SERPINB5 Human

What is SERPINB5 and what are its primary biological functions in human tissues?

SERPINB5, also known as Maspin or mammary serine protease inhibitor, is a member of the serpin peptidase inhibitor family initially characterized as a tumor suppressor gene. Despite its classification, SERPINB5 functions extend beyond protease inhibition, making it a pleiotropic protein primarily expressed in epithelial tissues.

The biological roles of SERPINB5 include:

• Regulation of cell adhesion, migration, and invasion processes

• Direct interaction with cytoskeletal components including microtubules and microfilaments

• Modulation of epithelial-mesenchymal transition (EMT)

• Regulation of desmoplakin membrane localization and intercellular adhesion

• Tumor suppression in certain contexts but potential oncogenic functions in others

Interestingly, SERPINB5's role appears to be context-dependent. While traditionally viewed as a tumor suppressor, recent studies indicate that in lung adenocarcinoma, SERPINB5 overexpression correlates with poor prognosis and promotes proliferation . Conversely, specific SERPINB5 haplotypes are associated with both increased and decreased hepatocellular carcinoma risk, demonstrating its complex genetic influences on cancer susceptibility .

How should researchers design experiments to investigate SERPINB5 expression patterns across cancer types?

When investigating SERPINB5 expression patterns across different cancer types, researchers should employ a multi-level approach that incorporates both in silico analysis and experimental validation:

Database Mining and In Silico Analysis:

• Utilize cancer genomics databases including TCGA (The Cancer Genome Atlas), GEO (Gene Expression Omnibus), GSCA (Gene Set Cancer Analysis), and cBioPortal to analyze expression, prognostic correlation, and genomic variation across multiple cancer types

• Examine methylation status data to determine epigenetic regulation patterns of SERPINB5

• Perform comprehensive transcriptome analysis across multiple cancer cohorts to identify expression patterns

Experimental Validation:

• Quantitative PCR (qPCR) to measure SERPINB5 mRNA expression levels in clinical samples

• Immunohistochemistry to evaluate protein expression patterns and subcellular localization in tissue sections

• Western blotting for semi-quantitative protein expression analysis

• Single-cell RNA sequencing to characterize cell type-specific expression in heterogeneous tumors

Prognostic Value Assessment:

• Construct Kaplan-Meier survival curves stratifying patients by SERPINB5 expression levels

• Perform univariate and multivariate Cox regression analyses to determine if SERPINB5 is an independent predictor of survival

• Validate findings in independent patient cohorts (e.g., validation with 106 clinical samples as reported for lung adenocarcinoma)

This comprehensive approach enables researchers to establish robust associations between SERPINB5 expression and clinical outcomes across different cancer types, providing a foundation for further functional studies.

What genetic polymorphisms of SERPINB5 are significant in cancer susceptibility, and how should researchers analyze them?

Several SERPINB5 single nucleotide polymorphisms (SNPs) and haplotypes have demonstrated significant associations with cancer susceptibility, particularly in hepatocellular carcinoma:

Key SERPINB5 Polymorphisms:

Promoter Region:

• Haplotype "C-C-C" (rs17071138 + rs3744941 + rs8089204) is associated with increased HCC risk (AOR = 1.450; P = 0.031)

Coding Region:

• Haplotype "T-C-A" (rs2289519 + rs2289520 + rs1455555) correlates with decreased HCC risk (AOR = 0.744; P = 0.031)

• Haplotype "C-C-C" (rs2289519 + rs2289520 + rs1455555) shows increased HCC risk (AOR = 1.981; P = 0.001)

• rs2289520 C allele carriers tend to exhibit better liver function than GG genotype carriers (Child-Pugh grade A vs. B or C; P = 0.047)

Methodological Approach for Polymorphism Analysis:

• Cohort Design:

  • Recruit well-matched case-control populations (e.g., 302 cases and 590 controls as in the HCC study)

  • Consider ethnicity and demographic factors to minimize population stratification

• Genotyping:

  • Select functionally relevant SNPs in promoter regions, exons, or splice sites

  • Employ appropriate genotyping techniques (PCR-RFLP, TaqMan assays, or next-generation sequencing)

• Haplotype Reconstruction:

  • Reconstruct haplotype blocks according to linkage disequilibrium structure of the SERPINB5 gene

  • Use software tools like Haploview for haplotype block visualization

• Statistical Analysis:

  • Calculate crude and adjusted odds ratios (AOR) with appropriate confidence intervals

  • Perform multivariate analysis controlling for confounding factors (age, gender, environmental exposures)

• Functional Validation:

  • Conduct in silico analysis to predict how polymorphisms affect SERPINB5 expression and protein stability

  • Perform reporter assays to assess effects on transcriptional activity

  • Examine expression levels in patient samples stratified by genotype

This systematic approach allows researchers to establish both statistical associations and potential functional mechanisms linking SERPINB5 polymorphisms to cancer susceptibility.

What methodologies are most effective for studying SERPINB5's interaction with the cytoskeleton?

SERPINB5 has been identified as a cytoskeleton-binding protein that regulates cell shape and adhesion. The following methodologies are most effective for investigating these interactions:

Biochemical Interaction Studies:

• Affinity purification-Mass spectrometry (AP/MS) to identify cytoskeletal binding partners comprehensively

• In vitro reconstitution assays with purified components to confirm direct binding to microtubules and microfilaments

• Co-immunoprecipitation to verify specific interactions with cytoskeletal components

Visualization Techniques:

• CRISPR/Cas9-mediated GFP tagging of endogenous SERPINB5 to visualize physiological localization without overexpression artifacts

• Immunofluorescence microscopy with co-staining of cytoskeletal markers (F-actin, tubulin) to demonstrate co-localization

• Super-resolution microscopy (STORM, PALM) for nanoscale localization precision

Functional Assays:

• Microtubule growth assays in vitro and in cells to assess SERPINB5's impact on cytoskeletal dynamics

• RNAi or CRISPR/Cas9-mediated depletion of SERPINB5 to observe effects on:

  • Cell-cell adhesion

  • Cytoskeletal organization

  • Epithelial-mesenchymal transition marker expression

• Analysis of cell rounding and cortical F-actin rearrangement in mitotic cells following SERPINB5 manipulation

Research has demonstrated that SERPINB5 localizes to the cortical cytoskeleton and mitotic spindle, and its depletion disrupts cell-cell adhesion, reorganizes the cytoskeleton, and upregulates mesenchymal markers . Furthermore, SERPINB5 suppresses microtubule growth both in vitro and in cells, indicating a direct regulatory effect on cytoskeletal dynamics .

How can researchers effectively analyze the role of SERPINB5 in cancer cell proliferation and migration?

To comprehensively analyze SERPINB5's role in cancer cell proliferation and migration, researchers should employ the following experimental approaches:

Gene Expression Manipulation:

• siRNA-mediated knockdown using validated sequences (e.g., 5′-CAAAGUGUGCUUAGAAAUAACTT-3′ and 5′-GUUAUUUCUAAGCACACUUUGTT-3′)

• CRISPR/Cas9 gene editing for complete knockout studies

• Overexpression using appropriate vectors (e.g., pCDNA3.1-MCS-EFla-copGFP containing SERPINB5 cDNA)

• Inducible expression systems to control timing and level of expression

Proliferation Assays:

• MTT or CCK-8 colorimetric assays to measure metabolic activity

• BrdU or EdU incorporation to quantify DNA synthesis

• Colony formation assays to assess long-term proliferative capacity

• Real-time cell analysis systems for continuous monitoring

Migration and Invasion Assays:

• Wound healing (scratch) assays with time-lapse imaging

• Transwell migration assays without matrix coating

• Invasion assays using Matrigel-coated transwell chambers

• 3D spheroid invasion assays in extracellular matrix

Molecular Mechanism Investigation:

• Western blotting to analyze EMT markers (E-cadherin, vimentin, N-cadherin) following SERPINB5 manipulation

• Immunofluorescence to visualize changes in cellular morphology and protein localization

• RNA-seq to identify global transcriptional changes induced by SERPINB5 modulation

Cell Line Validation:

• Use multiple cell lines to ensure reproducibility (e.g., A549 and PC9 lung adenocarcinoma cell lines)

• Include both high and low SERPINB5-expressing cell lines for comparison

What is the relationship between SERPINB5 and TGF-β signaling in epithelial cell adhesion, and how should it be investigated?

SERPINB5 has been identified as a regulator of TGF-β signaling in the context of epithelial cell adhesion. To investigate this relationship thoroughly:

Signaling Pathway Analysis:

• Western blotting for phosphorylated SMAD2/3 to assess canonical TGF-β pathway activation

• Luciferase reporter assays using SMAD-responsive elements to quantify TGF-β signaling activity

• Protein-protein interaction studies to determine if SERPINB5 directly interacts with TGF-β pathway components

Experimental Models:

• SERPINB5 knockdown or knockout models to assess effects on TGF-β signaling components

• Treatment with pemphigus vulgaris autoantibodies (PV-IgG) to induce loss of cell-cell adhesion as a model system

• Reconstitution experiments with wild-type or mutant SERPINB5 to identify domains required for TGF-β regulation

Functional Readouts:

• Immunofluorescence microscopy to assess desmoplakin (DSP) membrane localization following SERPINB5 manipulation

• Dispase assays to quantify intercellular adhesion strength

• Transepithelial/transendothelial electrical resistance (TEER) measurements to evaluate barrier function

TGF-β Pathway Manipulation:

• Small molecule inhibitors of TGF-β receptors to determine if they rescue adhesion defects caused by SERPINB5 deficiency

• TGF-β ligand stimulation to assess if it phenocopies SERPINB5 loss

• Genetic manipulation of downstream TGF-β effectors to identify specific mediators

Translational Models:

• Ex vivo human skin models to assess blister formation following PV-IgG treatment with or without TGF-β inhibition

• Analysis of skin biopsies from pemphigus patients to confirm TGF-β pathway activation in disease context

Research has demonstrated that SERPINB5 overexpression prevents PV-IgG-mediated loss of cell-cell adhesion and preserves DSP at the cell membrane . Mechanistically, SERPINB5 loss deregulates TGF-β signaling, which destabilizes DSP in keratinocytes. Importantly, inhibition of TGF-β signaling ameliorates PV-IgG-mediated loss of adhesion, increases DSP membrane expression, and prevents blister formation in human ex-vivo skin models .

How is SERPINB5 differentially regulated during viral infections, and what techniques should be used to study this phenomenon?

SERPINB5, along with other SERPIN family members, shows differential regulation during viral infections. To investigate this phenomenon comprehensively:

Expression Analysis in Clinical Samples:

• Single-cell RNA sequencing (scRNA-seq) of samples from virus-infected patients (e.g., bronchoalveolar lavage fluid from COVID-19 patients) to characterize cell type-specific expression patterns

• Correlation of SERPINB5 expression with markers of antiviral response and inflammation

• Immunohistochemistry of infected tissues to confirm protein-level changes

In Vitro Infection Models:

• Infection of relevant cell culture models, including human airway epithelial cultures (HAEC)

• Exposure to diverse respiratory viruses (adenovirus, reovirus, rhinovirus, parainfluenza, influenza, SARS-CoV-2) to identify virus-specific responses

• Time-course analysis (e.g., 24h and 72h post-infection) using RT-qPCR and western blotting

Regulatory Mechanism Investigation:

• Promoter analysis using reporter assays to identify virus-responsive elements

• ChIP-seq to identify transcription factors binding to the SERPINB5 promoter during infection

• Epigenetic profiling (methylation, histone modifications) to assess chromatin remodeling

• Treatment with pathway inhibitors to determine which signaling cascades mediate virus-induced SERPINB5 regulation

Functional Significance:

• SERPINB5 knockdown or overexpression followed by viral infection to assess impact on viral replication

• Protein-protein docking screens to predict interactions between SERPINB5 and viral proteases

• Biochemical assays to validate predicted SERPINB5-viral protease interactions

Research suggests that SERPINs, including SERPINB5, may be upregulated as part of the host antiviral response, potentially targeting viral proteases essential for viral replication and maturation . For example, SERPINB2 has been identified as potentially targeting Adpro, a viral protease . Understanding the differential regulation of SERPINB5 during viral infections may provide insights into novel antiviral mechanisms and therapeutic strategies.

What approaches should be used to establish SERPINB5 as a prognostic biomarker in cancer?

To robustly establish SERPINB5 as a clinically relevant prognostic biomarker in cancer:

Multi-Cohort Analysis:

• Analyze SERPINB5 expression across multiple independent patient cohorts to ensure reproducibility

• Utilize data from large cancer genomics databases (TCGA, GEO, cBioPortal) for initial discovery

• Stratify analyses by cancer type, stage, grade, and molecular subtypes

Survival Correlation Analysis:

• Generate Kaplan-Meier survival curves comparing outcomes between high and low SERPINB5 expression groups

• Perform univariate and multivariate Cox regression analyses to determine if SERPINB5 is an independent predictor when controlling for established prognostic factors

• Calculate hazard ratios with confidence intervals to quantify prognostic impact

Technical Validation:

• Confirm RNA-seq findings with qPCR in independent clinical samples

• Develop standardized immunohistochemistry protocols with validated antibodies

• Establish scoring systems for protein expression (H-score, Allred score, or percentage positive cells)

Clinical Parameter Correlation:

• Assess associations between SERPINB5 expression and:

  • Tumor stage and grade

  • Metastatic status

  • Response to therapy

  • Disease recurrence

Multivariate Biomarker Development:

• Combine SERPINB5 with other molecular markers to develop composite prognostic signatures

• Test signature performance using receiver operating characteristic (ROC) curves

• Calculate areas under the curve (AUC) to assess discriminatory ability

Integrated Analysis:

• Correlate SERPINB5 expression with methylation status

• Identify genetic alterations (mutations, CNVs) that may influence prognostic value

• Perform pathway analysis to contextualize SERPINB5's role within broader cancer biology

How should researchers design experiments to elucidate the molecular mechanisms by which SERPINB5 regulates desmosomal adhesion?

To elucidate the molecular mechanisms underlying SERPINB5's regulation of desmosomal adhesion:

Protein Interaction Studies:

• Co-immunoprecipitation of SERPINB5 with desmosomal components

• Proximity labeling techniques (BioID, APEX) to identify proteins in close proximity to SERPINB5 at cell-cell junctions

• Domain mapping to identify specific regions of SERPINB5 required for desmosomal regulation

• Direct binding assays with purified components to establish direct interactions

Localization Analysis:

• High-resolution imaging of SERPINB5 and desmosomal proteins (desmoplakin, desmoglein, desmocollin)

• FRAP (Fluorescence Recovery After Photobleaching) to assess dynamics of desmosomal components with and without SERPINB5

• Live-cell imaging with fluorescently tagged proteins to track real-time changes in localization

Adhesion Strength Measurement:

• Dispase-based dissociation assays to quantify intercellular adhesion following SERPINB5 manipulation

• Atomic force microscopy to measure cell-cell adhesion forces at single-cell resolution

• Shear stress assays to evaluate resistance to mechanical force

TGF-β Pathway Analysis:

• Assessment of TGF-β signaling components (SMAD phosphorylation, nuclear translocation) following SERPINB5 knockdown or overexpression

• TGF-β pathway inhibitor treatment to determine if it rescues adhesion defects in SERPINB5-deficient cells

• Analysis of desmoplakin membrane localization in relation to TGF-β signaling status

Disease Model Applications:

• Use of pemphigus vulgaris autoantibodies (PV-IgG) to induce loss of cell-cell adhesion

• Ex vivo human skin models to assess blister formation with manipulation of SERPINB5 or TGF-β signaling

• Analysis of patient skin biopsies to correlate SERPINB5 expression with desmosomal integrity in disease contexts

Research has demonstrated that SERPINB5 overexpression prevents PV-IgG-mediated loss of cell-cell adhesion and the loss of desmoplakin from the cell membrane . The mechanism involves SERPINB5 regulation of TGF-β signaling, which when activated (as occurs in SERPINB5 deficiency) leads to destabilization of desmoplakin at the membrane. Inhibition of TGF-β signaling ameliorates these effects, suggesting a direct mechanistic link .

What are the optimal methods for studying SERPINB5 involvement in epithelial-mesenchymal transition (EMT)?

Epithelial-mesenchymal transition (EMT) is a critical process in cancer progression, and SERPINB5 has been implicated in its regulation. To optimally study SERPINB5's role in EMT:

Expression Manipulation:

• Generate stable cell lines with SERPINB5 knockdown, knockout, or overexpression

• Use inducible systems to control timing and level of expression changes

• Create rescue lines expressing wild-type or mutant SERPINB5 in knockout backgrounds

EMT Marker Analysis:

• Western blotting to quantify canonical EMT markers:

  • Epithelial markers: E-cadherin, claudins, occludin, desmoplakin

  • Mesenchymal markers: N-cadherin, vimentin, fibronectin, α-SMA

  • EMT transcription factors: Snail, Slug, ZEB1/2, Twist

• Immunofluorescence microscopy to visualize changes in cellular localization of EMT markers

• qRT-PCR to measure changes in mRNA expression of EMT-related genes

Morphological Assessment:

• Phase-contrast microscopy to document changes in cell shape

• Quantitative image analysis of cell elongation, area, and aspect ratio

• Cytoskeletal staining (F-actin, microtubules) to visualize structural reorganization

Functional Assays:

• Migration assays (wound healing, transwell) to assess enhanced motility associated with EMT

• Invasion assays to evaluate increased invasive capacity

• Adhesion assays to quantify changes in cell-cell and cell-matrix interactions

• Resistance to anoikis (detachment-induced apoptosis)

Signaling Pathway Investigation:

• Analysis of EMT-related signaling pathways (TGF-β, Wnt, Notch)

• Pathway inhibitor treatments to determine which signaling cascades mediate SERPINB5's effects on EMT

• Epistasis experiments to position SERPINB5 within EMT regulatory hierarchies

In Vivo Validation:

• Xenograft models with SERPINB5-manipulated cells to assess effects on tumor growth and metastasis

• Analysis of circulating tumor cells for EMT characteristics

• Immunohistochemistry of primary tumors and metastases to evaluate EMT marker expression