Intracellular signalling by the insulin receptor

The insulin receptor transduces insulin's biological signal through the tyrosine kinase present in the receptor's B subunit. The activated insulin receptor kinase then phosphorylates a series of intracellular substrate including insulin receptor substrate 1 (IRS-1), which has been shown to be the pivotal substrate for insulin receptor signal transduction. The phosphorylated tyrosine residues in IRS-1 can bind and activate the downstream effectors, many of which are SH2 domain containing proteins such as phosphotidylinositol 3-kinase, growth factor binding protein 2, and SH2 phosphotyrosine phosphatase 2. Phosphorylated synthetic IRS-1 peptides with the corresponding sequences of the IRS-1 have been shown to associate and activate their respective SH2 domain containing proteins. Another important event happening during insulin binding with the insulin receptor is that the insulin receptor rapidly undergoes internalization. However, the insulin receptor signalling and the receptor endocytosis have been studied as two independent processes. The hypothesis of the present thesis is that the insulin receptor endocytosis is involved in insulin receptor signalling and signal termination. The results of the present investigation demonstrate that insulin receptors in the earliest stage of endocytosis contain significantly greater kinase activity towards IRS-1 peptides than the receptors localized at the plasma membrane, indicating that they are potentially more capable of transducing signals. On the other hand, insulin receptors in the middle and late stage of endocytosis lose their kinase activity, suggesting that insulin receptor kinase activity inactivation and signal termination might take place in the late phase of the insulin receptor internalization. In addition, this study also found that the increased insulin receptor kinase activity in the endosomes is related to the tyrosyl phosphorylation of the specific domains of the receptor's $\beta$ subunit. ^

Molecular mechanisms for the insulin sensitizing activity of rexinoids in skeletal muscle of diabetic (Db/Db) mice

Rexinoids are synthetic agonists for the retinoid X receptors (RXRs), a member of the nuclear receptor family of ligand-activated transcription factors. Rexinoids have been shown to lower serum glucose and insulin levels in animal models of type 2 diabetes. However the mechanisms that are responsible for the insulin-sensitizing action of rexinoids are largely unknown. Skeletal muscle accounts for the majority of insulin-regulated whole-body glucose disposal and impaired insulin action in muscle is an important contributor to the pathophysiology of type 2 diabetes. Glucose transport is a rate-limiting step in glucose utilization. The goal of these studies is to examine the mechanisms of the anti-diabetic activity of rexinoids in skeletal muscle of diabetic db/db mice. The results we have obtained showed that treatment of db/db mice with rexinoids for two weeks resulted in a significant increase in insulin-stimulated glucose transport activity in skeletal muscle. Insulin stimulates glucose transport in muscle via the regulation of both the insulin receptor substrate-1 (IRS-1)/Akt pathway and the Cbl-associated protein (CAP)/Cbl pathway. Rexinoids increased the insulin-stimulated IRS-1 tyrosine phosphorylation and Akt phosphorylation without effects on the activity of the CAP/Cbl pathway. The effects of rexinoids on the IRS-1/Akt pathway were associated with a decrease in the level of IRS-1 Serine 307 phosphorylation as well as qualitative and quantitative alterations in the fatty acyl-CoAs present within the muscle cells. In addition, rexinoids increased the expression of uncoupling protein 3 (UCP3) and activation of AMPK in diabetic muscle. This effect may also enhance the IRS-1/Akt signaling. We believe that it is the concerted activation of the IRS-1/Akt and AMPK signaling systems, a pharmacological mechanism that as far as we know, is unique to rexinoids, that results in the anti-diabetic effects of these drugs. Our results also suggest that the glucose-lowering mechanism of rexinoids is distinct from that of the thiazolidinediones (TZDs), peroxisome proliferator-activated receptor γ (PPARγ) agonists with well-characterized anti-diabetic activity. Rexinoids appear to represent a novel class of insulin sensitizers, with potential applications for the treatment of type 2 diabetes. ^

Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.

Insulin effects and receptor function in bovine aorta endothelial cells

The interaction of insulin with bovine aorta endothelial (BAE) cells has been studied to determine the effect of insulin on endothelial cells, and investigate the function of the insulin receptor in this cell type. BAE cell insulin receptor is similiar to insulin receptor in other cell types in the time to attain equilibrium binding, its physical properties in a solubilized assay system and affinity for insulin in the low nanomolar range. However, BAE cell insulin receptor has unusual properties in its interaction with insulin at 4$\sp\circ$C that include: (1) the inability to completely dissociate prebound $\sp{125}$I-insulin by dilution with excess insulin or acid rinse treatment, indicating that binding is not completely reversible (2) the inability to remove prebound insulin with trypsin and other proteases (3) the implication of disulfide complex formation during binding (4) the inability of pretreatment with trypsin to lower cell surface binding capacity and (5) the suppression of insulin binding by bacitracin. Interactions of insulin with the receptor at 37$\sp\circ$C showed that (1) BAE cells degrade insulin, but not as extensively as other cell types, and (2) an unusual biphasic interaction of insulin with the BAE cells is observed which is indicative of some regulatory mechanism which modulates binding affinity. Functional characterization of the BAE cell insulin receptor revealed that insulin-induced downregulation and phosphorylation of the receptor was observed, and the extent of these processes were comparable to that demonstrated in non-endothelial cell types. However, in contrast to other cell types, insulin did not stimulate deoxyglucose uptake in BAE cells. We were unable to confirm the receptor-mediated transport of insulin by the receptor across the endothelial cell monolayer as reported by a previous investigator. We could not demonstrate a role for the receptor to promote acute intracellular accumulation of insulin as postulated by several investigators. Thus, while BAE cell insulin receptor has many properties that are similiar to those in other cell types, it is distinctly different in its nondissociable binding at 4$\sp\circ$C, its interaction with insulin at 37$\sp\circ$C, and its functional role in the BAE cell. ^

Analyses of pp60$\sp{{\rm c}-src}$ and epidermal growth factor receptor protein tyrosine kinases in a human colon adenocarcinoma cell line following induction of goblet cell-like characteristics by tumor necrosis factor-alpha

Regulation of colonic epithelial cell proliferation and differentiation remains poorly understood due to the inability to design a model system which recapitulates these processes. Currently, properties of "differentiation" are studied in colon adenocarcinoma cell lines which can be induced to express some, but not all of the phenotypes of normal cells. In this thesis, the DiFi human colon adenocarcinoma cell line is utilized as an in vitro model system in which to study mucin production. In response to treatment with tumor necrosis factor-alpha, DiFi cells acquire some properties of mucin-producing goblet cells including altered morphology, increased reactivity to wheat germ agglutinin, and increased mucin production as determined by RNA expression as well as reactivity with the MUC-1 antibodies, HMFG-1 and SM-3. Thus, TNF-treated DiFi cells represent one of the few in vitro systems in which mucin expression can be induced.^ DiFi cells express an activated pp60$\sp{{\rm c}-src},$ as do most colon adenocarcinomas and derived cell lines, as well as an amplified epidermal growth factor (EGF) receptor. To assess potential changes in these enzymes during induction of differentiation characteristics, potential changes in the levels and activities of these enzymes were examined. For pp60$\sp{{\rm c}-src},$ no changes were observed in protein levels, specific activity of the kinase, cellular localization, or phosphorylation pattern as determined by Staphylococcus aureus V8 protease partial proteolytic mapping after induction of goblet cell-like phenotypic changes. These results suggest that pp60$\sp{{\rm c}-src}$ is regulated differentially in goblet cells than in absorptive cells, as down-modulation of pp60$\sp{{\rm c}-src}$ kinase occurs in the latter. Therefore, effects on pp60$\sp{{\rm c}-src}$ may be critical in colon regulation, and may be important in generating the various colonic epithelial cell types.^ In contrast to pp60$\sp{{\rm c}-src},$ EGF receptor tyrosine kinase activity decreased ($<$5-fold) after TNF treatment and at the time in which morphologic changes were observed. Similar decreases in tyrosine phosphorylation of EGF receptor were observed as assessed by immunoblotting with an anti-phosphotyrosine antibody. In addition, ($\sp{125}$I) -EGF cell surface binding was reduced approximately 3-fold following TNF treatment with a concomitant reduction in receptor affinity ($<$2-fold). These results suggest that modulation of EGF receptor may be important in goblet cell differentiation. In contrast, other published studies have demonstrated that increases in EGF receptor mRNA and in ($\sp{125}$I) -EGF binding accompany differentiation toward the absorptive cell phenotype. Therefore, differential regulation of both EGF receptor and pp60$\sp{{\rm c}-src}$ occur along the goblet cell and absorptive cell differentiation pathways. Thus, my results suggest that TNF-treated DiFi cells represent a unique system in which to study distinct patterns of regulation of pp60$\sp{{\rm c}-src}$ and EGF receptor in colonic cells, and to determine if increased MUC-1 expression is an early event in goblet cell differentiation. ^

Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

INTRODUCTION: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. METHODS: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. RESULTS: Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. CONCLUSION: We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.

Mechanotransduction pathway activation in familial thoracic aortic aneurysms and dissections

Thoracic aortic aneurysms and dissections (TAAD) are autosomal dominantly inherited in 19% of patients. Mapping studies determined that the disease is genetically heterogeneous with multiple loci and genetic mutations accounting for familial TAAD. However, regardless of the specific mutation, resulting pathology is consistently medial degeneration, characterized by increased proteoglycans and loss of elastic fibers. We tested the hypothesis that genetic mutations leading to familial TAAD alter common pathways in aortic smooth muscle cells (SMCs). Identification of mutations at R460 in TGFBR2 reveals a 5% contribution to TAAD, however downstream analysis of Smad2 phosphorylation in the TGF-β pathway is not commonly altered in familial or sporadic disease when compared to controls. Expression profiling using Illumina's Sentrix HumanRef 8 Expression Beadchip array was done on RNA isolated from SMCs explanted from 6 patients with inherited TAAD with no identified mutation and 3 healthy controls obtained from the International Institute for the Advancement of Medicine. Significant increases in expression of proteoglycan genes in patients' SMCs, specifically lumican, podocan, and decorin were confirmed using Q-PCR and tissue immunofluorescence. NCI's Ingenuity Pathway Analysis predicted alterations in the ERK, insulin receptor and SAPK/JNK pathways (p<0.001), which SMCs activate in response to cyclic stretch. Immunoblotting indicated increased phosphorylation of ERK and GSK-3β, a protein from the insulin receptor pathway, in explanted patient SMCs, also confirmed by increased immunoreactivity against phosphorylated ERK and GSK-3β in the sub-intimal SMCs from patient tissue compared to controls. To determine if mechanotransduction pathway activation was responsible for the medial degeneration a specific inhibitor of GSK-3β, SB216763 was incubated with control cells and significantly increased the expression levels of proteoglycans. Mechanical strain was also applied to control SMCs confirming pathways stimulation with stretch. Incubation with pathway inhibitors against insulin receptor and ERK pathways identify, for the first time that stretch induced GSK-3β phosphorylation may increase proteoglycan expression, and ERK phosphorylation may regulate the expression of MMP2, a protein known to degrade elastic fibers. Furthermore, specific mutations in SMC-specific β-myosin heavy chain and α-actin, in addition to upregulation of pathways activated by cyclic stretch suggest that SMC response to hemodynamic factors, play a role in this disease. ^

INDUCTION OF STRONGER AND LONG LASTING VACCINE IMMUNITY TO TUBERCULOSIS

Tuberculosis is a major cause of death due to an infection in mankind. BCG vaccine protects against childhood tuberculosis although, it fails to protect against adult tuberculosis. BCG vaccine localizes to immature phagosomes of macrophages, and avoids lysosomal fusion, which decreases peptide antigen production. Peptides are essential for macrophage-mediated priming of CD4 and CD8 T cells respectively through MHC-II and MHC-I pathways. Furthermore, BCG reduces the expression of MHC-II in macrophages of mice after infection, through Toll-like receptor-1/2 (TLR-1/2) mediated signaling. In my first aim, I hypothesized that BCG-induced reduction of MHC-II levels in macrophages can decrease CD4 T cell function, while activation of other surface Toll-like receptors (TLR) can enhance CD4 T cell function. An in vitro antigen presentation model was used where, TLR activated macrophages presented an epitope of Ag85B, a major immunogen of BCG to CD4 T cells, and T cell derived IL-2 was quantitated as a measure of antigen presentation. Macrophages with BCG were poor presenters of Ag85B while, TLR-7/9/5/4 and 1/2 activation led to an enhanced antigen presentation. Furthermore, TLR-7/9 activation was found to down-regulate the degradation of MHC-II through ubiquitin ligase MARCH1, and also stimulate MHC-II expression through activation of AP-1 and CREB transcription elements via p38 and ERK1/2 MAP kinases. I conclude from Aim-I studies that TLR-7/9 ligands can be used as more effective ‘adjuvants’ for BCG vaccine. In Aim-II, I evaluated the poor CD8 T cell function in BCG vaccinated mice thought to be due to a decreased leak of antigens into cytosol from immature phagosomes, which reduces the MHC-I mediated activation of CD8 T cells. I hypothesized that rapamycin co-treatment could boost CD8 T cell function since it was known to sort BCG vaccine into lysosomes increasing peptide generation, and it also enhanced the longevity of CD8 T cells. Since CD8 T cell function is a dynamic event better measurable in vivo, mice were given BCG vaccine with or without rapamycin injections and challenged with virulent Mycobacterium tuberculosis. Organs were analysed for tetramer or surface marker stained CD8 T cells using flow cytometry, and bacterial counts of organisms for evaluation of BCG-induced protection. Co-administration of rapamycin with BCG significantly increased the numbers of CD8 T cells in mice which developed into both short living effector- SLEC type of CD8 T cells, and memory precursor effector-MPEC type of longer-living CD8 T cells. Increased levels of tetramer specific-CD8 T cells correlated with a better protection against tuberculosis in rapamycin-BCG group compared to BCG vaccinated mice. When rapamycin-BCG mice were rested and re-challenged with M.tuberculosis, MPECs underwent stronger recall expansion and protected better against re-infection than mice vaccinated with BCG alone. Since BCG induced immunity wanes with time in humans, we made two novel observations in this study that adjuvant activation of BCG vaccine and rapamycin co-treatment both lead to a stronger and longer vaccine-mediated immunity to tuberculosis.

Axl -Gas6 signaling counteracts adenovirus type 5 E1A-mediated cell growth suppression and proapoptotic activity

The adenovirus type 5 E1A (abbreviated E1A) has previously been known as an immortalization oncogene because E1A is required for transforming oncogenes, such as ras and E1B, to transform cells in primary cultures. However, E1A has also been shown to downregulate the overexpression of the Her-2/neu oncogene, resulting in suppression of transformation and tumorigenesis induced by that oncogene. In addition, E1A is able to promote apoptosis induced by anticancer drugs, irradiation, and serum deprivation. Many tyrosine kinases, such as the EGF receptor, Her-2/Neu, Src, and Axl are known to play a role in oncogenic signals in transformed cells. To study the mechanism underlying the E1A-mediated tumor-suppressing function, we exploited a modified tyrosine kinase profile assay (Proc. Natl. Acad. Sci, 93, 5958–5962, 1996) to identify potential tyrosine kinases regulated by E1A. RT-PCR products were synthesized with two degenerate primers derived from the conserved motifs of various tyrosine kinases. A tyrosine kinase downregulated by E1A was identified as Axl by analyzing the Alu I-digested RT-PCR products. We isolated the DNA fragment of interest, and found that E1A negatively regulated the expression of the transforming receptor tyrosine kinase Axl at the transcriptional level. To study whether downregulation of the Axl receptor is involved in E1A-mediated growth suppression, we transfected axl cDNA into E1A-expressing cells (ip1-E1A) to establish cells that overexpressed Axl (ip1-E1A-Axl). The Axl ligand Gas6 triggered a greater mitogenic effect in these ip1-E1A-Axl cells than in the control cells ip1-E1A and protected the Axl-expressing cells from serum deprivation-induced apoptosis. Further study showed that Akt is required for Axl-Gas6 signaling to prevent ip1-E1A-Axl cells from serum deprivation-induced apoptosis. These results indicate that downregulation of the Axl receptor by E1A is involved in E1A-mediated growth suppression and E1A-induced apoptosis, and thereby contributes to E1A's anti-tumor activities. ^

TYPE I INSULIN-LIKE GROWTH FACTOR RECEPTOR TYROSINE KINASE AS A MOLECULAR TARGET IN MANTLE CELL LYMPHOMA

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoid malignancy representing 5-10% of all non-Hodgkin’s lymphomas. It is distinguished by the t(11;14)(q13;q32) chromosomal translocation that juxtaposes the proto-oncogene CCND1, which encodes cyclin D1 at 11q13 to the IgH gene at 14q32. MCL patients represent about 6% of all new cases of Non-Hodgkin’s lymphomas per year or about 3,500 new cases per year. MCL occurs more frequently in older adults – the average age at diagnosis is the mid-60s with a male-to-female ratio of 2-3:1. It is typically characterized by the proliferation of neoplastic B-lymphocytes in the mantle zone of the lymph node follicle that have a prominent inclination to disseminate to other lymphoid tissues, bone marrow, peripheral blood and other organs. MCL patients have a poor prognosis because they develop resistance/relapse to current non-specific therapeutic regimens. It is of note that the exact molecular mechanisms underlying the pathogenesis of MCL are not completely known. It is reasonable to anticipate that better characterization of these mechanisms could lead to the development of specific and likely more effective therapeutics to treat this aggressive disease. The type I insulin-like growth factor receptor (IGF-IR) is thought to be a key player in several different solid malignancies such as those of the prostate, breast, lung, ovary, skin and soft tissue. In addition, recent studies in our lab showed evidence to support a pathogenic role of IGF-IR in some types of T-cell lymphomas and chronic myeloid leukemia. Constitutively active IGF-IR induces its oncogenic effects through the inhibition of apoptosis and induction of transformation, metastasis, and angiogenesis. Previous studies have shown that signaling through IGF-IR leads to the vi activation of multiple signaling transduction pathways mediated by the receptor-associated tyrosine kinase domain. These pathways include PI3K/Akt, MAP kinase, and Jak/Stat. In the present study, we tested the possible role of IGF-IR in MCL. Our results demonstrate that IGF-IR is over-expressed in mantle cell lymphoma cell lines compared with normal peripheral blood B- lymphocytes. Furthermore, inhibition of IGF-IR by the cyclolignan picropodophyllin (PPP) decreased cell viability and cell proliferation in addition to induction of apoptosis and G2/M cell cycle arrest. Screening of downstream oncogenes and apoptotic proteins that are involved in both IGF-IR and MCL signaling after treatment with PPP or IGF-IR siRNA showed significant alterations that are consistent with the cellular changes observed after PPP treatment. Therefore, our findings suggest that IGF-IR signaling contributes to the survival of MCL and thus may prove to be a legitimate therapeutic target in the future.

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

Targeting dysregulated nuclear proteins androgen receptor and enhancer of zeste homolog 2: Clinical implication and mechanism underline

Cancer is the most devastating disease that has tremendous impacts on public health. Many efforts have been devoted to fighting cancer through either translational or basic researches for years. Nowadays, it emerges the importance to converge these two research directions and complement to each other for battling with cancer. Thus, our study aims at both translational and basic research directions. The first goal of our study is focus on translational research to search for new agents targeting prevention and therapy of advanced prostate cancer. Hormone refractory prostate cancer is incurable and lethal. Androgen receptor (AR) mediates androgen's effect not only on the tumor initiation but also plays the major role in the relapse transition of prostate cancer. Here we demonstrate that emodin, a natural compound, can directly target AR to suppress prostate cancer cell growth in vitro and prolong the survival of C3(1)/SV40 transgenic mice in vivo. Emodin treatment resulted in repressing androgen-dependent transactivation of AR by inhibiting AR nuclear translocation. Emodin decreased the association of AR and heat shock protein 90 and increased the association of AR and MDM2, which in turn, induces AR degradation through a proteasome-mediated pathway in a ligand independent manner. Our work indicates a new mechanism for the emodin-mediated anticancer effect and justifies further investigation of emodin as a therapeutic and preventive agent for prostate cancer. The second goal of our study is try to elucidate the fundamental tumor biology of cancer progression then provide the rationale to develop more efficient therapeutic strategy. Enhancer of zeste homologue 2 (EZH2) plays an important role in many biological processes through its intrinsic methyltransferase activity to trimethylate lysine 27 in histone H3. Although overexpression of EZH2 has been shown to be involved in cancer progression, the detailed mechanisms are elusive. Here, we show that Akt phosphorylates EZH2 at serine 21 and suppresses its methyltransferase activity by impeding the binding to its substrate histone H3, resulting in a decrease of lysine 27 trimethylation and derepression of silenced genes, thus promotes cell proliferation and tumorigenicity. Our results also show that histone methylation is not permanent but regulated in a dynamic manner and that the Akt signaling pathway is involved in the regulation of this epigenetic modification through phosphorylation of EZH2, thus contributing to oncogenic processes. ^

Insulin-like growth factor binding protein 2 (IGFBP2) promotes glioma development and progression

Overexpression of insulin-like growth factor binding protein 2 (IGFBP2) is associated with progression and poor survival in many types of human cancer (such as prostate, ovarian, adrenocortical, breast, colorectal carcinomas, leukemia, and high-grade gliomas). We therefore hypothesize that IGFBP2 is a key regulator of tumor progression. We tested our hypothesis in gliomas using the somatic gene transfer RCAS-tva mouse model system, which permits the introduction of specific genes into specific, cell lineages, in this case glial cells (RCAS: Replication competent avian sarcomavirus, tv-a: avian RCAS virus receptor). Mice are transgenic and harbor the tv-a receptor under the control of a glial-specific promoter and study genes are cloned into the RCAS vector for post-natal intracranial delivery. For these experiments, the study genes were IGFBP2, platelet-derived growth factor B (PDGFB), K-Ras, Akt, and IIp45 (invasion inhibitory protein 45 kDa; known to bind and block IGFBP2 activity), which were delivered separately and in combination. Our results show that PDGFB signaling leads exclusively to the formation of low-grade (WHO grade II) oligodendrogliomas. PDGFB delivered in combination with IGFBP2 results in the formation of anaplastic oligodendrogliomas (WHO grade III), which are characterized by increased cellularity, vascular proliferation, small regions of necrosis, increased mitotic activity, and increased activation of the Akt pathway. IIp45 injected in combination with PDGFB and IGFBP2 ablates IGFBP2-induced tumor progression, which results in formation of low-grade oligodendrogliomas, and an overall reduction in tumor incidence. K-Ras expression was required to form astrocytomas with either IGFBP2 or Akt, indicating the activation of two separate pathways is necessary for gliomagenesis. In ex vivo experiments, blockade of Akt by an inhibitor led to decreased viability of cells co-expressing IGFBP2 versus PDGFB expression alone. This study provides definitive evidence, for the first time, that: (1) IGFBP2 plays a role in activation of the Akt pathway, (2) IGFBP2 collaborates with K-Ras or PDGFB in the development and progression of two major types of glioma, and (3) IGFBP2-induced tumor progression can be ablated by IIp45 or by specific inhibition of the Akt pathway. ^

Body weight, insulin resistance, and serum adipokine levels 2 years after 2 types of bariatric surgery.

OBJECTIVE: Bariatric surgery reverses obesity-related comorbidities, including type 2 diabetes mellitus. Several studies have already described differences in anthropometrics and body composition in patients undergoing Roux-en-Y gastric bypass compared with laparoscopic adjustable gastric banding, but the role of adipokines in the outcomes after the different types of surgery is not known. Differences in weight loss and reversal of insulin resistance exist between the 2 groups and correlate with changes in adipokines. METHODS: Fifteen severely obese women (mean body mass index [BMI]: 46.7 kg/m(2)) underwent 2 types of laparoscopic weight loss surgery (Roux-en-Y gastric bypass=10, adjustable gastric banding=5). Weight, waist and hip circumference, body composition, plasma metabolic markers, and lipids were measured at set intervals during a 24-month period after surgery. RESULTS: At 24 months, patients who underwent Roux-en-Y were overweight (BMI 29.7 kg/m(2)), whereas patients who underwent gastric banding remained obese (BMI 36.3 kg/m(2)). Patients who underwent Roux-en-Y lost significantly more fat mass than patients who underwent gastric banding (mean difference 16.8 kg, P<.05). Likewise, leptin levels were lower in the patients who underwent Roux-en-Y (P=.003), and levels correlated with weight loss, loss of fat mass, insulin levels, and Homeostasis Model of Assessment 2. Adiponectin correlated with insulin levels and Homeostasis Model of Assessment 2 (r=-0.653, P=.04 and r=-0.674, P=.032, respectively) in the patients who underwent Roux-en-Y at 24 months. CONCLUSION: After 2 years, weight loss and normalization of metabolic parameters were less pronounced in patients who underwent gastric banding compared with patients who underwent Roux-en-Y gastric bypass. Our findings require confirmation in a prospective randomized trial.

Enterococcus faecalis PcfC, a spatially localized substrate receptor for type IV secretion of the pCF10 transfer intermediate.

Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCDelta N103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCDelta N103 bound pCF10 but not pcfG or Delta oriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCDelta N103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.