BACKGROUND:Tumor necrosis factor (TNF) is a pleiotropic cytokine that plays a critical role in the pathogenesis of immune-mediated diseases including inflammatory bowel disease (IBD). The stability of its mRNA transcript, determined in part by destabilizing sequences in its AAUU repeats (ARE) gene region, is an important regulator of its tissue and systemic levels. A deletion in the ARE region of the gene resulted in IBD and arthritis in mice and pigs, supporting a critical role for the cytokine in human IBD and several human arthritides. A mutation in the same area of the mouse genome by Genentech scientists (T.Y., M.K.) resulted in a similar but not identical phenotype. METHODS:Here, we compare histopathological, cellular, and molecular features of the strains and propose reasons for their distinct phenotypes. First, while homozygous TNFΔARE mice develop severe arthritis and die after weaning, homozygous Genentech TNFΔARE (ΔG/ΔG) mice have normal lifespans, and males are often fertile. RESULTS:We found that while the ileitic phenotype had peaked at 12 weeks of age in all mice, colitis progressed mostly after 20 weeks of age in heterozygous mice. Their variably penetrant arthritic phenotype progressed mostly after 20 weeks, also in heterozygous mice from both strains. There was expansion of central memory T and B cells in lymphoid organs of TNF-overproducing strains and their transcriptional profile shared well-known pathogenetic pathways with human IBD. Finally, we found differences in the mutated sequences within the ARE regions of the TNF gene and in their microbiota composition and genetic background. These differences likely explain their phenotypic differences. CONCLUSIONS:In summary, we describe a different strain of TNF-overproducing mice with an overlapping, yet not identical phenotype, which may have differential applications than the original strain.

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PDF file - 1.2MB, Nodal antibody used for IHC labels same cells as R&D antibody used in previous studies, but with less non-specific background staining

PDF file - 1MB, Nodal co-localizes with CD31 in human breast cancer

Aims: Though best known for its role in oxidative DNA damage repair, apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that regulates multiple host responses during oxidative stress, including the reductive activation of transcription factors. As knockout of the APE1-encoding gene, Apex1, is embryonically lethal, we sought to create a viable model with generalized inhibition of APE1 expression.Results: A hypomorphic (HM) mouse with decreased APE1 expression throughout the body was generated using a construct containing a neomycin resistance (NeoR) cassette knocked into the Apex1 site. Offspring were assessed for APE1 expression, breeding efficiency, and morphology with a focused examination of DNA damage in the stomach. Heterozygotic breeding pairs yielded 50% fewer HM mice than predicted by Mendelian genetics. APE1 expression was reduced up to 90% in the lungs, heart, stomach, and spleen. The HM offspring were typically smaller, and most had a malformed tail. Oxidative DNA damage was increased spontaneously in the stomachs of HM mice. Further, all changes were reversed when the NeoR cassette was removed. Primary gastric epithelial cells from HM mice differentiated more quickly and had more evidence of oxidative DNA damage after stimulation with Helicobacter pylori or a chemical carcinogen than control lines from wildtype mice.Innovation: A HM mouse with decreased APE1 expression throughout the body was generated and extensively characterized.Conclusion: The results suggest that HM mice enable studies of APE1's multiple functions throughout the body. The detailed characterization of the stomach showed that gastric epithelial cells from HM were more susceptible to DNA damage.

Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected with Salmonella enterica serovar Typhimurium or adherent-invasive Escherichia coli. The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function.

associated macrophage (TAM) and granulocytic tumor associated neutrophil (GTAN).Proinflammatory mediators and cytokines, including IL-10, IFN-g and NFkB were significantly increased (p<0.05) in P. anaerobius treated mice, indicating their involvement in regulating inflammatory response.In in vitro assays, P. anaerobius could attach and invade all three cell lines as determined by attachment/invasion assays and transmission electron microscopy (TEM).The level of attachment and invasion was stronger in CRC cell lines ( p<0.01) than in NCM460.Furthermore, far western blot showed that P. anaerobius surface proteins interacted directly with receptor proteins in colon cell lines.RNA-seq data revealed that P. anaerobius could mediate integrin b1/a2 signaling to activate PI3K/Akt pathway, thereby promoting cell proliferation.Integrin inhibitors suppressed P. anaerobius attachment/invasion and P. anaerobius-mediated PI3K/Akt signaling.Conclusions: P. anaerobius promotes CRC via: 1) direct interaction with colonic epithelial cells, leading to P. anaerobius adhesion, invasion and activation of PI3K/Akt oncogenic pathways; and 2) modulation of tumor microenvironment by activation of MDSCs, TAMs and GTANs.

Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.

Tumor cell extravasation is a key step during cancer metastasis, yet the precise mechanisms that regulate this dynamic process are unclear. We utilized a high-resolution time-lapse intravital imaging approach to visualize the dynamics of cancer cell extravasation in vivo. During intravascular migration, cancer cells form protrusive structures identified as invadopodia by their enrichment of MT1-MMP, cortactin, Tks4, and importantly Tks5, which localizes exclusively to invadopodia. Cancer cells extend invadopodia through the endothelium into the extravascular stroma prior to their extravasation at endothelial junctions. Genetic or pharmacological inhibition of invadopodia initiation (cortactin), maturation (Tks5), or function (Tks4) resulted in an abrogation of cancer cell extravasation and metastatic colony formation in an experimental mouse lung metastasis model. This provides direct evidence of a functional role for invadopodia during cancer cell extravasation and distant metastasis and reveals an opportunity for therapeutic intervention in this clinically important process.

Pannexin 1 (Panx1) is a channel‐forming glycoprotein expressed in different cell types of mammalian skin. We studied the role of Panx1 in melanoma tumorigenesis and metastasis since the mouse melanocyte line CRL‐2770 showed low levels of Panx1 expression, while Panx1 in isogenic melanoma cell lines (B16‐F0, ‐F10 and ‐BL6) was increasingly up‐regulated according to qPCR and Western blot assays. Panx1 shRNA knockdown generated stable BL6 cell lines (Panx1‐KD) that showed a marked increase in melanocyte‐like cell characteristics, including higher melanin production, decreased cell migration and enhanced formation of cellular projections, while compromising Panx1 dye uptake channel function. Western blotting and proteomic analyses using 2D‐SDS‐PAGE gels/mass spectroscopy identified vimentin and beta‐catenin as some of the markers of malignant melanoma that were down‐regulated in Panx1‐KD cells. In vivo, Panx1‐KD cells were implanted on chorioallantoic membranes of day 10 avian embryos. After seven days, the excised Panx1‐KD tumors were significantly smaller than controls and metastasis to the liver was significantly reduced as quantified by mouse‐Alu qPCR of the excised embryonic organs. These data suggests that Panx1 may play a role in skin melanocytes that is potentially dysregulated upon malignant transformation in metastatic melanomas. Supported by the Canadian Institutes of Health Research.

Nanomaterials with elongated architectures have been shown to possess differential tumor homing properties compared to their spherical counterparts. Here, we investigate whether this phenomenon is mirrored by plant viral nanoparticles that are filamentous (Potato virus X) or spherical (Cowpea mosaic virus). Our studies demonstrate that Potato virus X (PVX) and Cowpea mosaic virus (CPMV) show distinct biodistribution profiles and differ in their tumor homing and penetration efficiency. Analogous to what is seen with inorganic nanomaterials, PVX shows enhanced tumor homing and tissue penetration. Human tumor xenografts exhibit higher uptake of PEGylated filamentous PVX compared to CPMV, particularly in the core of the tumor. This is supported by immunohistochemical analysis of the tumor sections, which indicates greater penetration and accumulation of PVX within the tumor tissues. The enhanced tumor homing and retention properties of PVX along with its higher payload carrying capacity make it a potentially superior platform for applications in cancer drug delivery and imaging applications.

Abstract Tumor vascularization is requisite for breast cancer progression, and high microvascular density in tumors is a poor prognostic indicator. Patients bearing breast cancers expressing human embryonic stem cell (hESC)-associated genes similarly exhibit high mortality rates, and the expression of embryonic proteins is associated with tumor progression. Here, we show that Nodal, a hESC-associated protein, promotes breast cancer vascularization. We show that high levels of Nodal are positively correlated with high vascular densities in human breast lesions (P = 0.0078). In vitro, we show that Nodal facilitates breast cancer–induced endothelial cell migration and tube formation, largely by upregulating the expression and secretion of proangiogenic factors by breast cancer cells. Using a directed in vivo angiogenesis assay and a chick chorioallantoic membrane assay, we show that Nodal promotes vascular recruitment in vivo. In a clinically relevant in vivo model, whereby Nodal expression was inhibited following tumor formation, we found a significant reduction in tumor vascularization concomitant with elevated hypoxia and tumor necrosis. These findings establish Nodal as a potential target for the treatment of breast cancer angiogenesis and progression. Cancer Res; 72(15); 3851–63. ©2012 AACR.

Pannexin 1 (Panx1) is a channel-forming glycoprotein expressed in different cell types of mammalian skin. We examined the role of Panx1 in melanoma tumorigenesis and metastasis since qPCR and Western blots revealed that mouse melanocytes exhibited low levels of Panx1 while increased Panx1 expression was correlated with tumor cell aggressiveness in the isogenic melanoma cell lines (B16-F0, -F10, and -BL6). Panx1 shRNA knockdown (Panx1-KD) generated stable BL6 cell lines, with reduced dye uptake, that showed a marked increase in melanocyte-like cell characteristics including higher melanin production, decreased cell migration and enhanced formation of cellular projections. Western blotting and proteomic analyses using 2D-gel/mass spectroscopy identified vimentin and β-catenin as two of the markers of malignant melanoma that were down-regulated in Panx1-KD cells. Xenograft Panx1-KD cells grown within the chorioallantoic membrane of avian embryos developed tumors that were significantly smaller than controls. Mouse-Alu qPCR of the excised avian embryonic organs revealed that tumor metastasis to the liver was significantly reduced upon Panx1 knockdown. These data suggest that while Panx1 is present in skin melanocytes it is up-regulated during melanoma tumor progression, and tumorigenesis can be inhibited by the knockdown of Panx1 raising the possibility that Panx1 may be a viable target for the treatment of melanoma.

The analysis of dynamic events in the tumor microenvironment during cancer progression is limited by the complexity of current in vivo imaging models. This is coupled with an inability to rapidly modulate and visualize protein activity in real time and to understand the consequence of these perturbations in vivo. We developed an intravital imaging approach that allows the rapid induction and subsequent depletion of target protein levels within human cancer xenografts while assessing the impact on cell behavior and morphology in real time. A conditionally stabilized fluorescent E-cadherin chimera was expressed in metastatic breast cancer cells, and the impact of E-cadherin induction and depletion was visualized using real-time confocal microscopy in a xenograft avian embryo model. We demonstrate the assessment of protein localization, cell morphology and migration in cells undergoing epithelial-mesenchymal and mesenchymal-epithelial transitions in breast tumors. This technique allows for precise control over protein activity in vivo while permitting the temporal analysis of dynamic biophysical parameters.

BackgroundWe have shown that H. pylori (Hp) infection of gastric epithelial cells (GEC) increases reactive oxygen species (ROS) and induces APE1 expression. Rac1 and NAPDH oxidase contribute to Hp‐mediated oxidative stress. Thus, we hypothesized that APE1 regulates Rac1 and NADPH oxidase (Nox1).MethodsAfter Hp infection, superoxide was measured by luminol oxidation in stable APE1 deficient AGS cells (shRNA) and wild‐type (WT) cells. Diphenylene iodonium (DPI) and NSC23766 were used to inhibit NADPH oxidase and Rac1 respectively. Rac1 activity was assessed by GST‐PBD pulldown assay. Rac1‐APE1 interaction was measured by co‐immunoprecipitation and confocal microscopy. Nox1 and APE1 mRNA levels in patient gastric tissues were measured by RT‐ PCR.ResultsIncreased ROS, Rac1 activity and Nox1 were measured in shRNA cells compared to WT after Hp infection. APE1 overexpression decreased Hp induced Rac1 activation as well as Nox1 expression. N‐terminal Lys mutation of APE1 reduced the interaction with Rac1. Mutant APE1 overexpression resulted in increased ROS accumulation and Nox1 expression after Hp infection. Both Nox1 and APE1 mRNA levels were elevated in GEC from Hp infected patient tissues.ConclusionsAPE1 acts as a negative regulator of ROS accumulation due to Hp infection by inhibiting Rac1 and Nox1. (Research Support, R01DK061769‐09).

Abstract During the metastatic process, cancer cells must undergo trans-endothelial migration from the vessel lumen into underlying tissue in a process known as extravasation. Little is known about the dynamic mechanical aspects of this process. To address this, we performed real-time, sub-cellular resolution intravital imaging of human cancer cells during arrest, intravascular migration and extravasation using a shell-less avian embryo xenograft model. We find that extravasation occurs at endothelial junctions and that the majority of extravasation events occurred during the 12 hours subsequent to the intravenous injection of cancer cells. In the majority of extravasation events, long cytoplasmic extensions identified as invadopodia were observed prior to extravasation which breached underlying endothelium. We observed the release of microparticles during these invadopodial extension events prior to extravasation, and this resulted in a ∼40% reduction in cell volume post-extravasation. Based on this, we hypothesized that invadopodia are required for extravasation, and that cancer cell extravasation could be abrogated by inhibiting factors required for invadopodia function. We found that treatment with Src kinase inhibitors significantly reduced invadopodia formation in vivo and resulted in a ∼60% decrease in extravasation events when compared to vehicle treated controls. Furthermore, a higher proportion of cells in the Src kinase inhibitor-treated animals remained intravascular compared to vehicle control. In conclusion, we determined that 1) extravasation occurs at junctions between adjacent endothelial cells, 2) cancer cells form invadopodia that breach the endothelial layer prior to successful extravasation and 3) cancer cells undergoing extravasation release cancer microparticles into both the vessel lumen and tissue interstitium with a corresponding decrease in cell volume. Pharmacological inhibition of invadopodia by Src kinase inhibitors (Bosutinib, Dasatinib) reduces cancer cell extravasation, revealing a novel and potentially important mechanism of action against metastatic cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 968. doi:10.1158/1538-7445.AM2011-968