S100A6 Human

What is S100A6 and how is it characterized structurally?

S100A6 (S100 calcium-binding protein A6) belongs to the S100 family of proteins. It functions as an intracellular protein that modulates several cellular activities including proliferation, apoptosis, cytoskeleton dynamics, and responses to various stress factors. Structurally, S100A6 forms dimers and contains EF-hand calcium-binding domains that undergo conformational changes upon calcium binding. These conformational changes expose hydrophobic surfaces that facilitate interactions with target proteins .

How should researchers approach measuring S100A6 expression in different tissue types?

When measuring S100A6 expression across tissue types, researchers should employ multiple complementary techniques:

• RNA-level analysis: Utilize RNA-sequencing with standardized processing through packages like TCGAbiolinks for TCGA data and UCSCXenaTools for GTEx data, converting to TPM format for comparison . For differential expression analysis, employ DESeq2 with established parameters (fold change >2.0 or <−2.0, P<0.05) .

• Protein-level detection: Implement immunohistochemistry with standardized scoring systems to evaluate both intensity and localization of S100A6 expression .

• Statistical validation: Apply Wilcoxon rank-sum tests for comparing expression between sample groups and Kruskal-Wallis tests for relationships with multiple clinical parameters .

• Normalization controls: Include appropriate housekeeping genes or proteins and tissue-specific controls to account for baseline expression differences between tissues.

• Diagnostic performance assessment: Construct ROC curves using packages like pROC to calculate AUC values, which indicate diagnostic accuracy (0.5-0.7: low; 0.7-0.9: medium; >0.9: high) .

This multi-modal approach enables robust comparison between normal and pathological tissues while minimizing method-specific artifacts.

How does S100A6 expression correlate with glioma classification and established molecular markers?

S100A6 expression shows significant correlations with established glioma classifications and molecular markers, providing important stratification potential:

• WHO grade correlation: Higher S100A6 expression strongly correlates with higher WHO grades, suggesting association with more aggressive disease phenotypes .

• Histological associations: Expression varies significantly across different histological subtypes of glioma .

• Molecular marker correlations:

  • IDH status: S100A6 expression shows significant association with IDH mutation status, typically with higher expression in IDH-wildtype tumors .

  • 1p/19q codeletion: Expression levels significantly correlate with 1p/19q codeletion status .

• Clinical parameter associations:

  • Age and sex: Significant correlations exist with both parameters .

  • Primary treatment outcomes: Expression levels differ based on treatment response .

To analyze these correlations effectively, researchers should stratify patients into low and high S100A6 expression groups using median expression values as cutoffs. Univariate logistic regression confirms these relationships, though some studies report no significant association with primary treatment outcomes, sex, and race . These correlations suggest S100A6 may serve as an indicator for tumor grading and classification in gliomas.

What methodological approaches should be used to assess S100A6 as a prognostic biomarker in gliomas?

To rigorously evaluate S100A6 as a prognostic biomarker in gliomas, researchers should implement the following methodological approaches:

When implementing these approaches, researchers should acknowledge limitations including potential sampling bias, tumor heterogeneity effects, and the need for standardized quantification methods across studies.

How can researchers reconcile contradictory findings regarding S100A6 function in tumor biology?

Several approaches can help researchers reconcile contradictory findings regarding S100A6 function in tumor biology:

• Context-specific analysis:

  • Systematically evaluate S100A6 function across different cancer types, cell lines, and experimental conditions

  • Directly compare expression and function in primary patient-derived cells versus established cell lines

  • Assess temporal dynamics of S100A6 expression throughout disease progression

• Methodological standardization:

  • Implement multiple complementary techniques to confirm findings (RNA-seq, RT-qPCR, Western blot, immunohistochemistry)

  • Standardize quantification methods, scoring systems, and statistical approaches

  • Report detailed experimental conditions to facilitate reproducibility

• Interaction network mapping:

  • Identify cell type-specific binding partners using proteomics approaches

  • Characterize binding stoichiometry differences that might explain functional variation

  • Map downstream signaling networks using phosphoproteomics and transcriptomics

• Genetic manipulation studies:

  • Perform loss-of-function and gain-of-function experiments in multiple model systems

  • Use inducible expression systems to assess dose-dependent effects

  • Employ precise gene editing to evaluate specific domains or post-translational modifications

• Systematic review methodology:

  • Conduct meta-analyses with subgroup classification based on methodological approaches

  • Apply Bayesian analysis to reconcile seemingly contradictory results

  • Identify patterns in discrepancies that might reveal biological principles

Particularly in inflammatory processes, contradictory reports about S100A6's role highlight the importance of these systematic approaches to establish a coherent understanding of its function.

What cellular pathways and functions are influenced by S100A6 in cancer progression?

S100A6 influences multiple cellular pathways critical to cancer progression, which researchers can investigate through several methodological approaches:

• Proliferation and cell cycle regulation:

  • S100A6 enhances proliferation in multiple cancer types including breast, gastric, pancreatic, and colon cancers

  • Assessment methods: EdU incorporation, cell cycle analysis, Ki-67 quantification

• Migration and invasion mechanisms:

  • Promotes cell migration, invasiveness and adhesion in malignant cells

  • Analysis techniques: Wound healing assays, transwell migration/invasion assays, live cell imaging

• Apoptosis regulation:

  • Modulates apoptotic pathways affecting treatment resistance

  • Evaluation approaches: Annexin V/PI staining, caspase activity assays, TUNEL assays

• Cytoskeletal dynamics:

  • Affects cytoskeletal organization influencing cell morphology and motility

  • Investigation methods: Phalloidin staining, tubulin immunofluorescence, adhesion assays

• Tumor microenvironment interaction:

  • Communication between tumor cells and surrounding healthy cells

  • Study approaches: Co-culture systems, conditioned media experiments, immune cell infiltration analysis

To comprehensively map these pathways, researchers should employ:

• Gene set enrichment analysis (GSEA) using KEGG, GO, and PPI datasets

• Pearson's correlation analysis with the top 300 genes positively associated with S100A6

• Pathway visualization using Enrichplot package for the top five signaling pathways with highest enrichment significance

• Functional verification through gene manipulation experiments

These methodological approaches can delineate S100A6's complex role in cancer biology and identify potential therapeutic intervention points.

How does S100A6 interact with calcium, and what are the functional implications of these interactions?

The calcium-binding properties of S100A6 are central to its function and can be characterized through several experimental approaches:

• Structural aspects of calcium binding:

  • S100A6 contains EF-hand domains that undergo conformational changes upon calcium binding

  • Calcium binding exposes hydrophobic surfaces for target protein interactions

  • Typically forms dimers with canonical binding of two calcium ions per monomer

• Binding dynamics assessment methods:

  • Isothermal titration calorimetry (ITC) to measure binding affinities and thermodynamics

  • Microscale thermophoresis for interaction analysis

  • Fluorescence spectroscopy with calcium indicators

  • Circular dichroism to assess conformational changes

• Calcium-dependent interactions:

  • The canonical structure of S100 protein-ligand complexes involves the interacting domain of the ligand positioned in a cleft formed in a calcium-dependent manner

  • Co-immunoprecipitation under varying calcium concentrations

  • Yeast two-hybrid systems with calcium concentration modifications

  • Proximity ligation assays in cellular contexts

• Functional consequences:

  • Calcium binding alters S100A6's ability to interact with target proteins

  • Triggers signaling cascades affecting proliferation, apoptosis, and cytoskeleton dynamics

  • May mediate responses to calcium flux during cellular stress conditions

• Experimental considerations:

  • Control for calcium concentration in experimental buffers

  • Compare wildtype S100A6 with calcium-binding mutants

  • Assess function in calcium-depleted versus calcium-rich conditions

Understanding these calcium-dependent properties is crucial for interpreting S100A6's role in both normal physiology and pathological states like cancer.

What is the relationship between S100A6 and the immune microenvironment in gliomas?

S100A6 exhibits significant interactions with the immune microenvironment in gliomas, which can be investigated through several methodological approaches:

• Gene Ontology functional enrichment analysis reveals S100A6 implication in immune responses, with expression profiles linked to the immune microenvironment .

• Immune infiltrate characterization methods:

  • Computational deconvolution of bulk RNA-seq data to estimate immune cell proportions

  • Single-cell RNA sequencing to identify cell-specific expression patterns

  • Multiplex immunohistochemistry for spatial distribution of S100A6 and immune cells

  • Flow cytometry to quantify immune cell populations in relation to S100A6 expression

• Inflammatory context analysis:

  • S100A6 is frequently detected at sites of inflammation

  • Contradictory reports exist regarding its specific role in inflammatory processes

  • Cytokine/chemokine profiling in relation to S100A6 expression levels

  • NF-κB pathway activation assessment

• Tumor-immune interaction studies:

  • Co-culture experiments with tumor cells and immune components

  • Conditioned media experiments to assess secreted factors

  • S100A6 knockdown/overexpression effects on immune cell recruitment and function

  • Correlation with immune checkpoint molecule expression

• Clinical correlation approaches:

  • Stratification of patients based on both S100A6 expression and immune signature

  • Treatment response analysis in relation to combined S100A6/immune profiles

  • Survival analysis incorporating immune parameters alongside S100A6 expression

Understanding these interactions is crucial for developing innovative strategies targeting both S100A6 and the immune microenvironment in gliomas.

What approaches show promise for targeting S100A6 in cancer therapy?

Several therapeutic approaches targeting S100A6 show promise for cancer treatment, particularly for gliomas:

• Expression inhibition strategies:

  • RNA interference technologies (siRNA, shRNA) targeting S100A6 mRNA

  • CRISPR-Cas9 gene editing to knock out S100A6

  • Antisense oligonucleotides for targeted degradation

  • Promoter-targeting approaches to suppress transcription

  • Methodological considerations: delivery systems capable of crossing the blood-brain barrier, target cell specificity

• Protein function inhibition:

  • Small molecule inhibitors disrupting calcium binding

  • Peptide-based approaches to block interaction surfaces

  • Structure-based drug design leveraging known S100A6 binding modes

  • Screening approaches: in silico screening, high-throughput binding assays, functional verification

• Combination therapy approaches:

  • Integration with standard glioma treatments (temozolomide, radiation)

  • Synergistic targeting of multiple S100 family members

  • Combined targeting of S100A6 and immune checkpoints

  • Experimental design: factorial treatment designs, isobologram analysis for synergy

• S100A6 as a stratification biomarker:

  • Using expression levels to guide treatment intensity

  • Therapy selection based on S100A6-associated pathway activation

  • Monitoring expression during treatment as a response indicator

  • Implementation: standardized quantification protocols, clinically validated cutoffs

Inhibition of S100A6 expression represents a particularly promising therapeutic approach for treating gliomas , though substantial preclinical validation is required before clinical translation.

How can researchers design experiments to assess the efficacy of S100A6-targeted therapies?

To rigorously evaluate S100A6-targeted therapies, researchers should implement comprehensive experimental designs:

• In vitro efficacy assessment:

  • Establish dose-response relationships across multiple cell lines representing disease heterogeneity

  • Measure effects on proliferation, migration, invasion, and apoptosis

  • Evaluate target engagement through binding assays, thermal shift assays, CETSA

  • Assess on-target versus off-target effects using CRISPR knockout controls

  • Combination studies with standard-of-care treatments using Chou-Talalay method for synergy analysis

• Mechanism-of-action studies:

  • Transcriptomics to identify affected pathways

  • Phosphoproteomics to map signaling changes

  • Imaging technologies to visualize subcellular effects

  • Time-course analyses to determine primary versus secondary effects

  • Rescue experiments to confirm specificity

• In vivo efficacy models:

  • Orthotopic glioma models (particularly important given the blood-brain barrier)

  • Patient-derived xenograft models to capture tumor heterogeneity

  • Immunocompetent models when investigating immune interactions

  • Longitudinal monitoring: bioluminescence imaging, MRI, survival analysis

  • Pharmacokinetic/pharmacodynamic correlation studies

• Biomarker development:

  • Identify predictive biomarkers of response

  • Develop pharmacodynamic biomarkers for target engagement

  • Establish quantifiable endpoints for clinical translation

  • Correlation of biomarker changes with efficacy outcomes

• Translational readiness assessment:

  • Drug metabolism and pharmacokinetics studies

  • Toxicology in multiple species

  • Manufacturing feasibility assessment

  • Regulatory pathway planning

These methodological approaches provide a comprehensive framework for evaluating S100A6-targeted therapies from initial discovery through translational development.

What are the current limitations in S100A6 research and how might they be addressed?

Current S100A6 research faces several methodological and conceptual limitations that should be addressed through systematic approaches:

• Diagnostic and prognostic validation challenges:

  • Limited sample sizes in clinical validation studies

  • Potentially contaminated tissues in public databases leading to biased outcomes

  • Standardization issues in expression quantification across studies

  • Solution approaches: Multi-center validation studies, standardized protocols, meta-analyses of existing data

• Mechanistic understanding gaps:

  • Incomplete characterization of S100A6's binding partners in context-specific settings

  • Contradictory findings regarding function, particularly in inflammatory processes

  • Limited understanding of how S100A6 can bind structurally diverse proteins despite a rigid backbone

  • Methodological approaches: Comprehensive interactome mapping, structural studies, systematic context-dependent functional analyses

• Therapeutic development hurdles:

  • Blood-brain barrier penetration for glioma applications

  • Potential functional redundancy with other S100 family members

  • Target selectivity challenges due to structural similarities within the S100 family

  • Research directions: Drug delivery technology development, combination approaches targeting multiple family members, structure-based design for selectivity

• Translational challenges:

  • Communicating complex prognostic nomograms to patients and clinicians

  • Theoretical nature of nomograms not fully representing clinical outcomes

  • Integration of S100A6 biomarker with existing clinical decision frameworks

  • Solutions: Development of simplified clinical decision tools, prospective clinical validation, integration with digital health platforms

• Conceptual limitations:

  • Association versus causation in prognostic studies

  • Understanding S100A6's role within the broader context of calcium signaling

  • Disentangling direct versus indirect effects on cellular functions

  • Approaches: Genetic manipulation studies, systems biology modeling, temporal dynamics analysis

Addressing these limitations requires collaborative efforts combining expertise in molecular biology, structural biology, clinical research, and computational approaches to establish a more comprehensive understanding of S100A6 biology and its therapeutic potential.

What are the optimal methods for detecting and quantifying S100A6 in clinical samples?

Researchers should consider the following methodological approaches for optimal detection and quantification of S100A6 in clinical samples:

• Protein detection and quantification:

  • Immunohistochemistry: Use validated antibodies with standardized scoring systems considering both intensity and percentage of positive cells

  • Western blotting: Optimize protein extraction from clinical samples, include recombinant protein standards

  • ELISA: Develop high-sensitivity assays for biofluid analysis (serum, CSF)

  • Mass spectrometry: For absolute quantification and post-translational modification analysis

• mRNA quantification:

  • RT-qPCR: Design intron-spanning primers, validate efficiency, use multiple reference genes

  • RNA-seq: Apply consistent normalization methods (TPM format) for cross-sample comparison

  • NanoString: For targeted quantification from limited or degraded samples

  • In situ hybridization: For spatial localization in tissue sections

• Statistical considerations:

  • Establish appropriate cutoff values (median expression is commonly used)

  • Apply ROC curve analysis to determine optimal thresholds for clinical decision-making

  • Use AUC measurements to assess diagnostic accuracy (AUC=0.830 for S100A6 in glioma detection)

  • Compare with established markers (IDH has AUC=0.89 in validation studies)

• Quality control measures:

  • Include positive and negative tissue controls

  • Assess pre-analytical variables (fixation time, storage conditions)

  • Apply batch correction for multi-center studies

  • Document tumor cell content and necrosis percentage

• Reporting standards:

  • Follow REMARK guidelines for prognostic biomarker studies

  • Document detailed methodological parameters

  • Report analytical sensitivity and specificity

  • Specify antibody validation procedures

These methodological considerations ensure reliable detection and quantification of S100A6 in clinical samples, facilitating meaningful correlation with pathological features and clinical outcomes.

How can researchers effectively study S100A6 protein-protein interactions?

To effectively study S100A6 protein-protein interactions, researchers should employ multiple complementary techniques:

• Affinity-based methods:

  • Co-immunoprecipitation with specific anti-S100A6 antibodies

  • Pull-down assays using recombinant tagged S100A6

  • Proximity ligation assays for in situ detection

  • FRET/BRET approaches for live cell interaction monitoring

  • Methodological considerations: Calcium concentration control, detergent selection, washing stringency

• Direct binding characterization:

  • Surface plasmon resonance to determine binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

  • Microscale thermophoresis for interaction screening

  • AlphaScreen technology for high-throughput interaction analysis

  • Experimental controls: Calcium-free conditions, binding-deficient mutants

• Structural studies:

  • X-ray crystallography of S100A6-ligand complexes

  • NMR spectroscopy for dynamic interaction analysis

  • Hydrogen-deuterium exchange mass spectrometry for binding interface mapping

  • Cryo-EM for larger complexes

  • Computational considerations: Molecular dynamics simulations, docking studies

• Cellular context approaches:

  • BioID or APEX proximity labeling for in vivo interaction mapping

  • Mammalian two-hybrid systems

  • Split-protein complementation assays

  • Optogenetic approaches for controlled interaction studies

  • Validation strategy: Orthogonal methods confirmation, dose-response relationships

• Interaction specificity assessment:

  • Compare binding with other S100 family members

  • Evaluate stoichiometry (whether two ligands bind per S100A6 dimer or one ligand per dimer)

  • Map binding domains through truncation and point mutation analysis

  • Competitive binding studies with known interactors

These methodological approaches collectively provide a comprehensive framework for characterizing S100A6 protein-protein interactions, elucidating its functional networks, and identifying potential therapeutic intervention points.