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

<div><p>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 <i>in vivo</i> 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.</p></div

C1GALT1 modifies O-glycans on integrin β1.

<p>(<i>a</i>) Integrin β1 carried O-glycans in HCC cells. Lysates of HA22T cells (0.5 mg) were treated with neuraminidase (Neu) and/or PNGaseF and then pulled down (PD) by PNA agarose beads. The pulled down proteins were separated by 6% SDS-PAGE and analyzed by immunoblotting (IB) with anti-integrin β1 antibody. (<i>b</i>) C1GALT1 enhanced PNA binding to integrin β1 in HCC36 cells. (<i>c</i>) Knockdown of C1GALT1 suppressed PNA binding to integrin β1 in HA22T cells. Cell lysates (1.2 mg) were immunoprecipitated (IP) by anti-integrin β1 antibody, and were treated with (+) or without (−) neuraminidase. Proteins were separated by 8% SDS-PAGE, and then blotted with PNA or anti-integrin β1 antibody. Non-specific mouse IgG was used as control.</p

C1GALT1 regulates integrin β1 activity and signaling.

<p>(<i>a</i>) The activity of integrin β1 was enhanced in C1GALT1 overexpressing cells. Mock and C1GALT1 transfectants were analyzed by flow cytometry with anti-integrin β1 antibody (left) or anti-activated integrin β1 (HUTS-21) antibody (right). Non-specific mouse IgG was used as a background fluorescence control (dash line). The mean fluorescence intensities (MFI) are shown at the bottom. Results are represented as means ± SD from three independent experiments. * <i>P</i><0.05. (<i>b</i>) Knockdown of C1GALT1 suppressed integrin β1 activity. Control (Ctr sh) and C1GALT1 knockdown (C1GALT1 sh6 and C1GALT1 sh8) cells were analyzed by flow cytometry with the indicated antibodies. The average of MFI is shown at the bottom. Results are represented as means ± SD from three independent experiments. * <i>P</i><0.05. (<i>c</i>) C1GALT1 regulated integrin β1-induced FAK activation. p-FAK (Tyr397) and total FAK in C1GALT1 overexpressing cells (left) and knockdown cells (right) were analyzed by Western blotting. Transfectants were seeded on BSA or collagen IV (Col-IV)-coated plates for 0.5 h. The integrin β1 blocking antibody (P4C10, 2 µg/ml) was incubated with cells for 10 min before seeding on collagen IV-coated plates. GAPDH was used as a loading control.</p

Schematic diagram showing the NRG-erbB-FAK signaling in the repairing process following peripheral nerve injury (PNI).

<p>Following PNI [as created by end-to-side neurorraphy (ESN)], NRG would activate the erbB2-FAK pathway that promotes the Schwann cells to migrate to the distal stump for successful axonal guidance and nerve regeneration. Un: ulnar nerve; Mc: musculocutaneous nerve; ECM: extracellular matrix.</p

Immunoblotings (A) and histogram (B) showing the effects of neuregulin β1 (NRG β1) on focal adhesion kinase (FAK) phosphorylation in RSC96 cells seeding on poly-L-lysine (PLL) or laminin-coated plates.

<p>The physical association of erbB-FAK signaling was analyzed by the ratio expressed as phosphorylated FAK (p-FAK) over total FAK. Note that NRG β1 extensively increases erbB-FAK activation in both PLL and laminin-coated plates with the most significant effect observed in the later group. *<i>P</i><0.05 as compared to that of control value.</p

C1GALT1 induces HCC cell adhesion, migration and invasion through integrin β1.

<p>(<i>a</i>) The effects of the integrin β1-blocking antibody, P4C10, on C1GALT1 overexpressing cells. HCC cells were pre-treated with the indicated concentration of P4C10 or control IgG for 10 min. C1GALT1-induced cell-collagen IV adhesion (left), migration (middle), and invasion (right) were suppressed by the integrin β1-blocking antibody. Results are represented as means ± SD from three independent experiments. **<i>P</i><0.01. (<i>b</i>) The effects of the integrin β1-blocking antibody on C1GALT1 knockdown cells. P4C10 inhibited cell-collagen IV adhesion (left), migration (middle), and invasion (right) in cells transfected with control siRNA (Ctr si). Results are shown as means ± SD. * <i>P</i><0.05; **<i>P</i><0.01.</p

Compound muscle action potential recordings (CMAPs) of the biceps brachii muscle upon activation of the musculocutaneous nerve one month following end-to-side neurorraphy (ESN).

<p>Responses were recorded from normal rats (upper panel), phosphate buffer saline (PBS) treated rats (middle panel) and neuregulin β1 (NRG β1) treated rats (lower panel) with stimulus applied above the neurorrhaphy site. Note that NRG β1 effectively promotes nerve regeneration and functional recovery by triggering larger CMAP than that of PBS-treated ones.</p

Dermatan sulfate epimerase 1 (DSE) is frequently upregulated in human gliomas.

<p>(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 <i>DSE</i> gene expression in glioma subtypes and normal brain tissue. ****P<0.0001, n.s. = not statistically significant. These data are from the REMBRANDT database (<a href="http://www.betastasis.com/glioma/rembrandt/" target="_blank">http://www.betastasis.com/glioma/rembrandt/</a>). (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.</p

Dermatan sulfate epimerase 1 (DSE) regulates dermatan sulfate formation in glioma cells.

<p>(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.</p

Confocal photomicrographs showing erbB2 (red) and erbB3 (green) receptor expressions in the culture RSC96 cells following phosphate buffer saline (PBS) (A–D) and neuregulin β1 (NRG β1) treatment (E–H).

<p>The cell nucleus was stained by DAPI (blue). Note that in both PBS and NRG β1 treatment groups, the erbB2 and erbB3 immunoreactivities were present in cell membrane and cell processes (arrows in C, G). However, after NRG β1 treatment (10nM) for 15 minutes, more RSC96 cells (G) with erbB2 and erbB3 co-localization (yellow) in the perinuclear area [arrowhead in (H) showing high magnification of rectangles labeled in (G)] was observed than that of PBS group (C,D). Scale bar = 20 µm in (A, B, C, E, F, G) and represents 5 µm in (D, H).</p