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Background: Diabetes mellitus (DM) is associated with long-term damage, dysfunction, of various organs. Objectives: Study aims to assess the role of new Synthesized Flavonoid compound on experimentally induced diabetes. Methods: 50 adult male albino rats divided into 5 groups. Group 1 (control group, rats were orally administered with 1 ml saline daily). Group 2 (DMSO group, rats were orally administered with 0.2 % DMSO for 60 day orally). Group 3 (positive control, animals were injected intraperitoneally with 60 mg/kg b.wt streptozotocin followed by intraperitoneal injection with 120 mg/kg b.wt of Nicotinamide after 15 minute).Group 4 (standard group, diabetic animals treated with 100 mg/kg b.wt of metformin for 60 day orally). Group 5 (therapeutic group, diabetic rats treated with 50 mg /kg b.wt of Ethyl 2-amino-4-phenyl-4H-benzo(h)chromene-3-carboxylate for 60 day orally). At the end of experimental period blood serum & plasma samples, liver, kidney and pancreatic tissues were collected. Results: diabetic rats showed significant increase in plasma glucose, serum urea, creatinine, cholesterol and triglyceride . Also significant increase in mean level of Fetuin A and Netrin-1 in serum and different organs (Liver, kidney, Pancreas) in compared to control group. Oral administration of Ethyl 2-amino-4-phenyl-4H-benzo(h)chromene-3-carboxylate cause decrease in elevated biochemical parameters. Also, decrease Fetuin A and Netrin-1 levels when compared with diabetic rats. Molecular docking studies confirmed binding of compound with Fetuin A and Netrin-1 proteins in terms of energy and revealed of the existence of hydrogen bond ,hydrophobic interaction,Our results were confirmed by histopathological examination of pancreatic tissue. Conclusion: this study suggests that Ethyl 2-amino-4-phenyl-4H-benzo(h)chromene-3-carboxylate exihibits antihyperglycemic activity in streptozotocin- induced diabetic rats.

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 withSalmonella entericaserovar Typhimurium or adherent-invasiveEscherichia 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.

Summary Cells perceive and respond to the extracellular matrix via integrin receptors; their dysregulation has been implicated in inflammation and cancer metastasis. Here we show that a guanine nucleotide-exchange modulator of trimeric-GTPase Gαi, GIV (a.k.a Girdin), directly binds the integrin adaptor Kindlin-2. A non-canonical short linear motif within the C terminus of GIV binds Kindlin-2-FERM3 domain at a site that is distinct from the binding site for the canonical NPxY motif on the -integrin tail. Binding of GIV to Kindlin-2 allosterically enhances Kindlin-2's affinity for β1-integrin. Consequently, integrin activation and clustering are maximized, which augments cell adhesion, spreading, and invasion. Findings elucidate how the GIV•Kindlin-2 complex has a 2-fold impact: it allosterically synergizes integrin activation and enables β1-integrins to indirectly access and modulate trimeric GTPases via the complex. Furthermore, Cox proportional-hazard models on tumor transcriptomics provide trans-scale evidence of synergistic interactions between GIV•Kindlin-2•β1-integrin on time to progression to metastasis., Graphical Abstract, Highlights • GIV and Kindlin (K2), two integrin adaptors that promote metastasis, bind each other • Binding of GIV or integrin to K2 allosterically enhances GIV•K2•integrin complexes • Binding is required for the maximal recruitment of GIV and K2 to active integrins • Binding facilitates integrin clustering, activation, tumor cell adhesion, invasion, Biological Sciences; Molecular Biology; Cell Biology

CD98, which is required for the rapid proliferation of both normal and cancer cells, and MET, the hepatocyte growth factor receptor, are potential targets for therapeutic antitumor Abs. In this study, we report that the antiproliferative activity of a prototype anti-CD98 Ab, UM7F8, is due to Ab-induced membrane-associated ring CH (MARCH) E3 ubiquitin ligase-mediated ubiquitination and downregulation of cell surface CD98. MARCH1-mediated ubiquitination of CD98 is required for UM7F8’s capacity to reduce CD98 surface expression and its capacity to inhibit the proliferation of murine T cells. Similarly, CD98 ubiquitination is required for UM7F8’s capacity to block the colony-forming ability of murine leukemia–initiating cells. To test the potential generality of the paradigm that MARCH E3 ligases can mediate the antiproliferative response to antitumor Abs, we examined the potential effects of MARCH proteins on responses to emibetuzumab, an anti-MET Ab currently in clinical trials for various cancers. We report that MET surface expression is reduced by MARCH1, 4, or 8-mediated ubiquitination and that emibetuzumab-induced MET ubiquitination contributes to its capacity to downregulate MET and inhibit human tumor cell proliferation. Thus, MARCH E3 ligases can act as cofactors for antitumor Abs that target cell surface proteins, suggesting that the MARCH protein repertoire of cells is a determinant of their response to such Abs.

While chemotherapy is a key treatment strategy for many solid tumors, it is rarely curative as patients will eventually become resistant. In this study, we sought to develop an effective suicide gene therapy approach for solid tumors that specifically exploits their unique transcriptional activation state. The tumor suppressor p53 is frequently mutated or dysregulated in cancer, and as a result the upstream signaling pathways activating p53 transcription are strongly upregulated. RNA-seq analysis has demonstrated that p53 transcription is significantly upregulated in almost all forms of cancer. Additionally, HCT116 cells lacking functional p53 display a 6-fold increase in p53 promoter activity when compared to its wild type p53 parent cell line. To exploit this, we have developed a Fusogenix FAST-LNP formulation to deliver a p53-driven inducible suicide gene, iCasp9, to solid tumors and destroy them upon activation with small molecule dimerizer, Rapamycin. To establish a proof-of-concept, plasmid encoding iCasp9 and luciferase under control of the p53 promoter was constructed and evaluated in a panel of cancer cell lines. While LNPs administered without Rapamycin or Rapamycin administered alone had no impact on cell viability, we observed greater than 90% apoptotic cell death when both were employed in a wide range of cancer cell lines with p53 deletions or mutations, as measured using cell viability assays, imaging assays, as well as Annexin V and TUNEL flow cytometry. Induction of iCasp9 protein expression and caspase-mediated apoptosis was confirmed using Western blot. No cell death was observed in cells with intact p53 such as human umbilical vein endothelial cells or the fibroblast cell line IMR-90. Next, we assessed the efficacy of FAST-LNPs containing p53-iCasp9 in xenograft PC-3 and H1299 models of human prostate cancer and lung cancer respectively. In some experiments, tumors were implanted subcutaneously in the flanks of 30 mice and allowed to grow to 500 mm3 before treatment by intravenous doses of 100 µg LNP twice per week during continuous low dosing of Rapamycin. We observed a rapid and dramatic reduction in tumor volume averaging 87% over the following 48 hours, with durable response. Tumors in control mice continued to grow exponentially. Overall survival of mice was extended 250% in the PC-3 cohort and 300% in the H1299 cohort. Optimization of number and concentration of LNP doses should allow for long term control of both localized and systemic disease. In conclusion, we describe a novel LNP gene therapy approach for the treatment of cancer with high selectivity for tumors with dysregulated p53 transcriptional activation. This approach has the potential to provide a highly efficacious alternative to current therapies for localized and advanced solid tumors. Citation Format: Douglas Wilson Brown, Arun Raturi, Prakash Bhandari, Deborah Sosnowski, Liliya Grin, Ping Wee, Hector Vega, Jennifer Gyoba, Maryam Hejazi, Jailal Ablack, Matthew Scholz, John D. Lewis. Selective ablation of solid tumors using a p53-targeted FAST-LNP gene therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4069.

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and β subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact–mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell–specific loss of talin, a β integrin–binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin α4β1 led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.

Acute myelogenous leukemia (AML) is an aggressive disease associated with drug resistance and relapse. To improve therapeutic strategies, it is critical to better understand the mechanisms that underlie AML progression. Here we show that the integrin binding glycoprotein CD98 plays a central role in AML. CD98 promotes AML propagation and lethality by driving engagement of leukemia cells with their microenvironment and maintaining leukemic stem cells. Further, delivery of a humanized anti-CD98 antibody blocks growth of patient-derived AML, highlighting the importance of this pathway in human disease. These findings indicate that microenvironmental interactions are key regulators of AML and that disrupting these signals with targeted inhibitors such as CD98 antibodies may be a valuable therapeutic approach for adults and children with this disease.

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.

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.

SummaryOvercoming the cellular type I interferon (IFN) host defense response is critical for a virus to ensure successful infection. Investigating the effects of human adenovirus (HAdV) infection on global cellular histone posttranslational modification (hPTM), we discovered that virus infection-induced activation of IFN signaling triggers a global increase in the monoubiquitination of histone 2B (H2B) at lysine 120, which is a mark for transcriptionally active chromatin. This hPTM, catalyzed by the hBre1/RNF20 complex, is necessary for activation of the cellular IFN-stimulated gene (ISG) expression program in response to viruses. To establish effective infection, the HAdV E1A protein binds to and dissociates the hBre1 complex to block IFN-induced H2B monoubiquitination and associated ISG expression. Together, these data uncover a key role for H2B monoubiquitination in the type I IFN response and a viral mechanism of antagonizing this hPTM to evade the IFN response.

Maspin (mammary serine protease inhibitor or SerpinB5) acts as a tumor suppressor when overexpressed in aggressive cancer cell lines. However, its role in human cancer is controversial. Maspin expression has been associated with a poor prognosis in some studies, whereas in others, with favorable outcome. The clinical data suggest, however, that nuclear-localized maspin is associated with improved survival. We hypothesized that the tumor suppressor activity of maspin may require nuclear localization, and that the discordance between clinical and experimental reports is a consequence of the variable subcellular distribution of maspin. Furthermore, we surmized that nuclear maspin could function as a tumor suppressor through the regulation of genes involved in tumor growth and invasion. Maspin or maspin fused to a nuclear export signal were expressed in metastatic human breast and epidermoid carcinoma cell lines. We found that pan-cellular localized maspin inhibited in vivo tumor growth and metastasis when assessed in xenograft chicken embryo and murine mammary fat pad injection models. However, when maspin was excluded from the nucleus via a nuclear exclusion signal, it no longer functioned as a metastasis suppressor. Using chromatin immunoprecipitation, we show that nuclear maspin was enriched at the promoter of colony-stimulating factor-1 (CSF-1) and associated with diminished levels of CSF-1 mRNA. Our findings demonstrate that the nuclear localization of maspin is required for its tumor and metastasis suppressor functions in vivo, and suggest that its mechanism of action involves, in part, direct association of maspin with target genes.

CD98 heavy chain (SLC3A2) facilitates lymphocyte clonal expansion that enables adaptive immunity; however, increased expression of CD98 is also a feature of both lymphomas and leukemias and represents a potential therapeutic target in these diseases. CD98 is transcriptionally regulated and ectopic expression of the membrane-associated RING-CH (MARCH) E3 ubiquitin ligases MARCH1 or MARCH8 leads to ubiquitylation and lysosomal degradation of CD98. Here, we examined the potential role of ubiquitylation in regulating CD98 expression and cell proliferation. We report that blocking ubiquitylation by use of a catalytically inactive MARCH or by creating a ubiquitylation-resistant CD98 mutant, prevents MARCH-induced CD98 downregulation in HeLa cells. March1-null T cells display increased CD98 expression. Similarly, T cells expressing ubiquitylation-resistant CD98 manifest increased proliferation in vitro and clonal expansion in vivo. Thus, ubiquitylation and the resulting downregulation of CD98 can limit cell proliferation and clonal expansion.

Summary 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.

The imaging of molecular markers associated with disease offers the possibility for earlier detection and improved treatment monitoring. Receptors for gastrin-releasing peptide are overexpressed on prostate cancer cells offering a promising imaging target, and analogs of bombesin, an amphibian tetradecapeptide have been previously demonstrated to target these receptors. Therefore, the pan-bombesin analog [β-Ala11, Phe13, Nle14]bombesin-(7–14) was conjugated through a linker to dye-functionalized superparamagnetic iron oxide nanoparticles for the development of a new potential magnetic resonance imaging probe. The peptide was conjugated via click chemistry, demonstrating a complementary alternative methodology to conventional peptide-nanoparticle conjugation strategies. The peptide-functionalized nanoparticles were then demonstrated to be selectively taken up by PC-3 prostate cancer cells relative to unfunctionalized nanoparticles and this uptake was inhibited by the presence of free peptide, confirming the specificity of the interaction. This study suggests that these nanoparticles have the potential to serve as magnetic resonance imaging probes for the detection of prostate cancer.

Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130(Cas), which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1-Rho-GTPase interaction in metastasis, we disrupted Cav1-Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130(Cas) phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130(Cas)/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level(1,2,3,) highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM)( 6). The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) (7, 8, 9,10,11). Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor(7,12). PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect (7, 10,11). The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

Viral nanoparticles are a novel class of biomolecular agents that take advantage of the natural circulatory and targeting properties of viruses to allow the development of therapeutics, vaccines and imaging tools. We have developed a multivalent nanoparticle platform based on the cowpea mosaic virus (CPMV) that facilitates particle labeling at high density with fluorescent dyes and other functional groups. Compared with other technologies, CPMV-based viral nanoparticles are particularly suited for long-term intravital vascular imaging because of their biocompatibility and retention in the endothelium with minimal side effects. The stable, long-term labeling of the endothelium allows the identification of vasculature undergoing active remodeling in real time. In this study, we describe the synthesis, purification and fluorescent labeling of CPMV nanoparticles, along with their use for imaging of vascular structure and for intravital vascular mapping in developmental and tumor angiogenesis models. Dye-labeled viral nanoparticles can be synthesized and purified in a single day, and imaging studies can be conducted over hours, days or weeks, depending on the application.

Viruses are obligate intracellular parasites. Their genomes are not large enough to encode all the functions required to independently produce progeny; hence, viruses are absolutely dependent on host cell functions. Mechanistically, these host cell processes in eukaryotes are founded on an exquisitely complex series of molecular interactions. In particular, the execution of complex biological processes requires the precise interaction and regulation of thousands of proteins. The definition of cellular interactomes by systematic analysis of protein-protein interactions has revealed complex molecular networks (39, 82, 111, 122). Most cellular proteins interact with only one or two other proteins, making only one or two connections. However, the minority of proteins interact with tens, or even hundreds, of other proteins to form network hubs. Hub proteins play key roles in regulating and orchestrating the activity of the proteins they interact with, effectively creating functional modules within the cellular interactome (35, 48, 62). The central role served by cellular hub proteins in regulating cell functions makes them ideal targets during a viral infection. By targeting a single cellular hub, a viral regulatory protein can effectively gain control over an entire module, potentially comprised of hundreds of proteins. By targeting multiple cellular hubs, a virally encoded hub can transform the architecture of the cellular protein interaction network, reprogramming virtually all aspects of cell function and behavior. The viral oncogenes of the small DNA tumor viruses encode some of the most versatile and potent viral hub proteins. Among these, adenovirus E1A is one of the best characterized and is the subject of this review.

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., Author Summary Helicobacter pylori infection of the gastric mucosa is largely lifelong leading to continued stimulation of immune cells. This results in the generation of reactive oxygen species (ROS) which are produced to kill bacteria, but at the same time ROS regulate cellular events in the host. However, prolonged generation of ROS has been implicated in damage of DNA, which ultimately could lead to the development of cancer. We studied a molecule known as APE-1 in gastric and intestinal cells, which is activated upon encounter of ROS. Our results show that APE1 limits the production of ROS in cells that form the lining of the gastrointestinal tract. APE1 regulates ROS production by inhibiting activation of the molecule Rac1. Inhibition of ROS production by APE1 occurred after infection of gastric cells with Helicobacter pylori and after Salmonella infection of intestinal cells. These data demonstrate that APE1 inhibits production of ROS in cells that line the inside of the digestive tract.

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

The transformation of benign tumour cells to invasive, metastatic tumour cells involves the dynamic redeployment of factors involved in cell adhesion and motility collectively known as epithelial-mesenchymal transition (EMT). While the loss of E-cadherin localization at cell-cell junctions is a hallmark feature of EMT, the impact of E-cadherin re-expression and modulation in metastatic cancer cells at distinct steps of the metastatic cascade has not been explored. To address this, we developed an intravital imaging approach that allows us to visualize changes in behaviour of highly metastatic MDA-MB-231-LN breast cancer cells in real time using a dynamically tuneable form of E-cadherin. Here, we show that upon induction of E-cadherin in vivo, cell-cell adherens junctions begin to appear within 30 minutes, concurrent with the accumulation of plasma membrane-localized E-cadherin and the retraction of tumour cell protrusions and the induction of a rounded epithelial-like morphology. Furthermore, subsequent depletion of E-cadherin results in a rapid reversion to the mesenchymal morphology. These studies demonstrate that E-cadherin is sufficient to induce a rapid mesenchymal to epithelial transition in vivo, and that sustained E-cadherin expression is required to maintain this less invasive morphology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4329.