Syndecan-1 deficiency promotes tumor growth in a murine model of colitis-induced colon carcinoma

<div><p>Syndecan-1 (Sdc1) is an important member of the cell surface heparan sulfate proteoglycan family, highly expressed by epithelial cells in adult organisms. Sdc1 is involved in the regulation of cell migration, cell-cell and cell-matrix interactions, growth-factor, chemokine and integrin activity, and implicated in inflammatory responses and tumorigenesis. Gastrointestinal tract represents an important anatomic site where loss of Sdc1 expression was reported both in inflammation and malignancy. However, the biological significance of Sdc1 in chronic colitis-associated tumorigenesis has not been elucidated. To the best of our knowledge, this study is the first to test the effects of Sdc1 loss on colorectal tumor development in inflammation-driven colon tumorigenesis. Utilizing a mouse model of colitis-related colon carcinoma induced by the carcinogen azoxymethane (AOM), followed by the inflammatory agent dextran sodium sulfate (DSS), we found that Sdc1 deficiency results in increased susceptibility to colitis-associated tumorigenesis. Importantly, colitis-associated tumors developed in Sdc1-defficient mice were characterized by increased local production of IL-6, activation of STAT3, as well as induction of several STAT3 target genes that act as important effectors of colonic tumorigenesis. Altogether, our results highlight a previously unknown effect of Sdc1 loss in progression of inflammation-associated cancer and suggest that decreased levels of Sdc1 may serve as an indicator of colon carcinoma progression in the setting of chronic inflammation.</p></div

Syndecan-1 deficiency exacerbates colonic chronic inflammation in DSS treated mice.

<p><b>(A)</b> WT and Sdc1-KO mice (n = 14 mice) were treated with three cycles of DSS as described in Methods and weight changes were measured. <b>(B)</b> Blinded histological scoring of inflammation in colonic mucosa of mice was performed as described in Methods. Error bars represent mean ± SE. *<i>P</i> < 0.05, **P < 0.01, **P < 0.001 by Student’s t test for mass change and Mann-Whitney U test for inflammation score.<b>(C)</b> Representative histopathologic sections of colon from WT and Sdc1-KO mice. Hyperplastic/dysplastic glands (lower panel—black arrowheads) were more frequent in Sdc1-KO than WT mice. Scale bar, 100 μm. <b>(D)</b> Representative immunoreactive staining (brown) for IL-6 (top panel) and phospho-STAT3 (middle panel) in colons of WT and Sdc1-KO mice. Scale bar, 100 μm. Lower panel: Representative negative controls (NEG) in which normal IgG was used in place of primary antibody.</p

Elevated expression of pro-tumorigenic target genes of STAT3 in Syndecan-1 KO mice compared to WT.

<p><b>(A)</b> Quantitative RT-PCR analysis revealed increased levels of Cyclin D1 (left), CCL-2 (middle) and Myc (right) in AOM-DSS induced colonic tumors derived from Sdc1-KO as compared to WT mice (n = 5). <b>(B)</b> Representative immunoreactive staining (brown) for Cyclin D1 in AOM-DSS induced colonic tumors (day 61) of WT and Sdc1-KO mice. Scale bar, 100 μm. <b>(C)</b> Quantification of average numbers of Cyclin D1 positive cells per high power field (X400) in ≥ 12 fields of each slide from 3 mice of each group. Error bars represent mean ± SE. *P < 0.05, ***P < 0.001 by Student’s t test. (D) Immunostaining for cMyc (brown) revealed increased levels of cMyc protein in AOM-DSS induced colonic tumors derived from Sdc1-KO vs. WT mice. Of note, cytoplasmic localization of cMyc was previously reported in several pathophysiological settings, including tumors of diverse origins (reviewed in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174343#pone.0174343.ref070" target="_blank">70</a>]).</p

Increased activation of STAT3 in colonic tumors of Syndecan-1 KO mice compared to WT mice.

<p><b>(A)</b> Representative immunoreactive staining (brown) for phospho-STAT3 in AOM-DSS induced colonic tumors (day 61) of WT and Sdc1-KO mice. Scale bar, 100 μm. <b>(B)</b> Quantification of average numbers of phospho-STAT3 positive cells per high power field (X400) in ≥ 12 fields of each slide from 4 mice of each group. Error bars represent mean ± SE. **<i>P</i> < 0.01 by Student’s t test.</p

Syndecan-1 deficiency increases susceptibility to colitis-associated tumors in AOM/DSS-treated mice.

<p><b>(A)</b> Schematic representation of the mouse model of AOM/DSS-induced colitis–associated carcinoma performed as described in Methods. <b>(B)</b> Representative immunostaining (reddish) for Sdc-1 in normal colonic tissue and tumor tissue samples derived from the colons of WT and Sdc1-KO mice. Original magnification X200. <b>(C)</b> Quantification of average tumor size, and <b>(D)</b> tumor number/colon in WT and Sdc1-KO mice on day 61 of AOM-DSS–induced colon cancer (n = 6). Error bars represent mean ± SE. *<i>P</i> < 0.05 by Student’s t test. <b>(E)</b> Representative histopathologic sections of colon adenocarcinomas from WT and Sdc1-KO mice. (F) Representative immunostaining for beta-catenin in tumor tissue samples derived from the colons of WT and Sdc1-KO mice. Original magnification X1000.</p