STIP1 Human, His

What is STIP1 and what are its primary functions in human cells?

STIP1, also known as Heat Shock Protein (HSP) 70/90 organizing protein, is a 62.6-kDa adaptor protein that coordinates the functions of different chaperones to facilitate protein folding within cells . It serves as a co-chaperone of heat shock proteins and is ubiquitously expressed in various human tissues, including testis and epididymis .

STIP1 functions primarily as a scaffold protein that facilitates protein-protein interactions. For example, it promotes the interaction between lysine-specific demethylase 1 (LSD1) and glycogen synthase kinase-3 beta (GSK3β) through its TPR domains: the TPR1 and TPR2B domains bind to the AOL domain of LSD1, while the TPR2A and TPR2B domains interact with the kinase domain of GSK3β . This scaffolding function is critical for GSK3β-mediated LSD1 phosphorylation, which promotes LSD1 stability and enhances cell proliferation .

How is STIP1 expression regulated during cellular stress responses?

STIP1 expression shows a developmentally-regulated pattern and responds dynamically to various cellular stressors. In experimental models, STIP1 is up-regulated after heat shock in rat testis and epididymis, demonstrating its role in stress response . Conversely, under oxidative stress conditions, STIP1 expression is significantly down-regulated in the testis, with accompanying histomorphological changes .

To investigate STIP1 regulation under different stress conditions, researchers should design time-course experiments with various stressors (heat shock, oxidative stress, hypoxia) and measure STIP1 expression at both mRNA and protein levels. Western blot analysis for protein quantification and RT-qPCR for mRNA levels provide reliable data on expression changes, while immunohistochemistry can reveal altered tissue distribution patterns .

What are the structural domains of STIP1 and how do they contribute to its function?

STIP1 contains multiple tetratricopeptide repeat (TPR) domains that mediate protein-protein interactions. Specifically, STIP1 has TPR1, TPR2A, and TPR2B domains that interact with different protein partners. The TPR1 and TPR2B domains bind to the AOL domain of LSD1, while the TPR2A and TPR2B domains interact with the kinase domain of GSK3β .

To study the functional importance of each domain, researchers should employ domain deletion mutants or site-directed mutagenesis of key residues. Co-immunoprecipitation experiments can then determine how specific domains contribute to protein-protein interactions. For instance, expressing STIP1 constructs lacking individual TPR domains can reveal their differential contributions to interactions with LSD1 and GSK3β .

How does STIP1 contribute to cancer progression?

STIP1 promotes cancer progression through multiple mechanisms:

• JAK2/STAT3 signaling activation: STIP1 induces proliferation and growth in lung adenocarcinoma by activating the JAK2/STAT3 signaling pathway .

• Epithelial-mesenchymal transition (EMT) induction: STIP1 can enhance the migratory ability of cancer cells through EMT, as evidenced by decreased E-cadherin and increased vimentin expression in STIP1-expressing cells .

• Scaffold function for oncogenic pathways: STIP1 serves as a scaffold for GSK3β-mediated LSD1 phosphorylation, which enhances LSD1 stability and promotes cancer cell proliferation .

• Heat-induced metastasis: Intracellular STIP1 mediates heat-induced metastasis of hepatocellular carcinoma by facilitating EMT through increased nuclear shuttling of Snail1 .

• Inhibition of apoptosis: STIP1 suppression significantly increases cancer cell apoptosis, as demonstrated by increased cleaved caspase-3 and PARP levels .

Research approaches to study these mechanisms include knock-down/knock-out experiments, pathway inhibition studies, and in vivo metastasis models.

What experimental approaches can effectively inhibit STIP1 function in cancer cells?

Several experimental approaches have proven effective for STIP1 inhibition:

• RNA interference: STIP1 shRNA transfection significantly decreases cell proliferation, adhesion, and migration while increasing apoptosis in lung adenocarcinoma cells .

• Antibody-mediated inhibition: HEPES-mediated intracellular delivery of anti-STIP1 antibodies has been used to inhibit intracellular STIP1 function. This approach involves pre-incubating anti-STIP1 antibodies with 30 mM HEPES for 15 minutes before application to cells or in vivo administration .

• Cell-penetrating peptides: A cell-penetrating peptide (peptide 520) capable of inhibiting STIP1 function has been developed. This approach targets specific protein-protein interactions mediated by STIP1 .

• Combination therapy: Targeting STIP1-dependent pathways using combination therapy shows promise. For example, the LSD1 inhibitor SP2509 and the GSK3β inhibitor LY2090314 act synergistically to induce cancer cell death by disrupting pathways dependent on STIP1 scaffold function .

When designing inhibition experiments, researchers should include appropriate controls (non-targeting shRNA, control IgG antibodies, or scrambled peptides) and assess multiple cellular endpoints, including proliferation, migration, and apoptosis .

How does STIP1 expression correlate with clinical outcomes in different cancer types?

STIP1 is overexpressed in several malignancies, including liver, pancreatic, ovarian, colon, and breast tumors . The correlation between STIP1 expression and clinical outcomes varies by cancer type:

In lung adenocarcinoma, STIP1 expression is significantly higher in tumor tissue compared to adjacent normal tissue. Higher expression levels correlate with increased proliferative, adhesive, and migratory abilities of cancer cells, suggesting poorer prognosis .

To investigate STIP1's clinical relevance, researchers should:

• Analyze STIP1 expression in paired tumor and normal tissue samples using immunohistochemistry, RT-qPCR, and Western blot .

• Correlate expression levels with clinicopathological parameters (tumor stage, grade, metastasis) and survival data.

• Perform multivariate analysis to determine if STIP1 is an independent prognostic factor.

• Compare STIP1 expression patterns across different cancer types to identify cancer-specific functions.

Notably, immunohistochemical expression of STIP1 and LSD1 shows a positive correlation in human cancer specimens, suggesting a functional relationship that may influence patient outcomes .

What are the optimal methods for detecting and quantifying STIP1 expression?

For comprehensive STIP1 expression analysis, researchers should employ multiple complementary methods:

• Immunohistochemistry (IHC): Useful for visualizing STIP1 distribution in tissue sections and determining subcellular localization. IHC can reveal whether STIP1 is primarily cytoplasmic, nuclear, or membrane-associated in different cell types .

• Western blot analysis: Provides quantitative measurement of STIP1 protein levels. Antibodies against STIP1 (such as those from Santa Cruz Biotechnology) can be used with GAPDH or β-actin as loading controls .

• RT-qPCR: Measures STIP1 mRNA expression levels, which can reveal transcriptional regulation. This technique is particularly valuable for developmental studies and stress response experiments .

• Fluorescence microscopy: Using fluorescently-labeled antibodies allows visualization of STIP1's subcellular localization and co-localization with interaction partners .

When implementing these methods, researchers should include appropriate positive and negative controls, validate antibody specificity using STIP1 knockout cells, and normalize expression data to suitable reference genes or proteins .

How should experiments be designed to study STIP1's role as a scaffold protein?

To investigate STIP1's scaffold function, researchers should:

• Co-immunoprecipitation assays: To detect protein-protein interactions between STIP1 and its partners (such as LSD1 and GSK3β). Pull-down with anti-STIP1 antibodies followed by Western blot for interaction partners can confirm complex formation .

• Domain mapping experiments: Express truncated versions of STIP1 containing different TPR domains to determine which domains mediate specific interactions. For example, experiments have shown that TPR1 and TPR2B domains bind to LSD1, while TPR2A and TPR2B interact with GSK3β .

• Proximity ligation assays: This technique can visualize protein-protein interactions in situ, confirming that STIP1 brings its binding partners into close proximity within cells.

• Functional assays following disruption: After identifying key interaction domains, express dominant-negative STIP1 mutants that can bind one partner but not the other to determine how disrupting scaffold function affects downstream signaling.

• Phosphorylation assays: Since STIP1 facilitates GSK3β-mediated phosphorylation of LSD1, in vitro kinase assays with purified proteins can confirm direct effects on substrate phosphorylation .

Control experiments should include STIP1 knockout or knockdown cells to demonstrate specificity of observed interactions .

What approaches can differentiate between intracellular and extracellular effects of STIP1?

STIP1 functions both intracellularly and extracellularly, necessitating specific experimental approaches to distinguish these activities:

• For extracellular STIP1 studies:

  • Use recombinant STIP1 protein added to culture medium

  • Employ neutralizing antibodies against extracellular STIP1

  • Analyze conditioned media for secreted STIP1 levels

  • Examine activation of cell surface receptors and downstream pathways like ERK or ALK2-SMAD

• For intracellular STIP1 studies:

  • Use HEPES-mediated delivery of anti-STIP1 antibodies to target intracellular STIP1 specifically

  • Employ cell-penetrating peptides (like peptide 520) that inhibit intracellular interactions

  • Create cell lines with STIP1 knockdown/knockout, then rescue with STIP1 variants containing nuclear localization or export signals to control subcellular localization

  • Analyze intracellular signaling pathways like JAK2/STAT3 or GSK3β-LSD1

• Comparative studies:

  • Compare cellular responses to extracellular STIP1 neutralization versus intracellular STIP1 inhibition

  • Examine how blocking secretion affects intracellular STIP1 functions

  • Use subcellular fractionation to track STIP1 localization under different conditions

These approaches help distinguish whether observed phenotypes result from intracellular scaffolding functions or extracellular signaling activities of STIP1 .

How does STIP1 interact with the JAK2/STAT3 pathway to promote cancer progression?

STIP1 plays a critical role in activating the JAK2/STAT3 pathway in cancer cells:

• Complex formation: STIP1 promotes the formation of an HSP90-JAK2-STAT3 complex, facilitating JAK2 activation and subsequent STAT3 phosphorylation .

• Functional consequences: In lung adenocarcinoma cells, STIP1 knockdown significantly decreases levels of phosphorylated JAK2/STAT3, confirming STIP1's role in pathway activation .

• Downstream effects: The STIP1-mediated JAK2/STAT3 activation enhances cancer cell proliferation, adhesion, and migration, while reducing apoptosis .

To investigate this interaction, researchers should:

• Perform co-immunoprecipitation studies to detect physical interactions between STIP1, HSP90, JAK2, and STAT3

• Use phospho-specific antibodies to measure JAK2 and STAT3 activation states following STIP1 manipulation

• Employ JAK2 inhibitors to determine if they can reverse STIP1-mediated phenotypes

• Analyze nuclear translocation of phospho-STAT3 using subcellular fractionation or immunofluorescence

• Assess STAT3 target gene expression changes following STIP1 inhibition

What role does LSD1 phosphorylation play in STIP1-mediated oncogenic effects?

STIP1 facilitates GSK3β-mediated phosphorylation of LSD1, which has several important consequences:

• Enhanced LSD1 stability: Phosphorylation at residues S707 and S711 promotes LSD1 stability, preventing its degradation .

• Nuclear retention: Properly phosphorylated LSD1 remains in the nucleus where it can exert its epigenetic functions. When these phosphorylation sites are mutated (S707A/S711A), LSD1 is translocated from the nucleus to the cytoplasm .

• Increased cell proliferation: The stabilized, nuclear-localized LSD1 enhances cancer cell proliferation through its demethylase activity on histone and non-histone substrates .

To study this mechanism, researchers should:

• Use phospho-specific antibodies to monitor LSD1 phosphorylation status

• Express phospho-mimetic (S707D/S711D) or phospho-deficient (S707A/S711A) LSD1 mutants to assess functional consequences

• Perform cycloheximide chase experiments to measure LSD1 stability

• Use GSK3β inhibitors to confirm kinase specificity

• Analyze subcellular localization of wild-type and mutant LSD1 using immunofluorescence or subcellular fractionation

• Assess histone methylation status at LSD1 target genes following disruption of this pathway

How can contradictory data about STIP1 function be reconciled across different experimental systems?

When facing contradictory results regarding STIP1 function, researchers should:

• Consider tissue/cell type specificity: STIP1's effects may vary across different cell types. For example, STIP1 is up-regulated after heat shock in rat testis but down-regulated under oxidative stress conditions . Systematic comparison of STIP1 function across multiple cell lines can reveal tissue-specific roles.

• Examine experimental conditions: Different stress conditions (heat, oxidation, hypoxia) may trigger distinct STIP1 responses. Standardize experimental conditions and compare results using identical stressors and timepoints.

• Distinguish intracellular vs. extracellular effects: STIP1 has both intracellular and extracellular functions that may sometimes appear contradictory . Design experiments that specifically target one pool of STIP1 at a time.

• Account for compensatory mechanisms: In chronic STIP1 depletion models, compensatory pathways may emerge that mask acute effects. Compare acute inhibition (antibodies, peptides) with stable knockdown approaches.

• Validate key findings using multiple approaches: Confirm important results using complementary techniques (genetic knockdown, pharmacological inhibition, dominant-negative constructs) and multiple readouts (proliferation, apoptosis, signaling activation) .

What combination therapies targeting STIP1-dependent pathways show promise?

Combination approaches targeting STIP1-dependent pathways have demonstrated synergistic effects in preclinical studies:

• LSD1 and GSK3β inhibitors: The LSD1 inhibitor SP2509 and the GSK3β inhibitor LY2090314 act synergistically to induce cancer cell death by disrupting the STIP1 scaffold function that normally facilitates GSK3β-mediated LSD1 phosphorylation .

• Intracellular STIP1 targeting: HEPES-mediated intracellular delivery of anti-STIP1 antibodies or cell-penetrating peptides (peptide 520) shows promise in inhibiting cancer cell growth across multiple cancer cell lines .

• Pathway-specific combinations: Since STIP1 activates multiple oncogenic pathways, combining STIP1 inhibition with JAK2/STAT3 pathway inhibitors may provide enhanced anti-cancer effects .

When designing combination therapy experiments, researchers should:

• Determine optimal drug concentrations using dose-response curves

• Calculate combination indices to quantify synergistic, additive, or antagonistic effects

• Assess multiple cellular endpoints (proliferation, apoptosis, migration)

• Test combinations in both in vitro and in vivo models

• Evaluate potential toxicity to normal cells using non-cancerous cell lines

What are the potential off-target effects and toxicity concerns when targeting STIP1?

When considering STIP1 as a therapeutic target, researchers should address several potential concerns:

• Essential cellular functions: STIP1 is involved in protein folding and stress responses in normal cells. Complete inhibition might disrupt these essential functions, potentially causing toxicity in non-cancerous tissues .

• Developmental roles: STIP1 shows developmentally-regulated expression patterns, suggesting important roles in development and differentiation . Systemic inhibition could affect these processes.

• Immune system effects: As a stress-responsive protein, STIP1 may have roles in immune cell function that could be disrupted by therapeutic targeting.

• Specificity of targeting approaches: Methods like HEPES-mediated antibody delivery or cell-penetrating peptides may have variable efficacy across different tissues or off-target effects .

To address these concerns, researchers should:

• Compare effects of STIP1 inhibition on cancer cells versus non-cancerous cells (e.g., immortalized human endometrial stromal cells, HESC)

• Use inducible knockdown systems to determine if transient STIP1 inhibition is sufficient for therapeutic effects

• Develop tumor-targeted delivery systems for anti-STIP1 agents

• Monitor multiple organ systems in animal models during STIP1-targeted therapy

• Identify the minimum level of STIP1 inhibition needed for anti-cancer effects while preserving essential functions