Sulfatase 2 Research Articles

Cancer-Associated Fibroblasts Expressing Sulfatase 1 Facilitate VEGFA-Dependent Microenvironmental Remodeling to Support Colorectal Cancer.

Tumor stroma plays a critical role in fostering tumor progression and metastasis. Cancer-associated fibroblasts (CAF) are a major component of the tumor stroma. Identifying the key molecular determinants for the protumor properties of CAFs could enable the development of more effective treatment strategies. In this study, through analyses of single-cell sequencing data, we identified a population of CAFs expressing high levels of sulfatase 1 (SULF1), which was associated with poor prognosis in patients with colorectal cancer. Colorectal cancer models using mice with conditional SULF1 knockout in fibroblasts revealed the tumor-supportive function of SULF1+ CAFs. Mechanistically, SULF1+ CAFs enhanced the release of VEGFA from heparan sulfate proteoglycan. The increased bioavailability of VEGFA initiated the deposition of extracellular matrix and enhanced angiogenesis. In addition, intestinal microbiota-produced butyrate suppressed SULF1 expression in CAFs through its histone deacetylase (HDAC) inhibitory activity. The insufficient butyrate production in patients with colorectal cancer increased the abundance of SULF1+ CAFs, thereby promoting tumor progression. Importantly, tumor growth inhibition by HDAC was dependent on SULF1 expression in CAFs, and patients with colorectal cancer with more SULF1+ CAFs were more responsive to treatment with the HDAC inhibitor chidamide. Collectively, these findings unveil the critical role of SULF1+ CAFs in colorectal cancer and provide a strategy to stratify patients with colorectal cancer for HDAC inhibitor treatment. Significance: SULF1+ cancer-associated fibroblasts play a tumor-promoting role in colorectal cancer by stimulating extracellular matrix deposition and angiogenesis and can serve as a biomarker for the therapeutic response to HDAC inhibitors in patients.

Utility of an Untargeted Metabolomics Approach Using a 2D GC-GC-MS Platform to Distinguish Relapsing and Progressive Multiple Sclerosis.

Multiple sclerosis (MS) is the most common inflammatory neurodegenerative disease of the central nervous system (CNS) in young adults and results in progressive neurological defects. The relapsing-remitting phenotype (RRMS) is the most common disease course in MS, which ultimately progresses to secondary progressive MS (SPMS), while primary progressive MS (PPMS) is a type of MS that worsens gradually over time without remissions. There is a gap in knowledge regarding whether the relapsing form can be distinguished from the progressive course, or healthy subjects (HS) based on an altered serum metabolite profile. In this study, we performed global untargeted metabolomics with the 2D GC-GC-MS platform to identify altered metabolites between RRMS, PPMS, and HS. We profiled 235 metabolites in the serum of patients with RRMS (n = 41), PPMS (n = 31), and HS (n = 91). A comparison of RRMS and HS patients revealed 22 significantly altered metabolites at p < 0.05 (false-discovery rate [FDR] = 0.3). The PPMS and HS comparisons revealed 28 altered metabolites at p < 0.05 (FDR = 0.2). Pathway analysis using MetaboAnalyst revealed enrichment of four metabolic pathways in both RRMS and PPMS (hypergeometric test p < 0.05): (1) galactose metabolism; (2) amino sugar and nucleotide sugar metabolism; (3) phenylalanine, tyrosine, and tryptophan biosynthesis; and (4) aminoacyl-tRNA biosynthesis. The Qiagen IPA enrichment test identified the sulfatase 2 (SULF2) (p = 0.0033) and integrin subunit beta 1 binding protein 1 (ITGB1BP1) (p = 0.0067) genes as upstream regulators of altered metabolites in the RRMS vs. HS groups. However, in the PPMS vs. HS comparison, valine was enriched in the neurodegeneration of brain cells (p = 0.05), and heptadecanoic acid, alpha-ketoisocaproic acid, and glycerol participated in inflammation in the CNS (p = 0.03). Overall, our study suggests that RRMS and PPMS may contribute metabolic fingerprints in the form of unique altered metabolites for discriminating MS disease from HS, with the potential for constructing a metabolite panel for progressive autoimmune diseases such as MS.

SULF1 Activates the VEGFR2/PI3K/AKT Pathway to Promote the Development of Cervical Cancer.

Sulfatase 1 (SULF1) can regulate the binding of numerous signaling molecules by removing 6-O-sulfate from heparan sulfate proteoglycans (HSPGs) to affect numerous physiological and pathological processes. Our research aimed to investigate the effect of the SULF1-mediated VEGFR2/PI3K/AKT signaling pathway on tumorigenesis and development of cervical cancer (CC). The expression and prognostic values of SULF1 in patients with CC were analyzed through bioinformatics analysis, qRT-PCR, immunohistochemistry, and western blot. The function and regulatory mechanism of SULF1 in proliferation, migration, and invasion of cervical cancer cells were examined through lentivirus transduction, CCK8, flow cytometry analysis, plate colony formation assay, scratch assay, transwell assay, western blot, VEGFR2 inhibitor (Ki8751), and mouse models. SULF1 expression was significantly upregulated in CC tissues, which was significantly associated with poor prognosis of patients with CC. In vitro, the upregulation of SULF1 expression in HeLa cells promoted cell proliferation, colony formation, migration, and invasion while inhibiting apoptosis. Conversely, the downregulation of SULF1 expression had the opposite effect. In vivo, the upregulation of SULF1 expression resulted in a significant increase in both tumor growth and angiogenesis, while its downregulation had the opposite effect. Furthermore, western blot detection and cell function rescue assay confirmed that the upregulation of SULF1 in HeLa cells promoted the tumorigenic behaviors of cancer cells by activating the VEGFR2/PI3K/AKT signaling pathway. SULF1 plays an oncogenic role in the tumorigenesis and development of CC, indicating its potential as a novel molecular target for gene-targeted therapy in patients with CC.

Abstract 6525: Modulating the immune response in cholangiocarcinoma: Targeting sulfatase-2 for therapeutic intervention

Abstract Cholangiocarcinoma (CCA) is an aggressive epithelial malignancy of the bile ducts. Recent reports have suggested a rise in the incidence of CCA globally. Current treatments for CCA have limited effectiveness, with only 20-30% response rates in patients with advanced cancer. Thus, there is a growing need to investigate new therapeutic targets for CCA. The tumor microenvironment (TME) is a potential target for novel therapies. The TME plays an important role in progression and metastasis of cancer through facilitating immune escape. Recent studies have demonstrated potential roles for heparan sulfate proteoglycans (HSPGs) in tumorigenesis. Sulfatase 2 (SULF2) is a heparan sulfate editing sulfatase that removes the 6-O sulfate moiety within the heparan sulfate (HS) chains and alters the affinity of HS chains for growth factors and cytokines and their receptors, thus modulating HSPG function in cell signaling pathways. HSPG specific sulfatases have been recently identified as a potential therapeutic target in CCA. We aimed to assess the effects of anti-Sulfatase-2 antibody on immune cells in the tumor microenvironment of syngeneic CCA xenografts in C57BL6 mice. Mice bearing syngeneic CCA xenografts were treated with an anti-sulf2 antibody, and the tumors compared to untreated control xenografts. After treatment, the tumors were harvested and analyzed by Cytometry by time of flight (Cytof) to identify changes in the types and numbers of immune cells in the tumor xenografts. Treatment with anti-sulf2 antibody resulted in a decrease in immunosuppressive cells such as tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSC) compared to the control. There was also an increase in anti-tumor cells such as T-cells and NK cells, suggesting that suppressing sulf-2 in the microenvironment can shift the TME toward an anti-tumorigenic rather than a pro-tumorigenic environment. In summary, our work demonstrates the potential to target Sulfatase 2 as a therapeutic mechanism for immunomodulation of the tumor microenvironment. Future experiments to determine the relationship between specific clinical phenotypes and the number, activities and types of immune cells seen in the TME, and experiments in other hepatobiliary cancers models, such as hepatocellular carcinoma and gallbladder cancer, will be conducted. Citation Format: Tayla R. Brooks, Nellie Campbell, Melanie Keim, Lewis R. Roberts. Modulating the immune response in cholangiocarcinoma: Targeting sulfatase-2 for therapeutic intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6525.

Targeting carcinoma-associated mesothelial cells with antibody-drug conjugates in ovarian carcinomatosis.

Ovarian carcinomatosis is characterized by the accumulation of carcinoma-associated mesothelial cells (CAMs) in the peritoneal stroma and mainly originates through a mesothelial-to-mesenchymal transition (MMT) process. MMT has been proposed as a therapeutic target for peritoneal metastasis. Most ovarian cancer (OC) patients present at diagnosis with peritoneal seeding, which makes tumor progression control difficult by MMT modulation. An alternative approach is to use antibody-drug conjugates (ADCs) targeted directly to attack CAMs. This strategy could represent the cornerstone of precision-based medicine for peritoneal carcinomatosis. Here, we performed complete transcriptome analyses of ascitic fluid-isolated CAMs in advanced OC patients with primary-, high-, and low-grade, serous subtypes and following neoadjuvant chemotherapy. Our findings suggest that both cancer biological aggressiveness and chemotherapy-induced tumor mass reduction reflect the MMT-associated changes that take place in the tumor surrounding microenvironment. Accordingly, MMT-related genes, including fibroblast activation protein (FAP), mannose receptor C type 2 (MRC2), interleukin-11 receptor alpha (IL11RA), myristoylated alanine-rich C-kinase substrate (MARCKS), and sulfatase-1 (SULF1), were identified as specific actionable targets in CAMs of OC patients, which is a crucial step in the de novo design of ADCs. These cell surface target receptors were also validated in peritoneal CAMs of colorectal cancer peritoneal implants, indicating that ADC-based treatment could extend to other abdominal tumors that show peritoneal colonization. As proof of concept, a FAP-targeted ADC reduced tumor growth in an OC xenograft mouse model with peritoneal metastasis-associated fibroblasts. In summary, we propose MMT as a potential source of ADC-based therapeutic targets for peritoneal carcinomatosis. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The prognostic value and immunological role of SULF2 in adrenocortical carcinoma

BackgroundAdrenocortical carcinoma (ACC) represents the rare urological epithelial cancer of urinary tract, which has a large mass and is usually diagnosed at the advanced stage, thus inducing the poor prognosis. As a result, early detection and diagnosis are more important for the prognosis rather than the treatment of ACC. There is evidence supporting the association of Sulfatase2 (SULF2) with bladder cancer. However, the relationships of SULF2 with the clinical features and immune infiltration of ACC remain unclear. MethodsThis work comprehensively investigated the different expression levels of SULF2 within ACC and its prognostic significance through various databases including Gene Expression Profiling Interaction Analysis (GEPIA), Tumor Immune Estimation Resource (TIMER), The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Kaplan–Meier (KM) plotter and UALCAN. Besides, SULF2 levels within different tumor and paraneoplastic tissues were examined based on Human Protein Atlas (HPA) and TIMER. Afterwards, this study identified differentially expressed genes (DEGs) in high-compared with low-SULF2-expression groups. To predict the possible interaction between SULF2 and its targets, a protein-protein interaction (PPI) network was constructed based on relevant data collected in STRING database. Besides, the SULF2 functional annotation was carried out, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and GSEA. In addition, gene mutation analysis was also performed based on the cBioPortal database. The relation of SULF2 with immune infiltration was analyzed from various aspects by using the resources of various databases including TIMER, TISIDB, and GEPIA, which was first reported in this work. Finally, R package was utilized to plot the receiver operating characteristic (ROC) curves of diagnosis, time-dependent survival, and the association of SULF2 with cancer stage and the nomogram model. Finally, CellMiner dataset was adopted for SULF2 correlation as well as drug sensitivity analysis. ResultsRelative to healthy people, SULF2 level markedly elevated within ACC tissues. Besides, SULF2 up-regulation significantly predicted the dismal prognostic outcome, which may be an important prognostic factor. Afterwards, the PPI network was constructed, and the possible link of SULF2 with the corresponding targets was predicted. Besides, up-regulated SULF2 expression was tightly related to immune regulation and tumor-infiltration immune cell (TIICs), including CD8+, CD4+ and mast cells. Finally, SULF2 expression was speculated to help determine the sensitivity of certain drugs. ConclusionsSULF2 may offer a new therapeutic target for ACC patients and become an important potential prognostic biomarker.

EZH2/hSULF1 axis mediates receptor tyrosine kinase signaling to shape cartilage tumor progression.

Chondrosarcomas are primary cancers of cartilaginous tissue and capable of alteration to highly aggressive, metastatic, and treatment-refractory states, leading to a poor prognosis with a five-year survival rate at 11 months for dedifferentiated subtype. At present, the surgical resection of chondrosarcoma is the only effective treatment, and no other treatment options including targeted therapies, conventional chemotherapies, or immunotherapies are available for these patients. Here, we identify a signal pathway way involving EZH2/SULF1/cMET axis that contributes to malignancy of chondrosarcoma and provides a potential therapeutic option for the disease. A non-biased chromatin immunoprecipitation sequence, cDNA microarray analysis, and validation of chondrosarcoma cell lines identified sulfatase 1 (SULF1) as the top EZH2-targeted gene to regulate chondrosarcoma progression. Overexpressed EZH2 resulted in downregulation of SULF1 in chondrosarcoma cell lines, which in turn activated cMET pathway. Pharmaceutical inhibition of cMET or genetically silenced cMET pathway significantly retards the chondrosarcoma growth and extends mice survival. The regulation of EZH2/SULF1/cMET axis were further validated in patient samples with chondrosarcoma. The results not only established a signal pathway promoting malignancy of chondrosarcoma but also provided a therapeutic potential for further development of effective target therapy to treat chondrosarcoma.

The Cancer/Testis Antigen CT45A1 Promotes Transcription of Oncogenic Sulfatase-2 Gene in Breast Cancer Cells and Is Sensible Targets for Cancer Therapy.

Invasive breast carcinomas (BRCAs) are highly lethal. The molecular mechanisms underlying progression of invasive BRCAs are unclear, and effective therapies are highly desired. The cancer-testis antigen CT45A1 promotes overexpression of pro-metastatic sulfatase-2 (SULF2) and breast cancer metastasis to the lungs, but its mechanisms are largely unknown. In this study, we aimed to elucidate the mechanism of CT45A1-induced SULF2 overexpression and provide evidence for targeting CT45A1 and SULF2 for breast cancer therapy. The effect of CT45A1 on SULF2 expression was assessed using reverse transcription polymerase chain reaction and western blot. The mechanism of CT45A1-induced SULF2 gene transcription was studied using protein-DNA binding assay and a luciferase activity reporter system. The interaction between CT45A1 and SP1 proteins was assessed using immunoprecipitation and western blot. Additionally, the suppression of breast cancer cell motility by SP1 and SULF2 inhibitors was measured using cell migration and invasion assays. CT45A1 and SULF2 are aberrantly overexpressed in patients with BRCA; importantly, overexpression of CT45A1 is closely associated with poor prognosis. Mechanistically, gene promoter demethylation results in overexpression of both CT45A1 and SULF2. CT45A1 binds directly to the core sequence GCCCCC in the promoter region of SULF2 gene and activates the promoter. Additionally, CT45A1 interacts with the oncogenic master transcription factor SP1 to drive SULF2 gene transcription. Interestingly, SP1 and SULF2 inhibitors suppress breast cancer cell migration, invasion, and tumorigenicity. Overexpression of CT45A1 is associated with poor prognosis in patients with BRCA. CT45A1 promotes SULF2 overexpression by activating the promoter and interacting with SP1. Additionally, SP1 and SULF2 inhibitors suppress breast cancer cell migration, invasion, and tumorigenesis. Our findings provide new insight into the mechanisms of breast cancer metastasis and highlight CT45A1 and SULF2 as sensible targets for developing novel therapeutics against metastatic breast cancer.

Sulfatase 2 Affects Polarization of M2 Macrophages through the IL-8/JAK2/STAT3 Pathway in Bladder Cancer

Sulfatase 2 (SULF2) affects the occurrence and development of cancer by regulating HSPG-binding factors. However, the mechanism of SULF2 in bladder cancer (BCa) is unknown. To determine this, we analyzed the RNA sequencing of 90 patients with BCa. The results showed that the expression of SULF2 was closely related to the prognosis of BCa. Moreover, in vivo and in vitro experiments revealed that SULF2 promotes tumor proliferation and invasion. Furthermore, using a mouse orthotopic BCa model and flow cytometric analysis, we identified that SULF2 affects the polarization of macrophages. Mechanism studies clarified that SULF2 promoted the release of HSPG-binding factors, such as IL-8, in the microenvironment through β-catenin. Meanwhile, IL-8 activated the JAK2/STAT3 pathway of macrophages to promote the expression of CD163 and CD206, thereby regulating the polarization of macrophages to the M2-type. Conclusively, these results indicate that SULF2 plays an important role in regulating the microenvironment of BCa and promotes the polarization of macrophages to the M2-type by secreting IL-8, which further deepens the malignant progression of BCa.

Sulfatase 2 Along with Syndecan 1 and Glypican 3 Serum Levels are Associated with a Prognostic Value in Patients with Alcoholic Cirrhosis-Related Advanced Hepatocellular Carcinoma.

Sulfatase 2 (SULF2) is an enzyme related to heparan sulfate modifications. Its expression, as for some heparan sulfate proteoglycans expression, has been linked to hepatocellular carcinoma (HCC) at mRNA level and immunohistochemistry staining on biopsy samples. This study aims to evaluate the prognostic value of serum levels of SULF2 in patients with alcoholic cirrhosis with or without HCC. Two hundred and eighty-seven patients with alcoholic cirrhosis were enrolled in this study: 164 without HCC, 57 with early HCC, and 66 with advanced HCC at inclusion. We analyzed the association between SULF2 serum levels and prognosis using Kaplan-Meier method and univariate and multivariate analysis using a Cox model. Child-Pugh C Patients have higher serum levels of SULF2 than Child-Pugh A patients. Serum levels of SULF2 were also higher in patients with advanced HCC compared with the other groups. In patients with advanced HCC, high serum levels of SULF2 were associated with less favorable overall survival. Combination of SULF2 with Glypican 3 (GPC3) and Syndecan 1 (SDC1) serum levels enhanced the ability to discriminate worst prognostic in advanced HCC. SULF2 along with GPC3 and SDC1 serum levels have been shown to be associated with a prognostic value in advanced HCC.

Sulfatase-2 from Cancer Associated Fibroblasts: An Environmental Target for Hepatocellular Carcinoma?

Introduction: Heparin sulphate proteoglycans in the liver tumour microenvironment (TME) are key regulators of cell signalling, modulated by sulfatase-2 (SULF2). SULF2 overexpression occurs in hepatocellular carcinoma (HCC). Our aims were to define the nature and impact of SULF2 in the HCC TME. Methods: In liver biopsies from 60 patients with HCC, expression and localization of SULF2 were analysed associated with clinical parameters and outcome. Functional and mechanistic impacts were assessed with immunohistochemistry (IHC), in silico using The Cancer Genome Atlas (TGCA), in primary isolated cancer activated fibroblasts, in monocultures, in 3D spheroids, and in an independent cohort of 20 patients referred for sorafenib. IHC targets included αSMA, glypican-3, β-catenin, RelA-P-ser536, CD4, CD8, CD66b, CD45, CD68, and CD163. SULF2 impact of peripheral blood mononuclear cells was assessed by migration assays, with characterization of immune cell phenotype using fluorescent activated cell sorting. Results: We report that while SULF2 was expressed in tumour cells in 15% (9/60) of cases, associated with advanced tumour stage and type 2 diabetes, SULF2 was more commonly expressed in cancer-associated fibroblasts (CAFs) (52%) and independently associated with shorter survival (7.2 vs. 29.2 months, p = 0.003). Stromal SULF2 modulated glypican-3/β-catenin signalling in vitro, although in vivo associations suggested additional mechanisms underlying the CAF-SULF2 impact on prognosis. Stromal SULF2 was released by CAFS isolated from human HCC. It was induced by TGFβ1, promoted HCC proliferation and sorafenib resistance, with CAF-SULF2 linked to TGFβ1 and immune exhaustion in TGCA HCC patients. Autocrine activation of PDGFRβ/STAT3 signalling was evident in stromal cells, with the release of the potent monocyte/macrophage chemoattractant CCL2 in vitro. In human PBMCs, SULF2 preferentially induced the migration of macrophage precursors (monocytes), inducing a phenotypic change consistent with immune exhaustion. In human HCC tissues, CAF-SULF2 was associated with increased macrophage recruitment, with tumouroid studies showing stromal-derived SULF2-induced paracrine activation of the IKKβ/NF-κB pathway, tumour cell proliferation, invasion, and sorafenib resistance. Conclusion: SULF2 derived from CAFs modulates glypican-3/β-catenin signalling but also the HCC immune TME, associated with tumour progression and therapy resistance via activation of the TAK1/IKKβ/NF-κB pathway. It is an attractive target for combination therapies for patients with HCC.

Abstract 12577: Pulmonary Endothelial Sulfatase-1 is Regulated by NEDD9 and Increased in Pulmonary Arterial Hypertension

Introduction: Oxidation of the scaffolding protein NEDD9 by aldosterone (ALDO) increases NEDD9 bioactivity in human pulmonary artery endothelial cells (HPAECs), leading to pulmonary arterial hypertension (PAH). In tumor cells, oxidizing conditions alter Sulfatase-1 (SULF-1) activity. In turn, SULF-1 catalyzes 6-O-sulfate removal from heparan sulfate proteoglycans that releases cytokines implicated in the pathogenesis of PAH. Hypothesis: NEDD9 regulates SULF-1 expression in HPAECs, which is associated with pulmonary arterial remodeling in PAH. Methods: HPAECs were treated with ALDO (10 -7 mol/L) for 24 hr and then untransfected (Un-Tx) or transfected with siRNA against NEDD9, and cell lysates were used to isolate mRNA for RNA-Seq. Separately, SULF1 expression was measured by immunoblot in HPAECs transfected with si-NEDD9 or scrambled control siRNA. Paraffin-embedded lung specimens from donor controls (N=5), patients with PAH (N=5), or the monocrotaline (MCT) (inflammatory) and SU-5416/hypoxia (angioproliferative) experimental rat models of PAH were analyzed by immunofluorescence to quantify SULF1, NEDD9, and endothelial PECAM-1. Results: In ALDO-treated HPAECs, si-NEDD9 decreased SULF1 mRNA quantity vs. Un-Tx (logFC -6.39, N=3, P&lt;0.001, FDR&lt;0.001). Transfection with si-NEDD9 also decreased SULF1 protein compared to si-Scr (14 ± 2.0 vs. 2.7 ± 1.0 arb. units, N=3, P&lt;0.01) in vitro . In remodeled pulmonary arterioles from MCT-PAH and SU-5416/hypoxia-PAH, SULF1 was increased by 5.6-fold (P&lt;0.01) and 6.6-fold (P&lt;0.01), respectively, and correlated strongly with NEDD9 protein (r=0.91, N=9, P&lt;0.001). The translational relevance of these data was assessed next: compared to donor controls, pulmonary endothelial SULF1 was increased in remodeled pulmonary arterioles from PAH patients in situ (0.03 ± 0.01 vs. 0.14 ± 0.03 arb. units, P&lt;0.05). Conclusions: SULF1 is regulated by the scaffolding protein NEDD9 in HPAECs in vitro , and remodeled pulmonary arterioles from two experimental PAH models and PAH patients express increased NEDD9-SULF1. Identifying the molecular mechanism(s) regulating NEDD9-dependent regulation of SULF1 may have important implications for therapeutic target discovery to prevent vascular remodeling in PAH.

Sulfatase-2 Regulates Liver Fibrosis through the TGF-β Signaling Pathway.

Simple SummaryLiver fibrosis and/or cirrhosis is a major risk factor for hepatocellular carcinoma. Hepatic Fibrogenesis is the result of an excessive production and deposition of extracellular matrix by hepatic myofibroblasts, which are primarily formed from hepatic stellate cells. The heparan sulfate editing enzyme sulfatase-2 is known to be elevated in cirrhotic liver and hepatocellular carcinoma. Our aim in this study was to delineate the mechanistic role of sulfatase-2 in fibrotic liver disease using mouse and in vitro cell culture models of liver fibrosis. Our data here demonstrates that mice deficient in sulfatase-2 have reduced liver fibrosis. We also show that sulfatase-2 promotes cell proliferation, cell viability, the production of collagen, migration, and activation of hepatic stellate cells. Our findings highlight sulfatase-2 as a potential target for therapeutic intervention geared at reversing liver fibrosis.Transforming growth factor-β (TGF-β) activates hepatic stellate cells (HSCs), which drive liver fibrosis via the production and deposition of extracellular matrix (ECM). We aimed to elucidate the mechanistic role of sulfatase-2 (SULF2) in liver fibrosis. To this end, we induced liver fibrosis in wild-type (WT) and SULF2 knockout (Sulf2-KO) mice (6–8 weeks-old) via bile duct ligation (BDL), intraperitoneal injection of carbon tetrachloride (CCl4) or thioacetamide (TAA). The levels of fibrosis in the liver sections were assessed via Sirius red and Masson’s trichrome staining, immunohistochemistry and immunoblotting for α-smooth muscle actin (α-SMA) and hydroxyproline. To evaluate the interaction between TGF-β and SULF2, we transfected human HSCs with scrambled control shRNA and shRNA constructs targeting SULF2 and measured α-SMA expression following treatment with TGF-β1 ligand. We show here that knockout of SULF2 significantly decreases collagen content, as well as bands of bridging fibrosis, as demonstrated by Sirius red, Masson’s trichrome and α-SMA staining after BDL, CCl4 and TAA injection in Sulf2-KO versus WT mice. In all three models of liver fibrosis, we observed significantly lower levels of hydroxyproline in the Sulf2-KO mice compared to the WT mice. HSCs with reduced levels of SULF2 failed to significantly express α-SMA and collagen type I following treatment with TGF-β1. Furthermore, SULF2 co-localizes with TGFBR3 and the in vitro knockdown of SULF2 in HSCs decreases the release of TGF-β1 from TGFBR3. Together, these data suggest that SULF2 regulates liver fibrosis via the TGF-β signaling pathway. Pharmacologic inhibition of SULF2 may represent a novel therapeutic approach to improve liver fibrosis.

SULF2 is a novel diagnostic and prognostic marker for high-grade bladder cancer with lymphatic metastasis.

BackgroundSulfatase 2 (SULF2) is a member of the sulfatase family, and its expression and clinical significance in bladder cancer (BCa) are not currently known. In this study, we attempted to evaluate SULF2 expression in BCa patients who underwent radical cystectomy (RC) and the relationship between SULF2 expression and clinical-pathological characteristics.MethodsData on SULF2 expression in BCa tissues was obtained from the Oncomine database and the Gene Expression Omnibus (GEO). The expression of SULF2 and vascular endothelial growth factor-D (VEGF-D) in BCa was evaluated by immunohistochemistry (IHC) in tissues from 203 patients who had undergone RC. We also explored the value of the measurement of SULF2 and VEGF-D expression for diagnosis and prognosis in BCa patients with lymphatic metastasis.ResultsWe found an increase in SULF2 messenger RNA (mRNA) levels and gene amplification in BCa tissues from the Oncomine database. High expression of SULF2 was detected in 91/203 (44.8%) of BCa patients. Among these patients, 27 of 42 (64.3%) with lymphatic metastasis showed high SULF2 expression. Univariate analysis showed that tumor size, pathological stage, lymphatic metastasis, vascular infiltration, perineural infiltration, hydronephrosis, and VEGF-D and SULF2 expression were related to prognosis in BCa patients, and multivariable Cox regression analysis showed that SULF2 expression was an independent prognostic indicator. Receiver operating characteristic (ROC) analysis revealed that SULF2 expression resulted in an increased area under the curve (AUC) of 0.707, with a sensitivity of 71.4% and a specificity of 61.5%.ConclusionsThe upregulation of SULF2 is associated with poor prognosis in high-grade BCa patients. It might be a novel diagnostic marker for BCa patients with lymphatic metastasis.

Abstract LB137: OKN-007 suppresses uterine carcinosarcoma by inhibiting extracellular sulfatase 2

Abstract INTRODUCTION: Endometrial carcinoma (EMC) is the most common gynecologic malignancy and is highly lethal in the metastatic setting with an overall survival of 1.5-2 years due to chemotherapy resistance. Uterine carcinosarcoma (UCS) is a rare but aggressive biphasic subtype that comprises only 3-4% of EMCs but accounts for greater than 16% of uterine cancer deaths. Sulfatase 2 (SULF2) is an extracellular enzyme that activates pro-tumorigenic growth factor signaling, including transforming growth factor β (TGFβ). SULF2 is overexpressed in many cancers and is associated with tumor progression and poor prognosis. TCGA data reveal that the SULF2 gene is commonly amplified or mutated in UCS (17.5%) and may be a viable therapeutic target. OKN-007, a disulfonyl derivative of phenyl-tert-butyl nitrone, interrupts the TGFβ pathway and decreases downstream markers of epithelial mesenchymal transition (EMT) through inhibition of SULF2 in the tumor extracellular matrix. As such, assessment of OKN-007 was undertaken in UCS and EMC.METHODS: The anticancer effect of OKN-007 was evaluated in a UCS cell line (CS99) and EMC cell lines (Ishikawa, AN3CA, Hec1A, and RL95-2A). Cell viability, migration, colony-formation, and protein expression of SULF2, TGFβ, and EMT markers were evaluated in 2D and 3D culture conditions in vitro. The effect of OKN-007 alone or in combination with chemotherapy (carboplatin and paclitaxel) on tumor growth was evaluated in an intraperitoneal UCS xenograft mouse model. In vivo tumor growth was monitored using magnetic resonance imaging. TGFβ, SULF2, and EMT markers were investigated by western blot. RESULTS: SULF2 was highly expressed in UCS and EMC cells in 2D and 3D conditions. Decreased cell viability, migration, and colony-forming ability were observed with OKN-007 treatment with differences observed in IC50 values in 2D vs 3D conditions. Mice treated with OKN-007 demonstrated no observable toxicity, had decreased mean tumor volume (p&amp;lt;0.0001), and had prolonged survival compared to untreated mice (p&amp;lt;0.01). This effect was also observed when OKN-007 was given in combination with chemotherapy (p&amp;lt;0.05). Immunoblotting demonstrated a 30% reduction in SULF2 expression in OKN-007 treated tumors. Decreased expression of TGFβ and matrix metalloprotease 9 (MMP9) was also noted. CONCLUSION: OKN-007 is an effective agent in UCS and EMC. The anticancer activity of OKN-007 across histologic subtypes of EMC is associated with inhibition of the SULF2-TGFβ signaling pathway which decreases cellular proliferation, migration and viability in vitro and in vivo. Citation Format: Anjalika Gandhi, Samrita Dogra, Rheal Towner, Bethany N. Hannafon, Katherine Moxley. OKN-007 suppresses uterine carcinosarcoma by inhibiting extracellular sulfatase 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB137.

The upregulation of miR-204-3p in LPS-induced acute lung injury aggravated pulmonary endothelial cells apoptosis via targeting sulfatase 2.

Acute lung injury (ALI) results from the injury of alveolar epithelial cells and pulmonary capillary endothelial cells, with a high mortality rate ranging from 29% to 42%. Therefore, more efficient therapeutic strategies for ALI are necessary. Numerous studies revealed that miRNAs play a role in the regulation of ALI. Lipopolysaccharide (LPS) can induce an inflammatory response and has been widely applied in the establishment of the mouse ALI model. Here, we reported that miR-204-3p expression was upregulated by LPS treatment with increased cytokine secretion. LPS treatment promoted cell apoptosis, accompanied by abnormal cell structure and unobvious alveolar structure. These effects could be prevented by down-regulation of miR-204-3p, and promoted by miR-204-3p overexpression. Sulfatase 2 (SULF2) appeared to be the target of miR-204-3p predicted by TargetScan. Downregulation of miR-204-3p enhanced the protein level of SULF2, indicating that SULF2 was a target of miR-204-3p, which could negatively regulate the expression of SULF2. Thus, targeting miR-204-3p may be a potential therapeutic strategy for ALI.

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.

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. 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 (PDGFRβ)-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 PDGFRβ-YAP signaling and tumor growth in the mouse xenograft model. These results suggest that SULF2 monoclonal antibody 5D5 or related agents may be potentially promising therapeutic agents in CCA.

Rs3802278 in 3’-UTR of SULF1 associated with platinum resistance and survival in Chinese epithelial ovarian cancer patients

Ovarian cancer is the leading cause of death from gynecologic cancers, but platinum resistance remains a major obstacle in the chemotherapy of ovarian cancer. This study aims to examine the role of polymorphisms in sulfatase 1 (SULF1) in platinum resistance and survival in advanced epithelial ovarian cancer (EOC) patients. We genotyped 12 SNPs of SULF1 in 195 EOC patients treated with platinum using MassARRAY method and evaluated the association between the SNPs and platinum response. SULF1 rs3802278 was marginal significantly associated with platinum resistance in recessive model with p value of 0.055. The patients with SULF1 rs3802278 AA were more resistant to platinum-based chemotherapy comparing to those with AG/GG genotype (OR: 2.317, 95%CI: 0.982 ∼ 5.465). In survival analysis, rs3802278 was significantly associated with both of PFS and OS after adjusted by FIGO stage and age. Patients with AA genotypes showed a shorter PFS and OS than with AG/GG genotypes (median PFS: 15 months vs. 21 months, p = 0.010, HR = 1.876, 95%CI: 1.165-3.022; median OS: 42 months vs. 73 months, p = 0.031, HR = 1.928, 95%CI: 1.061-3.504). SULF1 rs3802278 may serve as a potential candidate biomarker for the prediction of platinum resistance and prognosis in Chinese EOC patients.

The Role of Heparan Sulfate and Neuropilin 2 in VEGFA Signaling in Human Endothelial Tip Cells and Non-Tip Cells during Angiogenesis In Vitro.

During angiogenesis, vascular endothelial growth factor A (VEGFA) regulates endothelial cell (EC) survival, tip cell formation, and stalk cell proliferation via VEGF receptor 2 (VEGFR2). VEGFR2 can interact with VEGFR2 co-receptors such as heparan sulfate proteoglycans (HSPGs) and neuropilin 2 (NRP2), but the exact roles of these co-receptors, or of sulfatase 2 (SULF2), an enzyme that removes sulfate groups from HSPGs and inhibits HSPG-mediated uptake of very low density lipoprotein (VLDL), in angiogenesis and tip cell biology are unknown. In the present study, we investigated whether the modulation of binding of VEGFA to VEGFR2 by knockdown of SULF2 or NRP2 affects sprouting angiogenesis, tip cell formation, proliferation of non-tip cells, and EC survival, or uptake of VLDL. To this end, we employed VEGFA splice variant 121, which lacks an HSPG binding domain, and VEGFA splice variant 165, which does have this domain, in in vitro models of angiogenic tip cells and vascular sprouting. We conclude that VEGFA165 and VEGFA121 have similar inducing effects on tip cells and sprouting in vitro, and that the binding of VEGFA165 to HSPGs in the extracellular matrix does not seem to play a role, as knockdown of SULF2 did not alter these effects. Co-binding of NRP2 appears to regulate VEGFA–VEGFR2-induced sprout initiation, but not tip cell formation. Finally, as the addition of VLDL increased sprout formation but not tip cell formation, and as VLDL uptake was limited to non-tip cells, our findings suggest that VLDL plays a role in sprout formation by providing biomass for stalk cell proliferation.

Sulfatase 2 Is Associated with Steroid Resistance in Childhood Nephrotic Syndrome.

Glucocorticoid (GC) resistance complicates the treatment of ~10–20% of children with nephrotic syndrome (NS), yet the molecular basis for resistance remains unclear. We used RNAseq analysis and in silico algorithm-based approaches on peripheral blood leukocytes from 12 children both at initial NS presentation and after ~7 weeks of GC therapy to identify a 12-gene panel able to differentiate steroid resistant NS (SRNS) from steroid-sensitive NS (SSNS). Among this panel, subsequent validation and analyses of one biologically relevant candidate, sulfatase 2 (SULF2), in up to a total of 66 children, revealed that both SULF2 leukocyte expression and plasma arylsulfatase activity Post/Pre therapy ratios were greater in SSNS vs. SRNS. However, neither plasma SULF2 endosulfatase activity (measured by VEGF binding activity) nor plasma VEGF levels, distinguished SSNS from SRNS, despite VEGF’s reported role as a downstream mediator of SULF2’s effects in glomeruli. Experimental studies of NS-related injury in both rat glomeruli and cultured podocytes also revealed decreased SULF2 expression, which were partially reversible by GC treatment of podocytes. These findings together suggest that SULF2 levels and activity are associated with GC resistance in NS, and that SULF2 may play a protective role in NS via the modulation of downstream mediators distinct from VEGF.