Functional characterization of a cystatin A from the bat Myotis davidii.

Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73-79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA's and reduced MdCSTA's inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat's stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.

An Integrated Approach to Protein Discovery and Detection From Complex Biofluids.

Ovarian cancer, a leading cause of cancer-related deaths among women, has been notoriously difficult to screen for and diagnose early, as early detection significantly improves survival. Researchers and clinicians seek routinely usable and noninvasive screening methods; however, available methods (i.e., biomarker screening) lack desirable sensitivity/specificity. The most fatal form, high-grade serous ovarian cancer, often originate in the fallopian tube; therefore, sampling from the vaginal environment provides more proximal sources for tumor detection. To address these shortcomings and leverage proximal sampling, we developed an untargeted mass spectrometry microprotein profiling method and identified cystatin A, which was validated in an animal model. To overcome the limits of detection inherent to mass spectrometry, we demonstrated that cystatin A is present at 100 pM concentrations using a label-free microtoroid resonator and translated our workflow to patient-derived clinical samples, highlighting the potential utility of early stage detection where biomarker levels would be low.

Correlated with better prognosis, CSTA inhibits metastasis of nasopharyngeal carcinoma cells via suppressing AKT signaling through promoting METTL3 degradation.

BACKGROUND: Metastasis is one of the main obstacles impeding the survival of nasopharyngeal carcinoma (NPC) patients, with the molecular mechanism underlying NPC metastasis still unclear. RESULTS: In this study, Cystatin A (CSTA) was found downregulated in NPC tissues with metastasis compared with those without metastasis. Shorter overall survival and distant metastasis-free survival were found in NPC patients with lower CSTA expression. Using functional assays, we found that CSTA prevented both the in vitro motility of NPC cells and their ability to metastasize in vivo. Transcriptome sequencing and western blot analysis revealed that CSTA inhibited the phosphorylation of AKT. Moreover, activating AKT using AKT agonist SG79 rescued the motility of CSTA-overexpressing NPC cells, whereas, treatment with AKT inhibitor MK2206 inhibited the motility of CSTA-knockdown NPC cells. Mechanically, immunoprecipitation coupled mass spectrometry found that CSTA interacted with the N6-adenosine-methyltransferase subunit METTL3 and promoted its ubiquitin-proteasome-mediated degradation following the upregulation of NKX3-1 and LHPP, which are negative regulators of AKT. Furthermore, knock-down of NKX3-1 and LHPP enhanced the motility of CSTA-overexpressing NPC cells. CONCLUSIONS: The inhibitory effect of CSTA upon NPC metastasis mainly depended on suppressing AKT signaling by the upregulation of NKX3-1 and LHPP expression resulting from the binding between CSTA and METLL3. Our study suggests that the CSTA-METLL3-NKX3-1/LHPP-AKT axis could be of therapeutic value for inhibiting NPC metastasis.

Functional analysis of the cystatin A gene response to SGIV infection in orange-spotted grouper, Epinephelus coioides.

Cystatin A (CyA), an inhibitor of cysteine protease, was widely studied in immune defense and cancer therapy. However, the function of CyA and its potential molecular mechanism during virus infection in fish remain unknown. In our study, we cloned the open reading frame (ORF) of CyA homology from orange-spotted grouper (Ec-CyA) consisting of 303 nucleotides and encoding a 101-amino acid protein. Ec-CyA included two conserved sequences containing one N-terminal glycine fragment and one QXVXG sequence (48aa-52aa) without the signal peptide. Tissue distribution analysis showed that Ec-CyA was highly expressed in spleen and head kidney. Moreover, further analysis indicated that the expression of Ec-CyA increased during SGIV simulation in grouper spleen (GS) cells. Subcellular localization assay demonstrated that Ec-CyA was mainly distributed in cytoplasm in GS cells. Overexpressed Ec-CyA promoted the mRNA level of viral genes MCP, VP19 and LITAF. Meanwhile, SGIV-induced apoptosis in fat head minnow (FHM) cells was facilitated, as well as the activation of caspase-3/7, caspase-9. In addition, Ec-CyA overexpression down-regulated the expression of interferon (IFN) related molecules including ISG15, IFN, IRF3, MAVS, MyD88, TRAF6 and up-regulated proinflammatory factors such as IL-1ß, IL-8 and TNF-α. At the same time, Ec-CyA-overexpressing inhibited the activity of IFN and ISRE promoter, but induced NF-κB promoter activity by luciferase reporter gene assay. In summary, our findings suggested that Ec-CyA was involved in innate immune response and played a key role in DNA virus infection.

CSTA plays a role in osteoclast formation and bone resorption by mediating the DAP12/TREM2 pathway.

Cystatin A (CSTA) is a cysteine protease inhibitor that is expressed highly during osteoporosis. However, the exact role of CSTA in osteoporosis remains unknown. In this study, we examined the role of CSTA in the formation, differentiation, and bone resorption of osteoclasts. We extracted bone marrow cells from 8-week-old wildtype mice to obtain RANKL and M-CSF-induced osteoclasts. We performed CSTA overexpression and knockdown experiments in the cells. We analyzed the role of CSTA in the process of osteoclasts by trap staining. In addition, we studied the contribution of CSTA to osteogenesis through the DAP12/TREM2 (DNAX-activating protein of 12 kDa/Triggering receptor expressed on myeloid cells-2) complex. We analyzed the role of CSTA in postmenopausal osteoporosis using OVX mouse models. We found that the silencing of CSTA inhibited the differentiation and formation of osteoclasts. The loss of CSTA weakened the expression of osteoclast marker genes. In contrast, overexpression of CSTA significantly increased differentiation and formation of osteoclasts and enhanced bone resorption. Immunofluorescence staining indicated that CSTA and DAP12 are co-expressed in osteoclasts, and the loss of either DAP12 or TREM2 inhibited osteoclast differentiation and bone resorption. Suppression of CSTA decreased DAP12 and TREM2 expression, whereas overexpression of CSTA rescued the loss of TREM2 expression caused by DAP12 knockdown. Co-immunoprecipitation and co-localization experiments indicated that CSTA interacted with DAP12. In addition, we found that injection of si-CSTA into OVX mice significantly improved bone parameters. Our research indicates that CSTA interacts with the DAP12/TREM2 complex and could be a potential targeted therapy for osteoporosis management.

In Silico, In Vitro, and Clinical Investigations of Cathepsin B and Stefin A mRNA Expression and a Correlation Analysis in Kidney Cancer.

The cysteine protease Cathepsin B (CtsB) plays a critical role in multiple signaling pathways, intracellular protein degradation, and processing. Endogenous inhibitors regulate its enzymatic activity, including stefins and other cystatins. Recent data proved that CtsB is implicated in tumor extracellular matrix remodeling, cell invasion, and metastasis: a misbalance between cathepsins and their natural inhibitors is often considered a sign of disease progression. In the present study, we investigated CtsB and stefin A (StfA) expression in renal cell carcinoma (RCC). mRNA analysis unveiled a significant CTSB and STFA increase in RCC tissues compared to adjacent non-cancerogenic tissues and a higher CtsB expression in malignant tumors than in benign renal neoplasms. Further analysis highlighted a positive correlation between CtsB and StfA expression as a function of patient sex, age, tumor size, grade, lymph node invasion, metastasis occurrence, and survival. Alternative overexpression and silencing of CtsB and StfA confirmed the correlation expression between these proteins in human RCC-derived cells through protein analysis and fluorescent microscopy. Finally, the ectopic expression of CtsB and StfA increased RCC cell proliferation. Our data strongly indicated that CtsB and StfA expression play an important role in RCC development by mutually stimulating their expression in RCC progression.

Patients With Atopic Dermatitis Sensitized to Pet Dander Mount IgE and T-Cell Responses to Mammalian Cystatins, Including the Human Self-Protein.

BACKGROUND: Immediate and delayed-type hypersensitivity reactions to pet-borne allergens are common in atopic diseases. In atopic dermatitis (AD), controversy surrounds the contribution to the disease of cross-reactivity to self-proteins. Human cystatin A and the cat allergen Fel d 3 belong to the cystatins, an evolutionary conserved protein family. The objective of the present study was to assess crossreactivity between mammalian cystatins and to analyze T-cell responses to cystatin in AD patients sensitized to pet dander. METHODS: cDNA coding for dog cystatin was cloned from dog skin. Sera from 245 patients with IgE-mediated sensitization to cat and dog dander were tested for IgE binding to recombinantly expressed feline, canine, and human cystatin. Of these, 141 were also diagnosed with AD. RESULTS: Cystatin-specific IgE was detected in 36 patients (14.7%), of whom 19 were considerably affected by AD. Within the AD patients, 9 had measurable IgE against all 3 cystatins. Cystatin-sensitized AD patients did not differ from non-cystatin-sensitized patients in terms of disease severity, age, or total IgE levels. T-cell cytokine measurements showed elevated IL-4 levels after stimulation with feline and human cystatin. CONCLUSIONS: The humoral response suggests that in addition to Fel d 3, the homologous protein from dog might play a role in allergy. Furthermore, human cystatin appears to be capable of driving a type 2 immune response in sensitized AD patients and may therefore be considered a so-called autoallergen, as proposed for other evolutionary conserved proteins.

Skin chronological aging drives age-related bone loss via secretion of cystatin-A.

Although clinical evidence has indicated an association between skin atrophy and bone loss during aging, their causal relationship and the underlying mechanisms are unknown. Here we show that premature skin aging drives bone loss in mice. We further identify that cystatin-A (Csta), a keratinocyte-enriched secreted factor, mediates the effect of skin on bone. Keratinocyte-derived Csta binds the receptor for activated C-kinase 1 in osteoblast and osteoclast progenitors, thus promoting their proliferation but inhibiting osteoclast differentiation. Csta secretion decreases with skin aging in both mice and humans, thereby causing senile osteoporosis by differentially decreasing the numbers of osteoblasts and osteoclasts. In contrast, topical application of calcipotriol stimulates Csta production in the epidermis and alleviates osteoporosis. These results reveal a mode of endocrine regulation of bone metabolism in the skin, and identify Csta as an epidermally derived hormone linking skin aging to age-related bone loss. Enhancers of skin Csta levels could serve as a potential topical drug for treatment of senile osteoporosis.

Decreased CSTA expression promotes lymphatic metastasis and predicts poor survival in oral squamous cell carcinoma.

OBJECTIVE: Herein, we aimed to identify biomarkers that affect lymphatic metastasis of oral squamous cell carcinoma (OSCC) through bioinformatic analysis, and clinicopathological and in vitro verifications. DESIGN: The OSCC-related gene expression dataset was retrieved from The Cancer Genome Atlas (TCGA) and analyzed to identify differentially expressed genes (DEGs), which were subjected to pathway analysis. Weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network analysis were performed to identify hub genes. Expression of potential biomarkers was examined using quantitative real-time polymerase chain reaction, immunohistochemistry, and western blotting. Statistical analyses were performed to determine the association between biomarker expression and clinicopathological characteristics of patients with OSCC. Effects of selected biomarkers on proliferation, migration, and invasion were evaluated using in vitro assays. RESULTS: For DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed potential lymphatic metastasis-related biological processes and signaling pathways. Eight hub genes - ALOXE3, CSTA, PLA2G4E, PPL, SPRR1A, SPRR2A, SPRR2D, and SPRR2E, were identified via WGCNA and PPI analyses. CSTA expression was markedly downregulated in primary OSCC tissues, and low CSTA expression significantly correlated with high tumor grade (P =  0.001), nodal metastasis (P =  0.028), and poor overall survival (P <  0.001). CTSA overexpression inhibited OSCC cell migration and invasion in vitro, with little effect on OSCC cell proliferation. CONCLUSIONS: Our study revealed that CSTA is a promising biomarker and therapeutic target with prognostic implications in patients with OSCC. CSTA may play an essential role in OSCC lymphatic metastasis and tumor differentiation.

Selection and characterisation of Affimers specific for CEA recognition.

Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications.

Grass carp (Ctenopharyngodon idella) stefin A: Systematic research on its cloning, expression, characterization and tissue distribution.

To fully understand the properties of piscine stefins (family I cystatins), the 294-bp stefinA gene from grass carp (Ctenopharyngodon idella, Ci) was cloned and expressed in E. coli BL21 (DE3). After purification by Ni2+-NTA agarose affinity chromatography, the CiStefin A protein was tested to have a molecular weight of 11.48 kDa and an isoelectric point of 8.7. The typical motif of the cystatins superfamily was characterized from CiStefin A (QVVQG). CiStefin A specifically inhibited the activity of papain and cathepsin B/L. The Ki value of CiStefin A against papain was 6.5 × 10-11 M. CiStefin A showed excellent heat and acid-base tolerance. StefinA gene transcription occurred in all tested tissues of grass carp, with the highest level in the hepatopancreas. Immunolocalization staining with an anti-CiStefinA antibody revealed the CiStefinA protein distribution in all tested tissues at various levels. Overall, these results clarified the physical and biochemical properties of grass carp stefin A.

Analysis of gene expression profiles of lung cancer subtypes with machine learning algorithms.

Lung cancer is one of the most common cancer types worldwide and causes more than one million deaths annually. Lung adenocarcinoma (AC) and lung squamous cell cancer (SCC) are two major lung cancer subtypes and have different characteristics in several aspects. Identifying their differentially expressed genes and different gene expression patterns can deepen our understanding of these two subtypes at the transcriptomic level. In this work, we used several machine learning algorithms to investigate the gene expression profiles of lung AC and lung SCC samples retrieved from Gene Expression Omnibus. First, the profiles were analyzed by using a powerful feature selection method, namely, Monte Carlo feature selection. A feature list, ranking all features according to their importance, and some informative features were obtained. Then, the feature list was used in the incremental feature selection method to extract optimal features, which can allow the support vector machine (SVM) to yield the best performance for classifying lung AC and lung SCC samples. Some top genes (CSTA, TP63, SERPINB13, CLCA2, BICD2, PERP, FAT2, BNC1, ATP11B, FAM83B, KRT5, PARD6G, PKP1) were extensively analyzed to prove that they can be differentially expressed genes between lung AC and lung SCC. Meanwhile, a rule learning procedure was applied on informative features to construct the classification rules. These rules provide a clear procedure of classification and show some different gene expression patterns between lung AC and lung SCC.

Interplay of ERα binding and DNA methylation in the intron-2 determines the expression and estrogen regulation of cystatin A in breast cancer cells.

Despite advances in early detection and treatment, invasion and metastasis of breast tumors remains a major hurdle. Cystatin A (CSTA, also called stefin A), an estrogen-regulated gene in breast cancer cells, is an inhibitor of cysteine cathepsins, and a purported tumor suppressor. Loss of CSTA expression in breast tumors evidently shifts the balance in favor of cysteine cathepsins, thereby promoting extracellular matrix remodeling, tumor invasion and metastasis. However, the underlying mechanism behind the loss of CSTA expression in breast tumors is not known. Here, we have analyzed CSTA expression, and methylation of upstream and intron-2 CpG sites within the CSTA locus in human breast cancer cell lines and breast tumors of the TCGA cohort. Results showed an inverse relationship between expression and methylation. Sequence analysis revealed a potential estrogen response element (ERE) in the intron-2. Analysis of ChIP-seq data (ERP000380) and our own ChIP experiments showed that 17ß-estradiol (E2) enhanced ERα binding to this ERE in MCF-7 cells. This ERE was located amidst the differentially methylated intron-2 CpG sites, which provoked us to examine the possible conflict between estrogen-regulation of CSTA and DNA methylation in the intron-2. We analyzed the expression of CSTA and its regulation by E2 in MDA-MB-231 and T47D cells subjected to global demethylation by 5-azacytidine (5-aza). 5-aza significantly demethylated intron-2 CpGs, and enhanced estrogen-induced ERα occupancy at the intron-2 ERE, leading to restoration of estrogen-regulation. Taken together, our results indicate that DNA methylation-dependent silencing could play a significant role in the loss of CSTA expression in breast tumors. The potential of DNA methylation as an indicator of CSTA expression or as a marker of tumor progression can be explored in future investigations. Furthermore, our results indicate the convergence of ERα-mediated estrogen regulation and DNA methylation in the intron-2, thereby offering a novel context to understand the role of estrogen-ERα signaling axis in breast tumor invasion and metastasis.

Increased levels of the megakaryocyte and platelet expressed cysteine proteases stefin A and cystatin A prevent thrombosis.

Increased platelet activity occurs in type 2 diabetes mellitus (T2DM) and such platelet dysregulation likely originates from altered megakaryopoiesis. We initiated identification of dysregulated pathways in megakaryocytes in the setting of T2DM. We evaluated through transcriptomic analysis, differential gene expressions in megakaryocytes from leptin receptor-deficient mice (db/db), exhibiting features of human T2DM, and control mice (db/+). Functional gene analysis revealed an upregulation of transcripts related to calcium signaling, coagulation cascade and platelet receptors in diabetic mouse megakaryocytes. We also evidenced an upregulation (7- to 9.7-fold) of genes encoding stefin A (StfA), the human ortholog of Cystatin A (CSTA), inhibitor of cathepsin B, H and L. StfA/CSTA was present in megakaryocytes and platelets and its expression increased during obesity and diabetes in rats and humans. StfA/CSTA was primarily localized at platelet membranes and granules and was released upon agonist stimulation and clot formation through a metalloprotease-dependent mechanism. StfA/CSTA did not affect platelet aggregation, but reduced platelet accumulation on immobilized collagen from flowing whole blood (1200 s-1). In-vivo, upon laser-induced vascular injury, platelet recruitment and thrombus formation were markedly reduced in StfA1-overexpressing mice without affecting bleeding time. The presence of CA-074Me, a cathepsin B specific inhibitor significantly reduced thrombus formation in-vitro and in-vivo in human and mouse, respectively. Our study identifies StfA/CSTA as a key contributor of platelet-dependent thrombus formation in both rodents and humans.

Stefin A-functionalized liposomes as a system for cathepsins S and L-targeted drug delivery.

Proteolytic activity in the tumor microenvironment is one of the key elements supporting tumor development and metastasis. One of the key families of proteases that are overexpressed in various types of cancer and implicated in different stages of tumor progression are cysteine cathepsins. Among them, cathepsins S and L can be secreted into the tumor microenvironment by tumor and/or immune cells, making them promising drug delivery targets. Here we present a new system for cathepsin S/L targeting using a liposomal drug carrier system functionalized with the endogenous cysteine cathepsin inhibitor, stefin A. The selective targeting of cathepsins by stefin A-conjugated liposomes was confirmed in vitro and in vivo, demonstrating the potential of this approach for cancer diagnosis and treatment.

Identification of CD37, cystatin A, and IL-23A gene expression in association with brain metastasis: analysis of a prospective trial.

PURPOSE/OBJECTIVES: We aimed to assess the predictive value of a lung cancer gene panel for the development of brain metastases. MATERIALS/METHODS: Between 2011 and 2015, 102 patients with lung cancer were prospectively enrolled in a clinical trial in which a diagnostic fine-needle aspirate was obtained. Gene expression was conducted on all samples that rendered a diagnosis of non-small cell lung cancer (NSCLC). Subsequent retrospective analysis of brain metastases-related outcomes was performed by reviewing patient electronic medical records. A competing risk multivariable regression was performed to estimate the adjusted hazard ratio for the development of brain metastases and non-brain metastases from NSCLC. RESULTS: A total of 49 of 102 patients had died by the last follow-up. Median time of follow-up was 13 months (range 0.23-67 months). A total of 17 patients developed brain metastases. Median survival time after diagnosis of brain metastases was 3.58 months (95% confidence interval (CI) 2.17, not available). A total of 30 patients developed metastases without any evidence of brain metastases until the time of death or last follow-up. Competing risk analysis identified three genes that were downregulated differentially in the patients with brain metastases versus non-brain metastatic disease: CD37 (0.017), cystatin A (0.022), and IL-23A (0.027). Other factors associated with brain metastases include: stage T ( P ⩽ 8.3e-6) and stage N ( P= 6.8e-4). CONCLUSIONS: We have identified three genes, CD37, cystatin A, and IL-23A, for which downregulation of gene expression was associated with a greater propensity for developing brain metastases. Validation of these biomarkers could have implications on surveillance patterns in patients with brain metastases from NSCLC.

Affimers as an Alternative to Antibodies in an Affinity LC-MS Assay for Quantification of the Soluble Receptor of Advanced Glycation End-Products (sRAGE) in Human Serum.

Antibodies are indispensable tools in biomedical research, but their size, complexity, and sometimes lack of reproducibility created a need for the development of alternative binders to overcome these limitations. Affimers are a novel class of affinity binders based on a structurally robust protease inhibitor scaffold (i.e., Cystatin A), which are selected by phage display and produced in a rapid and simple E. coli protein expression system. These binders have a defined amino acid sequence with defined binding regions and are versatile, thereby allowing for easy engineering. Here we present an affimer-based liquid chromatography-mass spectrometry (LC-MS) method for quantification of the soluble Receptor of Advanced Glycation End-products (sRAGE), a promising biomarker for chronic obstructive pulmonary disease. The method was validated according to European Medicines Agency and U.S. Food and Drug Administration guidelines and enabled quantitation of serum sRAGE between 0.2 and 10 ng/mL. Comparison between the affimer-based method and a previously developed, validated antibody-based method showed good correlation ( R2 = 0.88) and indicated that 25% lower sRAGE levels are reported by the affimer-based assay. In conclusion, we show the first-time application of affimers in a quantitative LC-MS method, which supports the potential of affimers as robust alternatives to antibodies.

Clinicopathological significance of cystatin A expression in progression of esophageal squamous cell carcinoma.

We previously conducted transcriptome analysis of a paired specimen of normal and esophageal squamous cell carcinoma (ESCC) tissues and found that mRNA expression of cystatin A (CSTA), a member of the cystatin superfamily, was perturbed in tumors compared with that in the background mucosa. However, little is known about the significance of CSTA expression in ESCC.The mRNA expression of CSTA was evaluated by qRT-PCR using 28 paired frozen samples of tumor and nontumor mucosae. The protein expression of CSTA was evaluated by the immunostaining of formalin-fixed, paraffin-embedded sections of ESCC samples from 59 patients who underwent surgery, and its relationship with clinical features was analyzed.The mRNA expression of CSTA was significantly decreased in ESCC compared with that in matched normal mucosa (P < .0001). The protein expression of CSTA was limited in stratum granulosum and stratum spinosum but not in stratum basal in normal esophageal mucosa. It was reduced in all ESCC tissue samples compared with normal tissues; however, CSTA expression levels in tumors showed considerable variation. Of the 59 samples, 20 did not express CSTA, whereas 39 clearly expressed it. The expression of CSTA in tumors was significantly associated with pT classification (deeper tumor invasions) (P = .0118) and advanced TNM stages (P = .0497). In CSTA-positive tumor samples, CSTA-expressing cancer cells often expressed Ki67, a proliferation marker, which was in sharp contrast to normal mucosa, where Ki67-expressing cells were limited to the basal layer and did not express CSTA. Furthermore, CSTA expression was observed in all 22 lymph node metastases analyzed.Relatively high levels of CSTA expression in tumors were correlated with tumor progression and advanced cancer stage, including lymph node metastasis.

Dual roles of cystatin A in the immune defense of the pacific oyster, Crassostrea gigas.

Cystatins are a large family of the proteins that function as reversible and tight-binding inhibitors of cysteine proteases, which consequently regulate multiple physiological activities including apoptosis and innate immunity. In the present study, we cloned a gene from Crassostrea gigas encoding cystatin, which is related to cystatin A superfamily. CgCytA was comprised of a cystatin-like domain with two conserved glycine residues (GG) near the N-terminal and a highly conserved glutamine-valine-glycine (Q-X-V-X-G) motif in the form of QVVAG loop. Transcription analysis of CgCytA indicated its constitutive expression in all tissues including mantle, gill, digestive tract, hemocytes, heart, adductor muscle, and gonads. Immune challenge with Vibrio alginolyticus, resulted in significant down-regulation of CgCytA expression at the initial stages of infection (till 12 h post infection) and the expression of cystatin increased 48 h post infection. Protease assay demonstrated the concentration of cystatin needed to inhibit half of the maximum biological response of cysteine protease is 14.4 µg/L (IC50). Furthermore, RNAi of CgCytA resulted in increase of apoptotic cell population in hemocytes of C. gigas, suggesting protection role of CgCytA from hemocytes apoptosis. Unexpectedly, knockdown of CgCytA leaded to enhancement of bacterial clearance in vivo, implying that CgCytA may negatively regulate immune defense by suppressing endogenous cysteine protease. Therefore, CgCytA plays dual roles in protection of host hemocytes from apoptosis and control of bacterial clearance, which may server as one of key endogenous balancer between apoptosis and innate immunity in oyster.