Sulfatase 2 (SULF2) Monoclonal Antibody 5D5 Suppresses Human Cholangiocarcinoma Xenograft Growth Through Regulation of a SULF2-Platelet-Derived Growth Factor Receptor Beta-Yes-Associated Protein Signaling Axis.

BACKGROUND AND AIMS: Existing therapeutic approaches to treat cholangiocarcinoma (CCA) have limited effectiveness, prompting further study to develop therapies for CCA. We report a mechanistic role for the heparan sulfate editing enzyme sulfatase 2 (SULF2) in CCA pathogenesis. APPROACH AND RESULTS: In silico analysis revealed elevated SULF2 expression in human CCA samples, occurring partly through gain of SULF2 copy number. We examined the effects of knockdown or overexpression of SULF2 on tumor growth, chemoresistance, and signaling pathway activity in human CCA cell lines in vitro. Up-regulation of SULF2 in CCA leads to increased platelet-derived growth factor receptor beta (PDGFRß)-Yes-associated protein (YAP) signaling activity, promoting tumor growth and chemotherapy resistance. To explore the utility of targeting SULF2 in the tumor microenvironment for CCA treatment, we tested an anti-SULF2 mouse monoclonal antibody, 5D5, in a mouse CCA xenograft model. Targeting SULF2 by monoclonal antibody 5D5 inhibited PDGFRß-YAP signaling and tumor growth in the mouse xenograft model. CONCLUSIONS: These results suggest that SULF2 monoclonal antibody 5D5 or related agents may be potentially promising therapeutic agents in CCA.

Expression of the extracellular sulfatase SULF2 is associated with squamous cell carcinoma of the head and neck.

Sulfatase 2 (SULF2), an extracellular sulfatase that alters sulfation on heparan sulfate proteoglycans, is involved in the tumorigenesis and progression of several carcinomas. SULF2 expression has not been evaluated in squamous cell carcinoma of the head and neck (HNSCC). Here we report results of IHC of SULF2 expression in HNSCC tissue. SULF2 was detected in 57% of tumors (n = 40) with a significant increase in intensity and number of stained cells compared to adjacent cancer-free tissue (p-value < 0.01), increasing with cancer stage when comparing stages 1 and 2 to stages 3 and 4 (p-value 0.01). SULF2 was not detected in epithelial cells of cancer-free controls, and expression was independent of patient demographics, tumor location and etiological factors, smoking and HPV infection by p16 IHC analysis. Sandwich ELISA was performed on serum of HNSCC patients (n = 28) and controls (n = 35), and although SULF2 was detectable, no change was observed in HNSCC. Saliva, collected by mouthwash, from HNSCC patients (n = 8) and controls (n = 8) was also tested by ELISA in a preliminary investigation and an increase in SULF2 was observed in HNSCC (p-value 0.041). Overall, this study shows that SULF2 is increased in HNSCC independent of tissue location (oral cavity, oropharynx, larynx and hypopharynx), patient demographics and etiology. Although no change in SULF2 was detected in HNSCC serum, its detection in saliva makes it worthy of further investigation as a potential HNSCC biomarker.

SULF2, a heparan sulfate endosulfatase, is present in the blood of healthy individuals and increases in cirrhosis.

BACKGROUND: SULF2 is an extracellular sulfatase that acts on heparan sulfate proteoglycans and modulates multiple signaling pathways. It is normally bound to the cell surface but can be released into the medium of cultured cells. SULF2 is known to be increased in cirrhotic liver compared to healthy liver. We asked whether SULF2 protein was present in the blood of healthy controls and increased in patients with liver cirrhosis. METHODS: We devised a sandwich ELISA for SULF2 using 2 novel monoclonal antibodies (mAbs) and measured its levels in sera of normal individuals and cirrhosis patients. RESULTS: SULF2 was higher in cirrhosis patients (1460 ± 1160 pg/ml, N=34) than in healthy individuals (728 ± 400 pg/ml, N=37). SULF2 levels increased with age in both healthy and patient groups. CONCLUSIONS: SULF2 may be a useful serologic biomarker for liver cirrhosis.

Leukotriene B4 amplifies eosinophil accumulation in response to nematodes.

Eosinophil accumulation is a defining feature of the immune response to parasitic worm infection. Tissue-resident cells, such as epithelial cells, are thought to initiate eosinophil recruitment. However, direct recognition of worms by eosinophils has not been explored as a mechanism for amplifying eosinophil accumulation. Here, we report that eosinophils rapidly migrate toward diverse nematode species in three-dimensional culture. These include the mammalian parasite Nippostrongylus brasiliensis and the free-living nematode Caenorhabditis elegans. Surprisingly, collective migration toward worms requires paracrine leukotriene B4 signaling between eosinophils. In contrast, neutrophils show a minimal response to nematodes, yet are able to undergo robust leukotriene-dependent migration toward IgG-coated beads. We further demonstrate that eosinophils accumulate around C. elegans in the lungs of mice. This response is not dependent on bacterial products, CCR3, or complement activation. However, mice deficient in leukotriene signaling show markedly attenuated eosinophil accumulation after injection of C. elegans or N. brasiliensis. Our findings establish that nematode-derived signals can directly induce leukotriene production by eosinophils and that leukotriene signaling is a major contributor to nematode-induced eosinophil accumulation in the lung. The similarity of the eosinophil responses to diverse nematode species suggests that conserved features of nematodes are recognized during parasite infection.

The SULFs, extracellular sulfatases for heparan sulfate, promote the migration of corneal epithelial cells during wound repair.

Corneal epithelial wound repair involves the migration of epithelial cells to cover the defect followed by the proliferation of the cells to restore thickness. Heparan sulfate proteoglycans (HSPGs) are ubiquitous extracellular molecules that bind to a plethora of growth factors, cytokines, and morphogens and thereby regulate their signaling functions. Ligand binding by HS chains depends on the pattern of four sulfation modifications, one of which is 6-O-sulfation of glucosamine (6OS). SULF1 and SULF2 are highly homologous, extracellular endosulfatases, which post-synthetically edit the sulfation status of HS by removing 6OS from intact chains. The SULFs thereby modulate multiple signaling pathways including the augmentation of Wnt/ß-catenin signaling. We found that wounding of mouse corneal epithelium stimulated SULF1 expression in superficial epithelial cells proximal to the wound edge. Sulf1⁻/⁻, but not Sulf2⁻/⁻, mice, exhibited a marked delay in healing. Furthermore, corneal epithelial cells derived from Sulf1⁻/⁻ mice exhibited a reduced rate of migration in repair of a scratched monolayer compared to wild-type cells. In contrast, human primary corneal epithelial cells expressed SULF2, as did a human corneal epithelial cell line (THCE). Knockdown of SULF2 in THCE cells also slowed migration, which was restored by overexpression of either mouse SULF2 or human SULF1. The interchangeability of the two SULFs establishes their capacity for functional redundancy. Knockdown of SULF2 decreased Wnt/ß-catenin signaling in THCE cells. Extracellular antagonists of Wnt signaling reduced migration of THCE cells. However in SULF2- knockdown cells, these antagonists exerted no further effects on migration, consistent with the SULF functioning as an upstream regulator of Wnt signaling. Further understanding of the mechanistic action of the SULFs in promoting corneal repair may lead to new therapeutic approaches for the treatment of corneal injuries.

Galactose 6-O-sulfotransferases are not required for the generation of Siglec-F ligands in leukocytes or lung tissue.

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.

KSGal6ST generates galactose-6-O-sulfate in high endothelial venules but does not contribute to L-selectin-dependent lymphocyte homing.

The addition of sulfate to glycan structures can regulate their ability to serve as ligands for glycan-binding proteins. Although sulfate groups present on the monosaccharides glucosamine, uronate, N-acetylglucosamine and N-acetylgalactosamine are recognized by defined receptors that mediate important functions, the functional significance of galactose-6-O-sulfate (Gal6S) is not known. However, in vitro studies using synthetic glycans and sulfotransferase overexpression implicate Gal6S as a binding determinant for the lymphocyte homing receptor, L-selectin. Only two sulfotransferases have been shown to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase-1 (C6ST-1). In the present study, we use mice deficient in KSGal6ST and C6ST-1 to test whether Gal6S contributes to ligand recognition by L-selectin in vivo. First, we establish that KSGal6ST is selectively expressed in high endothelial venules (HEVs) in lymph nodes and Peyer's patches. We also determine by mass spectrometry that KSGal6ST generates Gal6S on several classes of O-glycans in peripheral lymph nodes. Furthermore, KSGal6ST, but not C6ST-1, is required for the generation of the Gal6S-containing glycan, 6,6'-disulfo-3'sLN (Siaα2→3[6S]Galß1→4[6S]GlcNAc) or a closely related structure in lymph node HEVs. Nevertheless, L-selectin-dependent short-term homing of lymphocytes is normal in KSGal6ST-deficient mice, indicating that the Gal6S-containing structures we detected do not contribute to L-selectin ligand recognition in this setting. These results refine our understanding of the biological ligands for L-selectin and introduce a mouse model for investigating the functions of Gal6S in other contexts.

SULF2 expression by immunohistochemistry and overall survival in oesophageal cancer: a cohort study.

OBJECTIVES: Oesophageal cancer is the eighth most commonly diagnosed cancer worldwide, and there is a need for biomarkers to improve diagnosis, prognosis and treatment. Sulfatases 2 (SULF2) is an extracellular endosulphatase that regulates several signalling pathways in carcinogenesis and has been associated with poor prognosis. This study evaluates the relationship between SULF2 expression by immunohistochemistry and overall survival in patients with oesophageal cancer. DESIGN: Cohort study. SETTING: Single tertiary care centre. PARTICIPANTS: We included patients who underwent esophagectomy for invasive oesophageal adenocarcinoma and squamous cell carcinoma at a tertiary care centre from 1997 to 2006. We excluded patients with recurrent oesophageal cancer or less than 3 mm invasive tumour on H&E stained slide. A section from each paraffin-embedded tissue specimen was stained with an anti-SULF2 monoclonal antibody. OUTCOME MEASURES: A pathologist blinded to overall survival determined the percentage and intensity of tumour cells staining. Vital status was obtained through the Social Security Death Master File, and overall survival was calculated from the date of surgery. RESULTS: One-hundred patients with invasive oesophageal cancer were identified, including 75 patients with adenocarcinoma and 25 patients with squamous cell carcinoma. The squamous cell carcinoma samples had a higher mean percentage and intensity of tumour cells staining compared with the adenocarcinoma samples. After adjusting for age, sex, race, histological type, stage and neoadjuvant therapy, for every 10% increase in percentage of tumour cells staining for SULF2, the HR for death increased by 13% (95% CI 1.01 to 1.25; p=0.03). CONCLUSIONS: The majority of adenocarcinoma samples and all of the squamous cell carcinoma samples had SULF2 staining. The percentage of tumour cells staining for SULF2 was significantly associated with overall survival. Thus, SULF2 is a potential biomarker in oesophageal cancer and may have an important role in the management of patients with this disease.

Autotaxin through lysophosphatidic acid stimulates polarization, motility, and transendothelial migration of naive T cells.

Blood-borne lymphocytes home to lymph nodes by interacting with and crossing high endothelial venules (HEVs). The transendothelial migration (TEM) step is poorly understood. Autotaxin (ATX) is an ectoenzyme that catalyzes the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a bioactive lipid and a close relative of sphingosine 1-phosphate. HEVs produce and secrete ATX into the blood. A prior study implicated ATX in the overall homing process, but the step in which it functions and its mechanism of action have not been defined. In this article, we show that HA130, an inhibitor of the enzymatic activity of ATX, slows T cell migration across lymph node HEVs in vivo. Ex vivo, ATX plus LPC or LPA itself induces the polarization of mouse naive T cells and stimulates their motility on an ICAM-1 substratum. Under physiologic shear conditions in a flow chamber, LPA or ATX/LPC strongly enhances TEM of integrin-arrested T cells across an endothelial monolayer. HA130 blunts the TEM-promoting activity of ATX, paralleling its in vivo effects. T cells possess Mn(+2)-activatable receptors for ATX, which are localized at the leading edge of polarized cells. ATX must bind to these receptors to elicit a maximal TEM response, providing a mechanism to focus the action of LPA onto arrested lymphocytes in flowing blood. Our results indicate that LPA produced via ATX facilitates T cell entry into lymph nodes by stimulating TEM, substantiating an additional step in the homing cascade. This entry role for LPA complements the efflux function of sphingosine 1-phosphate.

Heparan sulfate sulfatase SULF2 regulates PDGFRα signaling and growth in human and mouse malignant glioma.

Glioblastoma (GBM), a uniformly lethal brain cancer, is characterized by diffuse invasion and abnormal activation of multiple receptor tyrosine kinase (RTK) signaling pathways, presenting a major challenge to effective therapy. The activation of many RTK pathways is regulated by extracellular heparan sulfate proteoglycans (HSPG), suggesting these molecules may be effective targets in the tumor microenvironment. In this study, we demonstrated that the extracellular sulfatase, SULF2, an enzyme that regulates multiple HSPG-dependent RTK signaling pathways, was expressed in primary human GBM tumors and cell lines. Knockdown of SULF2 in human GBM cell lines and generation of gliomas from Sulf2(-/-) tumorigenic neurospheres resulted in decreased growth in vivo in mice. We found a striking SULF2 dependence in activity of PDGFRα, a major signaling pathway in GBM. Ablation of SULF2 resulted in decreased PDGFRα phosphorylation and decreased downstream MAPK signaling activity. Interestingly, in a survey of SULF2 levels in different subtypes of GBM, the proneural subtype, characterized by aberrations in PDGFRα, demonstrated the strongest SULF2 expression. Therefore, in addition to its potential as an upstream target for therapy of GBM, SULF2 may help identify a subset of GBMs that are more dependent on exogenous growth factor-mediated signaling. Our results suggest the bioavailability of growth factors from the microenvironment is a significant contributor to tumor growth in a major subset of human GBM.

Functional contributions of N- and O-glycans to L-selectin ligands in murine and human lymphoid organs.

L-selectin initiates lymphocyte interactions with high endothelial venules (HEVs) of lymphoid organs through binding to ligands with specific glycosylation modifications. 6-Sulfo sLe(x), a sulfated carbohydrate determinant for L-selectin, is carried on core 2 and extended core 1 O-glycans of HEV-expressed glycoproteins. The MECA-79 monoclonal antibody recognizes sulfated extended core 1 O-glycans and partially blocks lymphocyte-HEV interactions in lymphoid organs. Recent evidence has identified the contribution of 6-sulfo sLe(x) carried on N-glycans to lymphocyte homing in mice. Here, we characterize CL40, a novel IgG monoclonal antibody. CL40 equaled or surpassed MECA-79 as a histochemical staining reagent for HEVs and HEV-like vessels in mouse and human. Using synthetic carbohydrates, we found that CL40 bound to 6-sulfo sLe(x) structures, on both core 2 and extended core 1 structures, with an absolute dependency on 6-O-sulfation. Using transfected CHO cells and gene-targeted mice, we observed that CL40 bound its epitope on both N-glycans and O-glycans. Consistent with its broader glycan-binding, CL40 was superior to MECA-79 in blocking lymphocyte-HEV interactions in both wild-type mice and mice deficient in forming O-glycans. This superiority was more marked in human, as CL40 completely blocked lymphocyte binding to tonsillar HEVs, whereas MECA-79 inhibited only 60%. These findings extend the evidence for the importance of N-glycans in lymphocyte homing in mouse and indicate that this dependency also applies to human lymphoid organs.

Use of a phage display antibody to measure the enzymatic activity of the Sulfs.

Sulf-1 and Sulf-2 are extracellular endoglucosamine 6-sulfatases, which selectively liberate the 6-O-sulfate groups on glucosamines present in N, 6-O, and 2-O trisulfated disaccharides of intact heparan sulfate (HS)/heparin chains. The Sulfs are known to regulate signaling of heparin/HS-binding protein ligands, such as morphogens and growth factors, presumably through their ability to decrease the association between the ligands and HS proteoglycans. These enzymes serve important roles in development and are dysregulated in many cancers. We previously described arylsulfatase and endoglucosamine 6-sulfatase assays for the Sulfs. RB4CD12 is a phage display anti-HS antibody. N-sulfation, 2-O-sulfation, and 6-O-sulfation are involved in its binding. In this chapter, we describe the application of RB4CD12 in ELISA, flow cytometry, and immunohistochemistry assays to measure the enzymatic activity of the Sulfs. These newly established methods should facilitate further investigation of the Sulfs in vitro and in vivo.

Sulf-2: an extracellular modulator of cell signaling and a cancer target candidate.

IMPORTANCE OF THE FIELD: Sulf-1 and Sulf-2 are sulfatases that edit the sulfation status of heparan sulfate proteoglycans (HSPGs) on the outside of cells and regulate a number of critical signaling pathways. The Sulfs are dysregulated in many cancers with Sulf-2 in particular implicated as a driver of carcinogenesis in NSCLC, pancreatic cancer and hepatocellular carcinoma. AREAS COVERED IN THIS REVIEW: This review describes the novel activity of the Sulfs in altering the sulfation pattern of HSPG chains on the outside of cells. Thereby, the Sulfs can change the binding of growth factors to these chains and can either promote (e.g., Wnt) or inhibit (e.g., fibroblast growth factor-2) signaling. The review focuses on the widespread upregulation of both Sulfs in cancers and summarizes the evidence that Sulf-2 promotes the transformed behavior of several types of cancer cells in vitro as well as their tumorigenicity in vivo. WHAT THE READER WILL GAIN: Sulf-2 is a bonafide candidate as a cancer-causing agent in NSCLC and other cancers in which it is upregulated. TAKE HOME MESSAGE: Sulf-2 is an extracellular enzyme and as such would be an attractive therapeutic target for the treatment of NSCLC and other cancers.

Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88.

Heparan sulfates (HS) bind a diversity of protein ligands on the cell surface and in the extracellular matrix and thus can modulate cell signaling. The state of sulfation in glucosamines and uronic acids within the chains strongly influences their binding. We have previously cloned and characterized two human extracellular endoglucosamine 6-sulfatases, HSulf-1 and HSulf-2, which selectively liberate the 6-O sulfate groups on glucosamines present in N, 6-O, and 2-O trisulfated disaccharides of intact HS and heparins. These enzymes serve important roles in development and are upregulated in a number of cancers. To determine whether the Sulfs act on the trisulfated disaccharides that exist on the cell surface, we expressed HSulfs in cultured cells and performed a flow cytometric analysis with the RB4CD12, an anti-HS antibody that recognizes N- and O-sulfated HS saccharides. The endogenously expressed level of the cell surface RB4CD12 epitope was greatly diminished in CHO, HEK293, and HeLa cells transfected with HSulf-1 or HSulf-2 cDNA. In correspondence with the RB4CD12 finding, the N, 6-O, and 2-O trisulfated disaccharides of the HS isolated from the cell surface/extracellular matrix were dramatically reduced in the Sulf-expressed HEK293 cells. We then developed an ELISA and confirmed that the RB4CD12 epitope in immobilized heparin was degraded by purified recombinant HSulf-1 and HSulf-2, and conditioned medium (CM) of MCF-7 breast carcinoma cells, which contain a native form of HSulf-2. Furthermore, HSulf-1 and HSulf-2 exerted activity against the epitope expressed on microvessels of mouse brains. Both HSulf activities were potently inhibited by PI-88, a sulfated heparin mimetic with anti-cancer activities. These findings provide new strategies for monitoring the extracellular remodeling of HS by Sulfs during normal and pathophysiological processes.

"Home sweet home" for lymphocytes.

In this issue of Blood, Hernandez Mir and colleagues provide the most detailed analysis to date of the glycans on an HEV-expressed ligand (CD34) isolated from a human lymphoid organ, (tonsils), adding to our understanding of how L-selectin mediates lymphocyte homing.

Functional consequences of the subdomain organization of the sulfs.

Sulf-1 and Sulf-2 are novel extracellular sulfatases that act on internal glucosamine 6-O-sulfate modifications within heparan sulfate proteoglycans and regulate their interactions with various signaling molecules, including Wnt ligands. Although the Sulfs are multidomain proteins, there is limited information available about how the subdomains contribute to their enzymatic and signaling activities. In this study, we found that both human Sulfs were synthesized as prepro-enzymes and cleaved by a furin-type proteinase to form disulfide-bond linked heterodimers of 75- and 50-kDa subunits. The mature Sulfs were secreted into conditioned medium, as well as retained on the cell membrane. Although the catalytic center resides in the N-terminal 75-kDa subunit, the C-terminal 50-kDa subunit was indispensable for both arylsufatase and glucosamine 6-O-sulfate-endosulfatase activity. We found that the hydrophilic regions of the Sulfs were essential for endosulfatase activity but not for arylsulfatase activity. Using Edman sequencing, we identified furin-type proteinase cleavage sites in Sulf-1 and Sulf-2. Deletion of these sequences resulted in uncleavable forms of Sulfs. The uncleavable Sulfs retained enzymatic activity. However, they were unable to potentiate Wnt signaling, which may be due to their defective localization into lipid rafts on the plasma membrane.

Endoglycan, a member of the CD34 family of sialomucins, is a ligand for the vascular selectins.

The interactions of the selectin family of adhesion molecules with their ligands are essential for the initial rolling stage of leukocyte trafficking. Under inflammatory conditions, the vascular selectins, E- and P-selectin, are expressed on activated vessels and interact with carbohydrate-based ligands on the leukocyte surface. While several ligands have been characterized on human T cells, monocytes and neutrophils, there is limited information concerning ligands on B cells. Endoglycan (EG) together with CD34 and podocalyxin comprise the CD34 family of sialomucins. We found that EG, previously implicated as an L-selectin ligand on endothelial cells, was present on human B cells, T cells and peripheral blood monocytes. Upon activation of B cells, EG increased with a concurrent decrease in PSGL-1. Expression of EG on T cells remained constant under the same conditions. We further found that native EG from several sources (a B cell line, a monocyte line and human tonsils) was reactive with HECA-452, a mAb that recognizes sialyl Lewis X and related structures. Moreover, immunopurified EG from these sources was able to bind to P-selectin and where tested E-selectin. This interaction was divalent cation-dependent and required sialylation of EG. Finally, an EG construct supported slow rolling of E- and P-selectin bearing cells in a sialic acid and fucose dependent manner, and the introduction of intact EG into a B cell line facilitated rolling interactions on a P-selectin substratum. These in vitro findings indicate that EG can function as a ligand for the vascular selectins.

Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs.

The extracellular lysophospholipase D autotaxin (ATX) and its product, lysophosphatidic acid, have diverse functions in development and cancer, but little is known about their functions in the immune system. Here we found that ATX had high expression in the high endothelial venules of lymphoid organs and was secreted. Chemokine-activated lymphocytes expressed receptors with enhanced affinity for ATX, which provides a mechanism for targeting the secreted ATX to lymphocytes undergoing recruitment. Lysophosphatidic acid induced chemokinesis in T cells. Intravenous injection of enzymatically inactive ATX attenuated the homing of T cells to lymphoid tissues, probably through competition with endogenous ATX and exertion of a dominant negative effect. Our results support the idea of a new and general step in the homing cascade in which the ectoenzyme ATX facilitates the entry of lymphocytes into lymphoid organs.

Extracellular sulfatases, elements of the Wnt signaling pathway, positively regulate growth and tumorigenicity of human pancreatic cancer cells.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are control elements in Wnt signaling, which bind extracellularly to Wnt ligands and regulate their ability to interact with signal transduction receptors on the cell surface. Sulf-1 and Sulf-2 are novel extracellular sulfatases that act on internal glucosamine-6-sulfate (6S) modifications within HSPGs and thereby modulate HSPG interactions with various signaling molecules, including Wnt ligands. Emerging evidence indicates the importance of reactivated Wnt signaling in a number of cancers, including pancreatic adenocarcinoma. PRINCIPLE FINDINGS: Both Sulf proteins were upregulated in human pancreatic adenocarcinoma tumors and were broadly expressed in human pancreatic adenocarcinoma cell lines. Expression of human extracellular sulfatases Sulf-1 and Sulf-2 enhanced Wnt signaling in a reconstituted system. Three of four pancreatic adenocarcinoma cell lines tested exhibited autocrine Wnt signaling, in that extracellular Wnt ligands were required to initiate downstream Wnt signaling. Exposure of these pancreatic adenocarcinoma cells to a catalytically inactive form of Sulf-2 or siRNA-mediated silencing of endogenous Sulf-2 inhibited both Wnt signaling and cell growth. Sulf-2 silencing in two of these lines resulted in markedly reduced tumorigenesis in immunocompromised mice. CONCLUSIONS/SIGNIFICANCE: We have identified the Sulfs as potentiators of autocrine Wnt signaling in pancreatic cancer cells and have demonstrated their contribution to the growth and tumorigenicity of these cells. Since the Sulfs are extracellular enzymes, they would be attractive targets for therapy of pancreatic cancer. Our results run counter to the prevailing view in the literature that the Sulfs are negative regulators of tumorigenesis.