SERPINB3 Expression Research Articles

SERPINB3 expression on B-cell surface in autoimmune diseases and hepatitis C virus-related chronic liver infection

SERPINB3 is a serine protease inhibitor with pleiotropic functions. It is involved in several physiological and pathological processes, where it appears to exert antiapoptotic effects. Little is known about its expression on immune system cells, the major players in mechanisms of viral defense and autoimmune disorders. The aim of this study was to characterize the expression of SERPINB3 on the surface of peripheral blood mononuclear cell subsets in both normal subjects and in patients with chronic viral infections and autoimmune diseases. Sixty-two patients were analyzed by flow cytometric analysis, including 45 with hepatitis C virus (HCV)-related chronic liver disease and 17 with systemic lupus erythematosus (SLE). SERPINB3 was expressed on B lymphocytes in 79% of the controls, in 32% of the HCV-infected patients and in none of the SLE patients. Surface localization of SERPINB3 was confirmed by confocal microscopy. SERPINB3 positivity was associated with CD27 reactivity (r = 0.98), but not to other activation molecules (CD69, CD71, CD86 and CXCR3). SERPINB3 is physiologically expressed on the surface of CD27(+) B lymphocytes, but its expression is reduced in HCV viral infection and not detectable in SLE patients. These results may suggest a role for SERPINB3 in B-cell defects typically found in viral infections and autoimmune disorders.

Tissue specific expression and estrogen regulation of SERPINB3 in the chicken oviduct

Serine protease inhibitors (SERPINs) comprise the largest superfamily of protease inhibitors and appear to be ubiquitously expressed in a variety of species. Of these, squamous cell carcinoma antigen 1 (SCCA1), also known as a SERPINB3, was first identified in squamous cell carcinoma tissue from the cervix of women. However, there is little known about the expression and hormonal regulation of SERPINB3 in chickens. Therefore, the avian SERPINB3 gene was compared with those of other species with respect to structure, phylogenetic evolution and tissue- and cell-specific expression in hens. Chicken SERPINB3 has moderate homology to mammalian SERPINB3 proteins (36–47%). Of particular note, SERPINB3 mRNA was most abundant in the chicken oviduct and cell-specific expression was in glandular (GE) and luminal (LE) epithelial cells of the oviduct of laying hens. Treatment of young chicks with DES (diethylstilbestrol, a synthetic nonsteroidal estrogen) induced SERPINB3 mRNA and protein in GE and LE, but not in other cell types of the oviduct. Western blot analyses determined that immunoreactive SERPINB3 protein was also increased by DES in LE and GE of the oviduct of chicks. Collectively, these results indicate that SERPINB3 is an estrogen-induced gene expressed only in LE and GE of the chicken oviduct and implicate SERPINB3 in regulation of oviduct development and egg formation.

SerpinB3, a new prognostic tool in breast cancer patients treated with neoadjuvant chemotherapy

It is unclear which patients with breast cancer benefit from anthracycline-based neoadjuvant chemotherapy and whether taxanes increase survival. Hsp70 and serpinB3 inhibit a lysosomal cell death pathway induced in anthracycline and taxane treated cells, which may be critical for breast cancer cell survival. Thus we evaluated serpinB3 and Hsp70 as putative prognostic biomarkers in breast cancer patients treated with neoadjuvant chemotherapy. SerpinB3 and Hsp70 were measured by immunohistochemistry in residual breast tumours of patients without a complete pathological response [pCR] (n = 250), from a retrospective cohort of 296 patients treated with anthracycline-based chemotherapy with or without sequential docetaxel prior to surgical resection. SerpinB3 (P = 0.02) and Hsp70 (P = 0.008) positivity in residual tumour were associated with a poor pathological response and serpinB3 was an independent prognostic biomarker (HR 2.1 (95% CI 1.2-3.8), P = 0.02). Docetaxel significantly improved overall survival of breast cancer patients treated with neoadjuvant chemotherapy. Furthermore, serpinB3 positivity predicted poor survival in patients treated with anthracycline-based chemotherapy alone (P = 0.02), but those with serpinB3 negative tumours had as equally good survival as those also treated with docetaxel (P = 0.7). Survival was independent of serpinB3 expression in patients who received sequential docetaxel. The Nottingham prognostic index (NPI), calculated at surgical resection, predicted overall survival in these neoadjuvantly treated patients (P < 0.001) and serpinB3 status segregated patients with a moderate NPI into distinct prognostic subgroups. The use of clinical (NPI) and molecular (serpinB3) biomarkers measured at surgical resection to provide accurate prognostication in patients who do not achieve a pCR following neoadjuvant chemotherapy could facilitate optimal post-operative clinical management of these patients and is of significant clinical value. Furthermore, serpinB3 status in residual tumour is a biomarker of neoadjuvant docetaxel benefit in patients not achieving a pCR and use of serpinB3 molecular subtyping for adjuvant docetaxel treatment planning warrants further investigation.

Overexpression of SERPIN B3 promotes epithelial proliferation and lung fibrosis in mice

SERPIN B3/B4, members of the serpin superfamily, are fundamental for the control of proteolysis through a known inhibitory function of different proteases. Several studies have documented an important role of SERPIN B3 in the modulation of inflammation, programmed cell death and fibrosis. To confirm the role of SERPIN B3 in lung fibrosis and overall investigate its influence on epithelial dysfunction, a stratified controlled trial randomly assigning bleomycin (BLM) treatment was performed on both SERPIN B3 transgenic (TG) and wild-type (WT) mice. TG and WT animals were killed 48 h (group T48 h) and 20 days (group T20d) after BLM treatment. Lung fibrosis was assessed by histology and hydroxyproline measurement. Architectural remodeling, inflammation, epithelial apoptosis and proliferation were quantified. Moreover, the profibrogenetic cytokine transforming growth factor (TGF)-β, cathepsin K, L and S were also investigated. No significant differences were observed between TG and WT mice of group T48 h in any parameters. In group T20d, less inflammation and a significant increase in epithelial proliferation were detected in treated TG than WT mice despite a similar apoptotic index, thus resulting in a different apoptosis/proliferation imbalance with a significant gain of epithelial proliferation. Moreover, TG mice showed higher TGF-β expression and more extended fibrosis. General linear model analysis, applied on morphological data, showed that interaction between SERPIN B3 expression and treatment was mainly significant for fibrosis. This study provides in vivo evidence for a role of SERPIN B3 in inhibiting inflammation and favoring epithelial proliferation with increased TGF-β secretion and thus the likelihood of consequent fibrogenesis.

SERPINB3 induces epithelial–mesenchymal transition

Epithelial-mesenchymal transition is believed to facilitate invasion and metastasis formation of epithelial tumour cells. SERPINB3 is a serine protease inhibitor, physiologically found in normal squamous epithelium but over-expressed in epithelial tumours and known to inhibit apoptosis. We tested the hypothesis that SERPINB3 has a role in invasion by modulating the epithelial-mesenchymal transition programme, using morphological, molecular and cell biology techniques on HepG2 cell clones transfected with the human SERPINB3 gene. The paracrine effect of this serpin was determined by the addition of exogenous recombinant SERPINB3 protein to HepG2 and MDCK cell line. SERPINB3 expression leads to changes in transfected cells morphology, characterized by clusters of loosely connected cells with elongated shape. Ultrastructural analysis confirmed the decrease of desmosomal junctions and widening of intercellular spaces. These alterations were associated with a reduction of E-cadherin and an increase of beta-catenin, with a parallel increase of cell proliferation. SERPINB3 clones, untransfected HepG2 and MDCK cells treated with exogenous SERPINB3 expressed vimentin, undetectable in controls. SERPINB3 induced significant cell scattering, migration and invasiveness in untransfected cells. These effects were not dependent on the anti-protease activity of the protein, as documented by the results obtained with an active loop-deleted recombinant SERPINB3 protein. Scatter activity was inhibited by an anti-SERPINB3 antibody in a dose-dependent manner and SERPINB3-transfected cells formed a significantly higher number of colonies on soft agar than controls. In conclusion, the observed results indicate that SERPINB3 induces deregulation of adhesion processes and increases the invasiveness potential supported by features of epithelial-mesenchymal transition, acting at both the autocrine and the paracrine level.

Serpin B3 up-regulation by hypoxia in HepG2 cells: A redox sensitive, Ras/ERK and PI3-K mediated event related to invasiveness and epithelial to mesenchymal transition

Background and aims. Hypoxia, very common in malignant solid tumours, can induce epithelial-to-mesenchymal transition (EMT) in human cancer cells. Poorly invasive HepG2 cells have been experimentally manipulated to investigate relationships between moderate hypoxia, EMT, invasiveness and expression of Serpin-B3 (formerly known as Squamous Cell Carcinoma Antigen), a serin protease inhibitor able to induce apoptosis resistance and involved in EMT and cell proliferation.

Chymotrypsin B Cached in Rat Liver Lysosomes and Involved in Apoptotic Regulation through a Mitochondrial Pathway

Lysosomes can trigger the mitochondrial apoptotic pathway by releasing proteases. Here we report that a 25-kDa protein purified from rat liver lysosomes possesses a long standing potent Bid cleavage activity at neutral pH, and the truncated Bid can in turn induce rapid mitochondrial release of cytochrome c. This protease was revealed as chymotrypsin B by biochemical and mass spectrometric analysis. Although it was long recognized as a digestive protease exclusively secreted by the exocrine pancreas, our data support that it also expresses and intracellularly resides in rat liver lysosomes. Translocation of lysosomal chymotrypsin B into cytosol was triggered by apoptotic stimuli such as tumor necrosis factor-alpha, and intracellular delivery of chymotrypsin B protein induced apoptotic cell death with a potency comparable with cathepsin B, suggestive of a lysosomal-mitochondrial pathway to apoptosis regulated by chymotrypsin B following its release. Noteworthily, either knockdown of chymotrypsin B expression by RNA interference or pretreatment with chymotrypsin B inhibitor N-p-tosyl-L-phenylalanine chloromethyl ketone significantly reduced tumor necrosis factor-alpha-induce apoptosis. These results demonstrate for the first time that chymotrypsin B is not only restricted to the pancreas but can function intracellularly as a pro-apoptotic protease.

Tumor Transcriptome Reveals the Predictive and Prognostic Impact of Lysosomal Protease Inhibitors in Non–Small-Cell Lung Cancer

Insight into clinical response to platinum-based chemotherapy (PBC) in non-small-cell lung cancer (NSCLC). Matched tumor and nontumor lung tissues from PBC-treated NSCLC patients (four nonresponders and four responders) and tumor tissue from an independent test set (four nonresponders and four responders), were profiled using microarrays. Lysosomal protease inhibitors SerpinB3 and cystatin C were highly correlated with clinical response and were further evaluated by immunohistochemistry in PBC-treated patients (36 prechemotherapy and 13 postchemotherapy). Investigation of the pathogenic and prognostic significance of SerpinB3 was performed in 251 primary tumors, with 64 regional lymph node pairs, from chemotherapy-naïve NSCLC patients using immunohistochemistry. Bioinformatic analyses of gene expression in the training set identified a gene set (n = 17) that separated all patients in the training and test sets (n = 16) according to response in hierarchical clustering. Transcriptome profiling revealed that SerpinB3 mRNA was highly correlated with degree of response (r = -0.978; P < .0001) and was a clear outlier (nonresponders:responders > 50-fold). SerpinB3 protein expression was correlated with clinical response in PBC-treated NSCLC patients (P = .045). Expression of SerpinB3 and cystatin C, relative to the target, protease cathepsin B, was independently predictive of response (odds ratio, 17.8; 95% CI, 2.0 to 162.4; P = .01), with an accuracy of 72%. High SerpinB3 expression levels, invariably associated with chemoresistance, had contrasting prognostic impact in untreated squamous cell carcinomas (hazard ratio [HR], 0.43; 95% CI, 0.18 to 0.93) or adenocarcinomas (HR, 2.09; 95% CI, 1.03 to 4.72). This provides the first comprehensive molecular characterization of clinical responsiveness to PBC in NSCLC and reveals the predictive and prognostic impact of two lysosomal protease inhibitors, potentially representing novel targets for NSCLC therapeutics.