disease setting.

What are the critical knowledge gaps in understanding TACC2 biology that require further investigation?

Despite significant advances in TACC2 research, several critical knowledge gaps remain that warrant targeted investigation:

• Isoform-Specific Functions: While multiple TACC2 isoforms have been identified, their distinct functions remain poorly characterized. Research is needed to determine how different isoforms contribute to normal cellular processes and cancer progression .

• Tissue-Specific Roles: TACC2 is expressed in various tissues, but tissue-specific functions are not well defined. Understanding these roles could explain seemingly contradictory findings across different cancer types .

• Post-Translational Modifications: Comprehensive mapping of TACC2 post-translational modifications (phosphorylation, acetylation, etc.) and their functional consequences would provide insights into regulatory mechanisms.

• Non-Centrosomal Functions: While TACC2's centrosomal roles are well-established, potential functions in transcriptional regulation, DNA damage response, and cellular metabolism require further exploration .

• Interaction Network: A comprehensive protein-protein interaction network for TACC2 across different cellular contexts would illuminate its diverse functions and identify potential therapeutic targets.

• Mechanistic Basis of Prognostic Value: The molecular mechanisms underlying TACC2's association with cancer prognosis remain incompletely understood and require mechanistic investigation .

• Therapeutic Resistance: The potential role of TACC2 in mediating resistance to conventional therapies, particularly in hormone-refractory prostate cancer, needs systematic evaluation .

• Biomarker Development: Standardized protocols for TACC2 assessment in clinical samples and validated cutpoints for prognostic stratification require development and validation in large cohorts.

• Animal Models: Sophisticated in vivo models with tissue-specific and inducible TACC2 modification would advance understanding of its role in tumorigenesis and treatment response.

• Therapeutic Targeting: Development and validation of specific TACC2 inhibitors or degraders would facilitate both mechanistic studies and therapeutic applications.

Addressing these knowledge gaps would significantly advance TACC2 biology and potentially yield novel diagnostic, prognostic, and therapeutic approaches for cancer management.