Recombinant Human Tumor necrosis factor receptor superfamily member 10B (TNFRSF10B),Partial (Active)

What is TNFRSF10B and what is its role in cellular processes?

TNFRSF10B, also known as Death Receptor 5 (DR5), TNF-Related Apoptosis-Inducing Ligand Receptor 2 (TRAIL-R2), CD262, or KILLER/DR5, is a member of the TNF receptor superfamily containing an intracellular death domain . The protein is encoded by the TNFRSF10B gene located on chromosome 8p21.3 . When activated by tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10/TRAIL), TNFRSF10B transduces an apoptosis signal by promoting the formation of the death-inducing signal complex (DISC) . This complex consists of death receptors, FADD, caspase-8 (CASP8), and c-FLIP (CFLAR) . The activation of CASP8 triggers downstream effector caspases like CASP3 and CASP7, leading to programmed cell death .

In cells with weak CASP8-CASP3 signaling, TNFRSF10B's apoptotic effect requires CASP8-mediated cleavage of the BH3-only BCL2 family member BID to activate the intrinsic apoptosis pathway . This dual pathway activation ensures efficient cell death induction under various cellular conditions. TNFRSF10B also promotes the activation of NF-kappa-B, suggesting its role extends beyond apoptosis induction .

How does TNFRSF10B differ from other members of the TRAIL receptor family?

TNFRSF10B is one of several receptors that can bind to TRAIL, but its structural and functional characteristics distinguish it from other family members. While TNFRSF10B (DR5) and TNFRSF10A (DR4) both contain functional death domains capable of transducing apoptotic signals, other TRAIL receptors like DcR1 and DcR2 lack functional death domains and act as decoy receptors . This diversity in receptor structure creates a complex regulatory system for TRAIL-induced apoptosis.

The TNFRSF10B gene produces two transcript variants encoding different isoforms and one non-coding transcript . These structural variations may contribute to differential responses to TRAIL stimulation across various cell types. TNFRSF10B's strong affinity for TRAIL and its efficient DISC formation capabilities make it a particularly important mediator of TRAIL-induced apoptosis, especially in cancer cells where it is often highly expressed .

What is the current understanding of TNFRSF10B expression patterns across normal and cancer tissues?

TNFRSF10B expression varies significantly across tissue types and is often dysregulated in cancer. Research indicates that TNFRSF10B is implicated in multiple cancer types, including breast, lung, colorectal, prostate, and head and neck cancers . This widespread involvement makes it an attractive target for cancer therapy development.

The table below summarizes the research focus on TNFRSF10B across different cancer types based on publication counts:

Data sourced from CancerIndex.org as of August 2019 .

Notably, triple-negative breast cancer has emerged as a particular area of interest for TNFRSF10B-targeted therapies, likely due to the limited treatment options available for this aggressive cancer subtype . Understanding the expression patterns and regulatory mechanisms of TNFRSF10B across different tissues is crucial for developing effective targeted therapies.

How should researchers optimize experimental protocols when working with recombinant TNFRSF10B proteins?

When working with recombinant human TNFRSF10B, researchers must implement rigorous quality control and experimental design strategies. High-quality recombinant protein should have purity >95% as determined by SDS-PAGE, with endotoxin levels <1.0 EU/μg as measured by the LAL method . The following protocol optimization steps are critical:

• Protein reconstitution and storage: Recombinant TNFRSF10B should be reconstituted according to manufacturer's instructions, typically in a buffer that maintains protein stability. Aliquot the protein to avoid repeated freeze-thaw cycles, which can compromise activity.

• Concentration determination: Establish appropriate working concentrations through dose-response experiments. The theoretical molecular weight of partial recombinant TNFRSF10B (expression region 56-182aa) is approximately 15.19 kDa , which should be considered when calculating molar concentrations.

• Validation of activity: Before using in complex experiments, verify the biological activity of the recombinant protein using established assays such as binding assays or functional tests measuring downstream signaling activation.

• Controls: Include proper controls in all experiments:

  • Negative control: untreated cells or cells treated with an irrelevant protein

  • Positive control: known inducer of the pathway being studied

  • Specificity control: blocking antibodies against TNFRSF10B

• Time course considerations: TRAIL/TNFRSF10B-mediated signaling is dynamic, with early events occurring within minutes to hours (receptor clustering, DISC formation) and late events over hours to days (apoptosis execution). Design experiments to capture this temporal complexity.

What experimental approaches can effectively investigate the crosstalk between TNFRSF10B-mediated apoptosis and autophagy?

Research has revealed important crosstalk between TNFRSF10B-mediated apoptosis and autophagy, with significant implications for cancer treatment strategies . To investigate this relationship, researchers should employ the following methodological approaches:

• Autophagy flux assays: Monitor changes in MAP1LC3B-II levels and SQSTM1/p62 degradation following TRAIL treatment . The use of lysosomal inhibitors like chloroquine (CQ) is crucial to distinguish between increased autophagosome formation and decreased degradation. In multiple cancer cell lines, TRAIL treatment combined with chloroquine further elevated MAP1LC3B-II expression compared to either treatment alone, indicating TRAIL induces autophagy .

• Pharmacological modulation: Use autophagy inhibitors such as 3-methyladenine (3MA) and wortmannin to block TNFSF10-induced autophagy . These inhibitors remarkably increased TRAIL-induced cell death in cancer cell lines, suggesting autophagy serves as a protective mechanism against TRAIL-induced apoptosis .

• Genetic manipulation: Employ siRNA targeting autophagy genes such as ATG7 or BECN1 to confirm the role of autophagy in modulating TRAIL sensitivity . Knockdown of either ATG7 or BECN1 effectively potentiated TNFSF10-induced cytotoxicity, providing genetic validation of the pharmacological studies .

• Signaling pathway analysis: Investigate key regulatory nodes such as MAPK8/JNK that may modulate both apoptosis and autophagy. MAPK8 activation following TRAIL treatment was shown to decrease expression of the anti-apoptotic protein BCL2L1, which also affects the BCL2L1-BECN1 complex involved in autophagy regulation .

• Co-immunoprecipitation studies: Examine protein-protein interactions critical for both pathways, such as the BCL2L1-BECN1 complex. TNFSF10 treatment reduced the binding of BECN1 to BCL2L1, which coincided with decreased BCL2L1 expression, linking apoptotic and autophagic mechanisms .

How can researchers effectively assess TNFRSF10B-mediated signaling dynamics in heterogeneous tumor samples?

Analyzing TNFRSF10B signaling in heterogeneous tumor samples presents unique challenges that require specialized methodological approaches:

How should researchers interpret variable responses to TNFRSF10B activation across different cancer cell lines?

Variable responses to TNFRSF10B activation across cancer cell lines reflect complex underlying biological mechanisms that researchers must systematically analyze. When interpreting such variability, consider the following methodological approach:

What statistical approaches are most appropriate for analyzing complex TNFRSF10B signaling data?

Analyzing TNFRSF10B signaling data requires sophisticated statistical methods to address the complexity and multivariate nature of the data:

How can researchers definitively distinguish TNFRSF10B-specific effects from other death receptor pathways?

Definitively attributing observed effects specifically to TNFRSF10B rather than other death receptors requires a systematic approach combining multiple complementary methods:

• Receptor-specific targeting: Utilize TNFRSF10B-specific agonistic antibodies that do not activate other death receptors. Compare responses to these specific agonists with those to TRAIL, which can activate multiple receptors including TNFRSF10A (DR4).

• Genetic knockdown/knockout experiments: Implement CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown specifically targeting TNFRSF10B. If effects persist after TNFRSF10B depletion, other pathways are likely involved. Consider creating cell lines with individual receptor knockouts to dissect specific contributions.

• Domain-specific mutants: Express mutant versions of TNFRSF10B with alterations in specific functional domains to determine which aspects of signaling are TNFRSF10B-dependent. This approach can reveal unique signaling properties of TNFRSF10B compared to other death receptors.

• Receptor dimerization/oligomerization analysis: As receptor clustering is critical for death receptor signaling, analyze TNFRSF10B oligomerization using techniques like proximity ligation assay or FRET. Compare oligomerization patterns with those of other death receptors.

• Pathway-specific inhibitors: Use inhibitors of specific downstream components that may differentially affect TNFRSF10B versus other death receptor pathways. While many downstream components overlap, there may be quantitative or kinetic differences in pathway utilization.

• Receptor expression correlation: Analyze whether the observed effects correlate with TNFRSF10B expression levels across multiple cell lines. If effects correlate specifically with TNFRSF10B levels but not with levels of other death receptors, this supports TNFRSF10B specificity.

What are common challenges in detecting TNFRSF10B-mediated DISC formation and how can they be overcome?

The death-inducing signaling complex (DISC) formation is a critical early event in TNFRSF10B-mediated apoptosis, but its detection presents several methodological challenges:

• Transient nature of the complex: The DISC forms rapidly and may be unstable, making timing critical for detection. Solution: Perform time-course experiments with multiple early time points (5-60 minutes after receptor engagement). Use chemical crosslinking approaches to stabilize transient protein-protein interactions before immunoprecipitation.

• Low abundance of assembled complexes: Only a fraction of cellular TNFRSF10B may be incorporated into active DISC complexes. Solution: Optimize cell lysis conditions to preserve membrane-associated complexes. Consider using epitope-tagged TRAIL ligands that can be used to pull down the entire receptor complex.

• Non-specific binding in co-immunoprecipitation: Background signals can obscure true interactions. Solution: Include appropriate negative controls (IgG, unstimulated cells) and use stringent washing conditions calibrated to maintain specific interactions while reducing background. Consider using tandem affinity purification approaches for improved specificity.

• Variable antibody performance: Antibodies against DISC components may have variable efficiency in different applications. Solution: Validate antibodies using positive and negative controls, including CRISPR knockout cells. Test multiple antibodies targeting different epitopes of the same protein.

• Western blot detection limitations: Traditional Western blot may lack sensitivity for detecting less abundant DISC components. Solution: Use more sensitive detection methods such as digital protein simple platforms (e.g., Wes) or enhanced chemiluminescence substrates. Consider mass spectrometry-based approaches for comprehensive DISC component identification.

• Heterogeneity in DISC composition: DISC composition may vary depending on cell type and context. Solution: Compare DISC formation across multiple cell lines to identify core components versus cell-type-specific factors. Use quantitative proteomics to assess stoichiometry of different components.

What methodological considerations are critical when studying autophagy induction in response to TNFRSF10B activation?

As TNFRSF10B activation has been shown to induce autophagy as a protective mechanism against apoptosis , proper methodological approaches for studying this process are essential:

• Autophagic flux measurement: Simply measuring MAP1LC3B-II levels is insufficient, as increased levels could result from either increased formation or decreased clearance of autophagosomes. Solution: Always include lysosomal inhibitors (e.g., chloroquine or bafilomycin A1) to assess flux . The research demonstrates that while either TNFSF10 or chloroquine alone caused moderate increases of MAP1LC3B-II, the combination further elevated MAP1LC3B-II expression, confirming autophagy induction rather than just blocked clearance .

• Multiple autophagy markers: Rely on multiple markers including MAP1LC3B-II formation and SQSTM1/p62 degradation . Solution: Track both markers via Western blot or immunofluorescence. SQSTM1 is particularly valuable as it is degraded during functional autophagy, providing complementary information to MAP1LC3B-II accumulation.

• Timing considerations: Autophagy induction following TNFRSF10B activation follows specific kinetics. Solution: Conduct detailed time-course experiments, as the research shows gradual reduction of SQSTM1/p62 following TRAIL treatment .

• Validation through genetic approaches: Pharmacological inhibitors may have off-target effects. Solution: Complement inhibitor studies with genetic approaches such as siRNA targeting ATG7 or BECN1, as demonstrated in the research where knockdown of either gene effectively potentiated TNFSF10-induced cytotoxicity .

• Signaling pathway integration: Consider the intersection between apoptotic and autophagic pathways. Solution: Investigate key nodes that regulate both processes, such as MAPK8/JNK which mediates TRAIL-induced BCL2L1 decrease and affects the BCL2L1-BECN1 complex involved in autophagy regulation .

• Cell type considerations: Autophagy regulation varies across cell types. Solution: Validate findings across multiple cell lines, as the research demonstrated consistent autophagy induction across different cancer cell lines .

What are effective approaches for assessing TNFRSF10B expression and functionality in primary patient samples?

Analyzing TNFRSF10B in primary patient samples presents unique challenges requiring specialized methodological approaches:

• Sample preservation considerations: TNFRSF10B protein conformation and cellular localization are critical for function and can be affected by sample handling. Solution: Optimize fixation and preservation protocols specifically for membrane proteins. For fresh samples, minimize time between collection and processing. Consider using specialized tissue preservation solutions designed to maintain receptor integrity.

• Multiplexed detection systems: Traditional single-marker IHC provides limited information. Solution: Implement multiplexed immunofluorescence or chromogenic detection systems to simultaneously visualize TNFRSF10B along with other relevant markers (e.g., proliferation markers, other death receptors, downstream signaling components). This provides valuable context regarding heterogeneity within the sample.

• Functional assays with patient-derived cells: Expression alone doesn't guarantee functionality. Solution: When possible, establish short-term cultures or organoids from patient samples to assess functional responses to TRAIL or TNFRSF10B-specific agonists. Include assays for both apoptosis (Annexin V/PI staining) and downstream signaling activation (phospho-specific antibodies for key pathway components).

• Transcript analysis with spatial context: mRNA levels provide complementary information to protein expression. Solution: Apply spatial transcriptomics or in situ hybridization techniques to assess TNFRSF10B transcript levels with spatial resolution. This can reveal expression patterns across different regions of heterogeneous samples.

• Quantitative assessment methods: Semi-quantitative scoring may miss subtle but important differences. Solution: Use digital pathology platforms with validated algorithms for quantitative assessment of TNFRSF10B expression. Consider developing an H-score or other composite measure that accounts for both intensity and percentage of positive cells.

• Reference standards: Interpretation requires appropriate comparisons. Solution: Include reference samples with known TNFRSF10B expression levels processed identically to patient samples. Consider using cell line microarrays with graduated expression levels as calibration standards.

What are promising strategies for overcoming resistance to TNFRSF10B-mediated apoptosis in cancer therapy?

Despite the theoretical promise of targeting TNFRSF10B for cancer therapy, resistance mechanisms limit clinical efficacy. Research suggests several promising strategies to address this challenge:

• Targeting the autophagy-apoptosis axis: Research has demonstrated that TNFSF10 induces protective autophagy in cancer cells, and inhibiting autophagy significantly potentiates TRAIL-induced cell death . Combining TRAIL or TNFRSF10B agonists with autophagy inhibitors represents a rational strategy, supported by data showing that both pharmacological inhibitors (3MA, wortmannin) and genetic approaches (ATG7 or BECN1 knockdown) enhance TRAIL sensitivity .

• Modulating BCL2 family proteins: TRAIL treatment decreases expression of the anti-apoptotic protein BCL2L1 through MAPK8/JNK activation . This suggests that combining TRAIL with BH3 mimetics that target BCL2 family proteins could further enhance apoptotic responses. The degradation of BCL2L1 was suppressed by blocking lysosomal degradation but not by blocking caspases, indicating a non-caspase mediated mechanism that could be therapeutically exploited .

• Developing improved TNFRSF10B agonists: The pipeline of TNFRSF10B-targeted molecules includes approximately 25 candidates in various stages of development . These include compounds in Phase III (2), Phase II (1), Phase I (5), Preclinical (11), and Discovery (4) stages . Next-generation agonists with improved pharmacokinetics, tissue penetration, and receptor selectivity could overcome limitations of earlier compounds.

• Cancer-specific targeting approaches: Different cancer types show varying levels of TNFRSF10B expression and pathway dysregulation . Triple-negative breast cancer has emerged as a particular focus area for TNFRSF10B-targeted therapies . Developing cancer type-specific targeting strategies based on molecular profiles could improve efficacy and reduce off-target effects.

• Combination with epigenetic modifiers: Epigenetic silencing can reduce TNFRSF10B expression in some cancers. Combining TRAIL therapy with epigenetic modifiers that upregulate TNFRSF10B could restore sensitivity in resistant tumors.

How might novel technologies advance our understanding of TNFRSF10B biology in the tumor microenvironment?

Emerging technologies offer unprecedented opportunities to understand TNFRSF10B biology within the complex tumor microenvironment:

• Single-cell multi-omics approaches: Integrating single-cell RNA sequencing with proteomics can reveal how TNFRSF10B expression and signaling vary across different cell populations within tumors. This technology can identify previously unrecognized cellular subsets with unique TNFRSF10B signaling characteristics and their interactions with immune and stromal cells.

• Advanced imaging technologies: Multiplexed imaging platforms allow simultaneous visualization of dozens of proteins in spatial context. These technologies can map TNFRSF10B expression and signaling relative to features like vasculature, immune infiltrates, and necrotic regions, providing insights into how the microenvironment influences receptor function.

• Organoid and microfluidic tumor models: These models can recreate tumor-stroma-immune interactions in controlled environments. Using these systems to study TNFRSF10B signaling can reveal how different cellular components of the tumor ecosystem affect receptor function and response to targeting agents.

• CRISPR-based functional genomics: Pooled CRISPR screens in complex tumor models can identify novel regulators of TNFRSF10B function that are specific to the tumor microenvironment. This approach can uncover unexpected interactions between cancer cells and their surroundings that impact TRAIL sensitivity.

• Structural biology advances: Cryo-electron microscopy and other structural techniques can reveal the molecular details of TNFRSF10B interactions with TRAIL and other binding partners at unprecedented resolution. These insights can guide the design of improved therapeutic agonists with enhanced selectivity and potency.

• In vivo imaging of receptor engagement: Developing tracers that can monitor TNFRSF10B engagement and subsequent signaling in living organisms would transform our ability to study receptor biology in authentic tissue contexts and optimize therapeutic dosing strategies.

What are critical research questions that must be addressed to advance TNFRSF10B-targeted therapies toward clinical success?

Despite substantial progress in understanding TNFRSF10B biology, several critical questions must be addressed to realize the therapeutic potential of targeting this receptor:

• Determinants of cancer-selective killing: Why are some cancer cells highly sensitive to TRAIL while others (and most normal cells) are resistant? Identifying the molecular basis for this selectivity is crucial for developing predictive biomarkers and rational combination strategies. Research should focus on comprehensive molecular profiling of sensitive versus resistant cells to identify consistent patterns.

• Optimal receptor engagement strategies: What is the ideal approach for activating TNFRSF10B - using the natural ligand TRAIL, receptor-specific antibodies, or novel small molecule agonists? Each approach has distinct advantages and limitations regarding specificity, pharmacokinetics, and tissue penetration. Comparative studies using standardized models are needed.

• Biomarker development: Which biomarkers reliably predict response to TNFRSF10B-targeted therapies? The current pipeline includes molecules in advanced clinical testing , highlighting the urgent need for companion diagnostics. Potential biomarkers may include not only TNFRSF10B expression levels but also autophagy capacity, BCL2 family protein profiles, and DISC component expression.

• Autophagy modulation strategies: Given the protective role of autophagy against TRAIL-induced apoptosis , what is the optimal approach for modulating autophagy in combination with TRAIL therapy? This requires determining which autophagy inhibitors work best with TRAIL, identifying the appropriate sequence and timing of administration, and developing methods to monitor autophagy modulation in vivo.

• Resistance mechanisms in vivo: Do resistance mechanisms observed in vitro translate to in vivo settings? The tumor microenvironment may introduce additional layers of regulation not captured in cell culture systems. Research using relevant animal models and patient-derived samples is needed to validate and extend in vitro findings.

• Rational combination approaches: Which therapeutic combinations most effectively overcome TRAIL resistance without introducing unacceptable toxicity? The research indicates that combining TRAIL with autophagy inhibitors enhances apoptotic effects , but other promising combinations involving immunotherapy, targeted agents, or conventional chemotherapy need systematic evaluation.