SerpinB3: A Multifaceted Player in Health and Disease-Review and Future Perspectives.

SerpinB3, a member of the serine-protease inhibitor family, has emerged as a crucial player in various physiological and pathological processes. Initially identified as an oncogenic factor in squamous cell carcinomas, SerpinB3's intricate involvement extends from fibrosis progression and cancer to cell protection in acute oxidative stress conditions. This review explores the multifaceted roles of SerpinB3, focusing on its implications in fibrosis, metabolic syndrome, carcinogenesis and immune system impairment. Furthermore, its involvement in tissue protection from oxidative stress and wound healing underscores its potential as diagnostic and therapeutic tool. Recent studies have described the therapeutic potential of targeting SerpinB3 through its upstream regulators, offering novel strategies for cancer treatment development. Overall, this review underscores the importance of further research to fully elucidate the mechanisms of action of SerpinB3 and to exploit its therapeutic potential across various medical conditions.

SERPINB3/B4 Is Increased in Psoriasis and Rosacea Lesions and Has Proinflammatory Effects in Mouse Models of these Diseases.

Psoriasis and rosacea are both chronic inflammatory skin disorders resulted from aberrant keratinocyte-immune cell crosstalk, but the common molecular foundations for these 2 conditions are poorly understood. In this study, we reveal that both patients with psoriasis and those with rosacea as well as their mouse models have significantly elevated expressions of SERPINB3/B4 (members of serine protease inhibitor) in the lesional skin. Skin inflammation in mice that resembles both psoriasis and rosacea is prevented by SERPINB3/B4 deficiency. Mechanistically, we demonstrate that SERPINB3/B4 positively induces NF-κB signaling activation, thereby stimulating disease-characteristic inflammatory chemokines and cytokines production in keratinocytes and promoting the chemotaxis of CD4+ T cells. Our results suggest that in keratinocytes, SERPINB3/B4 may be involved in the pathogenesis of both psoriasis and rosacea by stimulating NF-κB signaling, and they indicate a possible treatment overlap between these 2 diseases.

SerpinB3/4 Expression Is Associated with Poor Prognosis in Patients with Cholangiocarcinoma.

Cholangiocarcinoma (CCA), the second most common primary liver tumor, is associated with a dismal outcome, and useful prognostic markers are not currently available in clinical practice. SerpinB3, a serine protease inhibitor, was recently found to play a relevant role in malignant transformation in different cancers. The aim of the present study was to determine the expression of SerpinB3/4 in tissue and serum samples of patients with CCA in relation to clinical outcomes. SerpinB3/4 was assessed in the tissue microarrays (TMAs) of 123 surgically resected CCAs. ELISA assays were carried out in 188 patients with CCA to detect the free and IgM-linked forms of SerpinB3/4. Overall survival was analyzed in relation to SerpinB3/4 expression, and Cox models were used to identify the variables associated with survival. High levels of SerpinB3/4 (TMA score 2+/3+) were detected in 15 tumors (12.2%), characterized by a more advanced TNM stage (III/IV: 64.3% vs. 31.3%; p = 0.031) and lower overall patient survival, independently of CCA subclass (intrahepatic CCA: median 1.1 (0.8-Not Estimable, NE) vs. 2.4 (1.8-3.4) years; p = 0.0007; extrahepatic CCA: median 0.8 (0.2-NE) vs. 2.2 (1.5-5.4) years; p = 0.011). Vascular invasion (p = 0.027) and SerpinB3/4 scores (p = 0.0016) were independently associated with mortality in multivariate analysis. Patients who had detectable free or IgM-linked SerpinB3/4 in their serum showed poorer survival (1 vs. 2.4 years, p = 0.015, for free SerpinB3/4, and 1 vs. 2.6 years, p = 0.0026, for SerpinB3/4-IgM). In conclusion, high levels of SerpinB3/4 in tissue and serum in CCA are associated with poor outcomes after surgery, regardless of tumor subclass.

The polymorphic variant of SerpinB3 (SerpinB3-PD) is associated with faster cirrhosis decompensation.

BACKGROUND: SerpinB3 is a cysteine protease inhibitor involved in liver disease progression due to its proinflammatory and profibrogenic properties. The polymorphic variant SerpinB3-PD (SB3-PD), presents a substitution in its reactive centre loop, determining the gain of function. AIMS: To disclose the clinical characteristics of a cohort of patients with cirrhosis in relation to the presence of SB3-PD and to assess the effect of this genetic variant on fibrogenic and inflammatory cytokines in vitro. METHODS: We assessed SB3 polymorphism in 90 patients with cirrhosis, prospectively followed up in our referral centre. We used HepG2 and HuH-7 cells transfected to overexpress either wild-type SB3 (SB3-WT) or SB3-PD to assess their endogenous effect, while LX2 and THP-1 cells were treated with exogenous SB3-WT or SB3-PD proteins. RESULTS: Patients carrying SB3-PD had more severe portal hypertension and higher MELD scores, than patients carrying SB3-WT. In multivariate analysis, SB3-PD was an independent predictor of cirrhosis complications. Patients with SB3-PD polymorphism presented with more severe liver fibrosis and inflammatory features. Hepatoma cells overexpressing SB3-PD showed higher TGF-ß1 expression than controls. The addition of recombinant SB3-PD induced an up-regulation of TGF-ß1 in LX2 cells and a more prominent inflammatory profile in THP-1 cells, compared to the effect of SB3-WT protein. CONCLUSIONS: The polymorphic variant SB3-PD is highly effective in determining activation of TGF-ß1 and inflammation in vitro. Patients with cirrhosis who carry SB3-PD polymorphism may be more prone to develop severe liver disease progression. However, further validation studies are warranted to support the in vivo relevance of this polymorphism.

SERPINB3-MYC axis induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) exhibits distinct molecular subtypes: classical/progenitor and basal-like/squamous. Our study aimed to identify genes contributing to the development of the basal-like/squamous subtype, known for its aggressiveness. Transcriptome analyses revealed consistent upregulation of SERPINB3 in basal-like/squamous PDAC, correlating with reduced patient survival. SERPINB3 transgene expression in PDAC cells enhanced in vitro invasion and promoted lung metastasis in a mouse PDAC xenograft model. Metabolome analyses unveiled a metabolic signature linked to both SERPINB3 and the basal-like/squamous subtype, characterized by heightened carnitine/acylcarnitine and amino acid metabolism, associated with poor prognosis in patients with PDAC and elevated cellular invasiveness. Further analysis uncovered that SERPINB3 inhibited the cysteine protease calpain, a key enzyme in the MYC degradation pathway, and drove basal-like/squamous subtype and associated metabolic reprogramming through MYC activation. Our findings indicate that the SERPINB3-MYC axis induces the basal-like/squamous subtype, proposing SERPINB3 as a potential diagnostic and therapeutic target for this variant.

WTAP-Mediated N6-Methyladenosine of RNAs Facilitate the Pathophysiology of Atopic Dermatitis.

N6-methyladenosine (m6A) is the most abundant dynamic and reversible internal chemical modification of RNA in eukaryotic cells and is essential in multiple pathophysiological processes. However, it has not been reported in atopic dermatitis (AD). We used Arraystar m6A-mRNA epitranscriptomic microarray to screen for differentially expressed genes and their m6A levels and m6A-related enzymes in patients with AD. We confirmed that the m6A RNA methyltransferase WTAP and 2 candidate differentially expressed genes (S100A9 and SERPINB3) were significantly upregulated in keratinocytes in public data and epidermal lesions of patients with AD. In vitro cell experiments confirmed that WTAP influenced the expression of the 2 candidate differentially expressed genes and promoted primary human epidermal keratinocyte proliferation while inhibiting human epidermal keratinocyte differentiation. Furthermore, we showed that WTAP, S100A9, and SERPINB3 expression correlated with AD severity. Our findings revealed that WTAP-mediated m6A modification promoted the expression of S100A9 and SERPINB3 to aggravate human epidermal keratinocyte proliferation and dysdifferentiation contributing to the pathophysiological development of AD.

Novel Molecular Targets for Immune Surveillance of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common and aggressive cancer with a high mortality rate. The incidence of HCC is increasing worldwide, and the lack of effective screening programs often results in delayed diagnosis, making it a challenging disease to manage. Immunotherapy has emerged as a promising treatment option for different kinds of cancers, with the potential to stimulate the immune system to target cancer cells. However, the current immunotherapeutic approaches for HCC have shown limited efficacy. Since HCC arises within a complex tumour microenvironment (TME) characterized by the presence of various immune and stromal cell types, the understanding of this interaction is crucial for the identification of effective therapy. In this review, we highlight recent advances in our understanding of the TME of HCC and the immune cells involved in anti-tumour responses, including the identification of new possible targets for immunotherapy. We illustrate a possible classification of HCC based on the tumour immune infiltration and give evidence about the role of SerpinB3, a serine protease inhibitor involved in the regulation of the immune response in different cancers.

Epitope-Specific Anti-SerpinB3 Antibodies for SerpinB3 Recognition and Biological Activity Inhibition.

SerpinB3 is a serine protease inhibitor that plays a relevant role in disease progression and cancer by increasing fibrosis, cell proliferation, and invasion, besides conferring resistance to apoptosis. The mechanisms underlying these biological activities are not yet fully understood. The aim of this study was to generate antibodies directed against different SerpinB3 epitopes to better investigate their biological role. Five exposed epitopes were identified using the software DNASTAR Lasergene and the corresponding synthetic peptides were used for NZW rabbit immunization. Anti-P#2 and anti-P#4 antibodies were able to recognize both SerpinB3 and SerpinB4 by ELISA. Anti-P#5 antibody, produced against the reactive site loop of SerpinB3, showed the greatest specific reactivity for human SerpinB3. This antibody was able to recognize SerpinB3 at nuclear level, while anti-P#3 antibody recognized SerpinB3 only at cytoplasmic level, both by immunofluorescence and by immunohistochemistry. The biological activity of each antibody preparation was assessed in HepG2 cells overexpressing SerpinB3 and anti-P#5 antibody reduced proliferation by 12% cell and cell invasion by 75%, while trivial results were obtained with the other antibody preparations. These findings indicate that the reactive site loop of SerpinB3 is essential for the invasiveness features induced by this serpin and it could become a novel druggable target.

SerpinB3 as hepatic marker of post-resective shear stress.

Post-resective liver failure is a frequent complication of liver surgery and it is due to portal hyperperfusion of the remnant liver and to arterial vasoconstriction, as buffer response of the hepatic artery. In this context, splenectomy allows a reduction of portal flow and increases the survival chance in preclinical models. SerpinB3 is over-expressed in the liver in oxidative stress conditions, as a mechanism of cell defense to provide survival by apoptosis inhibition and cell proliferation. In this study, the expression of SerpinB3 was assessed as predictor of liver damage in in vivo models of major hepatic resection with or without splenectomy. Wistar male rats were divided into 4 groups: group A received 30% hepatic resection, group B > 60% resection, group C > 60% resection with splenectomy and group D sham-operated. Before and after surgery liver function tests, echo Doppler ultrasound and gene expression were assessed. Transaminase values and ammonium were significantly higher in groups that underwent major hepatic resection. Echo Doppler ultrasound showed the highest portal flow and resistance of the hepatic artery in the group with > 60% hepatectomy without splenectomy, while the association of splenectomy determined no increase in portal flow and hepatic artery resistance. Only the group of rats without splenectomy showed higher shear-stress conditions, reflected by higher levels of HO-1, Nox1 and of Serpinb3, the latter associated with an increase of IL-6. In conclusion, splenectomy controls inflammation and oxidative damage, preventing the expression of Serpinb3. Therefore, SerpinB3 can be considered as a marker of post-resective shear stress.

SERPINB3, Adult-Onset Immunodeficiency, and Generalized Pustular Psoriasis.

BACKGROUND: Generalized pustular psoriasis (GPP; MIM 614204) is a rare and severe pustular autoinflammatory skin disease in which acute generalized erythema and scaling develop with numerous sterile pustules. GPP shares skin manifestations, especially pustular skin reaction, with adult-onset immunodeficiency (AOID) with anti-interferon-γ autoantibodies, an autoimmune disease. METHODS: Clinical examinations and whole-exome sequencing (WES) were performed on 32 patients with pustular psoriasis phenotypes and 21 patients with AOID with pustular skin reaction. Immunohistochemical and histopathological studies were performed. RESULTS: WES identified three Thai patients presenting with similar pustular phenotypes-two with a diagnosis of AOID and the other with GPP. A heterozygous missense variant chr18:g.61325778C>A NM_006919.2: c.438G>T; NP_008850.1: p.Lys146Asn; rs193238900 in SERPINB3 was identified in two patients: one with GPP and the other with AOID. The other patient who had AOID carried a heterozygous missense variant chr18:g.61323147T>C NM_006919.2: c.917A>G; NP_008850.1: p.Asp306Gly in SERPINB3. Immunohistochemical studies showed overexpression of SERPINA1 and SERPINB3, a hallmark of psoriatic skin lesions. CONCLUSIONS: Genetic variants in SERPINB3 are associated with GPP and AOID with pustular skin reaction. The skin of patients with GPP and AOID carrying SERPINB3 mutations showed overexpression of SERPINB3 and SERPINA1. Clinically and genetically, GPP and AOID appear to share pathogenetic mechanisms.

Bile detection of squamous cell carcinoma antigen (SCCA) in extrahepatic cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is a rare biliary tract tumor with poor prognosis that often is challenging to diagnose and the majority of patients present with advanced stage. Squamous cell carcinoma antigen 1 (SCCA1) overexpression has been found in different tumors associated with poor prognosis and chemoresistance. AIMS: To assess the presence and possible prognostic role of SCCA1/2 isoforms in bile and serum of patients with CCA. METHODS: Forty seven surgical patients (36 with CCA and 11 with benign diseases) were prospectively included in the study. Serum and bile specimens were collected at the time of surgery and free and IgM-complexed SCCA was quantified by ELISA (Xeptagen, srl). RESULTS: Free or IgM linked SCCA was rarely found in serum, while SCCA was detectable in bile samples of patients with CCA, especially in those with extrahepatic form (43% vs 17%, p = 0.008), but not in controls. Despite similar tumor stage, these positive patients presented a trend toward a higher percentage of portal invasion (27% vs 15%) and of tumor recurrence than negative cases (62% vs 40%), although the difference was not statistically significant. CONCLUSION: These preliminary results indicate that bile testing for SCCA is a specific marker of extrahepatic CCA, with potential prognostic value.

HPV Enhances HNSCC Chemosensitization by Inhibiting SERPINB3 Expression to Disrupt the Fanconi Anemia Pathway.

Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor of the head and neck, and the prognosis of patients is poor due to chemotherapeutic resistance. Interestingly, patients with HNSCC induced by human papillomavirus (HPV) infection are more sensitive to chemotherapy and display a better prognosis than HPV-negative patients. The biological relevance of HPV infection and the mechanism underlying chemosensitivity to cisplatin remain unknown. Herein, SERPINB3 is identified as an important target for regulation of cisplatin sensitivity by HPV-E6/E7 in HNSCC. Downregulation of SERPINB3 inhibits cisplatin-induced DNA damage repair and enhances the cytotoxicity of cisplatin. In detail, decreasing SERPINB3 expression reduces the USP1-mediated deubiquitination of FANCD2-FANCI in the Fanconi anemia pathway, thereby interfering with cisplatin-induced DNA interstrand crosslinks repair and further contributing to HNSCC cell apoptosis. To translate this finding, pH-responsive nanoparticles are used to deliver SERPINB3 small interfering RNA in combination with cisplatin, and this treatment successfully reverses cisplatin chemotherapeutic resistance in a patient-derived xenograft model from HPV-negative HNSCC. Taken together, these findings suggest that targeting SERPINB3 based on HPV-positive HNSCC is a potential strategy to overcome cisplatin resistance in HPV-negative HNSCC and improves the prognosis of this disease.

Reactive Centre Loop Mutagenesis of SerpinB3 to Target TMPRSS2 and Furin: Inhibition of SARS-CoV-2 Cell Entry and Replication.

The SARS-CoV-2 virus can utilize host cell proteases to facilitate cell entry, whereby the Spike (S) protein is cleaved at two specific sites to enable membrane fusion. Furin, transmembrane protease serine 2 (TMPRSS2), and cathepsin L (CatL) are the major proteases implicated, and are thus targets for anti-viral therapy. The human serpin (serine protease inhibitor) alpha-1 antitrypsin (A1AT) shows inhibitory activity for TMPRSS2, and has previously been found to suppress cell infection with SARS-CoV-2. Here, we have generated modified serpin inhibitors with increased specificity for these cellular proteases. Using SerpinB3 (SCCA-1), a cross-class inhibitor of CatL, as a scaffold, we have designed and produced reactive centre loop (RCL) variants to more specifically target both furin and TMPRSS2. Two further variants were generated by substituting the RCL P7-P1 with the spike protein S1/S2 cleavage site from either SARS-CoV-2 alpha or delta (P681R) sequences. Altered inhibitory specificity of purified recombinant proteins was verified in protease assays, with attenuated CatL inhibition and gain of furin or TMPRSS2 inhibition, as predicted, and modified serpins were shown to block S protein cleavage in vitro. Furthermore, the serpin variants were able to inhibit S-pseudoparticle entry into A549-ACE2-TMPRSS2 cells and suppress SARS-CoV-2 replication in Vero E6 cells expressing TMPRSS2. The construct designed to inhibit TMPRSS2 (B3-TMP) was most potent. It was more effective than A1AT for TMPRSS2 enzyme inhibition (with an eighteen-fold improvement in the second order inhibition rate constant) and for blocking SARS-CoV-2 viral replication. These findings advance the potential for serpin RCL mutagenesis to generate new inhibitors, and may lead to novel anti-viral biological molecules.

Biological Pathway-Derived TMB Robustly Predicts the Outcome of Immune Checkpoint Blockade Therapy.

Although immune checkpoint blockade (ICB) therapies have achieved great progress, the patient response varies among cancers. In this study, we analyzed the potential genomic indicators contributing to ICB therapy response. The results showed that high tumor mutation burden (TMB) failed to predict response in anti-PD1 treated melanoma. SERPINB3 was the most significant response-related gene in melanoma and mutations in either SERPINB3 or PEG3 can serve as an independent risk factor in melanoma. Some recurrent mutations in CSMD3 were only in responders or non-responders, indicating their diverse impacts on patient response. Enrichment scores (ES) of gene mutations in 12 biological pathways were significantly higher in responders or non-responders. Next, the P-TMB calculated from genes in these pathways was significantly related to patient response with prediction AUC 0.74-0.82 in all collected datasets. In conclusion, our work provides new insights into the application of TMB in predicting patient response, which will benefit to immunotherapy research.

SerpinB3 drives cancer stem cell survival in glioblastoma.

Despite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. Knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown increased the efficacy of radiation in pre-clinical models. Taken together, our findings identify a GBM CSC-specific survival mechanism involving a cysteine protease inhibitor, SerpinB3, and provide a potential target to improve the efficacy of GBM therapies against therapeutically resistant CSCs.

SerpinB3 as a Pro-Inflammatory Mediator in the Progression of Experimental Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease worldwide. In 20-30% of patients, NAFLD can progress into non-alcoholic steatohepatitis (NASH), eventually leading to fibrosis, cirrhosis and hepatocellular carcinoma development. SerpinB3 (SB3), a hypoxia-inducible factor-2α dependent cysteine protease inhibitor, is up-regulated in hepatocytes during progressive NAFLD and proposed to contribute to disease progression. In this study we investigated the pro-inflammatory role of SB3 by employing phorbol-myristate acetate-differentiated human THP-1 macrophages exposed in vitro to human recombinant SB3 (hrSB3) along with mice overexpressing SB3 in hepatocytes (TG/SB3) or knockout for SB3 (KO/SB3) in which NASH was induced by feeding methionine/choline deficient (MCD) or a choline-deficient, L-amino acid defined (CDAA) diets. In vivo experiments showed that the induction of NASH in TG/SB3 mice was characterized by an impressive increase of liver infiltrating macrophages that formed crown-like aggregates and by an up-regulation of hepatic transcript levels of pro-inflammatory cytokines. All these parameters and the extent of liver damage were significantly blunted in KO/SB3 mice. In vitro experiments confirmed that hrSB3 stimulated macrophage production of M1-cytokines such as TNFα and IL-1ß and reactive oxygen species along with that of TGFß and VEGF through the activation of the NF-kB transcription factor. The opposite changes in liver macrophage activation observed in TG/SB3 or KO/SB3 mice with NASH were associated with a parallel modulation in the expression of triggering receptor expressed on myeloid cells-2 (TREM2), CD9 and galectin-3 markers, recently detected in NASH-associated macrophages. From these results we propose that SB3, produced by activated/injured hepatocytes, may operate as a pro-inflammatory mediator in NASH contributing to the disease progression.

The protease-inhibitor SerpinB3 as a critical modulator of the stem-like subset in human cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem-like compartment is available. METHODS: Enrichment of CCA stem-like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT-PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC-like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3-transfected MON (MONSB3+ ) cells in immune-deficient NOD-SCID/IL2Rgnull  (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients. RESULTS: SB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c-MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (ß-catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3-overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up-regulation of the proto-oncogene ß-catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients. CONCLUSION: These findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.

Engineered EVs for Oxidative Stress Protection.

Extracellular vesicles (EVs) are increasingly studied as vectors for drug delivery because they can transfer a variety of molecules across biological barriers. SerpinB3 is a serine protease inhibitor that has shown a protective anti-apoptotic function in a variety of stressful conditions. The aim of this study was to evaluate protection from oxidative stress-induced damage, using extracellular vesicles that overexpress SerpinB3 (EVs-SB3) in order to enhance the effect of extracellular vesicles on cellular homeostasis. EVs-SB3s were obtained from HepG2 cells engineered to overexpress SerpinB3 and they revealed significant proteomic changes, mostly characterized by a reduced expression of other proteins compared with EVs from non-engineered cells. These EV preparations showed a significantly higher protection from H2O2 induced oxidative stress in both the hepatoma cell line and in primary cardiomyocytes, compared to cells treated with naïve EVs or SerpinB3 alone, used at the same concentration. In conclusion, the induction of SerpinB3 transgene expression results in the secretion of EVs enriched with the protein product that exhibits enhanced cytoprotective activity, compared with naïve EVs or the nude SerpinB3 protein.

TEAD4 transcriptional regulates SERPINB3/4 and affect crosstalk between keratinocytes and T cells in psoriasis.

Psoriasis is a common chronic inflammatory disease with the prevalence rate of approximately 1-3 %. Currently, it is generally believed that the pathogenesis of psoriasis is a T-cell immune-mediated skin disease mediated by multiple genes and factors, and the interaction between keratinocytes and T cells. TEA domain family member 4 (TEAD4) is a transcription factor which regulates the expression of downstream genes in Hippo pathway and affects several biological processes, such as regulating cell differentiation and embryonic development. However, few studies have reported the role of TEAD4 in psoriasis and its possible regulatory mechanism. In this study, we found the expression level of TEAD4 in the skin of psoriasis was significantly higher than that of normal skin. In patients with the pathological keratinocytes, TEAD4 can transcriptionally regulate the expression of SERPINB3/4 and affect the secretion of chemokines, and the depletion of SERPINB3/4 inhibited the secretion of chemokines. In addition, the supernatant of keratinocytes of patients can significantly increase the migration ability of T cells, and the supernatant of T cells cultured by the supernatant of keratinocytes of patients can significantly enhance the proliferation ability of keratinocytes. Therefore, our results suggested that TEAD4 is a key regulatory factor in progression of psoriasis, and the crosstalk between keratinocytes and T cells mediated by TEAD4 plays a critical role in the psoriasis pathogenesis.

Study on the targeted therapy of oral squamous cell carcinoma with a plasmid expressing PE38KDEL toxin under control of the SERPINB3 promoter.

Oral squamous cell carcinoma (OSCC) has a poor prognosis and a high risk of recurrence. To improve the efficacy of OSCC therapy, it is of great significance to explore gene therapy for OSCC. The use of specific genes to regulate the targeted expression of suicide genes is a hot topic in gene therapy for cancer. The SERPINB3 gene is highly active in squamous cell carcinoma, but nearly undetectable or present at a low level in normal tissues. This specificity suggests that the SERPINB3 promoter can be used for targeted OSCC therapy. Pseudomonas aeruginosa secretes PE38KDEL, an exotoxin derivative, as a suicide gene used in gene therapy. A SERPINB3 promoter-mediated PE38KDEL expression vector was created. The SERPINB3 gene expression was tested in different cell lines by RT-qPCR and Western blotting, and the SERPINB3 promoter activity was detected by luciferase assay. The SERPINB3 promoter was more active in the TCA8113 cell line than in the other cell lines. The target therapeutic potential of the toxin vector pSERPINB3-PE38KDEL was tested in the SERPINB3-positive TCA8113 cell line, the SERPINB3-negative MG63 cell line, and normal L02 cell line. The SERPINB3 gene was expressed at a high level in TCA8113 cells but a low level in MG63 and L02 cells. Transfection of the pSERPINB3-PE38KDEL plasmid effectively inhibited the proliferation and invasion of TCA8113 cells and induced cell apoptosis, but no significant damage to MG63 and L02 cells was observed. The results of in vitro experiments indicated that the pSERPINB3-PE38KDEL plasmid could be a promising strategy for targeted OSCC gene therapy.