Acral peeling skin syndrome resulting from a novel homozygous mutation in the CSTA gene-A report of two cases.

Acral peeling skin syndrome is a rare genodermatosis characterized by asymptomatic peeling of the acral skin. It is usually caused by biallelic mutations in the gene TGM5. However, biallelic mutations in the CSTA gene have also been described to cause APSS with exfoliative ichthyosis, so far in only five pedigrees. Here, we report two new pedigrees, each with one patient having APSS, due to a novel CSTA mutation.

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.

Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion.

Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Salivary Cystatins: Exploring New Post-Translational Modifications and Polymorphisms by Top-Down High-Resolution Mass Spectrometry.

Cystatins are a complex family of cysteine peptidase inhibitors. In the present study, various proteoforms of cystatin A, cystatin B, cystatin S, cystatin SN, and cystatin SA were detected in the acid-soluble fraction of human saliva and characterized by a top-down HPLC-ESI-MS approach. Proteoforms of cystatin D were also detected and characterized by an integrated top-down and bottom-up strategy. The proteoforms derive from coding sequence polymorphisms and post-translational modifications, in particular, phosphorylation, N-terminal processing, and oxidation. This study increases the current knowledge of salivary cystatin proteoforms and provides the basis to evaluate possible qualitative/quantitative variations of these proteoforms in different pathological states and reveal new potential salivary biomarkers of disease. Data are available via ProteomeXchange with identifier PXD007170.

Clinical features of cystatin A expression in patients with pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy known, with an extremely poor prognosis due to the lack of an efficient diagnostic scheme and no radical treatment option, except surgery. Therefore, understanding the pathophysiology of, and finding a novel biomarker to detect, PDAC should be prioritized. We observed an increase in mRNA expression of the cysteine protease inhibitor cystatin A (CSTA) in CD4+ T cells in peripheral blood cells of nine patients with PDAC, compared with the expression in seven healthy volunteers. Moreover, we confirmed significantly higher CSTA mRNA expression in a larger cohort of 41 patients with PDAC compared with that in 20 healthy volunteers. Correspondingly, the serum CSTA concentrations in 36 patients with PDAC were higher than those in 37 healthy volunteers, and this increase was correlated with PDAC clinical stage. Furthermore, the expression of CSTA and cathepsin B, which is a lysosomal cysteine protease inhibited by CSTA, was observed in tumor tissues and tumor-infiltrating immune cells in 20 surgically resected PDAC tissues by immunohistochemical staining. Expression of CSTA was detected in some tumor tissues and many tumor-infiltrating immune cells. Cathepsin B expression was also observed in most tumor tissues and tumor-infiltrating immune cells. In conclusion, CSTA and its substrate cathepsin B are involved in PDAC-related inflammation. The increment of CSTA expression in peripheral blood of patients with PDAC may have a potential role as a PDAC immunopathologic biomarker.

Identification and validation of Cystatin A as a novel promising therapeutic target for gastric cancer.

Background: The effect of pharmacological treatment of gastric cancer (GC) is limited, thus, it holds significant scientific importance to thoroughly investigate the molecular mechanisms underlying GC development and identify novel molecules capable of substantially extending patients' survival. This study utilized bioinformatics techniques to identify 11 genes associated with recurrence-free survival (RFS) in GC patients and investigated the potential biological functions of these genes through single-cell transcriptomic analysis. Subsequently, a single gene Cystatin A (CSTA) was selected for further analysis to explore its impact on signaling pathways and treatment. Methods: Differentially expressed genes (DEGs) were identified and overlapped in the analysis of RFS to identify potential prognostic genes for GC patients, based on data from the Cancer Genome Atlas-stomach adenocarcinoma (TCGA-STAD) and GSE54129. Subsequently, a prognostic model based on RFS in GC patients was established. Single-cell sequencing data were employed to explore the potential functions of these model genes. CSTA, one of the RFS-related genes, was further investigated using immunohistochemistry (IHC), Cell Counting Kit 8 (CCK-8), transwell, scratch, colony formation assays, flow cytometry, and Western blotting methods. Results: Through bioinformatics analysis, we identified 23 RFS-related genes in GC. Using the least absolute shrinkage and selection operator (LASSO)-Cox method, an RFS prognostic model was developed which pinpointed 11 GC prognosis-related (GPR) genes as significant factors influencing RFS in GC patients. The single-cell analysis revealed their potential role in affecting differentiation and maturation of pre-fibroblasts thereby impacting RFS in GC patients. CSTA exhibited low expression levels in GC tissues. Overexpression of CSTA promoted apoptosis in GC cells through the caspase-dependent apoptotic pathway and enhanced their response to cisplatin via this same pathway. Conclusions: The 11 GPR genes are primarily enriched within a specific type of stromal cell exhibiting heightened communication, metabolism, and differentiation levels. The gene signature of these stromal cells has implications for patient prognosis. Additionally, CSTA, a gene related to prognosis, has been shown to influence apoptosis levels in GC cells.

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.

Transcriptional factor III A promotes colorectal cancer progression by upregulating cystatin A.

BACKGROUND: Advanced colorectal cancer (CRC) generally has poor outcomes and high mortality rates. Clarifying the molecular mechanisms underlying CRC progression is necessary to develop new diagnostic and therapeutic strategies to improve CRC outcome and decrease mortality. Transcriptional factor III A (GTF3A), an RNA polymerase III transcriptional factor, is a critical driver of tumorgenesis and aggravates CRC cell growth. AIM: To confirm whether GTF3A promotes CRC progression by regulating the expression of cystatin A (Csta) gene and investigate whether GTF3A can serve as a prognostic biomarker and therapeutic target for patients with CRC. METHODS: Human tissue microarrays containing 90 pairs of CRC tissues and adjacent non-tumor tissues, and human tissue microarrays containing 20 pairs of CRC tissues, adjacent non-tumor tissues, and metastatic tissues were examined for GTF3A expression using immunohistochemistry. The survival rates of patients were analyzed. Short hairpin GTF3As and CSTAs were designed and packaged into the virus to block the expression of Gtf3a and Csta genes, respectively. In vivo tumor growth assays were performed to confirm whether GTF3A promotes CRC cell proliferation in vivo. Electrophoretic mobility shift assay and fluorescence in situ hybridization assay were used to detect the interaction of GTF3A with Csta, whereas luciferase activity assay was used to evaluate the expression of the Gtf3a and Csta genes. RNA-Sequencing (RNA-Seq) and data analyses were used to screen for target genes of GTF3A. RESULTS: The expression of GTF3A was higher in CRC tissues and lymph node metastatic tissues than in the adjacent normal tissues. GTF3A was associated with CRC prognosis, and knockdown of the Gtf3a gene impaired CRC cell proliferation, invasion, and motility in vitro and in vivo. Moreover, RNA-Seq analysis revealed that GTF3A might upregulate the expression of Csta, whereas the luciferase activity assay showed that GTF3A bound to the promoter of Csta gene and increased Csta transcription. Furthermore, CSTA regulated the expression of epithelial-mesenchymal transition (EMT) markers. CONCLUSION: GTF3A increases CSTA expression by binding to the Csta promoter, and increased CSTA level promotes CRC progression by regulating the EMT. Inhibition of GTF3A prevents CRC progression. Therefore, GTF3A is a potential novel therapeutic target and biomarker for CRC.

Sequestration of Intestinal Acidic Toxins by Cationic Resin Attenuates Pancreatic Cancer Progression through Promoting Autophagic Flux for YAP Degradation.

Pancreatic cancer is driven by risk factors such as diabetes and chronic pancreatic injury, which are further associated with gut dysbiosis. Intestinal toxins such as bile acids and bacterial endotoxin (LPS), in excess and persistence, can provoke chronic inflammation and tumorigenesis. Of interest is that many intestinal toxins are negatively charged acidic components in essence, which prompted us to test whether oral administration of cationic resin can deplete intestinal toxins and ameliorate pancreatic cancer. Here, we found that increased plasma levels of endotoxin and bile acids in Pdx1-Cre: LSL-KrasG12D/+ mice were associated with the transformation of the pancreatic ductal carcinoma (PDAC) state. Common bile-duct-ligation or LPS injection impeded autolysosomal flux, leading to Yap accumulation and malignant transformation. Conversely, oral administration of cholestyramine to sequestrate intestinal endotoxin and bile acids resumed autolysosomal flux for Yap degradation and attenuated metastatic incidence. Conversely, chloroquine treatment impaired autolysosomal flux and exacerbated malignance, showing jeopardization of p62/ Sqxtm1 turnover, leading to Yap accumulation, which is also consistent with overexpression of cystatin A (CSTA) in situ with pancreatic cancer cells and metastatic tumor. At cellular levels, chenodeoxycholic acid or LPS treatment activated the ligand-receptor-mediated AKT-mTOR pathway, resulting in autophagy-lysosomal stress for YAP accumulation and cellular dissemination. Thus, this work indicates a potential new strategy for intervention of pancreatic metastasis through sequestration of intestinal acidic toxins by oral administration of cationic resins.

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.

Expression of Cathepsin B and Cystatin A in Oral Lichen Planus.

OBJECTIVE: Cathepsin B (Cat-B), a cysteine protease, and cystatin A (Cys-A), a protease inhibitor, are involved in the immune response. This study determined Cat-B and Cys-A expression in oral lichen planus (OLP) by immunohistochemistry. MATERIALS AND METHODS: Thirty specimens each of OLP and healthy gingiva (HG) were included. The expression pattern, the number of positive cells, the staining intensity, and the immunoreactive score (IRS) of Cat-B and Cys-A were investigated. The data were analyzed by using unpaired t-test, Chi-square, and Spearman's rank correlation. RESULTS: The Cat-B expression in OLP was observed as cytoplasmic staining in the epithelial cells, whereas Cys-A expression was exhibited in the nucleus and cytoplasm of the epithelium. An increase in Cat-B staining intensity was also observed in the basal cells. Conversely, the high staining intensity of Cys-A was observed in the stratum spinosum, but not the stratum basale. In HG, Cat-B expression demonstrated a relatively consistent intensity in the epithelial layer. The Cys-A expression in HG was similar to OLP with a lower staining intensity. The mean percentage of positive cells and the IRS score of Cat-B and Cys-A in OLP were significantly higher than HG (P < 0.05). There was no correlation between Cat-B and Cys-A levels in OLP. Interestingly, Cat-B expression in erosive OLP was greater than in non-erosive OLP (P < 0.05). CONCLUSION: The Cat-B and Cys-A expression in OLP was more outstanding than in HG, suggesting possible roles for the process of OLP pathogenesis. In addition, Cat-B expression may be an indicator of the disease severity.

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.

Cystatin A suppresses tumor cell growth through inhibiting epithelial to mesenchymal transition in human lung cancer.

Cystatin A (CSTA), belonging to type 1 cystatin super-family, is expressed primarily in epithelial and lymphoid tissues for protecting cells from proteolysis of cytoplasmic and cytoskeletal proteins by cathepsins B, H and L. CSTA acts as a tumor suppressor in esophageal cancer, however, its role in lung cancer has not yet been elucidated. Here we found that CSTA was down-regulated in all lung cancer cell lines compared to normal lung epithelial cells. CSTA was restored in most lung cancer cell lines after treatment with demethylation agent 5-aza-2-deoxycytidine and deacetylation agent Trichostatin. Bisulfite sequencing revealed that CSTA was partially methylated in the promoter and exon 1. In primary lung tumors, squamous cell carcinoma (SCC) significantly expressed more CSTA compared to adenocarcinoma (p<0.00001), and higher expression of CSTA was significantly associated with lower tumor grade (p<0.01). CSTA stable transfection reduced the activity of cathepsin B and inhibited the ability of colony formation, migration and invasion, and enhanced gemcitabine-induced apoptosis. CSTA overexpression resulted in reduced activity of ERK, p-38, and AKT. Additionally, CSTA overexpression led to a mesenchymal to epithelial transition (MET) and prevented the TGF-ß1-induced epithelial to mesenchymal transition (EMT) through inhibiting the ERK/MAPK pathway. In conclusion, our date indicate 1) epigenetic regulation is associated with CSTA gene silencing; 2) CSTA exerts tumor suppressive function through inhibiting MAPK and AKT pathways; 3) Overexpression of CSTA leads to MET and prevents TGF-ß1-induced EMT by modulating the MAPK pathway; 4) CSTA may be a potential biomarker for lung SCC and tumor differentiation.

Quantitative analysis of gene expression changes in response to genotoxic compounds.

Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n=18) chosen to represent compounds that are genotoxic (n=8), non-genotoxic non-carcinogenic (n=2) or have a less well defined mechanism of action with respect to genotoxicity (n=8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1fmol but often 10-50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology.