Membrane-anchored Serine Protease Research Articles

Matriptase-2 regulates iron homeostasis primarily by setting the basal levels of hepatic hepcidin expression through a nonproteolytic mechanism

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease. It plays a key role in iron homeostasis by suppressing the iron-regulatory hormone, hepcidin. Lack of functional MT2 results in an inappropriately high hepcidin and iron-refractory iron-deficiency anemia. Mt2 cleaves multiple components of the hepcidin-induction pathway in vitro. It is inhibited by the membrane-anchored serine protease inhibitor, Hai-2. Earlier in vivo studies show that Mt2 can suppress hepcidin expression independently of its proteolytic activity. In this study, our data indicate that hepatic Mt2 was a limiting factor in suppressing hepcidin. Studies in Tmprss6-/- mice revealed that increases in the dietary iron to ∼0.5% were sufficient to overcome the high hepcidin barrier and to correct the iron-deficiency anemia. Interestingly, the increased iron in Tmprss6-/- mice was able to further upregulate hepcidin expression to a similar magnitude as in wild-type mice. These results suggest that lack of Mt2 does not impact the iron induction of hepcidin. Additional studies of wild-type Mt2 and the proteolytic-dead form, fMt2S762A, indicated that the function of Mt2 is to lower the basal levels of hepcidin expression in a manner that primarily relies on its nonproteolytic role. This idea is supported by the studies in mice with the hepatocyte-specific ablation of Hai-2, which showed a marginal impact on iron homeostasis and no significant effects on iron regulation of hepcidin. Together, these observations suggest that the function of Mt2 is to set the basal levels of hepcidin expression and that this process is primarily accomplished through a nonproteolytic mechanism.

Elevated expression of the membrane-anchored serine protease TMPRSS11E in NSCLC progression.

TMPRSS11E was found to be upregulated in human nonsmall cell lung cancer samples (NSCLC) and cell lines, and high expression was associated with poor survival of NSCLC patients. The results of in vitro and in vivo experiments showed that overexpressing TMPRSS11E resulted in A549 cell proliferation and migration promotion, while the TMPRSS11E S372A mutant with the mutated catalytic domain lost the promoting function. In addition, in mouse xenograft models, silencing TMPRSS11E expression inhibited the growth of 95D cell-derived tumors. To explore the mechanism of marked upregulation of TMPRSS11E in NSCLC cells, promoter analysis, EMSA, and ChIP assays were performed. STAT3 was identified as the transcription factor responsible for TMPRSS11E transcription. Moreover, the purified recombinant TMPRSS11E catalytic domain exhibited enzymatic activity for the proteolytic cleavage of PAR2. Recombinant TMPRSS11E catalytic domain incubation further activated the PAR2-EGFR-STAT3 pathway. These findings established a mechanism of TMPRSS11E-PAR2-EGFR-STAT3 positive feedback, and the oncogenic role of TMPRSS11E as a PAR2 modulator in NSCLC was revealed.

Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several invivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

Matriptase-2/NR4A3 axis switches TGF-β action toward suppression of prostate cancer cell invasion, tumor growth, and metastasis.

Dysregulation of pericellular proteolysis is strongly implicated in cancer metastasis through alteration of cell invasion and the microenvironment. Matriptase-2 (MT-2) is a membrane-anchored serine protease which can suppress prostate cancer (PCa) cell invasion. In this study, we showed that MT-2 was down-regulated in PCa and could suppress PCa cell motility, tumor growth, and metastasis. Using microarray and biochemical analysis, we found that MT-2 shifted TGF-β action towards its tumor suppressor function by repressing epithelial-to-mesenchymal transition (EMT) and promoting Smad2 phosphorylation and nuclear accumulation to upregulate two TGF-β1 downstream effectors (p21 and PAI-1), culminating in hindrance of PCa cell motility and malignant growth. Mechanistically, MT-2 could dramatically up-regulate the expression of nuclear receptor NR4A3 via iron metabolism in PCa cells. MT-2-induced NR4A3 further coactivated Smad2 to activate p21 and PAI-1 expression. In addition, NR4A3 functioned as a suppressor of PCa and mediated MT-2 signaling to inhibit PCa tumorigenesis and metastasis. These results together indicate that NR4A3 sustains MT-2 signaling to suppress PCa cell invasion, tumor growth, and metastasis, and serves as a contextual factor for the TGF-β/Smad2 signaling pathway in favor of tumor suppression via promoting p21 and PAI-1 expression.

Insufficiency of hepatocyte growth factor activator inhibitor-1 confers lymphatic invasion of tongue carcinoma cells.

Hepatocyte growth factor (HGF) activator inhibitor type‐1 (HAI‐1), encoded by the SPINT1 gene, is a transmembrane protease inhibitor that regulates membrane‐anchored serine proteases, particularly matriptase. Here, we explored the role of HAI‐1 in tongue squamous cell carcinoma (TSCC) cells. An immunohistochemical study of HAI‐1 in surgically resected TSCC revealed the cell surface immunoreactivity of HAI‐1 in the main portion of the tumor. The immunoreactivity decreased in the infiltrative front, and this decrease correlated with enhanced lymphatic invasion as judged by podoplanin immunostaining. In vitro homozygous deletion of SPINT1 (HAI‐1KO) in TSCC cell lines (HSC3 and SAS) suppressed the cell growth rate but significantly enhanced invasion in vitro. The loss of HAI‐1 resulted in enhanced pericellular activities of proteases, such as matriptase and urokinase‐type plasminogen activator, which induced activation of HGF/MET signaling in the co‐culture with pro‐HGF‐expressing fibroblasts and plasminogen‐dependent plasmin generation, respectively. The enhanced plasminogen‐dependent plasmin generation was abrogated partly by matriptase silencing. Culture supernatants of HAI‐1KO cells had enhanced potency for converting the proform of vascular endothelial growth factor‐C (VEGF‐C), a lymphangiogenesis factor, into the mature form in a plasminogen‐dependent manner. Furthermore, HGF significantly stimulated VEGF‐C expression in TSCC cells. Orthotopic xenotransplantation into nude mouse tongue revealed enhanced lymphatic invasion of HAI‐1KO TSCC cells compared to control cells. Our results suggest that HAI‐1 insufficiency leads to dysregulated pericellular protease activity, which eventually orchestrates robust activation of protease‐dependent growth factors, such as HGF and VEGF‐C, in a tumor microenvironment to contribute to TSCC progression.

Sodium retention in nephrotic syndrome is independent of the activation of the membrane-anchored serine protease prostasin (CAP1/PRSS8) and its enzymatic activity

Experimental nephrotic syndrome leads to activation of the epithelial sodium channel (ENaC) by proteolysis and promotes renal sodium retention. The membrane-anchored serine protease prostasin (CAP1/PRSS8) is expressed in the distal nephron and participates in proteolytic ENaC regulation by serving as a scaffold for other serine proteases. However, it is unknown whether prostasin is also involved in ENaC-mediated sodium retention of experimental nephrotic syndrome. In this study, we used genetically modified knock-in mice with Prss8 mutations abolishing its proteolytic activity (Prss8-S238A) or prostasin activation (Prss8-R44Q) to investigate the development of sodium retention in doxorubicin-induced nephrotic syndrome. Healthy Prss8-S238A and Prss8-R44Q mice had normal ENaC activity as reflected by the natriuretic response to the ENaC blocker triamterene. After doxorubicin injection, all genotypes developed similar proteinuria. In all genotypes, urinary prostasin excretion increased while renal expression was not altered. In nephrotic mice of all genotypes, triamterene response was similarly increased, consistent with ENaC activation. As a consequence, urinary sodium excretion dropped in all genotypes and mice similarly gained body weight by + 25 ± 3% in Prss8-wt, + 20 ± 2% in Prss8-S238A and + 28 ± 3% in Prss8-R44Q mice (p = 0.16). In Western blots, expression of fully cleaved α- and γ-ENaC was similarly increased in nephrotic mice of all genotypes. In conclusion, proteolytic ENaC activation and sodium retention in experimental nephrotic syndrome are independent of the activation of prostasin and its enzymatic activity and are consistent with the action of aberrantly filtered serine proteases or proteasuria.

Evolutionary history of type II transmembrane serine proteases involved in viral priming.

Type II transmembrane serine proteases (TTSPs) are a family of trypsin-like membrane-anchored serine proteases that play key roles in the regulation of some crucial processes in physiological conditions, including cardiac function, digestion, cellular iron homeostasis, epidermal differentiation, and immune responses. However, some of them, in particular TTSPs expressed in the human airways, were identified as host factors that promote the proteolytic activation and spread of respiratory viruses such as influenza virus, human metapneumovirus, and coronaviruses, including SARS-CoV-2. Given their involvement in viral priming, we hypothesized that members of the TTSP family may represent targets of positive selection, possibly as the result of virus-driven pressure. Thus, we investigated the evolutionary history of sixteen TTSP genes in mammals. Evolutionary analyses indicate that most of the TTSP genes that have a verified role in viral proteolytic activation present signals of pervasive positive selection, suggesting that viral infections represent a selective pressure driving the evolution of these proteases. We also evaluated genetic diversity in human populations and we identified targets of balancing selection in TMPRSS2 and TMPRSS4. This scenario may be the result of an ancestral and still ongoing host–pathogen arms race. Overall, our results provide evolutionary information about candidate functional sites and polymorphic positions in TTSP genes.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00439-022-02435-y.

The ectodomain of matriptase-2 plays an important nonproteolytic role in suppressing hepcidin expression in mice

AbstractMatriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease that plays a key role in suppressing hepatic hepcidin expression. MT2 is synthesized as a zymogen and undergoes autocleavage for activation. Previous studies suggest that MT2 suppresses hepcidin by cleaving hemojuvelin and other components of the bone morphogenetic protein–signaling pathway. However, the underlying mechanism is still debatable. Here we dissected the contributions of the nonproteolytic and proteolytic activities of Mt2 by taking advantage of Mt2 mutants and Tmprss6−/− mice. Studies of the protease-dead full-length Mt2 (Mt2S762A) and the truncated Mt2 that lacks the catalytic domain (Mt2mask) indicate that the catalytic domain, but not its proteolytic activity, was required for Mt2 to suppress hepcidin expression. This process was likely accomplished by the binding of Mt2 ectodomain to Hjv and Hfe. We found that Mt2 specifically cleaved the key components of the hepcidin-induction pathway, including Hjv, Alk3, ActRIIA, and Hfe, when overexpressed in hepatoma cells. Nevertheless, studies of a murine iron-refractory iron-deficiency anemia–causing mutant (Mt2I286F) in the complement protein subcomponents C1r/C1s, urchin embryonic growth factor, and bone morphogenetic protein 1 domain indicate that Mt2I286F can be activated, but it exhibited a largely compromised ability to suppress hepcidin expression. Coimmunoprecipitation analysis revealed that Mt2I286F, but not Mt2S762A, had reduced interactions with Hjv, ActRIIA, and Hfe. In addition, increased expression of a serine protease inhibitor, the hepatocyte growth factor activator inhibitor-2, in the liver failed to alter hepcidin. Together, these observations support the idea that the substrate interaction with Mt2 plays a determinant role and suggest that the proteolytic activity is not an appropriate target to modulate the function of MT2 for clinical applications.

Inhibition of TMPRSS2 by HAI-2 reduces prostate cancer cell invasion and metastasis.

TMPRSS2 is an important membrane-anchored serine protease involved in human prostate cancer progression and metastasis. A serine protease physiologically often comes together with a cognate inhibitor for execution of proteolytically biologic function; however, TMPRSS2’s cognate inhibitor is still elusive. To identify the cognate inhibitor of TMPRSS2, in this study, we applied co-immunoprecipitation and LC/MS/MS analysis and isolated hepatocyte growth factor activator inhibitors (HAIs) to be potential inhibitor candidates for TMPRSS2. Moreover, the recombinant HAI-2 proteins exhibited a better inhibitory effect on TMPRSS2 proteolytic activity than HAI-1, and recombinant HAI-2 proteins had a high affinity to form a complex with TMPRSS2. The immunofluorescence images further showed that TMPRSS2 was co-localized to HAI-2. Both KD1 and KD2 domain of HAI-2 showed comparable inhibitory effects on TMPRSS2 proteolytic activity. In addition, HAI-2 overexpression could suppress the induction effect of TMPRSS2 on pro-HGF activation, extracellular matrix degradation and prostate cancer cell invasion. We further determined that the expression levels of TMPRSS2 were inversely correlated with HAI-2 levels during prostate cancer progression. In orthotopic xenograft animal model, TMPRSS2 overexpression promoted prostate cancer metastasis, and HAI-2 overexpression efficiently blocked TMPRSS2-induced metastasis. In summary, the results together indicate that HAI-2 can function as a cognate inhibitor for TMPRSS2 in human prostate cancer cells and may serve as a potential factor to suppress TMPRSS2-mediated malignancy.

Testisin/Prss21 deficiency causes increased vascular permeability and a hemorrhagic phenotype during luteal angiogenesis.

Testisin (encoded by PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. While testisin is found in abundance in spermatozoa, it is also expressed in microvascular endothelial cells where its function is unknown. Here we identify testisin as a novel regulator of physiological hormone-induced angiogenesis and microvascular endothelial permeability. Using a murine model of rapid physiological angiogenesis during corpus luteal development in the ovary, we found that mice genetically deficient in testisin (Prss21-/-) show a substantially increased incidence of hemorrhages which are significantly more severe than in littermate control Prss21+/+ mice. This phenotype was associated with increased vascular leakiness, demonstrated by a greater accumulation of extravasated Evans blue dye in Prss21-/- ovaries. Live cell imaging of in vitro cultured microvascular endothelial cells depleted of testisin by siRNA knockdown revealed that loss of testisin markedly impaired reorganization and tubule-like formation on Matrigel basement membranes. Moreover testisin siRNA knockdown increased the paracellular permeability to FITC-albumin across endothelial cell monolayers, which was associated with decreased expression of the adherens junction protein VE-cadherin and increased levels of phospho(Tyr658)-VE-cadherin, without affecting the levels of the tight junction proteins occludin and claudin-5, or ZO-1. Decreased expression of VE-cadherin in the neovasculature of Prss21-/- ovaries was also observed without marked differences in endothelial cell content, vascular claudin-5 expression or pericyte recruitment. Together, these data identify testisin as a novel regulator of VE-cadherin adhesions during angiogenesis and indicate a potential new target for regulating neovascular integrity and associated pathologies.

COVID-19 pandemic; transmembrane protease serine 2 (TMPRSS2) inhibitors as potential drugs.

Ongoing search for treatment to ease COVID-19 pandemic concentrate on development of a vaccine or medication to prevent and treat this disease. One of the possibilities is developing new antiviral drugs that are aiming at both a virus replication or the host factor(s) that are critical to virus’s replication. Serine proteases, which activate the viral spike glycoproteins and facilitate virus-cell membrane fusions for host cell entry, its replication and spread, are proposed as the potential targets for antiviral drug design. Existing literature is already providing evidence that transmembrane protease serine 2 (TMPRSS2) is one of the promising targets. When inhibited it can slow or stop replication of viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One of the convincing evidences of the critical role of TMPRSS2 in the coronavirus’s replication was provided by animal study. The replication of influenza viruses was inhibited in TMPRSS2(-/-) knockout mice in comparison to wild type (WT) mice, which developed high mortality rate. Existing inhibitors of TMPRSS2 can be divided into two groups. The first include drugs already approved by FDA or other organizations for treatment of different diseases. That include: Camostat (from Japan, produced by Ono Pharmaceutical), aprotinin (Trasylol, produced by Nordic Group Pharmaceuticals) and rimantadine (Flumadine, produced by Forest Pharmaceuticals, Inc.). Existing in vitro, in vivo and some limited human studies show that this type of drugs limit reproduction of coronaviruses and/or prevented the development of viral pneumonia. One study indicated that combined treatment by aprotinin and rimantadine prevented the development of fatal hemorrhagic viral pneumonia, and protected about 75% animals, when the separate administration of aprotinin or rimantadine induced less protection. The second group includes potential drugs not approved for the human use yet. That include plasminogen activator inhibitor type 1 (PAI-1) and recently developed small molecular inhibitors. PAI-1 is a serine protease inhibitor that regulates physiological breakdown of blood clots by inhibiting of tissue (tPA) and urokinase (uPA) plasminogen activators. But PAI-1 is also an effective inhibitor of various membrane-anchored serine proteases including TMPRSS2. It was reported that PAI-1 inhibited trypsin- and TMPRSS2-mediated cleavage of hemagglutinin and suppressed influenza virus in animals. PAI-1 is human in origin and engineered forms with extended half-life were developed and could be an attractive addition to the existing TMPRSS2 inhibitors. And finally, derivatives of sulfonylated 3-amindinophenylalanylamide were found to inhibit TMPRSS2 with a high affinity and efficiently block the influenza virus propagation in human cells. This paper is intended to provide review on possible or hypothetical beneficial effects of (TMPRSS2) inhibitors as one of options to fight infection with Covid-19.

Membrane-anchored serine proteases as regulators of epithelial function.

Cleavage of proteins in the extracellular milieu, including hormones, growth factors and their receptors, ion channels, and various cell adhesion and extracellular matrix molecules, plays a key role in the regulation of cell behavior. Among more than 500 proteolytic enzymes encoded by mammalian genomes, membrane-anchored serine proteases (MASPs), which are expressed on the surface of epithelial cells of all major organs, are excellently suited to mediate signal transduction across the epithelia and are increasingly being recognized as important regulators of epithelial development, function, and disease [ 1-3]. In this minireview, we summarize current knowledge of the in vivo roles of MASPs in acquisition and maintenance of some of the defining functions of epithelial tissues, such as barrier formation, ion transport, and sensory perception.

Iterative, multiplexed CRISPR-mediated gene editing for functional analysis of complex protease gene clusters

Elucidation of gene function by reverse genetics in animal models frequently is complicated by the functional redundancy of homologous genes. This obstacle often is compounded by the tight clustering of homologous genes, which precludes the generation of multigene-deficient animals through standard interbreeding of single-deficient animals. Here, we describe an iterative, multiplexed CRISPR-based approach for simultaneous gene editing in the complex seven-member human airway trypsin-like protease/differentially expressed in a squamous cell carcinoma (HAT/DESC) cluster of membrane-anchored serine proteases. Through four cycles of targeting, we generated a library of 18 unique congenic mouse strains lacking combinations of HAT/DESC proteases, including a mouse strain deficient in all seven proteases. Using this library, we demonstrate that HAT/DESC proteases are dispensable for term development, postnatal health, and fertility and that the recently described function of the HAT-like 4 protease in epidermal barrier formation is unique among all HAT/DESC proteases. The study demonstrates the potential of iterative, multiplexed CRISPR-mediated gene editing for functional analysis of multigene clusters, and it provides a large array of new congenic mouse strains for the study of HAT/DESC proteases in physiological and in pathophysiological processes.

Defective Tmprss3-Associated Hair Cell Degeneration in Inner Ear Organoids.

SummaryMutations in the gene encoding the type II transmembrane protease 3 (TMPRSS3) cause human hearing loss, although the underlying mechanisms that result in TMPRSS3-related hearing loss are still unclear. We combined the use of stem cell-derived inner ear organoids with single-cell RNA sequencing to investigate the role of TMPRSS3. Defective Tmprss3 leads to hair cell apoptosis without altering the development of hair cells and the formation of the mechanotransduction apparatus. Prior to degeneration, Tmprss3-KO hair cells demonstrate reduced numbers of BK channels and lower expressions of genes encoding calcium ion-binding proteins, suggesting a disruption in intracellular homeostasis. A proteolytically active TMPRSS3 was detected on cell membranes in addition to ER of cells in inner ear organoids. Our in vitro model recapitulated salient features of genetically associated inner ear abnormalities and will serve as a powerful tool for studying inner ear disorders.

Activation of sphingosine kinase by lipopolysaccharide promotes prostate cancer cell invasion and metastasis via SphK1/S1PR4/matriptase.

Gram-negative bacteria have been found to be a major population in prostatitis and prostate cancer (PCa) tissues. Lipopolysaccharide (LPS), a major compound of Gram-negative bacteria, with stimulatory activities in some cancer types, but has not been fully studied in PCa. In this study, we examined the effect of LPS on the invasion of PCa cells. Interestingly, LPS can enhance the invasiveness of PCa, but had no significant effect on PCa cell viability. Using protease inhibitor screening and biochemical analyses, matriptase, a member of the membrane-anchored serine protease family, is found to play a key role in LPS-induced PCa cell invasion. Mechanistically, Toll-like receptor 4 (TLR4, LPS receptor)-sphingosine kinase 1 (SphK1) signaling underlies LPS-induced matriptase activation and PCa cell invasion. Specifically, LPS induced the S225 phosphorylation of SphK1 and the translocation of SphK1 to plasma membrane, leading to the production of sphingosine 1-phosphate (S1P), ERK1/2 and matriptase activation via S1P receptor 4 (S1PR4). This phenomenon is further validated using the patient-derived explant (PDE) model. Indeed, there is a significant correlation among the elevated SphK1 levels, the Gleason grades of PCa specimens, and the poor survival of PCa patients. Taken together, this study demonstrates a potential impact of LPS on PCa progression. Our results provide not only a new finding of the role of bacterial infection in PCa progression but also potential therapeutic target(s) associated with PCa metastasis.

Membrane-Anchored Serine Proteases and Protease-Activated Receptor-2-Mediated Signaling: Co-Conspirators in Cancer Progression.

Pericellular proteolysis provides a significant advantage to developing tumors through the ability to remodel the extracellular matrix, promote cell invasion and migration, and facilitate angiogenesis. Recent advances demonstrate that pericellular proteases can also communicate directly to cells by activation of a unique group of transmembrane G-protein-coupled receptors (GPCR) known as protease-activated receptors (PAR). In this review, we discuss the specific roles of one of four mammalian PARs, namely PAR-2, which is overexpressed in advanced stage tumors and is activated by trypsin-like serine proteases that are highly expressed or otherwise dysregulated in many cancers. We highlight recent insights into the ability of different protease agonists to bias PAR-2 signaling and the newly emerging evidence for an interplay between PAR-2 and membrane-anchored serine proteases, which may co-conspire to promote tumor progression and metastasis. Interfering with these pathways might provide unique opportunities for the development of new mechanism-based strategies for the treatment of advanced and metastatic cancers.

Abstract A081: Activation of sphingosine kinase 1 by lipopolysaccharide promotes prostate cancer cell invasion via matriptase

Abstract A 15-year cohort study indicates that bacterial-induced prostatitis is correlated to prostate cancer (PCa) incidence. Several gram-negative bacteria have been identified in PCa patient specimens and inflammatory cytokines derived from bacterial infection have been involved in PCa tumorigenesis. However, the direct effect of lipopolysaccharide (LPS), a potent inflammatory substance from gram-negative bacteria, on PCa development is not well studied. In this study, we examine the effect of LPS on the growth (MTT assay) and invasion (Transwell assay) of several PCa cells lines including LNCaP, PC3, and DU145. Although LPS does not change the cell growth, our results reveal that LPS can enhance the PCa cell invasiveness and increase pericellular proteolytic activities. Using protease inhibitor screening and proteolytic analyses, we have identified that matriptase (MTX), a member of the membrane-anchored serine protease family, plays a key role in LPS-induced PCa cell invasiveness. The activated levels of MTX can be detected in the cells and the conditioned medium (CM) from LPS-treated cells. Indeed, the elevation of MTX expression is associated with high-grade PCa in archival specimens. Knowing LPS as a ligand for toll-like receptor 4 (TLR4), specific antagonists and shRNA knockdown methodology were used to delineate the mechanism by which TLR4/sphingosine kinase 1 (SphK1) signaling cascade plays a role in LPS-induced PCa cell invasion. The results show that SphK1 was important for MTX activation and PCa cell invasion. Moreover, we found that LPS could induce the activation of SphK1 based on an elevated phosphorylation level and SphK1 translocation to plasma membrane for the production of sphingosine 1-phosphate (S1P). S1P can subsequently induce MTX activation and PCa cell invasion through S1P receptor 4 (S1PR4) in a paracrine or autocrine manner. S1PR4 is further identified as an important receptor for S1P to mediate MAPK signaling. Furthermore, there is a significant correlation between the elevated expression of SphK1 and the Gleason scores of PCa specimens. The phenomenon was further corroborated by the fact that LPS could induce SphK1 activation in patient-derived explant model (PDeX). Taken together, we have unveiled a novel LPS-elicited pathway leading to PCa cell invasion. Our results provide not only the potential impact of bacterial infection on PCa progression but also several therapeutic target(s) against PCa metastasis. Citation Format: Cheng-Fan Lee, Benjamin Chen, Jer-Tsong Hsieh, Ming-Shyue Lee. Activation of sphingosine kinase 1 by lipopolysaccharide promotes prostate cancer cell invasion via matriptase [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A081.

Intestinal regulation of suppression of tumorigenicity 14 (ST14) and serine peptidase inhibitor, Kunitz type -1 (SPINT1) by transcription factor CDX2

The type II membrane-anchored serine protease, matriptase, encoded by suppression of tumorgenicity-14 (ST14) regulates the integrity of the intestinal epithelial barrier in concert with its inhibitor, HAI-1 encoded by serine peptidase inhibitor, Kunitz type -1 (SPINT1). The balance of the protease/inhibitor gene expression ratio is vital in preventing the oncogenic potential of matriptase. The intestinal cell lineage is regulated by a transcriptional regulatory network where the tumor suppressor, Caudal homeobox 2 (CDX2) is considered to be an intestinal master transcription factor. In this study, we show that CDX2 has a dual function in regulating both ST14 and SPINT1, gene expression in intestinal cells. We find that CDX2 is not required for the basal ST14 and SPINT1 gene expression; however changes in CDX2 expression affects the ST14/SPINT1 mRNA ratio. Exploring CDX2 ChIP-seq data from intestinal cell lines, we identified genomic CDX2-enriched enhancer elements for both ST14 and SPINT1, which regulate their corresponding gene promoter activity. We show that CDX2 displays both repressive and enhancing regulatory abilities in a cell specific manner. Together, these data reveal new insight into transcriptional mechanisms controlling the intestinal matriptase/inhibitor balance.

PO-179 Breast cancer cells and macrophages in a paracrine-juxtacrine loop

IntroductionCancer metastasis is a main cause for mortality. Dysregulation of pericellular proteolysis usually accounts for cancer cell invasion and metastasis. In this study, we are interested in delineating the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) in prostate and lung cancer cell invasion and metastasis.Material and methodsWe used different prostate and lung cancer cells and animal models to examine the role of HAI-2 in prostate and lung cancer cell invasion and metastasis.Results and discussionsIn this study, we identified a membrane-anchored serine protease inhibitor, hepatocyte growth factor activator inhibitor-2 (HAI-2), was down-regulated in human prostate and lung cancer. The decreased levels of HAI-2 were related to the progression of human prostate and lung adenocarcinoma. HAI-2 overexpression can repress both cancer cell migration and invasion. Recombinant HAI-2 proteins can inhibit both cancer cell motility. In addition, HAI-2 overexpression reduced the tumour growth and metastasis of prostate cancer cells, while down-regulation of HAI-2 can increase the metastatic ability of lung adenocarcinoma cells in xenografted animal models.ConclusionHAI-2 functions as a suppressor to inhibit the cell invasion and metastasis of human prostate and lung cancer.

Design, Synthesis, and Testing of Potent, Selective Hepsin Inhibitors via Application of an Automated Closed-Loop Optimization Platform

Hepsin is a membrane-anchored serine protease whose role in hepatocyte growth factor (HGF) signaling and epithelial integrity makes it a target of therapeutic interest in carcinogenesis and metastasis. Using an integrated design, synthesis, and screening platform, we were able to rapidly develop potent and selective inhibitors of hepsin. In progressing from the initial hit 7 to compound 53, the IC50 value against hepsin was improved from ∼1 μM to 22 nM, and the selectivity over urokinase-type plasminogen activator (uPA) was increased from 30-fold to >6000-fold. Subsequent in vitro ADMET profiling and cellular studies confirmed that the leading compounds are useful tools for interrogating the role of hepsin in breast tumorigenesis.