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 5

HSAT is expressed in early pancreatic intraepithelial neoplasia lesions leading to PDAC and correlates with favorable outcome.

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HSAT is expressed in early pancreatic intraepithelial neoplasia lesions ...
(A) Representative cores from a TMA containing FFPE triple cores from 150 patients with PDAC, including dissected pancreatic intraepithelial neoplasia (PanIN) lesions and healthy adjacent pancreatic tissue, stained for HSAT. AT staining was intense along basolateral side of tumor lesions (red arrowheads). Tissue was scored based on staining intensity: 0, less than 10% membrane staining; 1, faint/barely perceptible basolateral or complete membrane staining in 10% or more of tumor core; 2, weak basolateral or complete membrane staining in 10% or more of tumor core; 3, moderate/strong basolateral or complete membrane staining in 10% or more of tumor core. In representative images, intensities 0 and 1 are low-grade PanIN lesions; 2 and 3 are high-grade PanIN lesions. Pie charts show cumulative grading intensity 0 (green), 1 (blue), 2 (orange), or 3 (red) of the entire TMA (individual cores included separately). Removal of HS with heparin lyase diminished staining. . Numbers represent percentage of cores with a given intensity stain. Scale bar: 100 μm. (B) Quantification of data in A. An increase in HSAT seen during PanIN progression compared with healthy ducts. No significant increase seen in PDAC versus healthy pancreas. Median (red line) and quartiles (dotted lines) are illustrated; 1-way ANOVA with Tukey’s multiple-comparison correction. *P ≤ 0.05; **P ≤ 0.01; NS, not significant. (C) Correlation between HS3ST1 expression and tumor grade in PDAC reference single-cell RNA-Seq data set (44). (D) Association between HSAT expression in PDAC samples and patient survival (PanIN lesions excluded and intensity of HSAT staining included as average of 3 patient-specific tissue cores). A stepwise improvement in survival with increased HSAT was evident but did not achieve significance. (E) Association between HSAT expression and patient survival for high versus low HSAT stain. Presence of HSAT was significantly associated with better outcome; log-rank (Mantel-Cox) test.