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

Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation, but Not for Hypertrophy, Aneuploidy and Hyperfunction of Pancreatic β-Cells

Constitutive expression of active Akt (Akttg) drives hyperplasia and hypertrophy of pancreatic β-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in β-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6P-/-). rpS6 phosphorylation deficiency failed to block Akttg-induced hypertrophy and aneuploidy in β-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akttg-driven hyperplasia and tumor formation in β-cells. In vitro experiments with Akttg and rpS6P-/-;Akttg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akttg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation.Fil: Wittenberg, Avigail Dreazen. The Hebrew University Of Jerusalem; IsraelFil: Azar, Shahar. The Hebrew University Of Jerusalem; IsraelFil: Klochendler, Agnes. The Hebrew University Of Jerusalem; IsraelFil: Stolovich-Rain, Miri. The Hebrew University Of Jerusalem; IsraelFil: Avraham, Shlomit. The Hebrew University Of Jerusalem; IsraelFil: Birnbaum, Lea. The Hebrew University Of Jerusalem; IsraelFil: Binder Gallimidi, Adi. The Hebrew University Of Jerusalem; IsraelFil: Katz, Maximiliano Javier. The Hebrew University Of Jerusalem; Israel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Dor, Yuval. The Hebrew University Of Jerusalem; IsraelFil: Meyuhas, Oded. The Hebrew University Of Jerusalem; Israe

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

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

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

rpS6 phosphorylation deficiency renders Ras^G12D MEFs more sensitive to both genotoxic and proteotoxic stresses.

<p>(A) Immortalized WT, rpS6^P-/-, Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (B) Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were seeded in 96-well plates at a density of 4 x10³ per well. Proliferation was monitored by measuring the A₆₅₀ of the methylene-blue dye extracted from stained cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149995#pone.0149995.ref032" target="_blank">32</a>]. Absorbance measured 24 h after platting, was set arbitrarily at 1 and absorbance measured at later time points (average ± SEM [n = 6]) for each time point) was normalized to that value. t_d, population-doubling time. (C) Immortalized Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were incubated with the indicated concentrations of etoposide for 24 h, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (D) Immortalized Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were incubated with the indicated concentrations of MG132 for 24 h, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies.</p

List of proteins that selectively interact with unphosphorylatable form of rpS6.

<p>Whole cell extract from HEK293 cells infected with pS6^[5S]-GFP, pS6^[5A]-GFP, pS6^[5D]-GFP or pEGFP-N1, was subjected to GFP pull-down, and the bound proteins were size fractionated by SDS-polyacrlamide gel electrophoresis. Mass spectrometric analysis of proteins in each lane was performed as described in “material and Methods” and proteins, selectively bound to pS6^[5A]-GFP in two independent experiments, are presented (numbers separated by slash [/] represent results obtained in each of the two individual analyses).</p

rpS6 phosphorylation deficiency increases proliferation and decreases translational fidelity in fibroblasts.

<p>(A) Immortalized WT, rpS6^P-/-, Akt^tg and rpS6^P-/-;Akt^tg MEFs were harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (B) MEFs described in (A) were seeded in 96-well plates at a density of 4 x10³ per well. The proliferation of MEFs described in (A) was monitored as described in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149995#pone.0149995.g004" target="_blank">Fig 4B</a>). t_d, population doubling time. (C) WT and rpS6^P-/- immortalized MEFs were incubated for 1 h with 1 mM puromycin, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (D) Quantification of signals obtained in 6 experiments similar to that described in (C). (E) WT, rpS6^P-/-, Akt^tg, and rpS6^P-/-;Akt^tg MEFs were cotransfected with Renilla luciferase expression vector and any one of the following firefly luciferase expression vectors: Fluc(WT), Fluc(Stop) and Fluc(R218S). The transfection efficiency of any of the Fluc constructs was normalized to that of the Renilla luciferase. The relative Fluc activity of each mutant was normalized to Fluc (WT). The result obtained for each mutant in WT MEFs (n = 24) was arbitrarily set at 1 and the relative luciferase activity obtained for each Fluc mutant in rpS6^P-/- MEFs (n = 24) was normalized to that value. Similarly, results obtained for each mutant in Akt^tg MEFs (n = 12) were arbitrarily set at 1 and the relative luciferase activity obtained for each Fluc mutant in rpS6^P-/-;Akt^tg MEFs (n = 12) was normalized to that value.</p

Constitutive expression of Akt induces polyploidy regardless of the rpS6 phosphorylation status.

<p>A. rpS6 deficiency does not affect ploidy in β-cells. Islets were isolated from wild-type (WT) and 2 month-old Akt^tg mice. Dissociated islets were fixed, permeabilized, immunostained for Insulin, HA, Ki67 and Hoechst before FACS analysis. The DNA content of 5,000 to 10,000 quiescent β-cells (insulin-positive, Ki67-negative) was measured by FACS analysis. B. 5,000 quiescent (Ki67-negative) β-cells (insulin-positive) that express myr-Akt (HA-positive) are polyploidy, whereas their HA-negative counterpart shows mostly a diploid profile as wild-type β-cells. C. rpS6 deficiency does not affect the ploidy profile of myr-Akt expressing β-cells. Islets were isolated from 2 month-old mice (three wild-type (WT), two Akt^tg and three rpS6^P-/-;Akt^tg mice) and 5,000 to 10,000 HA-positive β-cells processed as in (A) for FACS analysis.</p