Quiescin Sulfhydryl Oxidase 1 Research Articles

QSOX1 Modulates Glioblastoma Cell Proliferation and Migration In Vitro and Invasion In Vivo.

Background: Quiescin Sulfhydryl Oxidase 1 (QSOX1) is an enzyme that catalyzes the oxidation of free thiols to generate disulfide bonds in a variety of proteins, including the cell surface and extracellular matrix. QSOX1 has been reported to be upregulated in a number of cancers, and the overexpression of QSOX1 has been correlated with aggressive cancers and poor patient prognosis. Glioblastoma (GBM) brain cancer has been practically impossible to treat effectively, with cells that rapidly invade normal brain tissue and escape surgery and other treatment. Thus, there is a crucial need to understand the multiple mechanisms that facilitate GBM cell invasion and to determine if QSOX1 is involved. Methods and Results: Here, we investigated the function of QSOX1 in human glioblastoma cells using two cell lines derived from T98G cells, whose proliferation, motility, and invasiveness has been shown by us to be dependent on disulfide bond-containing adhesion and receptor proteins, such as L1CAM and the FGFR. We lentivirally introduced shRNA to attenuate the QSOX1 protein expression in one cell line, and a Western blot analysis confirmed the decreased QSOX1 expression. A DNA content/cell cycle analysis using flow cytometry revealed 27% fewer knockdown cells in the S-phase of the cell cycle, indicating a reduced proliferation. A cell motility analysis utilizing our highly quantitative SuperScratch time-lapse microscopy assay revealed that knockdown cells migrated more slowly, with a 45% decrease in migration velocity. Motility was partly rescued by the co-culture of knockdown cells with control cells, indicating a paracrine effect. Surprisingly, knockdown cells exhibited increased motility when assayed using a Transwell migration assay. Our novel chick embryo orthotopic xenograft model was used to assess the in vivo invasiveness of knockdown vs. control cells, and tumors developed from both cell types. However, fewer invasive knockdown cells were observed after about a week. Conclusions: Our results indicate that an experimental reduction in QSOX1 expression in GBM cells leads to decreased cell proliferation, altered in vitro migration, and decreased in vivo invasion.

QSOX1 facilitates dormant esophageal cancer stem cells to evade immune elimination via PD-L1 upregulation and CD8 T cell exclusion

Dormant cancer stem cells (DCSCs) exhibit characteristics of chemotherapy resistance and immune escape, and they are a crucial source of tumor recurrence and metastasis. However, the underlying mechanisms remain unrevealed. We demonstrate that enriched Gzmk+ CD8+ T cells within the niche of esophageal DCSCs restrict the outgrowth of tumor mass. Nonetheless, DCSCs can escape immune elimination by enhancing PD-L1 signaling, thereby maintaining immune equilibrium. Quiescent fibroblast-derived quiescin sulfhydryl oxidase 1 (QSOX1) promotes the expression of PD-L1 and its own expression in DCSCs by elevating the level of reactive oxygen species. Additionally, high QSOX1 in the dormant tumor niche contributes to the exclusion of CD8+ T cells. Conversely, blocking QSOX1 with Ebselen in combination with anti-PD-1 and chemotherapy can effectively eradicate residual DCSCs by reducing PD-L1 expression and promoting CD8+ T cell infiltration. Clinically, high expression of QSOX1 predicts a poor response to anti-PD-1 treatment in patients with esophageal cancer. Thus, our findings reveal a mechanism whereby QSOX1 promotes PD-L1 upregulation and T cell exclusion, facilitating the immune escape of DCSCs, and QSOX1 inhibition, combined with immunotherapy and chemotherapy, represents a promising therapeutic approach for eliminating DCSCs and preventing recurrence.

S-nitrosylation switches the Arabidopsis redox sensor protein, QSOX1, from an oxidoreductase to a molecular chaperone under heat stress

The Arabidopsis quiescin sulfhydryl oxidase 1 (QSOX1) thiol-based redox sensor has been identified as a negative regulator of plant immunity. Here, we have found that small molecular weight proteins of QSOX1 were converted to high molecular weight (HMW) complexes upon exposure to heat stress and that this was accompanied by a switch in QSOX1 function from a thiol-reductase to a molecular chaperone. Plant treatment with S-nitrosoglutathione (GSNO), which causes nitrosylation of cysteine residues (S-nitrosylation), but not with H2O2, induced HMW QSOX1 complexes. Thus, functional switching of QSOX1 is induced by GSNO treatment. Accordingly, simultaneous treatment of plants with heat shock and GSNO led to a significant increase in QSOX1 chaperone activity by increasing its oligomerization. Consequently, transgenic Arabidopsis overexpressing QSOX1 (QSOX1OE) showed strong resistance to heat shock, whereas qsox1 knockout plants exhibited high sensitivity to heat stress. Plant treatment with GSNO under heat stress conditions increased their resistance to heat shock. We conclude that S-nitrosylation allows the thiol-based redox sensor, QSOX1, to respond to various external stresses in multiple ways.

Aberrant m5C hypermethylation mediates intrinsic resistance to gefitinib through NSUN2/YBX1/QSOX1 axis in EGFR-mutant non-small-cell lung cancer

BackgroundRNA 5-methylcytosine (m5C) modification plays critical roles in the pathogenesis of various tumors. However, the function and molecular mechanism of RNA m5C modification in tumor drug resistance remain unclear.MethodsThe correlation between RNA m5C methylation, m5C writer NOP2/Sun RNA methyltransferase family member 2 (NSUN2) and EGFR-TKIs resistance was determined in non-small-cell lung cancer (NSCLC) cell lines and patient samples. The effects of NSUN2 on EGFR-TKIs resistance were investigated by gain- and loss-of-function assays in vitro and in vivo. RNA-sequencing (RNA-seq), RNA bisulfite sequencing (RNA-BisSeq) and m5C methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the target gene of NSUN2 involved in EGFR-TKIs resistance. Furthermore, the regulatory mechanism of NSUN2 modulating the target gene expression was investigated by functional rescue and puromycin incorporation assays.ResultsRNA m5C hypermethylation and NSUN2 were significantly correlated with intrinsic resistance to EGFR-TKIs. Overexpression of NSUN2 resulted in gefitinib resistance and tumor recurrence, while genetic inhibition of NSUN2 led to tumor regression and overcame intrinsic resistance to gefitinib in vitro and in vivo. Integrated RNA-seq and m5C-BisSeq analyses identified quiescin sulfhydryl oxidase 1 (QSOX1) as a potential target of aberrant m5C modification. NSUN2 methylated QSOX1 coding sequence region, leading to enhanced QSOX1 translation through m5C reader Y-box binding protein 1 (YBX1).ConclusionsOur study reveals a critical function of aberrant RNA m5C modification via the NSUN2-YBX1-QSOX1 axis in mediating intrinsic resistance to gefitinib in EGFR-mutant NSCLC.

A Golgi oxygen sensor controls intestinal mucin glycosylation.

Intestinal mucin glycosylation is important for mucus-bacterial homeostasis and is altered in disease. In this issue of The EMBO Journal, Ilani etal (2022) identify the Golgi enzyme quiescin sulfhydryl oxidase 1 (QSOX1) as a novel mucus regulator by controlling mucin sialylation.

Secretion of the disulfide bond generating catalyst QSOX1 from pancreatic tumor cells into the extracellular matrix: association with extracellular vesicles and matrix proteins.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a disulfide bond generating catalyst that is overexpressed in solid tumors. Expression of QSOX1 is linked to cancer cell invasion, tumor grade, and extracellular matrix (ECM) protein deposition. While the secreted version of QSOX1 is known to be present in various fluids and secretory tissues, its presence in the ECM of cancer is less understood. To characterize secreted QSOX1, we separated conditioned media based on size and density. We discovered that the majority of secreted QSOX1 resides in the EV-depleted fraction and in the soluble protein fraction. Very little QSOX1 could be detected in the EVP fraction. We used immunofluorescence to image subpopulations of EVs and found QSOX1 in Golgi-derived vesicles and medium/large vesicles, but in general, most extracellular QSOX1 was not attributed to these vesicles. Next, we quantified QSOX1 co-localization with the EV marker Alix. For the medium/large EVs, ~98% contained QSOX1 when fibronectin was used as a coating. However, on collagen coatings, only ~60% of these vesicles contained QSOX1, suggesting differences in EV cargo based on ECM coated surfaces. About 10% of small EVs co-localized with QSOX1 on every ECM protein surface except for collagen (0.64%). We next investigated adhesion of QSOX1 to ECM proteins in vitro and in situ and found that QSOX1 preferentially adheres to fibronectin, laminins, and Matrigel compared to gelatin and collagen. This mechanism was found to be, in part, mediated by the formation of mixed disulfides between QSOX1 and cysteine-rich ECM proteins. In summary, we found that QSOX1 (1) is in subpopulations of medium/large EVs, (2) seems to interact with small Alix+ EVs, and (3) adheres to cysteine-rich ECM proteins, potentially through the formation of intermediate disulfides. These observations offer significant insight into how enzymes, such as QSOX1, can facilitate matrix remodeling events in solid tumor progression.

Polarized epithelium-sperm co-culture system reveals stimulatory factors for the secretion of mouse epididymal quiescin sulfhydryl oxidase 1.

Spermatozoa acquire fertilization ability through post-translational modifications. These membrane surface alterations occur in various segments of the epididymis. Quiescin sulfhydryl oxidases, which catalyze thiol-oxidation reactions, are involved in disulfide bond formation, which is essential for sperm maturation, upon transition and migration in the epididymis. Using castration and azoospermia transgenic mouse models, in the present study, we showed that quiescin sulfhydryl oxidase 1 (QSOX1) protein expression and secretion are positively correlated with the presence of testosterone and sperm cells. A two-dimensional in vitro epithelium-sperm co-culture system provided further evidence in support of the notion that both testosterone and its dominant metabolite, 5α-dihydrotestosterone, promote epididymal QSOX1 secretion. We also demonstrated that immature caput spermatozoa, but not mature cauda sperm cells, exhibited great potential to stimulate QSOX1 secretion in vitro, suggesting that sperm maturation is a key regulatory factor for mouse epididymal QSOX1 secretion. Proteomic analysis identified 582 secretory proteins from the co-culture supernatant, of which 258 were sperm-specific and 154 were of epididymal epithelium-origin. Gene Ontology analysis indicated that these secreted proteins exhibit functions known to facilitate sperm membrane organization, cellular activity, and sperm-egg recognition. Taken together, our data demonstrated that testosterone and sperm maturation status are key regulators of mouse epididymal QSOX1 protein expression and secretion.

Quiescin sulfhydryl oxidase 1 promotes sorafenib-induced ferroptosis in hepatocellular carcinoma by driving EGFR endosomal trafficking and inhibiting NRF2 activation

Sorafenib is a first-line molecular-target drug for advanced hepatocellular carcinoma (HCC), but its clinical effects are still limited. In this study we identify Quiescin sulfhydryl oxidase 1 (QSOX1) acting as a cellular pro-oxidant, specifically in the context of sorafenib treatment of HCC. QSOX1 disrupts redox homoeostasis and sensitizes HCC cells to oxidative stress by inhibiting activation of the master antioxidant transcription factor NRF2. A negative correlation between QSOX1 and NRF2 expression was validated in tumor tissues from 151 HCC patients. Mechanistically, QSOX1 restrains EGF-induced EGFR activation by promoting ubiquitination-mediated degradation of EGFR and accelerating its intracellular endosomal trafficking, leading to suppression of NRF2 activity. Additionally, QSOX1 potentiates sorafenib-induced ferroptosis by suppressing NRF2 in vitro and in vivo. In conclusion, the data presented identify QSOX1 as a novel candidate target for sorafenib-based combination therapeutic strategies in HCC or other EGFR-dependent tumor types.

Abstract 5323: Chemical inhibitors of quiescin sulfhydryl oxidase (QSOX1) suppress tumor invasion and metastasis

Abstract Purpose: Quiescin Sulfhydryl Oxidase 1 (QSOX1) is an enzyme over-expressed by tumor cells. Our group and others have shown that QSOX1 is involved in invasion and metastasis. We previously reported that a small molecule, SBI-183, inhibited the enzymatic activity of QSOX1 and suppressed tumor cell invasion in vitro and metastasis of MDA-MB-231 breast tumor cells in vivo. In our current work we tested SPX-009, an analog of SBI-183 with increased potency, to suppress invasion and metastasis in vitro and in vivo. Methods: Commercially available analogs for SBI-183 were screened in a cell-free fluorescent screening assay for the ability to inhibit QSOX1. Analog compounds that inhibited QSOX1 in a concentration-dependent manner were then tested for the ability to inhibit growth and invasion of triple negative breast cancer (TNBC) cells (MDA-MB-231), sarcomatoid kidney cancer cells (RCJ-41T2) and pancreatic adenocarcinoma cells (MIAPaCa2). MTT assays were used to measure the effect of the compounds on tumor growth. 2D invasion assays and 3D tumor spheroid assays were employed to measure the effect of the analogs on invasion. One potent analog (SPX-009) was evaluated for the ability to suppress tumor growth and metastasis in mice bearing MDA-MB-231-luc breast cancer orthotopic xenografts. Results: Three of 53 analogs inhibited the enzymatic activity of QSOX1 at levels similar to the parent compound. However, one of those compounds, SPX-009, suppressed invasion of breast, kidney and pancreas tumor cells in 2D and 3D invasion assays at >10-fold lower concentrations than the parent compound (SBI-183). In a non-metastatic xenograft of kidney cancer SBI-183 suppressed primary kidney tumor growth by 51%, but in a metastatic xenograft breast cancer model (MDA-MB-231-luc TNBC), SBI-183 reduced lung metastasis by 76% compared to control. Human-mouse xenograft studies are ongoing with the more potent analog (SPX-009) and will also be presented. Conclusions: Chemical inhibitors of QSOX1 have a minor effect on tumor cell viability, but a major effect on tumor cell invasion in vitro and metastasis in vivo. This finding suggests that combining a QSOX1 inhibitor with a cytotoxic agent might provide a means to inhibit metastasis of cells that are resistant to cytotoxic agents. Citation Format: Amber Fifield, Thai H. Ho, Douglas O. Faigel, Sergei Svarovsky, Eduard Sergienko, Thomas C. Chung, Douglas F. Lake. Chemical inhibitors of quiescin sulfhydryl oxidase (QSOX1) suppress tumor invasion and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5323.

Quiescin Sulfhydryl Oxidase 1 Regulates the Proliferation, Migration and Invasion of Human Glioblastoma Cells via PI3K/Akt Pathway.

BackgroundQuiescin sulfhydryl oxidase 1 (QSOX1) involves in the formation of disulfide bonds and participates in the protein folding process. In recent years, accumulating evidences have shown that QSOX1 is a biomarker for tumor development and prognosis. However, the biological function of QSOX1 in glioblastoma (GBM) remains unclear.Materials and MethodsQSOX1 expression in glioma and overall survival of glioma patients were analyzed through The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) databases. shRNAs were used to decrease the expression of QSOX1 in U87 and U251 cells. Celltiter-Glo and colony formation assays were used to assess cell proliferation. Transwell and scratch assays were utilized to determine cell migration and invasion, the xenograft mouse models were established to evaluate the effect of QSOX1 knockdown in vivo. Western blot assays were used to detect the changes of E-cadherin/N-cadherin/vimentin and PI3K/Akt pathway.ResultsWe found that QSOX1 was upregulated in glioma, especially in GBM. Upregulation of QSOX1 was correlated with poor prognosis in glioma patients. We discovered for the first time that suppression of QSOX1 expression inhibited proliferation, migration and invasion, as well as epithelial-mesenchymal transition (EMT) in GBM cell lines. In addition, phosphorylated PI3K and Akt were downregulated in the QSOX1-knockdown groups. Moreover, QSOX1 knockdown-impaired cell growth was partially rescued by Akt activator.ConclusionOur findings revealed that QSOX1 was a novel biomarker for GBM patients and QSOX1 promoted cell proliferation, migration and invasion through regulating PI3K/Akt pathway in GBM.

Inhibition of fibroblast secreted QSOX1 perturbs extracellular matrix in the tumor microenvironment and decreases tumor growth and metastasis in murine cancer models.

Extracellular matrix (ECM) plays an important role in tumor development and dissemination, but few points of therapeutic intervention targeting ECM of the tumor microenvironment have been exploited to date. Recent observations suggest that the enzymatic introduction of disulfide bond cross-links into the ECM may be modulated to affect cancer progression. Specifically, the disulfide bond-forming activity of the enzyme Quiescin sulfhydryl oxidase 1 (QSOX1) is required by fibroblasts to assemble ECM components for adhesion and migration of cancer cells. Based on this finding and the increased QSOX1 expression in the stroma of aggressive breast carcinomas, we developed monoclonal antibody inhibitors with the aim of preventing QSOX1 from participating in pro-metastatic ECM remodeling. Here we show that QSOX1 inhibitory antibodies decreased tumor growth and metastasis in murine cancer models and had added benefits when provided together with chemotherapy. Mechanistically, the inhibitors dampened stromal participation in tumor development, as the tumors of treated animals showed fewer myofibroblasts and poorer ECM organization. Thus, our findings demonstrate that specifically targeting excess stromal QSOX1 secreted in response to tumor-cell signaling provides a means to modulate the tumor microenvironment and may complement other therapeutic approaches in cancer.

Quiescin/sulfhydryl oxidase 1b (QSOX1b) induces migration and proliferation of vascular smooth muscle cells by distinct redox pathways

Quiescent and contractile VSMC can switch to proliferative and migratory phenotype in response to growth factors and cytokines, an effect underscored by Nox family NADPH oxidases, particularly Nox1. We previously showed that quiescin/sulfhydryl oxidase 1 (QSOX1) has a role in neointima formation in balloon-injured rat carotid. Here, we investigated the intracellular redox mechanisms underlying these effects in primary VSMC. Our results show that exogenous incubation with wild type QSOX1b (wt QSOX), or with secreted QSOX1, but not with the inactive C452S QSOX 1b (C452S QSOX) or secreted inactive C455S QSOX1, induces VSMC migration and chemotaxis. PEG-catalase (PEG-CAT) prevented, while PEG-superoxide dismutase (PEG-SOD) increased migration induced by wt QSOX. Moreover, wt QSOX-induced migration was abrogated in NOX1-null VSMC. In contrast, both wt QSOX and C452S QSOX, and both secreted QSOX1 and C455S QSOX1, induce cell proliferation. Such effect was unaltered by PEG-CAT, while being inhibited by PEG-SOD. However, QSOX1-induced proliferation was not significantly affected in NOX1-null VSMC, compared with WT VSMC. These results indicate that hydrogen peroxide and superoxide mediate, respectively, migration and proliferation. However, Nox1 was required only for QSOX1-induced migration. In parallel, QSOX1-induced proliferation was independent of its redox activity, although mediated by intracellular superoxide.

Cis-Proline mutants of quiescin sulfhydryl oxidase 1 with altered redox properties undermine extracellular matrix integrity and cell adhesion in fibroblast cultures.

The thioredoxin superfamily has expanded and diverged extensively throughout evolution such that distant members no longer show appreciable sequence homology. Nevertheless, redox‐active thioredoxin‐fold proteins functioning in diverse physiological contexts often share canonical amino acids near the active‐site (di‐)cysteine motif. Quiescin sulfhydryl oxidase 1 (QSOX1), a catalyst of disulfide bond formation secreted by fibroblasts, is a multi‐domain thioredoxin superfamily enzyme with certain similarities to the protein disulfide isomerase (PDI) enzymes. Among other potential functions, QSOX1 supports extracellular matrix assembly in fibroblast cultures. We introduced mutations at a cis‐proline in QSOX1 that is conserved across the thioredoxin superfamily and was previously observed to modulate redox interactions of the bacterial enzyme DsbA. The resulting QSOX1 variants showed a striking detrimental effect when added exogenously to fibroblasts: they severely disrupted the extracellular matrix and cell adhesion, even in the presence of naturally secreted, wild‐type QSOX1. The specificity of this phenomenon for particular QSOX1 mutants inspired an investigation of the effects of mutation on catalytic and redox properties. For a series of QSOX1 mutants, the detrimental effect correlated with the redox potential of the first redox‐active site, and an X‐ray crystal structure of one of the mutants revealed the reorganization of the cis‐proline loop caused by the mutations. Due to the conservation of the mutated residues across the PDI family and beyond, insights obtained in this study may be broadly applicable to a variety of physiologically important redox‐active enzymes.Impact statementWe show that mutation of a conserved cis‐proline amino acid, analogous to a mutation used to trap substrates of a bacterial disulfide catalyst, has a dramatic effect on the physiological function of the mammalian disulfide catalyst QSOX1. As the active‐site region of QSOX1 is shared with the large family of protein disulfide isomerases in humans, the effects of such mutations on redox properties, enzymatic activity, and biological targeting may be relevant across the family.

Quiescin Sulfhydryl Oxidase 1 (QSOX1) Secreted by Lung Cancer Cells Promotes Cancer Metastasis.

As lung cancer shows the highest mortality in cancer-related death, serum biomarkers are demanded for lung cancer diagnosis and its treatment. To discover lung cancer protein biomarkers, secreted proteins from primary cultured lung cancer and adjacent normal tissues from patients were subjected to LC/MS–MS proteomic analysis. Quiescin sulfhydryl oxidase (QSOX1) was selected as a biomarker candidate from the enriched proteins in the secretion of lung cancer cells. QSOX1 levels were higher in 82% (51 of 62 tissues) of lung cancer tissues compared to adjacent normal tissues. Importantly, QSOX1 serum levels were significantly higher in cancer patients (p < 0.05, Area Under curve (AUC) = 0.89) when measured by multiple reaction monitoring (MRM). Higher levels of QSOX1 were also uniquely detected in lung cancer tissues, among several other solid cancers, by immunohistochemistry. QSOX1-knock-downed Lewis lung cancer (LLC) cells were less viable from oxidative stress and reduced migration and invasion. In addition, LLC mouse models with QSOX1 knock-down also proved that QSOX1 functions in promoting cancer metastasis. In conclusion, QSOX1 might be a lung cancer tissue-derived biomarker and be involved in the promotion of lung cancers, and thus can be a therapeutic target for lung cancers.

QSOX1 regulates trophoblastic apoptosis in preeclampsia through hydrogen peroxide production

Objective: Oxidative stress plays a significant role in the pathogenesis of preeclampsia (PE), by inducing trophoblast cell death and consequent placental dysfunction. Quiescin sulfhydryl oxidase 1 (QSOX1) is upregulated in many types of cancer cells; it promotes disulfide bond formation as well as hydrogen peroxide (H2O2) production. The aims of present study are to investigate the expression pattern of QSOX1 in placentae of pregnancies complicated by PE and the role of QSOX1 in the regulation of trophoblastic function, thus providing in-depth understanding of the putative involvement of QSOX1 in the development of PE.Methods: Human term placenta from normal pregnancies and from pregnancies complicated by PE was collected to measure QSOX1 expression and H2O2 levels. Down-regulation of QSOX1 in HTR-8/SVneo cells was achieved by siRNA interference. An in vitro cellular PE model was generated by hypoxic incubation. Protein expression levels were assessed by Western blotting, and H2O2 levels were determined in the cell culture medium as well as in the cell lysate. Trophoblast apoptosis was evaluated by TUNEL staining.Results: QSOX1 was overexpressed in the PE placenta. Inhibition of QSOX1 expression in HTR-8/SVneo cells attenuated cell apoptosis and intracellular H2O2 levels. Hypoxia-induced QSOX1 expression in HTR-8/SVneo cells and led to apoptosis of HTR-8/SVneo cells, and knock-down of QSOX1 rescued hypoxia-induced trophoblast apoptosis.Conclusions: Hypoxia-induced upregulation of QSOX1 and a consequent elevation in intracellular H2O2 increased apoptosis in placentae of pregnancies complicated by PE.

Quiescin sulfhydryl oxidase 1 (QSOX1) glycosite mutation perturbs secretion but not Golgi localization

Quiescin sulfhydryl oxidase 1 (QSOX1) catalyzes the formation of disulfide bonds in protein substrates. Unlike other enzymes with related activities, which are commonly found in the endoplasmic reticulum, QSOX1 is localized to the Golgi apparatus or secreted. QSOX1 is upregulated in quiescent fibroblast cells and secreted into the extracellular environment, where it contributes to extracellular matrix assembly. QSOX1 is also upregulated in adenocarcinomas, though the extent to which it is secreted in this context is currently unknown. To achieve a better understanding of factors that dictate QSOX1 localization and function, we aimed to determine how post-translational modifications affect QSOX1 trafficking and activity. We found a highly conserved N-linked glycosylation site to be required for QSOX1 secretion from fibroblasts and other cell types. Notably, QSOX1 lacking a glycan at this site arrives at the Golgi, suggesting that it passes endoplasmic reticulum quality control but is not further transported to the cell surface for secretion. The QSOX1 transmembrane segment is dispensable for Golgi localization and secretion, as fully luminal and transmembrane variants displayed the same trafficking behavior. This study provides a key example of the effect of glycosylation on Golgi exit and contributes to an understanding of late secretory sorting and quality control.

High expression of QSOX1 is associated with tumor invasiveness and high grades groups in prostate cancer

Prostate cancer is the most common malignancy in men, and biologically shows highly heterogeneous clinical outcomes, despite early detection. Therefore, the identification of novel molecular markers that are associated with biological aggressiveness is very important for prostatic cancer clinical outcome predictions and treatment choices.Here, we investigate quiescin sulfhydryl oxidase 1 (QSOX1) expression and evaluate its clinicopathological significance and prognostic impact in prostate cancers, with immunohistochemistry on tissue microarrays.QSOX1 over-expression was observed in 12 (11.2%) of prostate cancers. High QSOX1 expression significantly associated with prostate cancer with vascular invasion, neural invasion, extra prostatic extension, higher pT stage, higher pathological tumor stage, higher prognostic grouping, and higher grades groups, but did not associated with worse overall survival. High QSOX1 expression correlates with tumor invasiveness and Gleason grade, reflects aggressive tumor features, and could be an important biomarker and therapeutic target.

Endoplasmic reticulum–resident protein 57 (ERp57) oxidatively inactivates human transglutaminase 2

Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370-Cys-371 that renders the enzyme inactive in the extracellular matrix. Although recent studies have established that extracellular TG2 is switched "on" by the redox cofactor protein thioredoxin-1 (TRX), it is unclear how TG2 is switched "off." Here, we demonstrate that TG2 oxidation by small-molecule biological oxidants, including glutathione, cystine, and hydrogen peroxide, is unlikely to be the inactivation mechanism. Instead, endoplasmic reticulum (ER)-resident protein 57 (ERp57), a protein in the ER that promotes folding of nascent proteins and is also present in the extracellular environment, has the cellular and biochemical characteristics for inactivating TG2. We found that ERp57 colocalizes with extracellular TG2 in cultured human umbilical vein endothelial cells (HUVECs). ERp57 oxidized TG2 with a rate constant that was 400-2000-fold higher than those of the aforementioned small molecule oxidants. Moreover, its specificity for TG2 was also markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). Lastly, siRNA-mediated ERp57 knockdown in HUVECs increased TG2-catalyzed transamidation in the extracellular environment. We conclude that, to the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX).

Effect of QSOX1 on cattle carcass traits as well as apoptosis and triglyceride production in bovine fetal fibroblasts and mammary epithelial cells.

QSOX1 (quiescin-sulfhydryl oxidase 1) is involved in various processes, including apoptosis and the development of breast diseases. Here, we investigated the effect of QSOX1 on the meat quality of Simmental cattle by analyzing the correlation between QSOX1 single nucleotide polymorphisms (SNPs), I2 204 C>T and I2 378 C>T, and certain meat quality traits. The effects of QSOX1 on triglyceride synthesis and cell apoptosis were further validated by gene silencing or overexpression in bovine fetal fibroblasts and mammary epithelial cells. The results showed that I2 204 C>T and I2 378 C>T had significant correlations with loin thickness, hind hoof weight, fat coverage, liver weight, heart weight, marbling and back fat thickness (P<0.05). QSOX1 overexpression also increased triglyceride production and suppressed apoptosis. In summary, QSOX1 is an important factor for meat quality, lipid metabolism, and cell apoptosis, indicating that QSOX1 could be used as a biomarker to assist in breeding cattle with superior meat.

QSOX1 expression is associated with aggressive tumor features and reduced survival in breast carcinomas

The biological role of quiescin sulfhydryl oxidase 1 (QSOX1) in tumor development is not well known, and its relation to breast cancer progression and prognosis is controversial. Here, our aim was to study the expression pattern and prognostic impact of QSOX1 in breast cancer, in relation to molecular subgroups and tumor cell proliferation. We examined a population-based series as part of the prospective Norwegian Breast Cancer Screening Program, including all women (50–69 years) diagnosed with breast cancer in one county of Norway during 1996–2003. QSOX1 expression was assessed by immunohistochemistry on tissue microarrays (n=458). Median follow-up time was 13 years. High expression of QSOX1 protein was associated with features of poor prognosis including high histologic grade, hormone receptor negativity, HER2 positivity, and increased tumor cell proliferation. High QSOX1 expression was further associated with reduced breast cancer-specific survival in both univariate and multivariate analysis, independent of molecular subtypes. High QSOX1 expression is a strong and independent factor of reduced survival in breast cancer, also reflected by elevated levels in more aggressive molecular subgroups. QSOX1 expression may represent a biomarker for aggressive disease and a potential treatment target.