CTU2 Antibody

What is CTU2 and why are CTU2 antibodies important in cancer research?

CTU2 (Cytosolic thiouridylase 2) is an enzyme involved in the post-transcriptional modification of transfer RNAs (tRNAs). It forms a complex with CTU1 to participate in the 2-thiolation of cytosolic tRNAs, which improves codon reading accuracy during protein translation . CTU2 antibodies are crucial research tools because CTU2 has been implicated in multiple cancer types, including breast cancer, melanoma, and hepatocellular carcinoma (HCC) . Recent research has identified CTU2 as a Liver X receptor (LXR) target gene that promotes lipogenesis and cell proliferation in HCC, making CTU2 antibodies essential for studying these oncogenic mechanisms .

How does CTU2 function in normal cellular processes versus cancer development?

In normal cellular processes, CTU2 partners with CTU1 to mediate tRNA thiolation, which helps maintain genome integrity and supports growth under nutritionally challenging environments . This modification regulates codon-anticodon interactions and enhances translational fidelity.

In cancer contexts, CTU2 becomes dysregulated and contributes to malignancy through several mechanisms:

• Enhances proliferation of cancer cells, particularly in HCC

• Promotes metastasis in breast cancer by supporting translation of the oncogenic factor LEF1 through internal ribosome entry site (IRES)-dependent mechanisms

• Facilitates melanoma growth by regulating HIF1α codon-dependent translation

• Activates lipogenesis pathways, which are critical for cancer cell proliferation

What validation methods should be employed when using CTU2 antibodies in research?

When validating CTU2 antibodies for research, several complementary approaches should be implemented:

• Western blotting with positive and negative controls: Use HepG2 cells with CTU2 knockdown (shCTU2) as negative controls and wild-type HepG2 cells as positive controls .

• Immunofluorescence correlation: Compare protein detection with mRNA expression data through parallel qRT-PCR analysis, as demonstrated in studies showing CTU2 knockdown effects on both protein and mRNA levels .

• Specificity testing: Conduct pre-absorption tests with recombinant CTU2 protein to confirm antibody specificity.

• Cross-validation: Employ multiple antibodies targeting different CTU2 epitopes to confirm consistent detection patterns.

• Immunohistochemistry (IHC) controls: When using CTU2 antibodies for IHC in tumor sections, include both high-expressing and low-expressing tissues as demonstrated in xenograft studies .

How can researchers optimize immunostaining protocols for CTU2 detection in hepatocellular carcinoma tissues?

Optimizing immunostaining for CTU2 in HCC tissues requires several specialized considerations:

Protocol Optimization Table for CTU2 Immunostaining in HCC Tissues:

Additionally, researchers should perform double immunofluorescence staining with Ki67 to correlate CTU2 expression with proliferation markers, as studies have demonstrated a significant relationship between CTU2 levels and Ki67 expression in tumor tissues .

What are the key considerations when selecting CTU2 antibodies for studying its relationship with the LXR pathway?

When investigating CTU2's relationship with the Liver X Receptor (LXR) pathway, researchers should consider several critical factors:

• Epitope selection: Choose antibodies targeting CTU2 epitopes that aren't masked by potential post-translational modifications occurring after LXR activation, as research has shown that LXR can transcriptionally activate CTU2 expression .

• Cross-reactivity assessment: Verify the antibody doesn't cross-react with other proteins in the LXR signaling pathway, particularly those containing similar structural domains.

• Compatibility with chromatin immunoprecipitation (ChIP): If studying direct LXR binding to the CTU2 promoter, select antibodies validated for ChIP applications to detect the typical LXR element identified in the CTU2 promoter region .

• Sensitivity to expression changes: Choose antibodies sensitive enough to detect the activation of CTU2 expression by LXR agonists and depression by LXR knockout, as observed in research models .

• Application versatility: Select antibodies validated for multiple applications (Western blot, immunofluorescence, IHC) to enable comprehensive pathway analysis across different experimental approaches.

How can CTU2 antibodies be utilized to investigate the relationship between CTU2 and lipogenic pathways in cancer?

Research has revealed that CTU2 participates in lipogenesis by directly enhancing the synthesis of lipogenic proteins, providing a novel mechanism for LXR-regulated lipid synthesis . To investigate this relationship using CTU2 antibodies:

Methodological Approach:

• Co-immunoprecipitation (Co-IP) studies:

  • Use CTU2 antibodies to pull down CTU2 protein complexes

  • Analyze binding partners related to lipogenic pathways (SREBP1, FASN, ACC1)

  • Western blot these samples to quantify associations between CTU2 and lipogenic proteins

• Proximity ligation assays (PLA):

  • Employ CTU2 antibodies together with antibodies against key lipogenic factors

  • Visualize and quantify protein-protein interactions in situ

  • Compare interaction patterns between normal and cancer tissues

• Translational profiling:

  • Combine CTU2 immunoprecipitation with polysome profiling

  • Identify specific mRNAs of lipogenic proteins associated with CTU2-modified tRNAs

  • This approach can elucidate how CTU2 enhances the synthesis of lipogenic proteins

• Metabolic labeling experiments:

  • Use CTU2 antibodies to deplete CTU2 via immunoprecipitation

  • Measure incorporation of labeled fatty acid precursors

  • Quantify changes in lipid synthesis when CTU2 is depleted

Research Finding: CTU2 knockdown in HepG2 cells reduced triglyceride levels and lipid droplets in tumor tissues, particularly under T0901317 (LXR agonist) treatment. CTU2 inhibition antagonized T0901317-induced elevation of FASN protein and reduced SREBP1, FASN, and ACC1 mRNA levels .

How should researchers design experiments to investigate CTU2's role in therapeutic resistance using CTU2 antibodies?

Designing experiments to investigate CTU2's role in therapeutic resistance requires a multi-faceted approach:

Experimental Design Framework:

• Baseline CTU2 expression analysis:

  • Use CTU2 antibodies for immunoblotting and IHC to establish baseline expression in sensitive and resistant cell lines/tissues

  • Correlate CTU2 levels with resistance phenotypes across a panel of cancer cell lines

• Manipulate CTU2 expression:

  • Generate paired cell models (CTU2-knockdown, CTU2-overexpression, and controls) using techniques validated in published research

  • Confirm expression changes via Western blot with CTU2 antibodies

  • Test drug sensitivity using dose-response assays and calculate IC50 shifts

• Temporal dynamics analysis:

  • Expose cancer cells to therapeutic agents and track CTU2 expression changes over time using antibody-based detection methods

  • Determine whether CTU2 upregulation precedes or follows resistance development

• Mechanistic investigation:

  • Combine CTU2 antibody-based detection with analyses of known resistance pathways

  • Focus on LXR-dependent pathways, as CTU2 is an LXR target gene

  • Examine whether combining LXR agonists with CTU2 inhibition enhances therapeutic efficacy, as suggested by research showing that inhibition of CTU2 expression synergizes with LXR ligands in HCC treatment

• Clinical correlation:

  • Apply CTU2 immunostaining to patient samples before and after treatment

  • Correlate CTU2 expression patterns with treatment outcomes

  • Consider that high CTU2 levels correlate with poor survival in HCC patients

What controls and validation steps are necessary when using CTU2 antibodies in xenograft tumor models?

When applying CTU2 antibodies in xenograft tumor models, rigorous controls and validation steps are essential:

Critical Controls and Validation Steps:

• Genetic validation controls:

  • Include xenografts derived from both wild-type and CTU2-knockdown cells (e.g., shCTU2 HepG2 cells)

  • Verify retained knockdown in harvested tumors using both mRNA (qRT-PCR) and protein (Western blot) analyses

• Antibody validation in tumor tissue:

  • Perform peptide competition assays to confirm specificity in the complex tumor microenvironment

  • Include isotype controls to identify non-specific binding

• Spatial expression analysis:

  • Use CTU2 antibodies for IHC to analyze expression patterns across different tumor regions

  • Compare with adjacent normal tissue to establish differential expression patterns

• Correlation with functional markers:

  • Co-stain with proliferation markers (Ki67), as research has shown CTU2 knockdown reduces Ki67-positive staining in tumors

  • Analyze apoptosis markers, as CTU2 inhibition enhances tumor cell apoptosis

  • Examine cancer-associated fibroblast (CAF) markers (αSMA) and angiogenesis factors (VEGFA), as CTU2 affects CAF presentation and tumor vascular development

• Treatment response monitoring:

  • Track CTU2 expression changes during treatment with agents like T0901317 (LXR agonist)

  • Correlate expression with lipid accumulation (using Nile red staining) and lipogenic gene expression (FASN, SREBP1)

Research Finding: In xenograft models, inhibition of CTU2 expression synergistically enhanced the anti-tumor effects of LXR ligands by promoting apoptosis and inhibiting proliferation, with corresponding changes in Ki67 expression, CAF presentation, and VEGFA levels .

How can researchers use CTU2 antibodies to investigate the relationship between tRNA modifications and translational control in cancer?

CTU2 forms a complex with CTU1 for the 2-thiolation of cytosolic tRNAs, which improves codon reading accuracy during translation . Researchers can use CTU2 antibodies to explore this process in cancer:

Methodological Framework:

• Ribosome profiling coupled with CTU2 immunoprecipitation:

  • Isolate CTU2-associated tRNAs using CTU2 antibodies

  • Identify mRNAs being actively translated using ribosome-protected fragments

  • Analyze codon usage patterns in translationally active mRNAs

• CTU2 antibody-based RNA immunoprecipitation (RIP):

  • Pull down CTU2-associated RNAs

  • Perform high-throughput sequencing to identify specific tRNA species modified by CTU2

  • Compare modification patterns between normal and cancer cells

• Translation efficiency assays:

  • Generate reporter constructs with cancer-relevant genes (e.g., SREBP1, FASN, ACC1)

  • Compare translation efficiency in CTU2-normal versus CTU2-depleted conditions

  • Use CTU2 antibodies to confirm knockdown or overexpression

• Polysome profiling with CTU2 antibody validation:

  • Fractionate polysomes from cells with normal or altered CTU2 levels

  • Analyze mRNA distribution across polysome fractions to identify transcripts with CTU2-dependent translation

  • Focus on lipogenic proteins, as research shows CTU2 can directly enhance their synthesis

Data from Research:
CTU2 influences specific translational programs rather than global protein synthesis. In breast cancer, CTU2 promotes translation of the oncogenic factor LEF1 through IRES-dependent mechanisms . In melanoma, CTU2-linked tRNA modification regulates HIF1α codon-dependent translation . In HCC, CTU2 enhances the synthesis of lipogenic proteins .

What are the considerations for developing CTU2 as a prognostic biomarker in hepatocellular carcinoma?

CTU2 shows potential as a prognostic biomarker in HCC based on UALCAN database analysis, which revealed that:

• CTU2 is up-regulated in HCC tumor compared to normal tissue

• There is a negative correlation between CTU2 expression and HCC patient survival

• CTU2 levels positively correlate with tumor progression (stages 1-4)

• CTU2 levels positively correlate with poor differentiation (grades 1-4)

Key Considerations for CTU2 Biomarker Development:

• Antibody selection criteria:

  • High specificity and sensitivity for CTU2 detection in human tissues

  • Consistent performance across different sample preparation methods

  • Compatibility with standard clinical immunohistochemistry protocols

• Sample processing standardization:

  • Optimal fixation conditions to preserve CTU2 epitopes

  • Antigen retrieval methods specific for CTU2 detection

  • Scoring systems to quantify CTU2 expression levels

• Validation requirements:

  • Large, diverse patient cohorts representing different HCC stages and etiologies

  • Multivariate analysis to determine independence from established prognostic factors

  • Comparison with existing HCC biomarkers (AFP, GPC3, etc.)

• Clinical implementation considerations:

  • Development of standardized CTU2 immunohistochemistry protocols

  • Establishment of clear cutoff values for high versus low expression

  • Correlation with treatment response to LXR agonists, as CTU2 is an LXR target gene

Prognostic Value Data from UALCAN Database:

How can CTU2 antibodies be utilized in developing targeted therapies for hepatocellular carcinoma?

Research has identified CTU2 as a promising target for HCC treatment, with inhibition of CTU2 expression enhancing the anti-tumor effect of LXR ligands . CTU2 antibodies can play critical roles in developing targeted therapies:

Therapeutic Development Applications:

• Target validation and mechanism elucidation:

  • Use CTU2 antibodies to confirm target engagement of potential CTU2 inhibitors

  • Validate downstream effects on lipogenic pathways and proliferation markers

  • Establish pharmacodynamic biomarkers for clinical development

• Development of antibody-drug conjugates (ADCs):

  • Evaluate CTU2 surface expression and internalization using fluorescently-labeled antibodies

  • If CTU2 shows appropriate localization patterns, develop ADCs targeting CTU2

  • Test efficacy in preclinical models, particularly in combination with LXR agonists

• Combination therapy development:

  • Use CTU2 antibodies to monitor expression changes during treatment with various agents

  • Identify synergistic combinations based on CTU2 regulation

  • Focus on combinations with LXR agonists, as research shows CTU2 inhibition synergizes with LXR ligands

• Patient stratification for clinical trials:

  • Develop immunohistochemistry assays using validated CTU2 antibodies

  • Select patients with high CTU2 expression for targeted therapy trials

  • Monitor CTU2 levels during treatment as a pharmacodynamic biomarker

Research Findings Supporting Therapeutic Development:

• CTU2 knockdown synergistically enhanced T0901317 (LXR agonist)-inhibited tumor growth in xenograft models

• CTU2 inhibition reduced tumor burden and enhanced the anti-tumor effect of LXR ligands by inducing tumor cell apoptosis and inhibiting cell proliferation

• CTU2 knockdown attenuated the lipogenic effects of LXR in tumor tissues

What methodological approaches can resolve contradictory findings about CTU2 function across different cancer types?

Researchers have observed varied roles of CTU2 across different cancer types, including breast cancer, melanoma, and HCC . To resolve potential contradictions, several methodological approaches using CTU2 antibodies can be employed:

Reconciliation Methodologies:

• Tissue-specific interactome analysis:

  • Use CTU2 antibodies for immunoprecipitation across different cancer types

  • Identify tissue-specific binding partners through mass spectrometry

  • Compare interaction networks to identify common and unique pathways

• Codon usage and translation efficiency analysis:

  • Apply ribosome profiling in CTU2-normal and CTU2-depleted conditions across cancer types

  • Determine if CTU2 affects different codons or mRNAs in a tissue-specific manner

  • Use validated CTU2 antibodies to confirm knockdown efficiency

• Context-dependent post-translational modifications:

  • Develop and apply modification-specific CTU2 antibodies

  • Compare PTM patterns across cancer types

  • Correlate modifications with functional differences

• Cancer microenvironment considerations:

  • Examine CTU2 effects on tumor-stroma interactions across cancer types

  • Analyze cancer-associated fibroblasts (CAFs) and angiogenesis markers (VEGFA)

  • Use multiplex immunofluorescence with CTU2 antibodies to assess spatial relationships

Contextual Differences Table:

These methodological approaches using CTU2 antibodies can help resolve apparent contradictions by revealing tissue-specific mechanisms while identifying conserved functions across cancer types.

How can researchers investigate the bidirectional relationship between CTU2 and metabolic reprogramming in cancer using CTU2 antibodies?

CTU2 has been implicated in lipogenesis and metabolic pathways critical for cancer cell proliferation . Investigating the complex bidirectional relationship between CTU2 and cancer metabolism requires sophisticated approaches:

Investigative Framework:

• Metabolic flux analysis with CTU2 manipulation:

  • Use stable isotope-labeled metabolites (e.g., 13C-glucose, 13C-glutamine)

  • Compare metabolic pathways in CTU2-normal vs. CTU2-depleted conditions

  • Validate CTU2 levels with antibodies via Western blot and immunofluorescence

• Nutrient-dependent CTU2 regulation:

  • Examine how different nutrient availability affects CTU2 expression and localization

  • Use CTU2 antibodies for immunofluorescence under various metabolic stresses

  • Correlate with tRNA thiolation levels and translational outcomes

• CTU2-dependent translatome under metabolic stress:

  • Combine polysome profiling with RNA-seq in CTU2-manipulated cells

  • Subject cells to metabolic stressors (glucose deprivation, hypoxia)

  • Use CTU2 antibodies to confirm manipulation efficiency

  • Focus on translation of metabolic enzymes, particularly lipogenic proteins

• In vivo metabolic imaging with CTU2 correlation:

  • Use techniques like hyperpolarized MRI to track tumor metabolism in vivo

  • Correlate with CTU2 expression in tumor sections using immunohistochemistry

  • Compare metabolic profiles between tumors with high and low CTU2 expression

Research Findings on CTU2-Metabolism Relationship:

• CTU2 participates in lipogenesis by enhancing synthesis of lipogenic proteins

• Lipogenesis is particularly active during cell proliferation in tumor cells

• CTU2 knockdown reduced triglyceride levels in tumor tissues under T0901317 treatment

• CTU2 inhibition decreased lipid accumulation in tumor tissues as visualized by Nile red staining

What are the most rigorous approaches to determine if CTU2 could serve as a therapeutic target across multiple cancer types?

Given CTU2's involvement in breast cancer, melanoma, and HCC , evaluating its potential as a pan-cancer therapeutic target requires rigorous investigation:

Multi-cancer Evaluation Framework:

• Large-scale expression profiling:

  • Use CTU2 antibodies for tissue microarray analysis across multiple cancer types

  • Correlate expression with clinical outcomes in each cancer

  • Establish tissue-specific expression thresholds for potential therapeutic relevance

• Genetic dependency screening:

  • Perform CTU2 knockdown/knockout in cell line panels representing multiple cancers

  • Validate manipulation using CTU2 antibodies

  • Identify cancer types with highest dependency on CTU2 for survival

  • Correlate with genetic and metabolic features to identify biomarkers of sensitivity

• Pharmacologic inhibition studies:

  • Develop small molecule inhibitors of CTU2 or CTU1-CTU2 complex formation

  • Validate target engagement using competitive binding assays with CTU2 antibodies

  • Test efficacy across cancer types in vitro and in vivo

  • Identify synthetic lethal interactions to enhance efficacy

• Combination strategy evaluation:

  • Test CTU2 inhibition in combination with standard therapies for each cancer type

  • Focus on combinations with LXR agonists, as CTU2 inhibition synergizes with these agents in HCC

  • Use CTU2 antibodies to monitor target modulation during treatment

Predictive Biomarkers of CTU2 Dependency:

How can researchers develop spatially-resolved methods to study CTU2 function in the tumor microenvironment?

CTU2 affects not only cancer cells but also influences the tumor microenvironment, including cancer-associated fibroblasts (CAFs) and angiogenesis . Developing spatially-resolved methods to study these interactions requires sophisticated approaches:

Spatial Analysis Methodologies:

• Multiplex immunofluorescence imaging:

  • Combine CTU2 antibodies with markers for different cell populations (epithelial cells, CAFs, endothelial cells)

  • Include functional markers like Ki67 (proliferation), αSMA (CAFs), and VEGFA (angiogenesis)

  • Apply machine learning algorithms to identify spatial relationships

  • Compare patterns between CTU2-high and CTU2-low regions

• Laser capture microdissection with CTU2 immunostaining:

  • Use CTU2 antibodies to identify CTU2-high and CTU2-low regions

  • Microdissect these regions for molecular analysis

  • Compare transcriptomes, proteomes, and metabolomes

  • Focus on lipogenic programs and angiogenesis factors

• Spatial transcriptomics with protein validation:

  • Apply spatial transcriptomics technologies to map gene expression across tumor sections

  • Validate CTU2 protein expression in sequential sections using immunohistochemistry

  • Correlate CTU2 expression with spatially-resolved transcriptomic signatures

  • Identify microenvironmental niches with distinct CTU2-associated functions

• In situ proximity ligation assays:

  • Detect protein-protein interactions involving CTU2 in tissue sections

  • Map spatial distribution of CTU2 interaction networks

  • Compare interactions in different tumor regions and microenvironmental contexts

Research Findings on CTU2 in Tumor Microenvironment:

• Higher CTU2 expression correlates with more CAFs (fibroblast-like cells) in tumor sections

• CTU2 inhibition decreased αSMA-positive cells (marker of CAFs)

• CTU2 knockdown reduced VEGFA expression, suggesting effects on tumor angiogenesis

• These findings indicate CTU2 may influence tumor growth through effects on the microenvironment beyond cancer cell-intrinsic mechanisms