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. 2018 Jun 4;13(6):e0198364.
doi: 10.1371/journal.pone.0198364. eCollection 2018.

DSE promotes aggressive glioma cell phenotypes by enhancing HB-EGF/ErbB signaling

Wen-Chieh Liao  1   2 , Chih-Kai Liao  1   2 , You-Huan Tsai  1 , To-Jung Tseng  1   2 , Li-Ching Chuang  1 , Chyn-Tair Lan  1   2 , Hung-Ming Chang  3 , Chiung-Hui Liu  1   2
Affiliations

DSE promotes aggressive glioma cell phenotypes by enhancing HB-EGF/ErbB signaling

Wen-Chieh Liao et al. PLoS One. .

Abstract

Remodeling of the extracellular matrix (ECM) in the tumor microenvironment promotes glioma progression. Chondroitin sulfate (CS) proteoglycans appear in the ECM and on the cell surface, and can be catalyzed by dermatan sulfate epimerase to form chondroitin sulfate/dermatan sulfate (CS/DS) hybrid chains. Dermatan sulfate epimerase 1 (DSE) is overexpressed in many types of cancer, and CS/DS chains mediate several growth factor signals. However, the role of DSE in gliomas has never been explored. In the present study, we determined the expression of DSE in gliomas by consulting a public database and conducting immunohistochemistry on a tissue array. Our investigation revealed that DSE was upregulated in gliomas compared with normal brain tissue. Furthermore, high DSE expression was associated with advanced tumor grade and poor survival. We found high DSE expression in several glioblastoma cell lines, and DSE expression directly mediated DS chain formation in glioblastoma cells. Knockdown of DSE suppressed the proliferation, migration, and invasion of glioblastoma cells. In contrast, overexpression of DSE in GL261 cells enhanced these malignant phenotypes and in vivo tumor growth. Interestingly, we found that DSE selectively regulated heparin-binding EGF-like growth factor (HB-EGF)-induced signaling in glioblastoma cells. Inhibiting epidermal growth factor receptor (EGFR) and ErbB2 with afatinib suppressed DSE-enhanced malignant phenotypes, establishing the critical role of the ErbB pathway in regulating the effects of DSE expression. This evidence indicates that upregulation of DSE in gliomas contributes to malignant behavior in cancer cells. We provide novel insight into the significance of DS chains in ErbB signaling and glioma pathogenesis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Dermatan sulfate epimerase 1 (DSE) is frequently upregulated in human gliomas.
(A) Expression of DSE in the ONCOMINE cancer microarray database. Four independent representative datasets showed that DSE is significantly upregulated in glioma tissue. (B) High expression of DSE is associated with worse overall survival in glioma patients. The high and low expression groups are divided by median expression levels of DSE in 329 cases. (C) Comparison of DSE gene expression in glioma subtypes and normal brain tissue. ****P<0.0001, n.s. = not statistically significant. These data are from the REMBRANDT database (http://www.betastasis.com/glioma/rembrandt/). (D) Immunohistochemistry of DSE on a tissue array comprising 77 primary glioma samples and 5 normal human brain tissue samples. All sections were counterstained with hematoxylin. Representative images of normal brain tissue; one DSE low expression case (upper panel), and two DSE high expression cases (lower panel) are shown. Amplified images are shown at the bottom right of each image. Scale bars, 100 μm. (E) Expression of DSE in glioma cell lines and mouse brain tissue. Protein expression was analyzed by western blotting. Actin was used as an internal control. Relative expression levels in U118 cells from three independent blots are shown at the right.
Fig 2
Fig 2. Dermatan sulfate epimerase 1 (DSE) regulates dermatan sulfate formation in glioma cells.
(A) Stable knockdown of DSE in U118 cells. U118 cells were stably transfected with control short hairpin RNA (shRNA) (Ctr sh) or DSE-shRNA (DSE sh). The protein expression levels of DSE were analyzed by Western blotting. Immunofluorescence microscopy showed decrease of DSE (green) in stable knockdown cells. Nuclei were counterstained with Hoechst (blue). (B) Blotting of DS chains on proteoglycans was evaluated by horseradish peroxidase (HRP)-conjugated DS-binding protein. Total protein is shown on the right as loading control. (C) Overexpression of DSE in GL261 cells increased DS chain formation. GL261 cells were stably transfected with empty vectors (mock) or DSE-expressing plasmids (DSE). (D) Quantify DS in total protein lysate by DS ELISA assay. Average among of DS in cell lysate was shown. **P < 0.01.
Fig 3
Fig 3. Dermatan sulfate epimerase 1 (DSE) regulates malignant phenotypes in glioma cells.
(A) DSE modulated cell viability in vitro. The cell viability of U118 and GL261 cells was determined using a CCK-8 assay at the indicted time-points. Data represent means ± SD from three independent experiments. *P < 0.05; **P < 0.01. (B) Effects of DSE on anchorage-dependent colony formation. Representative images of colonies are shown at the bottom. Results are presented as the mean ± SD from three independent experiments. **P < 0.01. (C) Effects of DSE on Transwell cell migration, and (D) Matrigel invasion. Representative images are shown at the bottom. All results are represented as means ± SD from three independent experiments. **P < 0.01. (E) DSE enhanced tumor growth in vivo. GL261 transfectants were subcutaneously injected into C57BL/6 mice. The size of the tumors was measured at the indicated time-points, and is represented as the mean ± SD. Tumors were excised and weighted on the 14th day. **P < 0.01, n = 6. Scale bars, 0.5 cm. (F) Expression of DSE in excised tumors. The protein lysate was analyzed by western blotting (top). Actin was used as a loading control. Immunohistochemistry of DSE in tumors (bottom). Representative images are shown. Scale bars, 50 μm.
Fig 4
Fig 4. Dermatan sulfate epimerase 1 (DSE) modulates heparin-binding EGF-like growth factor (HB-EGF)/ErbB signaling in glioma cells.
(A) Knockdown of DSE suppressed HB-EGF-induced downstream signaling. U118 transfectants were treated without (−)/with (+) HB-EGF for 5 and 15 min. Phosphorylation levels of ERK, AKT, total ERK, and AKT were measured by western blotting. Signals were quantified by Image J, and represented as means ± SD from three independent experiments. **P < 0.01 (B) Knockdown of DSE suppressed epidermal growth factor receptor (EGFR) and ErbB2 activation. The protein expression and phosphorylation of EGFR and ErbB2 were analyzed by western blotting with the indicated antibodies. Actin was used as a loading control. (C) Overexpression of DSE enhanced HB-EGF-induced signaling. GL261 transfectants were treated without (−)/with (+) HB-EGF or EGF. Cell lysates were analyzed by western blotting with various antibodies, as indicated. (D) The correlation of DSE expression with EGFR, ERBB2, and ERBB4. Expression of DSE and ERBB2 were positively correlated in glioma patients. Data were analyzed using the REMBRANDT database (http://www.betastasis.com/glioma/rembrandt/).
Fig 5
Fig 5. Effects of afatinib on dermatan sulfate epimerase 1 (DSE)-mediated malignant phenotypes.
U118 and GL261 transfectants were treated with ethanol (EtOH, solvent control) or the indicated concentrations of afatinib. (A) Cell viability was measured using a CCK8 assay. (B) Cell invasion was analyzed using a Transwell assay. All results are represented as means ± SD from three independent experiments. **P < 0.01.
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Grants and funding

This study was supported by the following grants from the Ministry of Science and Technology, Taiwan: MOST-105-2320-B-040-033-MY2 and MOST-106-2320-B-040-009-MY3 (Chiung-Hui Liu), MOST-105-2320-B-040-029-MY3 (Wen-Chieh Liao). TissueFAX Plus Cytometer was performed in the Instrument Center of Chung Shan Medical University, which is supported by Ministry of Science and Technology, Ministry of Education, and Chung Shan Medical University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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