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2013 Research Showcase
IVCB Abstracts
Abstract Title : Role for AMPK-activators in treatment of cryopyrin-associated periodic syndromes (CAPS)
Abstract : Cryopyrin-associated periodic syndromes (CAPS) are rare inherited autoinflammatory disorders characterized by over activation of the cryopyrin inflammasome which results in a release of the proinflammatory cytokine interleukin-1β (IL-1β). Current therapeutic drugs for CAPS are costly, require frequent injections, and are ineffective in some patients; thus, more effective treatments for CAPS are needed. Preliminary in-vitro experiments have suggested a role for AMP-activated protein kinase (AMPK), a key metabolic regulator of energy homeostasis, in inflammasome function. To investigate the role of AMPK in the pathogenesis of CAPS and the potential therapeutic use of AMPK-activators in disease treatment, the effects of specific AMPK-activators A-769662 and Berberine were studied in mouse and human in vitro models of CAPS. Bone marrow derived cells (BMDCs) from mutant mouse models of CAPS following treatment with AMPK-activators were performed to analyze IL-1β release and protein levels. Results in mutant BMDCs show that A-769662 significantly inhibits IL-1β release in a dose dependent manner in a pattern consistent with direct inflammasome inhibition without a measurable effect on cell survival. However, only minimal effect was observed with the less potent inhibitor, Berberine. These results suggest a role for AMPK in the pathogenesis of CAPS and a potential therapeutic use of the small molecule AMPK-activator A-769662 in the treatment of CAPS.
Abstract Title : Characterization of the interaction between caspase-8 and p85
Abstract : Caspase-8 is a cytosolic cysteine protease that induces programmed cell death by processing a variety of apoptotic substrates [1]. Conversely, it has also been found to promote cell survival and migration, which are hallmarks of cancer cells. In fact, caspase-8 is upregulated in many cancers, including ovarian cancer [2, 3]. Post-translational modification of caspase-8 and/or association with specific proteins may toggle caspse-8 between these two opposing outcomes. In particular, modification of caspase-8 by tyrosine phosphorylation has been shown to promote cell migration [2, 4]. Phosphorylated tyrosine residues may bind to SH2 (src homology 2) domains, which are present in ~140 human proteins, including those involved in caspase-8 dependent migration [5]. Here, I characterize the interaction between caspase-8 and p85, the regulatory subunit of PI3K (phosphoinositide-3 kinase). Preliminary confocal microscopy data indicates that both proteins colocalize along the periphery of spreading cells. P85 contains two SH2 domains and has been previously suggested to be involved in migration [2, 4]. Pull down experiments using these SH2 domains as GST fusion protiens and lysates from human ovarian cancer cell lines reveal phosphorylated caspase-8 and p85 to be present within the same complex. Additional biochemical studies demonstrate a direct interaction between the two proteins. In particular, I identify that phosphorylation of caspase-8 on Tyr380 is necessary for this interaction, and that Arg358 of the n-terminal SH2 domain of p85 is critical for binding. The direct interaction between caspase-8 and p85 may further clarify the mechanisms by which caspase-8's dual roles are regulated, providing insight into the paradoxical retention of the caspase during tumor progression, as well as for therapies directed at optimizing tumor cell death. 1. Fulda, S., Caspase-8 in cancer biology and therapy. Cancer Lett, 2009. 281(2): p. 128-33. 2. Senft, J., B. Helfer, and S.M. Frisch, Caspase-8 interacts with the p85 subunit of phosphatidylinositol 3-kinase to regulate cell adhesion and motility. Cancer Res, 2007. 67(24): p. 11505-9. 3. Stupack, D.G., Caspase-8 as a therapeutic target in cancer. Cancer Lett, 2010. 4. Barbero, S., et al., Identification of a critical tyrosine residue in caspase 8 that promotes cell migration. J Biol Chem, 2008. 283(19): p. 13031-4. 5. Liu, B.A. and P.D. Nash, Evolution of SH2 domains and phosphotyrosine signalling networks. Philos Trans R Soc Lond B Biol Sci, 2012. 367(1602): p. 2556-73.
Abstract Title : Severity of Murine Arthritis Modulated by Neutrophil Cell Death via Interferon and IKKe Pathways
Abstract : Although interferon beta is a successful treatment in multiple sclerosis it had no effect and even resulted in worse arthritis in some rheumatoid arthritis trials. To investigate this disparity in autoimmune disease response to treatment genetically targeted mice were used in a serum transfer model of arthritis. From those investigated IKKepsilon (IKKe) null mice were protected from severe antibody induced arthritis; whereas interferon receptor (IFNR) deficient mice had accelerated arthritis. The double deficient mice paradoxically had even worse arthritis. Experimental results suggest parallel mechanisms contributing to disease severity include: 1) Increased neutrophil recruiting chemokine secretion in IFNR mice that is not compensated by IKKe deficiency. 2) Resultant neutrophil recruitment to inflammatory sites are comparable in the IFNR and double knockout mice, and 3) neutrophil cell death is lower in the double mice than the IFNR null mice. Hence perturbation in neutrophil cell death accounts for the clinically worse arthritis.
Abstract Title : Paracrine Wnt Signaling Both Promotes and Inhibits Human Breast Tumor Growth
Abstract : Wnt proteins are a family of highly conserved signaling molecules that are found to have a role in both oncogenesis and normal developmental processes, such as regulation of cell fate and embryogenesis. While these proteins are thought to have a role in mammary tumor development, a specific role for Wnt proteins in human breast cancer has not yet been identified due in part to the instability of these molecules at physiological conditions and their difficulty to produce using traditional methods. Our lab addressed this problem by engineering cells to produce these proteins and creating reporters to assess their activity. Using these tools, this study examined the effects of various Wnt proteins on oncogenesis in specific breast cancer cell lines and primary patient derived xenograft (PDX) models using in vivo analysis in mice. We developed an heterotypic system that involved injecting Wnt3A overexpressing human mammary fibroblasts with either human breast cancer cell lines or PDX lines into mouse mammary fat pads. In human breast cancer cell lines, we found that elevated paracrine Wnt activity was correlated with accelerated tumor growth. In PDX models, exogenous Wnt3A promoted tumor growth in one human epidermal growth factor receptor 2-overexpressing line, but inhibited tumor growth in a second PDX model derived from a patient with triple negative human breast cancer. Thus, our research demonstrates varying effects of Wnt activity on breast tumor growth.
Abstract Title : Regulation of microRNAs by mTOR in head and neck squamous cell carcinoma
Abstract : Head and neck squamous cell carcinoma (HNSCC) is the 6th leading cancer in the world and despite advances in the field of cancer, there has been very little change in the survival of patients with this disease. The Akt/mTOR signaling pathway is commonly dysregulated in HNSCC and inhibition of the mTOR complex has been shown to induce a rapid regression and prevent recurrence of tumors in xenograft mouse models. Using established HNSCC cell lines and a putative cancer stem cell (CSC) culture, we show that inhibition of mTOR by rapamycin results in a decrease in cellular proliferation. Furthermore, treatment with rapamycin also caused a decrease in the side population, which has been demonstrated to be more stem cell-like. However, the molecular mechanism by which Akt/mTOR is able to promote HNSCC pathogenesis is not well defined. In this study, we propose that the oncogenic properties of Akt/mTOR are mediated by specific mTOR-regulated microRNAs (miRs). Using the same cell lines, a microarray was performed to determine differentially expressed miRs when treated with an mTOR inhibitor. We show that miRs 145 and 15a were up-regulated, while miRs 106a, 106b, 10b and 136 were down-regulated in all HNSCC cell lines. Although further verification is necessary, our data suggests that these miRs may play a role in the pathogenic properties of the Akt/mTOR pathway, and can potentially be targets for more effective treatments.
Abstract Title : Characterization of cell cycle progression using a novel bi-cistronic lentiviral FUCCI reporter
Abstract : Currently, there are few methods to visualize cell cycle progression in leukemia stem cells (LSC). Existing techniques require sequential transduction and clonal selection, which is not feasible to study primary patient samples and LSC. To alleviate this challenge, we generated a lentiviral biscistronic vector encoding FUCCI (fluorescent ubiquitination-based cell-cycle indicator) probes. Time-lapse confocal imaging of transduced 293 and SKNO-1 cells exhibited normal cell division and distinct nuclear staining of either green or red fluorescence. Further analysis by flow cytometry of 293 cells, in comparison to FACS plots using traditional Ki67 and 7AAD staining, confirms that S/G2/M and G1/G0 phases of cell cycle correlate to expression of fluorescent reporters expressed from the bicistronic lentiviral vector. The development of this new reagent offers a new, more effective method to track cell cycle progression in limited mammalian cell populations, and in the future, in primary patient CD34+CD38- selected leukemic stem cell populations.
Abstract Title : Effects of Streptolysin O in Drosophila development and roles of HIF1 in toxin-mediated apoptosis
Abstract : Group A Streptococcus (GAS) is a leading human bacterial pathogen responsible for a wide spectrum of local and systemic infections. Our laboratory has shown that the potent GAS pore-forming cytolysin called streptolysin O (SLO) accelerates apoptosis of host macrophages and neutrophils. However, the mechanisms involved in mediating these effects of SLO on host tissues are still incompletely understood. The fruitfly model (Drosophila melanogaster) offers a powerful genetic system for dissecting potential signal pathways and developmental consequences of endogenous SLO in the host. Using germline transformation and organ specific drivers we are engineering expression of SLO in different Drosophila tissues during development, discovering that expression of SLO in the fly midgut epithelium results in a high frequency of lethality. In contrast, SLO expression in other epithelial cell types does not seem to have much effect suggesting that gut epithelial cells are particularly susceptible to the action of this toxin. Morphological analysis of the intestinal tissues of SLO-expressing flies and controls is ongoing. In parallel studies, we are examining potential approaches to limit SLO damage to host cells, by exploring the role of a transcriptional activator hypoxia-inducible factor-1 (HIF-1) that serves as a master regulator of the inflammatory and innate immune function of white blood cells. Pharmacological approaches to modulate HIF-1 are being analyzed to define their contribution to the apoptosis phenotype in response to the bacterial pore-forming toxin. Since GAS can potentially produce severe, life-threatening infections, even in previously healthy individuals, our findings may contribute to our understanding on the mechanisms through which bacterial virulence factors like SLO interact with the multifaceted constituents of the host's innate immunity.
Advisor : ANDREW M. LOWY, MD
Abstract Title : The Role of Interleukin-11 in Pancreatic Cancer Progression
Abstract : Pancreatic cancer is the fourth leading cause of cancer related deaths in the United States, with a five-year survival rate of less than five percent. Clearly a greater understanding of the multiple signaling pathways involved in pancreatic cancer progression is needed to develop more effective treatment. Interleukin-11 is a member of the IL-6 class of cytokines and has been implicated in the development and/or progression of multiple human cancers, including breast (9), colorectal (2), gastric (11), and endometrial (7) carcinomas. IL-11 promotes inflammation involved in tumor progression (12), cancer cell migration and proliferation (7), differentiation of progenitor cells in the tumor microenvironment (9), and invasiveness (11). IL-11 binds to the α-subunit of the receptor IL-11Rα, and interacts with gp130 to form a homodimer composed of two subunits each involving IL-11, IL-11Rα, and gp130. This activates a signaling cascade that results in STAT3 phosphorylation and translocation to the nucleus, where phospho-STAT3 binds to DNA and exhibits transcriptional activation of target genes that promote angiogenesis, cell-cycle progression, and cell survival (4). In addition, IL-11 has been shown to promote differentiation of osteoclastic progenitor cells in breast cancer (9) and in bone marrow transplantation (5). The role of IL-11 in pancreatic cancer is largely unknown. Through my work in the Lowy laboratory, we have developed evidence that IL-11 is secreted by pancreatic cancer cells, often at high levels. My research project will address the relevance of IL-11 in pancreatic cancer by characterizing the expression of IL-11 signaling components in human pancreatic cancer cell lines and primary tumor tissues and by examining the effects of IL-11 overexpression on epithelial cell proliferation and differentiation of various stromal progenitor cells. In pancreatic cancer, epithelial cell proliferation is essential for tumorigenesis. In human colorectal carcinoma cells IL-11 overexpression promotes cell proliferation (15). IL-11 binding to gp130 to signal in the STAT3 pathway promotes proliferation in hepatocellular adenoma (14). Furthermore, a hallmark feature of pancreatic cancer is an immense desmoplastic response in the tumor microenvironment. I will interrogate the effects of IL-11 on stromal cell recruitment and differentiation of stromal cell component progenitor cells. This work is highly relevant as the stromal component of pancreatic tumors is intimately involved in both tumor progression and therapeutic response. Studies on the role of IL-11 in pancreatic cancer are necessary to determine if therapeutic targeting of IL-11 may be a useful treatment for pancreatic cancer progression.
Advisor : DR. DWAYNE
Abstract Title : Drug Strategies to Enhance Autophagic Death in Ovarian Cancers
Abstract : In the United States ovarian cancer is diagnosed in more than 22,000 women each year. Aside from ubiquitous loss of the p53 tumor suppressor, no single driver mutation has been identified in ovarian cancer. Autophagy is a highly regulated catabolic mechanism required for cell survival. Our studies have shown that autophagy-regulating genes are deleted in 95% of ovarian cancer cases. In studies presented here we test drug combinations which manipulate autophagy, evaluating their impact on tumor cell survival. Two adjuvant drugs were used to form these drug combinations. Metformin is a commonly used as a diabetes treatment recently associated with an increase in progression-free survival in ovarian cancer patients. Chloroquine is a drug originally used for the treatment and prevention of malaria. Both display anti-tumor effects by altering autophagy and inducing cell death in tumors. These drugs target separate events in autopahgy and may prove useful in dose-controlled combinations to enhance ovarian cancer cell death. We examined the effects of these drugs in combination with preclinical (thapsigargin, ABT-737) approved (rapamycin), as well as standard of care (cisplatin) on several ovarian cancer lines. While some variability was observed, results indicated that the combined use of chloroquine or metformin with standard of care or experimental drugs more efficiently effected cancer cell death. With chemotherapy stagnant and needing more dramatic improvement, options to low-toxicity and increase efficacy may provide a more offer patients improved options for healthcare.
Abstract Title : In vitro model for fat-resident regulatory T cells
Abstract : Regulatory T cells (Tregs) are a type of immunosuppressive T cell that is indispensable for life. Many autoimmune diseases including rheumatoid arthritis, type-1 diabetes, multiple sclerosis, and asthma have been linked to dysfunctional Tregs. Recently, a unique population of Tregs found in fat (fTregs) has been linked in suppressing the low-grade chronic inflammation that exists in fat in many diseases of obesity like type-2 diabetes. Interestingly, the gene expression signature of fTregs differs from Tregs found in spleen or lymph nodes. This different gene expression signature is largely driven by the unique expression of peroxisome proliferator-activated receptor-gamma (PPARg). fTregs do not exist at numbers high enough to conduct a biochemical characterization of the cell-type. Therefore, we have developed a prototype in vitro model of fTregs through viral transduction of PPARg in splenic Tregs. Using next-generation RNA sequencing, we will show key pathways and genes up- and down-regulated by PPARg in this in vitro model and compare it to in vivo fTregs to determine if PPARg virally transduced Tregs are an applicable in vitro model to study fTregs.
Advisor : DAVID M. SMITH
Abstract Title : Gut Lactobacillales are Associated with Higher CD4 and Less Microbial Translocation during HIV Infection
Abstract : Early HIV infection is characterized by a dramatic depletion of CD4 T-cells in the gastrointestinal tract and translocation of bacterial products from the gut into the blood. In this study, we evaluated how gut bacterial profiles were associated with immune status before and after starting antiretroviral therapy (ART). We followed and assessed the gut microbiota of men recently infected with HIV who were participating in a randomized, double-blind controlled trial of combination ART plus maraviroc versus placebo for 48 weeks. We pyrosequenced the bacterial populations from anal swabs collected before and longitudinally after the initiation of ART. Associations of the gut flora with clinical variables (lymphocyte profiles, viral loads), activation and proliferation markers in peripheral blood mononuclear cells and gut biopsies (measured by flow cytometry) and markers of microbial translocation (lipopolysaccharide and soluble CD14) were performed by regression analyses using R statistical software. We identified that higher proportions of Lactobacillales in the distal gut of recently HIV-infected individuals were associated with lower markers of microbial translocation, higher CD4% and lower viral loads before ART was started. Similarly, during ART, higher proportions of gut Lactobacillales were associated with higher CD4%, less microbial translocation, less systemic immune activation, less gut T lymphocyte proliferation, and higher CD4% in the gut. Shaping the gut microbiome, especially proportions of Lactobacillales, could help to preserve immune function during HIV infection.
Abstract Title : Characterizing the Role of NETs in the K/BxN Mouse Model of Rheumatoid Arthritis
Abstract : Neutrophils expunge their nuclear DNA as a protective mechanism to ensnare invading microbes. These neutrophil extracellular traps (NET)s have been implicated as an antigenic reservoir for autoimmune diseases such as lupus and vasculitis, which have autoantibodies to DNA and myeloperoxidase respectively. The role of NETosis in the pathogenesis of rheumatoid arthritis has yet to be defined, however the essential activity of PAD4 in NETosis and in citrullinating key antigens in RA potentially links these two. In a neutrophil dependent murine model of arthritis, induction of arthritis was associated with increased levels of circulating DNA. The driving antigen in this model co-localized by immunohistochemistry with NETs. PAD4 deficiency did not ameliorate arthritis; however PAD4 deficiency in combination with Caspase-1 deficiency dramatically reduced arthritis. In this model the presence of PAD4 and NETs did not initiate arthritis but might play a role in a self perpetuating cascade.