Humanized Mouse Model of Ovarian Cancer Recapitulates Patient Solid Tumor Progression, Ascites Formation, and Metastasis

Ovarian cancer is the most common cause of death from gynecological cancer. Understanding the biology of this disease, particularly how tumor-associated lymphocytes and fibroblasts contribute to the progression and metastasis of the tumor, has been impeded by the lack of a suitable tumor xenograft model. We report a simple and reproducible system in which the tumor and tumor stroma are successfully engrafted into NOD-scid IL2Rγnull (NSG) mice. This is achieved by injecting tumor cell aggregates derived from fresh ovarian tumor biopsy tissues (including tumor cells, and tumor-associated lymphocytes and fibroblasts) i.p. into NSG mice. Tumor progression in these mice closely parallels many of the events that are observed in ovarian cancer patients. Tumors establish in the omentum, ovaries, liver, spleen, uterus, and pancreas. Tumor growth is initially very slow and progressive within the peritoneal cavity with an ultimate development of tumor ascites, spontaneous metastasis to the lung, increasing serum and ascites levels of CA125, and the retention of tumor-associated human fibroblasts and lymphocytes that remain functional and responsive to cytokines for prolonged periods. With this model one will be able to determine how fibroblasts and lymphocytes within the tumor microenvironment may contribute to tumor growth and metastasis, and will make it possible to evaluate the efficacy of therapies that are designed to target these cells in the tumor stroma

The anaphase-promoting complex protein 5 (AnapC5) associates with A20 and inhibits IL-17-mediated signal transduction.

IL-17 is the founding member of a family of cytokines and receptors with unique structures and signaling properties. IL-17 is the signature cytokine of Th17 cells, a relatively new T cell population that promotes inflammation in settings of infection and autoimmunity. Despite advances in understanding Th17 cells, mechanisms of IL-17-mediated signal transduction are less well defined. IL-17 signaling requires contributions from two receptor subunits, IL-17RA and IL-17RC. Mutants of IL-17RC lacking the cytoplasmic domain are nonfunctional, indicating that IL-17RC provides essential but poorly understood signaling contributions to IL-17-mediated signaling. To better understand the role of IL-17RC in signaling, we performed a yeast 2-hybrid screen to identify novel proteins associated with the IL-17RC cytoplasmic tail. One of the most frequent candidates was the anaphase promoting complex protein 7 (APC7 or AnapC7), which interacted with both IL-17RC and IL-17RA. Knockdown of AnapC7 by siRNA silencing exerted no detectable impact on IL-17 signaling. However, AnapC5, which associates with AnapC7, was also able to bind IL-17RA and IL-17RC. Moreover, AnapC5 silencing enhanced IL-17-induced gene expression, suggesting an inhibitory activity. Strikingly, AnapC5 also associated with A20 (TNFAIP3), a recently-identified negative feedback regulator of IL-17 signal transduction. IL-17 signaling was not impacted by knockdown of Itch or TAXBP1, scaffolding proteins that mediate A20 inhibition in the TNFα and IL-1 signaling pathways. These data suggest a model in which AnapC5, rather than TAX1BP1 and Itch, is a novel adaptor and negative regulator of IL-17 signaling pathways

Schematic model of AnapC5 and AnapC7 in IL-17R signaling.

<p>The data in this paper support a model in which AnapC5 serves an adaptor or scaffold protein to facilitate A20 recruitment to the CBAD domain of IL-17RA. Although AnapC7 binds to both IL-17RA and IL-17RC, its functional role is still unclear.</p

Neither Rrad nor CFD mediate IL-17 signal transduction. A. Rrad-deficient fibroblasts mediate normal IL-17 induction of IL-6 secretion.

<p>Multiple Rrad^−/− cell lines (derived from adult tail biopsies of Rrad^−/− mice) or ST2 stromal cell lines were treated with IL-17 (200 /ng/ml), IL-17F (200/ ng/ml) or suboptimal TNFα (2 ng/ml) for 24 h. IL-6 was measured in culture supernatants in triplicate by ELISA. *<i>p</i><0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ^‡<i>p</i><0.05 by Chi Square comparing experimental replicates. B. Rrad-deficient fibroblasts mediate normal IL-17 induction of target gene expression. A representative Rrad^−/− cell line or IL-17RC^−/− fibroblasts were treated with IL-17 or TNFα as outlined in panel A for 24 h. Expression of the indicated genes was assessed by qPCR in triplicate. *<i>p</i><0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls of each cell line. ^‡<i>p</i><0.05 by Chi Square comparing experimental replicates. C. Complement deficient cell lines mediate normal IL-17 signaling. Fibroblast cell lines from CFD^−/−, C3^−/− or IL-17RC^−/− mice were treated with IL-17 and TNFα as described in panel A, and IL-6 concentrations in culture supernatants were assessed by ELISA. *<i>p</i><0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ^‡<i>p</i><0.05 by Chi Square comparing experimental replicates. ^¶concentration values above standard curve for ELISA detection. All data are representative of at least two independent experiments.</p

AnapC5 binds to the IL-17 receptor and restricts IL-17-mediated signal transduction. A. AnapC5 associates with IL-17RA, IL-17RC and AnapC7.

<p>HEK293T cells were transfected with AnapC5 (tagged with HA) together with IL-17RA, IL-17RC or AnapC7 (tagged with Myc), as indicated. Lysates were subjected to IP with anti-Myc Abs and immunoblotted with anti-HA or anti-Myc. Arrows indicate identity of each Myc-tagged protein. Whole cell lysates (WCL) were verified for AnapC7 by staining with anti-HA. Migration of protein size markers is indicated. B. AnapC5 associates with IL-17RA through the inhibitory CBAD domain. HEK293T cells were transfected with AnapC5 and the indicated IL-17RA deletion constructs. Lysates were subjected to co-IP with anti-Myc and blotted for HA or Myc. Whole cell lysates (WCL) were verified for AnapC7 by staining with anti-Myc. Migration of protein size markers is indicated. C. AnapC5 associates with IL-17RC in the SEFIR domain. HEK293T cells were transfected with AnapC5 and the indicated IL-17RC deletion constructs. Lysates were subjected to co-IP with anti-Myc and blotted for HA or Myc. Whole cell lysates (WCL) were verified for AnapC7 by staining with anti-HA. Migration of protein size markers is indicated. D. Knockdown of AnapC5 enhances IL-17 signaling. ST2 cells were transfected with the indicated siRNAs, stimulated with IL-17 for 24 h, and IL-6 in culture supernatants assessed by ELISA. <i>n.s.,</i> not significant. *<i>p</i><0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ^‡<i>p</i><0.05 by Chi Square comparing experimental replicates. E. Efficient knockdown of AnapC5 and AnapC7. mRNA from the samples in panel D were assessed for AnapC5 and AnapC7 expression by qPCR. *<i>p</i><0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ^‡<i>p</i><0.05 by Chi Square comparing experimental replicates. Data are representative of at least 2 independent experiments.</p

AnapC7 binds to IL-17R but does not impact IL-17 signaling.

<p><b>A. Schematic diagram of IL-17RA and IL-17RC mutants.</b> ECD, extracellular domain. SEFIR and SEFEX domain approximate boundaries are indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070168#pone.0070168-Novatchkova1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070168#pone.0070168-Maitra1" target="_blank">[24]</a>. CBAD, C/EBPβ activation domain. <b>B. AnapC7 associates with IL-17RA and IL-17RC.</b> HEK293T cells were transfected with AnapC7 tagged with HA and IL-17RA or IL-17RC tagged with Myc, as indicated. Lysates were immunoprecipitated with anti-Myc Abs and immunoblotted for HA or Myc. Whole cell lysates (WCL) were verified for AnapC7 by staining with anti-HA. Migration of protein size markers is indicated. <b>C. RNA silencing of AnapC7 does not alter IL-17-dependent signaling.</b> ST2 cells were transfected with the indicated siRNAs, treated with IL-17 (black bars) for 24 h and IL-6 in culture supernatants assessed by ELISA. <i>n.s.,</i> not significant. *<i>p</i><0.05 by ANOVA and post-hoc Tukey’s test relative to unstimulated controls. ^‡<i>p</i><0.05 by Chi Square comparing experimental replicates.</p

C/EBPβ^-/- mice exhibit increased susceptibility to oral candidiasis in the context of cortisone-induced immunosuppression.

<p><b>(A)</b> C65BL/6 mice (“WT”) were treated with the indicated doses of cortisone acetate at days -1, +1 and +2 relative to infection. After 5 d, fungal loads in tongue were assessed by CFU enumeration of tongue tissue homogenates. SHAM (n = 3), No cortisone control (n = 5), 60mg/kg (n = 9), 120/112mg/kg (n = 8), and 225mg/kg (n = 8). Data are pooled from 2 independent experiments. Bars indicate geometric mean with 95% CI. P<0.05 by t-test with Mann-Whitney correction: * vs NO CORT, ≠ vs 60mg/kg, # vs 120/112mg/kg and π vs 225mg/kg. (<b>B</b>) The indicated mice were infected orally as described in panel A. Cortisone acetate was administered subcutaneously on days -1, +1 and +2 relative to infection. C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ (n = 5), C/EBPβ^+/+ 60 mg/kg (n = 16), C/EBPβ^+/- 60mg/kg (n = 16), C/EBPβ^-/- 60mg/kg (n = 10) and C/EBPβ^+/+ 225mg/kg (n = 4). <i>P</i><0.05 by t-test with Mann-Whitney correction: * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg. Data are pooled from two independent experiments. <b>C.</b> Representative tongue sections from the indicated mice were stained with H&E or Periodic-acid Schiff (PAS). Scale bar indicates 200 μM. White arrows indicate hyphae.</p

Susceptibility of C/EBPβ^-/- mice to OPC correlates with expression of BD3.

<p><b>(A)</b> mRNA from tongue was isolated from the indicated mice 5 days after oral <i>C</i>. <i>albicans</i> infection [C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ NO CORT (n = 3), C/EBPβ^+/+ 60mg/kg (n = 5), C/EBPβ^+/- 60mg/kg (n = 6), and C/EBPβ^-/- 60mg/kg (n = 5)]. Complementary DNA was prepared and subjected to qPCR analysis to detect the indicated genes. Results are presented as fold induction over SHAM treated mice and normalized to expression of <i>Gapdh</i>. Data are pooled from two independent experiments. <i>P</i><0.05 by student unpaired t-test. * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg. (<b>B)</b> mRNA from tongue was isolated from the indicated mice 5 days after oral <i>C</i>. <i>albicans</i> infection and analyzed as in panel A. C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ (n = 2), C/EBPβ^+/- (n = 2), and C/EBPβ^-/- (n = 2). Data are from one experiment. * <i>P</i><0.05 by student unpaired t-test. vs C/EBPβ^+/+, ≠ vs C/EBPβ^+/- and π vs C/EBPβ^-/-. (<b>C)</b> OKF6/TERT2 human oral keratinocytes were treated with 200 ng/ml IL-17 plus 2ng/ml TNFα or with 2×10⁶ HK <i>C</i>. <i>albicans</i> for 24 h. Complementary DNA was prepared and subjected to qPCR analysis to detect <i>DEFB4A</i>. Data are normalized to expression of <i>GAPDH</i> and represent absolute levels. Data are representative of 2 independent experiments. *<i>P</i><0.05 compared to unstimulated OKF6/TERT2 cells.</p

The susceptibility of C/EBPβ^-/- mice to OPC does not correlate with expression of prototypical IL-17-regulated genes.

<p>mRNA from tongue was isolated from the indicated mice 5 days after oral <i>C</i>. <i>albicans</i> infection [C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ NO CORT (n = 3), C/EBPβ^+/+ 60mg/kg (n = 5), C/EBPβ^+/- 60mg/kg (n = 6), and C/EBPβ^-/- 60mg/kg (n = 5)]. Complementary DNA was prepared and subjected to qPCR analysis to detect the indicated genes. Results are presented as fold induction over SHAM treated mice and normalized to expression of <i>Gapdh</i>. Data are pooled from 2 independent experiments. <i>P</i><0.05 by student unpaired t-test: * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg.</p