The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation.

Human gene MAGE-1 encodes tumor-specific antigens that are recognized on melanoma cells by autologous cytolytic T lymphocytes. This gene is expressed in a significant proportion of tumors of various histological types, but not in normal tissues except male germ-line cells. We reported previously that reporter genes driven by the MAGE-1 promoter are active not only in the tumor cell lines that express MAGE-1 but also in those that do not. This suggests that the critical factor causing the activation of MAGE-1 in certain tumors is not the presence of the appropriate transcription factors. The two major MAGE-1 promoter elements have an Ets binding site, which contains a CpG dinucleotide. We report here that these CpG are demethylated in the tumor cell lines that express MAGE-1, and are methylated in those that do not express the gene. Methylation of these CpG inhibits the binding of transcription factors, as seen by mobility shift assay. Treatment with the demethylating agent 5-aza-2'-deoxycytidine activated gene MAGE-1 not only in tumor cell lines but also in primary fibroblasts. Finally, the overall level of CpG methylation was evaluated in 20 different tumor cell lines. It was inversely correlated with the expression of MAGE-1. We conclude that the activation of MAGE-1 in cancer cells is due to the demethylation of the promoter. This appears to be a consequence of a genome-wide demethylation process that occurs in many cancers and is correlated with tumor progression.

Antiangiogenic therapy of transgenic mice impairs de novo tumor growth.

Angiogenesis is activated during multistage tumorigenesis prior to the emergence of solid tumors. Using a transgenic mouse model, we have tested the proposition that treatment with angiogenesis inhibitors can inhibit the progression of tumorigenesis after the switch to the angiogenic phenotype. In this model, islet cell carcinomas develop from multifocal, hyperproliferative nodules that show the histological hallmarks of human carcinoma in situ. Mice were treated with a combination of the angiogenesis inhibitor AGM-1470 (TNP-470), the antibiotic minocycline, and interferon alpha/beta. The treatment regimen markedly attenuated tumor growth but did not prevent tumor formation; tumor volume was reduced to 11% and capillary density to 40% of controls. The proliferation index of tumor cells in treated and control mice was similar, whereas the apoptotic index was doubled in treated tumors. This study shows that de novo tumor progression can be restricted solely by antiangiogenic therapy. The results suggest that angiogenesis inhibitors represent a valid component of anticancer strategies aimed at progression from discrete stages of tumorigenesis and demonstrate that transgenic mouse models can be used to evaluate efficacy of candidate antiangiogenic agents.

Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase.

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in a variety of cellular functions, although its activation mechanism in vivo is not yet fully understood. To evaluate the signaling pathways involved in the activation of PKC epsilon upon stimulation by platelet-derived growth factor (PDGF) receptor (PDGFR), we used a series of PDGFR "add-back" mutants. Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3-kinase (PI 3-kinase) caused translocation of PKC epsilon from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase C gamma (PLC gamma), but not PI 3-kinase, also caused the PDGF-dependent translocation of PKC epsilon. The translocation of PKC epsilon upon stimulation of PDGFR (Y40/51) was inhibited by wortmannin, an inhibitor of PI 3-kinase. Activation of PKC epsilon was further confirmed in terms of PKC epsilon-dependent expression of a phorbol 12-tetradecanoate 13-acetate response element (TRE)-luciferase reporter. Further, purified PKC epsilon was activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-trisphosphate. These results clearly demonstrate that PKC epsilon is activated through redundant and independent signaling pathways which most likely involve PLC gamma or PI 3-kinase in vivo and that PKC epsilon is one of the downstream mediators of PI 3-kinase whose downstream targets remain to be identified.

Synergy between T-cell immunity and inhibition of paracrine stimulation causes tumor rejection.

During tumor progression, variants may arise that grow more vigorously. The fate of such variants depends upon the balance between aggressiveness of the variant and the strength of the host immunity. Although enhancing host immunity to cancer is a logical objective, eliminating host factors necessary for aggressive growth of the variant should also be considered. The present study illustrates this concept in the model of a spontaneously occurring, progressively growing variant of an ultraviolet light-induced tumor. The variant produces chemotactic factors that attract host leukocytes and is stimulated in vitro by defined growth factors that can be produced or induced by leukocytes. This study also shows that CD8+ T-cell immunity reduces the rate of tumor growth; however, the variant continues to grow and kills the host. Treatment with a monoclonal anti-granulocyte antibody that counteracts the infiltration of the tumor cell inoculum by non-T-cell leukocytes did not interfere with the CD8+ T-cell-mediated immune response but resulted in rejection of the tumor challenge, indicating a synergy between CD8+ T-cell-mediated immunity and the inhibition of paracrine stimulation.

Interações moleculares no mecanismo de ação da galectina-4 humana

A galectina-4 humana (HGal-4), pertencente à família das galectinas, possui dois domínios de reconhecimento de carboidratos (CRDs) com alta afinidade para β-galactosídeos e se encontra amplamente distribuída em células normais e neoplásicas de diferentes organismos. Suas funções snglobam uma grande variedade de eventos celulares, tais como processos inflamatórios, neoplásicos, progressão tumoral e metástase. Entretanto, muitas perguntas sobre suas interações com diferentes carboidratos, a especificidade destas interações e o papel específico das galectinas permanecem ainda sem resposta. No presente trabalho, propomos a investigação das interações galectina-glicano da galectina-4 humana e de seus domínios CRDs independentes (CRD-I e CRD-II) através de um conjunto de métodos biofísicos. Através do método de dicroísmo circular (CD), usando várias regiões espectrais, e fluorescência fomos capazes de entender mudanças ocorrentes na estrutura secundária e terciária das protéinas quando da interação com lactose/sacarose. Estes dados, juntamente com testes de hemaglutinação, mostraram que a glectina-4 e os CRDs respondem de forma distinta à ligação com açúcar. Por diferentes técnicas (fluorescência, ITC e MST) determinamos as constantes de dissociação para os domínios CRDs (Kd ~0,5 mM) e para HGal-4 e, de forma qualitativa, os valores obtidos indicaram possíveis estados oligoméricos dessas proteínas. A investigação da interação proteína-membrana da HGal-4 foi feita, primeiramente, com miméticos de membranas e monitorada pela técnica de RPE em crescente complexidade de composição de tais miméticos, indo desde composições mais simples, passando por lipid rafts na presença de diferentes glicolipídeos (GM1, LPS) e chegando-se à interação com células tumorais (U87MG, T98G e HT-29). Tais experimentos mostraram que galectina-4 reconhece e se liga naqueles modelos onde existem glicanos complexos na superfície. Investigamos também a participação de HGal-4 endógena e exógena no tratamento quimioterápico de células tumorais e verificamos um papel importante de HGal-4 para células HT-29. Finalizando esta tese, apresentamos o trabalho realizado em um ano de estágio na University of Oxford, durante o qual, investigamos a estrutura da região C-terminal de um receptor da família GPCR, qual seja o receptor de neurotensina NTS1. Aqui, mais uma vez, foi empregada a técnica de RPE que aliada à produção/marcação de mutantes do receptor, permitiu determinar que a hélice H8 se estabiliza quando em proteolipossomos.

Caracterização do envolvimento do gene RECKna proliferação celular e progressão tumoral: inversa correlação com a expressão do oncogene c-myc

Este trabalho mostra o envolvimento do gene RECK no processo de progressão do ciclo celular. Foi verificado que a expressão endógena de RECK é modulada durante a progressão do ciclo celular. A superexpressão de RECK em fibroblastos normais de camundongo promove uma diminuição da capacidade proliferativa das células e um retardo da transição das fases G0/G1-S do ciclo celular. Além disso, os resultados sugerem que um dos possíveis mecanismos de ação de RECK, que promovem este processo, envolve a indução da expressão de um inibidor de CDK, especificamente de p21, e retardo da fosforilação de pRb. Os resultados indicam, ainda, que durante a progressão do ciclo celular a expressão do gene RECK apresenta uma correlação inversa com a expressão do proto-oncogene c-myc. Estes dados corroboram os dados da literatura que mostram RECK como um alvo para o produto de diversos oncogenes, como ras e c-myc. A caracterização da repressão de RECK por c-Myc mostrou que a mesma ocorre ao nível transcricional e que sítios Sp1, presentes no promotor de RECK, são essenciais para a ação de Myc. Dados adicionais sugerem que a repressão de RECK por c-Myc parece envolver mecanismos de desacetilação de histonas. A modulação da expressão de RECK também foi avaliada durante a progressão maligna de tumores do sistema nervoso central (especificamente, gliomas). Foi verificado que a expressão de RECK não é alterada com a progressão deste tipo de tumor. Porém, foi verificado que os pacientes que manifestaram um maior tempo de sobrevida apresentaram tumores com uma significativa maior expressão do gene RECK. Estes dados sugerem que RECK possa ser um possível marcador prognóstico. A caracterização da regulação da expressão de RECK, tanto em células normais como em diferentes tipos de tumores, assim como os alvos moleculares da sua ação, são pontos muito importantes para o entendimento dos mecanismos que controlam a proliferação celular e podem contribuir para o desenvolvimento de novas formas de terapia anti-tumoral.

Anlaysis of complementary expression profiles following WT1 induction versus repression reveals the cholesterol/fatty acid synthetic pathways as a possible major target of WT1

The Wilms' tumour suppressor gene, WT1, encodes a zinc-finger protein that is mutated in Wilms' tumours and other malignancies. WT1 is one of the earliest genes expressed during kidney development. WT1 proteins can activate and repress putative target genes in vitro, although the in vivo relevance of such target genes often remains unverified. To better understand the role of WT1 in tumorigenesis and kidney development, we need to identify downstream target genes. In this study, we have expression pro. led human embryonic kidney 293 cells stably transfected to allow inducible WT1 expression and mouse mesonephric M15 cells transfected with a WT1 antisense construct to abolish endogenous expression of all WT1 isoforms to identify WT1-responsive genes. The complementary overlap between the two cell lines revealed a pronounced repression of genes involved in cholesterol biosynthesis by WT1. This pathway is transcriptionally regulated by the sterol responsive element-binding proteins (SREBPs). Here, we provide evidence that the C-terminal end of the WT1 protein can directly interact with SREBP, suggesting that WT1 may modify the transcriptional function of SREBPs via a direct protein-protein interaction. Therefore, the tumour suppressor activities of WT1 may be achieved by repressing the mevalonate pathway, thereby controlling cellular proliferation and promoting terminal differentiation.

Over-expression of Eph and ephrin genes in advanced ovarian cancer: ephrin gene expression correlates with shortened survival

Background: Increased expression of Eph receptor tyrosine kinases and their ephrin ligands has been implicated in tumor progression in a number of malignancies. This report describes aberrant expression of these genes in ovarian cancer, the commonest cause of death amongst gynaecological malignancies. Methods: Eph and ephrin expression was determined using quantitative real time RT-PCR. Correlation of gene expression was measured using Spearman's rho statistic. Survival was analysed using log-rank analysis and ( was visualised by) Kaplan-Meier survival curves. Results: Greater than 10 fold over-expression of EphA1 and a more modest over-expression of EphA2 were observed in partially overlapping subsets of tumors. Over-expression of EphA1 strongly correlated ( r = 0.801; p < 0.01) with the high affinity ligand ephrin A1. A similar trend was observed between EphA2 and ephrin A1 ( r = 0.387; p = 0.06). A striking correlation of both ephrin A1 and ephrin A5 expression with poor survival ( r = - 0.470; p = 0.02 and r = - 0.562; p < 0.01) was observed. Intriguingly, there was no correlation between survival and other clinical parameters or Eph expression. Conclusion: These data imply that increased levels of ephrins A1 and A5 in the presence of high expression of Ephs A1 and A2 lead to a more aggressive tumor phenotype. The known functions of Eph/ephrin signalling in cell de-adhesion and movement may explain the observed correlation of ephrin expression with poor prognosis.

Patched1 functions as a gatekeeper by promoting cell cycle progression

Mutations in the Hedgehog receptor, Patched 1 (Ptch1), have been linked to both familial and sporadic forms of basal cell carcinoma (BCC), leading to the hypothesis that loss of Ptch1 function is sufficient for tumor progression. By combining conditional knockout technology with the inducible activity of the Keratin6 promoter, we provide in vivo evidence that loss of Ptch1 function from the basal cell population of mouse skin is sufficient to induce rapid skin tumor formation, reminiscent of human BCC. Elimination of Ptch1 does not promote the nuclear translocation of beta-catenin and does not induce ectopic activation or expression of Notch pathway constituents. In the absence of Ptch1, however, a large proportion of basal cells exhibit nuclear accumulation of the cell cycle regulators cyclin D1 and B1. Collectively, our data suggest that Ptch1 likely functions as a tumor suppressor by inhibiting G(1)-S phase and G(2)-M phase cell cycle progression, and the rapid onset of tumor progression clearly indicates Ptch1 functions as a gatekeeper. In addition, we note the high frequency and rapid onset of tumors in this mouse model makes it an ideal system for testing therapeutic strategies, such as Patched pathway inhibitors.

Dramatic extension of tumor latency and correction of neurobehavioral phenotype in Atm-mutant mice with a nitroxide antioxidant

Mutations in the ATM gene (mutated in ataxia telangiectasia) in both humans and mice predispose to lymphoid tumors. A defect in this gene also causes neurodegeneration in humans and a less severe neurological phenotype in mice. There is some evidence that oxidative stress contributes to these defects, suggesting that antioxidants could alleviate the phenotype. We demonstrate here that the antioxidant 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO) dramatically delays the onset of thymic lymphomas in Atm(-/-) mice which is not due to an enhancement of apoptosis by CTMIO. We also show that this compound corrects neurobehavioral deficits in these mice and reduces oxidative damage to Purkinje cells. The likely mechanism of action of CTMIO is due to a reduction in oxidative stress, which is protective against both the tumor progression and the development of neurological abnormalities. These data suggest that antioxidant therapy has considerable potential in the management of ataxia telangiectasia and possibly other neurodegenerative disorders where oxidative stress is implicated. (c) 2006 Elsevier Inc. All rights reserved.

Matrix changes induced by transglutaminase 2 lead to inhibition of angiogenesis and tumor growth

Administration of active TG2 to two different in vitro angiogenesis assays resulted in the accumulation of a complex extracellular matrix (ECM) leading to the suppression of endothelial tube formation without causing cell death. Matrix accumulation was accompanied by a decreased rate of ECM turnover, with increased resistance to matrix metalloproteinase-1. Intratumor injection of TG2 into mice bearing CT26 colon carcinoma tumors demonstrated a reduction in tumor growth, and in some cases tumor regression. In TG2 knockout mice, tumor progression was increased and survival rate reduced compared to wild-type mice. In wild-type mice, an increased presence of TG2 was detectable in the host tissue around the tumor. Analysis of CT26 tumors injected with TG2 revealed fibrotic-like tissue containing increased collagen, TG2-mediated crosslink and reduced organized vasculature. TG2-mediated modulation of cell behavior via changes in the ECM may provide a new approach to solid tumor therapy.

A Model of Lung Tumor Angiogenesis in a Biomimetic Poly(ethylene glycol)-based Hydrogel System

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.

In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.

Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.

Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.

A bioavailable cathepsin S nitrile inhibitor abrogates tumor development

BACKGROUND: Cathepsin S has been implicated in a variety of malignancies with genetic ablation studies demonstrating a key role in tumor invasion and neo-angiogenesis. Thus, the application of cathepsin S inhibitors may have clinical utility in the treatment of cancer. In this investigation, we applied a cell-permeable dipeptidyl nitrile inhibitor of cathepsin S, originally developed to target cathepsin S in inflammatory diseases, in both in vitro and in vivo tumor models.

METHODS: Validation of cathepsin S selectivity was carried out by assaying fluorogenic substrate turnover using recombinant cathepsin protease. Complete kinetic analysis was carried out and true K i values calculated. Abrogation of tumour invasion using murine MC38 and human MCF7 cell lines were carried out in vitro using a transwell migration assay. Effect on endothelial tube formation was evaluated using primary HUVEC cells. The effect of inhibitor in vivo on MC38 and MCF7 tumor progression was evaluated using cells propagated in C57BL/6 and BALB/c mice respectively. Subsequent immunohistochemical staining of proliferation (Ki67) and apoptosis (TUNEL) was carried out on MCF7 tumors.

RESULTS: We confirmed that this inhibitor was able to selectively target cathepsin S over family members K, V, L and B. The inhibitor also significantly reduced MC38 and MCF7 cell invasion and furthermore, significantly reduced HUVEC endothelial tubule formation in vitro. In vivo analysis revealed that the compound could significantly reduce tumor volume in murine MC38 syngeneic and MCF7 xenograft models. Immunohistochemical analysis of MCF7 tumors revealed cathepsin S inhibitor treatment significantly reduced proliferation and increased apoptosis.

CONCLUSIONS: In summary, these results highlight the characterisation of this nitrile cathepsin S inhibitor using in vitro and in vivo tumor models, presenting a compound which may be used to further dissect the role of cathepsin S in cancer progression and may hold therapeutic potential.