Multiple Signaling Networks Research Articles

AMG925, a Dual FLT3-CDK4/6 Inhibitor, Disrupts Survival Signaling and Triggers Apoptosis in AML Progenitor/Stem Cells

FLT3 mutations are detected in one third of patients diagnosed with acute myeloid leukemia (AML) and confer poor outcomes. Selective FLT3 inhibitors have been developed and are being tested at different stages of clinical development. Drug resistance triggered by inhibitor-induced secondary mutations is often observed, and is a major cause of treatment failure. AMG925 (FLX925) is a small molecule dual FLT3-CDK4/6 inhibitor, currently in clinical development. Because AMG925 concomitantly inhibits both FLT3 and CDK4/6, AMG925 treatment rarely causes development of secondary drug-resistant FLT3 mutations (Li C et. al Mol. Cancer Ther. 2015). In this study, we examined the anti-leukemic effect of AMG925 on AML, focusing on AML progenitor/stem cells. Utilizing the high-throughput technology of time-of-flight mass cytometry (CyTOF) and reverse phase protein array (RPPA), we investigated alterations of the FLT3-mediated signaling network trigged by AMG925 at the single cell level. While AMG925 caused cell growth inhibition and apoptosis in both, FLT3-mutated (MOLM13, MV4;11, Ba/F3-ITD, Ba/F3-D835Y and Ba/F3-ITD/D835Y) and -wild type (Ba/F3-wt, OCI-AML3 and U937) cell lines and primary AML blasts, it elicited maximal response in cells carrying ITD, D835Y and ITD/D835Y double mutations. Mechanistically, AMG925 attenuates CDK4 phosphorylation of retinoblastoma protein (Rb), inhibits FLT3 activation and abrogates FLT3-dependent and independent AKT/mTOR, MEK/ERK and STAT5 signaling. AMG925 at 10nM effectively inhibited colony formation of PDX-derived leukemic stem cells originating from 2 AML patients carrying FLT3-ITD mutation (54±8.7% inhibition, n=4). CyTOF analysis of cell surface and intracellular molecules measured by a panel of 36 antibodies revealed that the inhibitory effect of AMG925 on p-AKT, p-ERK and p-STAT5 is operational primarily in subpopulations expressing the stem cell markers CD123, CD117, CD34, CD133 and CD47. Using RPPA, we studied the expression and phosphorylation changes of 273 proteins from the same 2 AML-ITD xenograft samples in response to AMG925 ex-vivo. Heterogeneous expression signatures were observed in all samples isolated from bone marrows (n=1) and spleens (n=4). AMG925 treatment altered multiple signaling networks by inhibiting key molecules in the AKT/mTOR, cyclin D family, MEK/ERK and Src pathways, and triggered a stress-response by up-regulating STAT3 and TSC1 in all samples examined. The combinational consequences of targeting FLT3-CDK4/6 and the proteins that contribute to resistance are under investigation. Conclusions: combined FLT3-CDK4/6 inhibition by AMG925 results in growth inhibition and apoptosis of AML cells with or without FLT3 mutations and selectively inhibits signaling within PDX and phenotypically defined AML stem/progenitor cell populations. CyTOF analysis confirmed on-target effects of AMG925 on FLT3-regulated signaling in AML stem cell subsets. RPPA profiling of AML-ITD xenografts exposed ex-vivo to AMG925 revealed alterations in multiple pro-survival signaling pathways and identified the upregulation of alternative pathways potentially conferring resistance to AMG925. While our results indicate efficacy of combined FLT3-CDK4/6 inhibition it identifies unexpected resistance mechanisms that provide rationale for the additional mechanism-based combinatorial approaches to increase the therapeutic efficacy in AML DisclosuresShah:ARIAD: Research Funding; BMS: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Research Funding; Plexxikon: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

Abstract 2809: ROS signaling by NADPH oxidase 5 (Nox5) modulates proliferation of human melanoma UACC-257 cells and prolongs growth in the absence of serum or growth factors

Abstract Reactive oxygen species (ROS) produced by the NADPH oxidase (Noxs) homologs participate in signaling cascades regulating proliferation. Recently, we have reported substantial overexpression of Nox5 in several human cancers including those of prostate, ovary and melanomas. Despite being upregulated in many human cancers and implicated in promoting cell proliferation, the molecular mechanisms modulated by Nox5 are poorly understood. In this study, the functional significance of Nox5 was assessed in human UACC-257 and WM-852 melanoma cells by generating Nox5-overexpressing and knockdown cells. Stable overexpression of Nox5 in UACC-257 cells resulted in enhanced cell growth, increased BrdU positive cells and decreased protein tyrosine phosphatase activity. Additionally, these cells had increased normoxic Hif-1α expression and decreased p27Kip1 expression. Conversely, knockdown of endogenous Nox5 in UACC-257 cells resulted in decreased cell growth, decreased BrdU positive cells, decreased Hif-1α expression and increased p27Kip1 expression. Likewise, in human WM-852 melanoma cells, transient overexpression of Nox5 decreased p27Kip1 expression and knockdown of endogenous Nox5 resulted in increased p27Kip1 and gamma-H2AX expression with decreased cell growth. Cadherin switch, loss of E-cadherin expression and upregulation of N-cadherin was observed in UACC-257 Nox5 overexpressing cells indicative of an invasive phenotype; conversely, an upregulation of E-cadherin expression in Nox5 knockdown cells was noted. Cell invasion assay through matrigel-coated membranes also demonstrated enhanced invasion by Nox5 overexpressing cells. Additionally, 3D cultures of Nox5 overexpressing UACC-257 cells exhibit an amoeboid morphology compared to that of the Nox5 knockdown cells that were mesenchymal, suggestive of an amoeboid - mesenchymal (AMT) transition. Strikingly, UACC-257 cells that overexpress Nox5 were able to proliferate in serum-free conditions for over a month. In summary, our findings suggest that ROS signaling by Nox5 in human melanoma could modulate multiple signaling networks that regulate Hif-1α and p27Kip1 expression, contributing in part, to cell proliferation and the ability to grow in the absence of serum or growth factors. Citation Format: Smitha Antony, Yongzhong Wu, Diana C. Haines, Guojian Jiang, Jennifer L. Meitzler, Agnes Juhasz, Jiamo Lu, Krishnendu K. Roy, James H. Doroshow. ROS signaling by NADPH oxidase 5 (Nox5) modulates proliferation of human melanoma UACC-257 cells and prolongs growth in the absence of serum or growth factors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2809.

Class I PI-3-Kinase Signaling Is Critical for Bone Formation Through Regulation of SMAD1 Activity in Osteoblasts.

Bone formation and homeostasis is carried out by osteoblasts, whose differentiation and activity are regulated by osteogenic signaling networks. A central mediator of these inputs is the lipid kinase phosphatidylinositol 3-kinase (PI3K). However, at present, there are no data on the specific role of distinct class IA PI3K isoforms in bone biology. Here, we performed osteoblast-specific deletion in mice to show that both p110α and p110β isoforms are required for survival and differentiation and function of osteoblasts and thereby control bone formation and postnatal homeostasis. Impaired osteogenesis arises from increased GSK3 activity and a depletion of SMAD1 protein levels in PI3K-deficient osteoblasts. Accordingly, pharmacological inhibition of GSK3 activity or ectopic expression of SMAD1 or SMAD5 normalizes bone morphogenetic protein (BMP) transduction and osteoblast differentiation. Together, these results identify the PI3K-GSK3-SMAD1 axis as a central node integrating multiple signaling networks that govern bone formation and homeostasis. © 2016 American Society for Bone and Mineral Research.

Raf Kinase Inhibitor Protein (RKIP) expression and function in human myometrium and leiomyoma

Many growth factors been identified in human myometrium and leiomyoma and activate multiple signaling pathways in order to regulate major cellular processes, including proliferation and fibrosis which are linked to uterine leiomyoma development and growth. The Raf kinase inhibitor protein (RKIP) has emerging roles as regulator of multiple signaling networks including mitogen activated protein (MAP) kinase cascade, as well as interaction with glycogen synthase kinase 3 (GSK3). In our study, we aimed to investigate the presence of RKIP in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation and migration in human myometrial and leiomyoma cells. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and western blotting. Myometrial and leiomyoma cells were treated with locostatin to measure ECM expression by real time PCR, GSK3b expression by western blotting, cell migration by wound-healing assay and cell proliferation by MTT assay. We found that RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the MAPK signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells.

Abstract 1251: Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells

Abstract NADPH oxidase 5 (Nox5), one of the seven members of the NADPH oxidase gene family that generate reactive oxygen species (ROS), has been implicated in physiological and pathophysiological processes. Recently, we have reported substantial overexpression of Nox5 in several human cancers including those of prostate, ovary and melanomas; expression in most non-malignant tissues was weak to modest. Despite being upregulated in many human cancers and implicated in promoting cell proliferation, the molecular mechanisms regulated by Nox5 are poorly understood. In this study, the functional significance of Nox5 was assessed in human UACC-257 melanoma cells by generating stable Nox5-overexpressing clones and knockdown clones. Overexpression of Nox5 in UACC-257 cells resulted in enhanced cell growth, increased BrdU positive cells and decreased phosphatase activity. Additionally, these cells had increased normoxic Hif-1α expression and decreased p27Kip1 expression. Conversely, knockdown of endogenous Nox5 in UACC-257 cells resulted in decreased cell growth, decreased BrdU positive cells, decreased Hif-1α expression and increased p27Kip1 expression. Cadherin switch, loss of E-cadherin expression and upregulation of N-cadherin was observed in Nox5 overexpressing cells indicative of an invasive phenotype; conversely, an upregulation of E-cadherin expression in Nox5 knockdown cells was noted. Cell invasion assay through matrigel-coated membranes also demonstrated enhanced invasion by Nox5 overexpressing cells. Additionally, 3D cultures of Nox5 overexpressing cells exhibit an amoeboid morphology compared to that of the Nox5 knockdown cells that were mesenchymal, suggestive of an amoeboid - mesenchymal (AMT) transition. Strikingly, cells that overexpress Nox5 were able to proliferate in serum-free conditions for over a month. In summary, our findings suggest that Nox5 expression in human melanoma UACC-257 cells could contribute to cell proliferation, invasion, amoeboid morphology, and the ability to grow in the absence of serum or growth factors in part due to the generation of high local concentrations of extracellular ROS that modulate multiple signaling networks that regulate Hif-1α, p27Kip1, and the cadherin switch. Citation Format: Smitha Antony, Yongzhong Wu, Guojian Jiang, Jennifer L. Meitzler, Han Liu, Agnes Juhasz, Jiamo Lu, Miriam R. Anver, Krishnendu K. Roy, James H. Doroshow. Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1251. doi:10.1158/1538-7445.AM2015-1251

Natural product inspired HSP90 inhibitors

Natural products continue to play a key role in drug discovery, an example of which is highlighted by Hsp90 inhibitors. The 90 kDa heat shock proteins (Hsp90) are responsible for the conformational maturation of nascent polypeptides, many of which are critical to the maintenance of cell signaling networks that contribute to the six hallmarks of cancer. Thus, through Hsp90 inhibition, one can simultaneously derail multiple signaling networks through inhibition of a single biological target. The natural products, geldanamycin and radicicol, have served as lead compounds to develop multiple inhibitors that have entered clinical trials for the treatment of cancer that manifest their activity through inhibition of the Hsp90 N-terminal ATPase domain. In contrast, the natural products, novobiocin, EGCG, and silybin have served as lead compounds that regulate Hsp90 protein folding activity through allosteric modulation of the chaperone via the C-terminal dimerization domain. In addition, the natural products, cruentaren A, celastrol and gedunin, have shown to provide a unique opportunity to modulate Hsp90 activity through disruption of co-chaperone interactions with Hsp90. In this presentation, the utilization of natural products to modulate the Hsp90 protein folding machinery will be discussed along with their potential therapeutic applications.

IRF-1 inhibits NF-κB activity, suppresses TRAF2 and cIAP1 and induces breast cancer cell specific growth inhibition

Interferon Regulatory Factor (IRF)-1, originally identified as a transcription factor of the human interferon (IFN)-β gene, mediates tumor suppression and may inhibit oncogenesis. We have shown that IRF-1 in human breast cancer cells results in the down-regulation of survivin, tumor cell death, and the inhibition of tumor growth in vivo in xenogeneic mouse models. In this current report, we initiate studies comparing the effect of IRF-1 in human nonmalignant breast cell and breast cancer cell lines. While IRF-1 in breast cancer cells results in growth inhibition and cell death, profound growth inhibition and cell death are not observed in nonmalignant human breast cells. We show that TNF-α or IFN-γ induces IRF-1 in breast cancer cells and results in enhanced cell death. Abrogation of IRF-1 diminishes TNF-α and IFN-γ-induced apoptosis. We test the hypothesis that IRF-1 augments TNF-α-induced apoptosis in breast cancer cells. Potential signaling networks elicited by IRF-1 are investigated by evaluating the NF-κB pathway. TNF-α and/or IFN-γ results in decreased presence of NF-κB p65 in the nucleus of breast cancer cells. While TNF-α and/or IFN-γ can induce IRF-1 in nonmalignant breast cells, a marked change in NF-κB p65 is not observed. Moreover, the ectopic expression of IRF-1 in breast cancer cells results in caspase-3, -7, -8 cleavage, inhibits NF-κB activity, and suppresses the expression of molecules involved in the NF-κB pathway. These data show that IRF-1 in human breast cancer cells elicits multiple signaling networks including intrinsic and extrinsic cell death and down-regulates molecules involved in the NF-κB pathway.

Silencing LRH-1 in colon cancer cell lines impairs proliferation and alters gene expression programs

Colorectal cancers (CRCs) account for nearly 10% of all cancer deaths in industrialized countries. Recent evidence points to a central role for the nuclear receptor liver receptor homolog-1 (LRH-1) in intestinal tumorigenesis. Interaction of LRH-1 with the Wnt/β-catenin pathway, highly active in a critical subpopulation of CRC cells, underscores the importance of elucidating LRH-1's role in this disease. Reduction of LRH-1 diminishes tumor burden in murine models of CRC; however, it is not known whether LRH-1 is required for tumorigenesis, for proliferation, or for both. In this work, we address this question through shRNA-mediated silencing of LRH-1 in established CRC cell lines. LRH-1 mRNA knockdown results in significantly impaired proliferation in a cell line highly expressing the receptor and more modest impairment in a cell line with moderate LRH-1 expression. Cell-cycle analysis shows prolongation of G0/G1 with LRH-1 silencing, consistent with LRH-1 cell-cycle influences in other tissues. Cluster analysis of microarray gene expression demonstrates significant genome wide alterations with major effects in cell-cycle regulation, signal transduction, bile acid and cholesterol metabolism, and control of apoptosis. This study demonstrates a critical proproliferative role for LRH-1 in established colon cancer cell lines. LRH-1 exerts its effects via multiple signaling networks. Our results suggest that selected CRC patients could benefit from LRH-1 inhibitors.

Analysis of platelet function and dysfunction.

Although platelets act as central players of haemostasis only their cross-talk with other blood cells, plasma factors and the vascular compartment enables the formation of a stable thrombus. Multiple activation processes and complex signalling networks are responsible for appropriate platelet function. Thus, a variety of platelet function tests are available for platelet research and diagnosis of platelet dysfunction. However, universal platelet function tests that are sensitive to all platelet function defects do not exist and therefore diagnostic algorithms for suspected platelet function disorders are still recommended in clinical practice. Based on the current knowledge of human platelet activation this review evaluates point-of-care related screening tests in comparison with specific platelet function assays and focuses on their diagnostic utility in relation to severity of platelet dysfunction. Further, systems biology-based platelet function methods that integrate global and specific analysis of platelet vessel wall interaction (advanced flow chamber devices) and post-translational modifications (platelet proteomics) are presented and their diagnostic potential is addressed.

Deteriorated stress response in stationary-phase yeast: Sir2 and Yap1 are essential for Hsf1 activation by heat shock and oxidative stress, respectively.

Stationary-phase cultures have been used as an important model of aging, a complex process involving multiple pathways and signaling networks. However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging. The budding yeast Saccharomyces cerevisiae is a valuable model organism to study the genetics of aging, because yeast ages within days and are amenable to genetic manipulations. As a unicellular organism, yeast has evolved robust systems to respond to environmental challenges. This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses. Here we demonstrate that Hsf1 response to heat shock and oxidative stress deteriorates during yeast transition from exponential growth to stationary-phase, whereas Hsf1 activation by glucose starvation is maintained. Overexpressing Hsf1 does not significantly improve heat shock response, indicating that Hsf1 dwindling is not the major cause for Hsf1 attenuated response in stationary-phase yeast. Rather, factors that participate in Hsf1 activation appear to be compromised. We uncover two factors, Yap1 and Sir2, which discretely function in Hsf1 activation by oxidative stress and heat shock. In Δyap1 mutant, Hsf1 does not respond to oxidative stress, while in Δsir2 mutant, Hsf1 does not respond to heat shock. Moreover, excess Sir2 mimics the heat shock response. This role of the NAD+-dependent Sir2 is supported by our finding that supplementing NAD+ precursors improves Hsf1 heat shock response in stationary-phase yeast, especially when combined with expression of excess Sir2. Finally, the combination of excess Hsf1, excess Sir2 and NAD+ precursors rejuvenates the heat shock response.

Systems-Level G Protein-Coupled Receptor Therapy Across a Neurodegenerative Continuum by the GLP-1 Receptor System.

With our increasing appreciation of the true complexity of diseases and pathophysiologies, it is clear that this knowledge needs to inform the future development of pharmacotherapeutics. For many disorders, the disease mechanism itself is a complex process spanning multiple signaling networks, tissues, and organ systems. Identifying the precise nature and locations of the pathophysiology is crucial for the creation of systemically effective drugs. Diseases once considered constrained to a limited range of organ systems, e.g., central neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’ disease (HD), the role of multiple central and peripheral organ systems in the etiology of such diseases is now widely accepted. With this knowledge, it is increasingly clear that these seemingly distinct neurodegenerative disorders (AD, PD, and HD) possess multiple pathophysiological similarities thereby demonstrating an inter-related continuum of disease-related molecular alterations. With this systems-level appreciation of neurodegenerative diseases, it is now imperative to consider that pharmacotherapeutics should be developed specifically to address the systemic imbalances that create the disorders. Identification of potential systems-level signaling axes may facilitate the generation of therapeutic agents with synergistic remedial activity across multiple tissues, organ systems, and even diseases. Here, we discuss the potentially therapeutic systems-level interaction of the glucagon-like peptide 1 (GLP-1) ligand–receptor axis with multiple aspects of the AD, PD, and HD neurodegenerative continuum.

Tsukushi is involved in the wound healing by regulating the expression of cytokines and growth factors

During the wound-healing process, macrophages, fibroblasts, and myofibroblasts play a leading role in shifting from the inflammation phase to the proliferation phase, although little is known about the cell differentiation and molecular control mechanisms underlying these processes. Previously, we reported that Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan family, functions as a key extracellular coordinator of multiple signalling networks. In this study, we investigated the contribution of TSK to wound healing. Analysis of wound tissue in heterozygous TSK-lacZ knock-in mice revealed a pattern of sequential TSK expression from macrophages to myofibroblasts. Quantitative PCR and in vitro cell induction experiments showed that TSK controls macrophage function and myofibroblast differentiation by inhibiting TGF-β1 secreted from macrophages. Our results suggest TSK facilitates wound healing by maintaining inflammatory cell quiescence.

Regulation of mitochondrial apoptosis by Pin1 in cancer and neurodegeneration

Mitochondria are sensitive and efficient organelles that regulate essential biological processes including: energy metabolism, decoding and transduction of intracellular signals, and balance between cell death and survival. Of note, dysfunctions in mitochondrial physiology are a general hallmark of cancer cells, leading to transformation-related features such as altered cellular metabolism, survival under stress conditions and reduced apoptotic response to chemotherapy. Mitochondrial apoptosis is a finely regulated process that derives from activation of multiple signaling networks. A crucial biochemical requirement for transducing pro-apoptotic stimuli is represented by kinase-dependent phosphorylation cascades. In this context a pivotal role is played by the prolyl-isomerase Pin1, which translates Ser/Thr-Pro phosphorylation into conformational changes able to modify the activities of its substrates. In this review we will discuss the impact of Pin1 in regulating various aspects of apoptosis in different biological contexts with particular emphasis on cancer and neurodegenerative diseases.

Multiple signaling networks of extracellular ATP, hydrogen peroxide, calcium, and nitric oxide in the mediation of root ion fluxes in secretor and non-secretor mangroves under salt stress

Using 3-month-old seedlings of Kandelia obovata and Aegiceras corniculatum, we compared multiple signaling networks controlling K+/Na+ homeostasis in salt-secreting and non-secreting mangroves. K. obovata roots exhibited a higher capacity to extrude Na+ and take up H+ after exposure to 100mM NaCl for 12h compared to A. corniculatum. However, the salt-induced Na+/H+ exchange was inhibited by amiloride, a Na+/H+ antiporter inhibitor, and sodium orthovanadate, a H+-ATPase inhibitor, indicating that Na+ efflux resulted from active Na+ exclusion. Ca2+, H2O2, NO, and extracellular ATP (eATP) enhanced Na+ extrusion by 27–152% in the salinized roots of K. obovata and A. corniculatum. Moreover, eATP, H2O2, and Ca2+ interacted with each other to increase Na+ extrusion in the two species. However, the interactions between NO and other signals differed in the secretor and non-secretor mangroves. Under a saline environment, NO enhanced the Na+ efflux elicited by Ca2+ in A. corniculatum. In contrast, NO reduced the Na+ efflux elicited by eATP, H2O2, and Ca2+ in K. obovata. In the two mangrove species, salt caused a net K+ efflux, but it was markedly restricted by the K+ channel blocker TEA, indicating that the K+ efflux is mediated by K+-permeable channels. In A. corniculatum, Ca2+ interacted with H2O2, NO, and eATP to reduce the K+ loss during salt treatment, whereas eATP enhanced the inhibitory effect of H2O2, Ca2+, and NO on K+ efflux in the non-secretor K. obovata. Thus, species-specific salt signaling networks regulating root K+ and Na+ flux were established in the secreting and non-secreting mangroves.

ITCH E3 ligase in ATM network.

ITCH E3 ligase in ATM network.

Shear stress-initiated signaling and its regulation of endothelial function.

Atherosclerosis develops preferentially at branches and curvatures of the arterial tree, where blood flow pattern is disturbed rather than being laminar, and wall shear stress has an irregular distribution without defined directions. The endothelium in the atherosusceptible regions, in comparison to that in atheroresistant regions, shows activation of proproliferative and proinflammatory gene expressions, reduced production of nitric oxide (NO), increased leukocyte adhesion, and permeability, as well as other atheroprone phenotypes. Differences in gene expressions and cell phenotypes have been detected in endothelia residing in native atherosusceptible and atheroresistant regions of the arteries, or in arteries from animal models with artificial creation of disturbed flow. Similar results have also been shown in in vitro systems that apply controlled shear stresses with or without clear directions to cultured endothelial cells in fluid-dynamically designed flow-loading devices. The available evidence indicates that the coordination of multiple signaling networks, rather than individual separate pathways, links the mechanical signals to specific genetic circuitries in orchestrating the mechanoresponsive networks to evoke comprehensive genetic and functional responses.

Synthesis of Butein Analogues and their Anti-proliferative Activity Against Gefitinib-resistant Non-small Cell Lung Cancer (NSCLC) through Hsp90 Inhibition

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer representing 85% of lung cancer patients. Despite several EGFR-targeted drugs have been developed in the treatment of NSCLC, the clinical efficacy of these EGFR-targeted therapies is being challenged by the occurrence of drug resistance. In this regard, Hsp90 represents great promise as a therapeutic target of cancerous diseases due to its role in modulating and stabilizing numerous oncogenic proteins. Accordingly, inhibition of single Hsp90 protein simultaneously disables multiple signaling networks so as to overcome drug resistance in cancer. In this study, we synthesized a series of 11 butein analogues and evaluated their biological activities against gefitinibresistant NSCLC cells (H1975). Our study indicated that analogue 1h inhibited the proliferation of H1975 cells, down-regulated the expression of Hsp90 client proteins, including EGFR, Met, Her2, Akt and Cdk4, and upregulated the expression of Hsp70. The result suggested that compound 1h disrupted Hsp90 chaperoning function and could serve a potential lead compound to overcome the drug resistance in cancer chemotherapy.

Sa1805 An mTOR-Sirtuin Axis in the Intestinal Cell Differentiation Network

BACKGROUND. Colorectal cancers (CRC) account for nearly 10% of cancer deaths in industrialized countries. Recent evidence points to a central role for the nuclear receptor Liver Receptor Homolog-1 (LRH-1) in intestinal tumorigenesis. Interaction of LRH-1 with the Wnt/β-catenin pathway, highly active in a critical subpopulation of CRC cancer cells, underscores the importance of elucidating LRH-1's role in this disease. Reduction of LRH1 diminishes tumor burden in murine models of CRC. The central role of receptor LRH-1 in CRC pathogenesis and a wealth of structural data at atomic resolution make LRH-1 an attractive target. Methods: To evaluate the contributions of LRH-1 to CRC growth, we constructed stably transduced CRC cell lines expressing Tet-inducible shRNA directed against LRH-1. Cell growth of the LRH-1 knockdown lines was compared with growth of cells expressing a non-silencing control RNA. To explore alterations in gene expression due to LRH-1 suppression, we performed a microarray analysis of our knockdown cell lines. Additionally, we sought to develop and characterize specific, non-cytotoxic inhibitors of LRH-1 function for use as both potential therapeutic agents and more broadly as research tools to study organogenesis and tumorigenesis. We applied differential scanning fluorimetry (DSF), used to measure qualitative binding, to screen over 200 compounds for association with the LRH-1 ligand binding domain. Results: LRH-1mRNA knockdown results in impaired cell proliferation. Cluster analysis of microarray gene expression demonstrates significant alterations in signal transduction, bile acid and cholesterol metabolism, and control of apoptosis. Moreover, we discovered 9 unique compounds that interact with LRH-1, but not the closely related receptor SF-1. We have further demonstrated that four of these compounds have antiproliferative effects with IC50 values ranging from 8.6 to 30 μM. Conclusions: Silencing of LRH-1 expression in CRC cell lines leads to impaired cell growth, demonstrating that LRH-1 is a viable target in CRC. LRH-1 may exert its effects via multiple signaling networks, with inhibition leading to cell cycle arrest or apoptosis. General features for binding shared between all compounds include a linear array of hydrophobic groups with polar atoms likely allowing for registration within the hormone pocket. Interestingly, two compounds showing antiproliferative activity share a cholesterol motif which may be related to LRH-1's function in bile acid and cholesterol homeostasis. Support: K12 HD07222, F32 CA163092, T32 DK007762

Sa1804 Dysregulation of WNT5A/ROR2 Signaling Pathway Characterizes the Progression of Barrett's-Associated Esophageal Adenocarcinoma

BACKGROUND. Colorectal cancers (CRC) account for nearly 10% of cancer deaths in industrialized countries. Recent evidence points to a central role for the nuclear receptor Liver Receptor Homolog-1 (LRH-1) in intestinal tumorigenesis. Interaction of LRH-1 with the Wnt/β-catenin pathway, highly active in a critical subpopulation of CRC cancer cells, underscores the importance of elucidating LRH-1's role in this disease. Reduction of LRH1 diminishes tumor burden in murine models of CRC. The central role of receptor LRH-1 in CRC pathogenesis and a wealth of structural data at atomic resolution make LRH-1 an attractive target. Methods: To evaluate the contributions of LRH-1 to CRC growth, we constructed stably transduced CRC cell lines expressing Tet-inducible shRNA directed against LRH-1. Cell growth of the LRH-1 knockdown lines was compared with growth of cells expressing a non-silencing control RNA. To explore alterations in gene expression due to LRH-1 suppression, we performed a microarray analysis of our knockdown cell lines. Additionally, we sought to develop and characterize specific, non-cytotoxic inhibitors of LRH-1 function for use as both potential therapeutic agents and more broadly as research tools to study organogenesis and tumorigenesis. We applied differential scanning fluorimetry (DSF), used to measure qualitative binding, to screen over 200 compounds for association with the LRH-1 ligand binding domain. Results: LRH-1mRNA knockdown results in impaired cell proliferation. Cluster analysis of microarray gene expression demonstrates significant alterations in signal transduction, bile acid and cholesterol metabolism, and control of apoptosis. Moreover, we discovered 9 unique compounds that interact with LRH-1, but not the closely related receptor SF-1. We have further demonstrated that four of these compounds have antiproliferative effects with IC50 values ranging from 8.6 to 30 μM. Conclusions: Silencing of LRH-1 expression in CRC cell lines leads to impaired cell growth, demonstrating that LRH-1 is a viable target in CRC. LRH-1 may exert its effects via multiple signaling networks, with inhibition leading to cell cycle arrest or apoptosis. General features for binding shared between all compounds include a linear array of hydrophobic groups with polar atoms likely allowing for registration within the hormone pocket. Interestingly, two compounds showing antiproliferative activity share a cholesterol motif which may be related to LRH-1's function in bile acid and cholesterol homeostasis. Support: K12 HD07222, F32 CA163092, T32 DK007762

Sa1803 Dissecting the Oncogenic Activity of the Nuclear Receptor LRH-1 in the Colon

BACKGROUND. Colorectal cancers (CRC) account for nearly 10% of cancer deaths in industrialized countries. Recent evidence points to a central role for the nuclear receptor Liver Receptor Homolog-1 (LRH-1) in intestinal tumorigenesis. Interaction of LRH-1 with the Wnt/β-catenin pathway, highly active in a critical subpopulation of CRC cancer cells, underscores the importance of elucidating LRH-1's role in this disease. Reduction of LRH1 diminishes tumor burden in murine models of CRC. The central role of receptor LRH-1 in CRC pathogenesis and a wealth of structural data at atomic resolution make LRH-1 an attractive target. Methods: To evaluate the contributions of LRH-1 to CRC growth, we constructed stably transduced CRC cell lines expressing Tet-inducible shRNA directed against LRH-1. Cell growth of the LRH-1 knockdown lines was compared with growth of cells expressing a non-silencing control RNA. To explore alterations in gene expression due to LRH-1 suppression, we performed a microarray analysis of our knockdown cell lines. Additionally, we sought to develop and characterize specific, non-cytotoxic inhibitors of LRH-1 function for use as both potential therapeutic agents and more broadly as research tools to study organogenesis and tumorigenesis. We applied differential scanning fluorimetry (DSF), used to measure qualitative binding, to screen over 200 compounds for association with the LRH-1 ligand binding domain. Results: LRH-1mRNA knockdown results in impaired cell proliferation. Cluster analysis of microarray gene expression demonstrates significant alterations in signal transduction, bile acid and cholesterol metabolism, and control of apoptosis. Moreover, we discovered 9 unique compounds that interact with LRH-1, but not the closely related receptor SF-1. We have further demonstrated that four of these compounds have antiproliferative effects with IC50 values ranging from 8.6 to 30 μM. Conclusions: Silencing of LRH-1 expression in CRC cell lines leads to impaired cell growth, demonstrating that LRH-1 is a viable target in CRC. LRH-1 may exert its effects via multiple signaling networks, with inhibition leading to cell cycle arrest or apoptosis. General features for binding shared between all compounds include a linear array of hydrophobic groups with polar atoms likely allowing for registration within the hormone pocket. Interestingly, two compounds showing antiproliferative activity share a cholesterol motif which may be related to LRH-1's function in bile acid and cholesterol homeostasis. Support: K12 HD07222, F32 CA163092, T32 DK007762