Antithrombin-binding heparan sulfate is ubiquitously expressed in epithelial cells and suppresses pancreatic tumorigenesis
Thomas Mandel Clausen, Ryan J. Weiss, Jacob R. Tremblay, Benjamin P. Kellman, Joanna Coker, Leo A. Dworkin, Jessica P. Rodriguez, Ivy M. Chang, Timothy Chen, Vikram Padala, Richard Karlsson, Hyemin Song, Kristina L. Peck, Satoshi Ogawa, Daniel R. Sandoval, Hiren J. Joshi, Gaowei Wang, L. Paige Ferguson, Nikita Bhalerao, Allison Moores, Tannishtha Reya, Maike Sander, Thomas C. Caffrey, Jean L. Grem, Alexandra Aicher, Christopher Heeschen, Dzung Le, Nathan E. Lewis, Michael A. Hollingsworth, Paul M. Grandgenett, Susan L. Bellis, Rebecca L. Miller, Mark M. Fuster, David W. Dawson, Dannielle D. Engle, Jeffrey D. Esko
Thomas Mandel Clausen, Ryan J. Weiss, Jacob R. Tremblay, Benjamin P. Kellman, Joanna Coker, Leo A. Dworkin, Jessica P. Rodriguez, Ivy M. Chang, Timothy Chen, Vikram Padala, Richard Karlsson, Hyemin Song, Kristina L. Peck, Satoshi Ogawa, Daniel R. Sandoval, Hiren J. Joshi, Gaowei Wang, L. Paige Ferguson, Nikita Bhalerao, Allison Moores, Tannishtha Reya, Maike Sander, Thomas C. Caffrey, Jean L. Grem, Alexandra Aicher, Christopher Heeschen, Dzung Le, Nathan E. Lewis, Michael A. Hollingsworth, Paul M. Grandgenett, Susan L. Bellis, Rebecca L. Miller, Mark M. Fuster, David W. Dawson, Dannielle D. Engle, Jeffrey D. Esko
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Research Article Cell biology Oncology

Antithrombin-binding heparan sulfate is ubiquitously expressed in epithelial cells and suppresses pancreatic tumorigenesis

Abstract

3-O-sulfation of heparan sulfate (HS) is the key determinant for binding and activation of antithrombin III (AT). This interaction is the basis of heparin treatment to prevent thrombotic events and excess coagulation. Antithrombin-binding HS (HSAT) is expressed in human tissues but is thought to be expressed in the subendothelial space, mast cells, and follicular fluid. Here, we show that HSAT is ubiquitously expressed in the basement membranes of epithelial cells in multiple tissues. In the pancreas, HSAT is expressed by healthy ductal cells, and its expression is increased in premalignant pancreatic intraepithelial neoplasia lesions but not in pancreatic ductal adenocarcinoma (PDAC). Inactivation of HS3ST1, a key enzyme in HSAT synthesis, in PDAC cells eliminated HSAT expression, induced an inflammatory phenotype, suppressed markers of apoptosis, and increased metastasis in an experimental mouse PDAC model. HSAT-positive PDAC cells bind AT, which inhibits the generation of active thrombin by tissue factor and factor VIIa. Furthermore, plasma from patients with PDAC showed accumulation of HSAT, suggesting its potential as a marker of tumor formation. These findings suggest that HSAT exerts a tumor-suppressing function through recruitment of AT and that the decrease in HSAT during progression of pancreatic tumorigenesis increases inflammation and metastatic potential.

Authors

Thomas Mandel Clausen, Ryan J. Weiss, Jacob R. Tremblay, Benjamin P. Kellman, Joanna Coker, Leo A. Dworkin, Jessica P. Rodriguez, Ivy M. Chang, Timothy Chen, Vikram Padala, Richard Karlsson, Hyemin Song, Kristina L. Peck, Satoshi Ogawa, Daniel R. Sandoval, Hiren J. Joshi, Gaowei Wang, L. Paige Ferguson, Nikita Bhalerao, Allison Moores, Tannishtha Reya, Maike Sander, Thomas C. Caffrey, Jean L. Grem, Alexandra Aicher, Christopher Heeschen, Dzung Le, Nathan E. Lewis, Michael A. Hollingsworth, Paul M. Grandgenett, Susan L. Bellis, Rebecca L. Miller, Mark M. Fuster, David W. Dawson, Dannielle D. Engle, Jeffrey D. Esko

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Figure 6

Murine PDAC cells derived from the KrasLSL.G12D/+ p53R172H/+ PdxCre (KPC) mouse express HSAT.

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Murine PDAC cells derived from the KrasLSL.G12D/+ p53R172H/+ PdxCre (KPC...
(A) AT bound to FC1199, FC1242, and FC1245 as measured by flow cytometry. Treatment with heparin lyase (HSase) abrogated AT binding. Graph shows mean ± SEM. (B) mAB 10E4 (anti-HS) binding to FC1199, FC1242, and FC1245 by flow cytometry. HSase treatment diminished mAb binding. Graph shows mean ± SEM. (C) Genetic inactivation of HS3ST1 in FC1242 and FC1245 abolished AT binding but had no effect on binding of mAb 10E4. Graph shows mean ± SEM. (D) Disaccharide analysis of purified HS from WT FC1242 and HS3ST1–/– cells. (E) 3-O-sulfated disaccharides (D0S3 and D0S9) were present in FC1242 cells. No 3-O-sulfation was detected in HS3ST1–/– cells. (F) Anti-FXa assay was used to determine the presence of HSAT in purified HS from WT and HS3ST1–/– FC1242 cells compared with unfractionated heparin (UFH). (G) Binding of [35S]HS to AT and FGF-2 as measured by filtration across nitrocellulose. Approximately 5% of input [35S]HS from WT FC1242 HS cells bound AT, whereas [35S]HS from HS3ST1–/– cells did not. BSA served as a negative control. Statistical analysis by 2-tailed t test in A–C. ***P ≤ 0.001; ****P ≤ 0.0001; NS, not significant.