Rozprawy doktorskie na temat „MEK/ERK Signaling”

Transkriptionelle Netzwerke (Transkriptionsfaktoren, epigenetische Modulatoren und spezifische Zielgene) stellen die unterste Ebene der zellinternen Signalübertragung dar. Eingebettet in verschiedene stimulusabhängige Signalwege bedienen sich ihre Komponenten genetischer und epigenetischer Mechanismen, um Zielgene transkrip-tionell zu regulieren und Veränderungen der Chromatinstruktur hervorzurufen. In der vorliegenden Arbeit wurde die hierarchische Organisation und Zusam-mensetzung des MEK-ERK-abhängig gesteuerten transkriptionellen Netzwerks und seine Veränderung im Zuge der HRAS-vermittelten onkogenen Transformation von HA1-Zellen untersucht. Viele Arbeiten haben sich bereits eingehend mit der Charak-terisierung einzelner Komponenten und Zielgene beschäftigt (Wagner et al. 2005, Reddy et al. 2002, Sun et al. 2006, Kapitel 1). Im Unterschied zu den zitierten Studien wurde in der vorliegenden Arbeit ein umfassendes Protokoll zur genomweiten De-chiffrierung transkriptioneller Netzwerke unter Kombination von experimentellen und bioinformatischen Methoden entwickelt und durchgeführt. Die Analyse ge-nomweiter Expressionsprofile un- und U0126-behandelter immortaler und HRASV12-transformierter humaner Nierenepithelzellen (HA1EB, HA1ER) erlaubte die Identifi-zierung von 138 auf- und 103 abregulierten genspezifischen IDs der RAS-ERK-abhängig gesteuerten Signalkaskade. Regulierte Transkriptionsfaktoren wurden i-dentifiziert und im Westernblot, sowie zum Teil mittels Durchflusszytometrie und RT-PCR validiert und nachfolgend transienten siRNA-Experimenten unterzogen. Für die Transkriptionsfaktoren ELK3, SRF und den hierarisch darunter liegenden Faktor FRA1 wurden Expressionsprofile der spezifischen siRNA-vermittelten Hemmung in beiden Zelllinien erstell und mit bioinformatischen Methoden (TRAP, GSEA-GO) a-nalysiert um direkte und indirekte sowie gemeinsame Zielgene zu ermitteln. Zusätz-lich wurde der Effekt auf phänotypischer Ebene (Softagar, MTT) überprüft. In der vorliegenden Arbeit ließ sich keine direkte Hierarchie der drei Transkrip-tionsfaktoren SRF, FRA1 und ELK3 bestätigen. Allerdings konnte zum ersten Mal eine gemeinsam regulierte Gruppe von Genen identifiziert werden, die darauf schließen lässt, dass die drei Transkriptionsfaktoren sowohl in HA1EB, als auch in HA1ER Teile eines gemeinsam regulierenden Netzwerks sind. Aus den Proliferationsexperimenten wurde zudem bestätigt, dass jeder Transkriptionsfaktor individuell eine essentielle Rolle bei der Promotion maligner Eigenschaften spielt. Für alle drei Transkriptionsfak-toren konnte eine RAS-abhängige starke Verschiebung der spezifisch angesteuerten Gene nachgewiesen werden. Diese Verschiebung wurde mittels TRAP und GSEA auch für alternative Regulatoren der spezifischen Zielgene festgestellt. Die nähere Analyse der FRA1-abhängigen Zielgene führte zu neuen Erkenntnis-sen zur Umordnung des Transkriptoms im Zuge der onkogenen Transformation. Die FRA1-spezifischen Zielgene in HA1EB und HA1ER weisen unterschiedliche Funktio-nalitäten auf. So wurden in HA1EB viele Gene identifiziert, die im Rahmen der Im-munantwort eine Rolle spielen und in HA1ER nicht reguliert werden. In den RAS-transformierten HA1ER konnten dagegen Gene identifiziert werden, die in der Tu-morprogression eine Rolle spielen (FRA1, STAT3, MTA1, TCFL5). Die Verifizierungen mittels qPCR und ChIP bestätigten 5 der 38 möglichen FRA1-Zielgene. Von diesen, FRA1, AEBP1, FRA1, TCFL5, NPAS2 und YWHAZ ist lediglich FRA1 bereits als FRA1-Zielgen beschrieben. Die Funktionen der neu identifizierten RAS-abhängigen FRA1-Zielgene untermauerten bereits bekannte Funktionen der FRA1-vermittelten Transkription (Differenzierung, Proliferation, zirkadiane Rhythmen, Apoptose) und erweitern sie um verschiedene Aspekte wie Metabolismus und Rückkopplungen in die Signaltransduktion, die noch nicht für die RAS-abhängige FRA1-vermittelte Transktiption beschrieben worden sind. Dazu gehören unter anderem Interaktionen mit TGFbeta, WNT, JAK/STAT und JNK. Daneben sind in den HA1ER eine Vielzahl von Regulatoren des RHO-Signalwegs identifiziert worden, was für FRA1 auf bisher unbekannte Interaktionen mit RAC/RHO-Signalwegen schließen lässt.
Transcriptional networks represent the final level of internal signal transmission. They are embedded in different signalling pathways and use genetic as well as epi-genetic mechanisms to regulate their according target genes. During oncogenic trans-formation they are undergoing massive rearrangements in composition, regulation and interaction. This leads to radical changes in the transcriptome and drives the on-cogenic phenotype of the according cells. My thesis employs the composition of the MEK-ERK-dependent transcriptional net-work and its alteration during the HRAS-oncogene-mediated transformation in HA1-cells. By commencing from already known components: SRF, Ternary Complex Fac-tors (TCF: SAP1, SAP2/ELK3, ELK1) and members of the AP1-complex (JUN, FOS-proteins) I analyzed the alteration in expression of secondary targets and their inter-action as well as their relation to the superior factors. Therefore I compared genome wide expression profiles (Affymetrix, HG-U133A) of immortal HA1EB and HRASV12-oncogene-transformed HA1ER-cells with and without U0126-induced MEK/ERK-inhibition and extracted several MEK/ERK-dependent transcription factors. Among them where FRA1 and ELK3, two transcription factors already known to be involved in oncogenesis and proliferation associated processes. ELK3 needs SRF as crucial binding partner to function. Therefor I also included SRF into the subsequent analysis. The three transcription factors function in different time-dependent hierarchy states so we supposed a putative hierachical network be-tween them. I established transient knockdown cells deriving from HA1EB and HA1ER for all three transcription factors and generated further expression profiles from them. Additionally I verified the importance of these transcription factors on survival and proliferation via MTT and Softagar experiments. Using different statis-tically and bioinformatical methods (GSEA, TRAP) in collaboration with the Max-Planck-Institute for molecular Genetics Berlin, several direct and indirect targets of these transcription factors were predicted. These were partially overlapping in all transcription factors. Also, in comparison of the immortal and the transformed cell line, a shift of functionalities and composition of the different target gene populations and collaborating factors could be detected for all three transcription factors. It was found that in HA1EB FRA1 seems more likely to regulate immunresponsive genes as well as genes associated with the cytoskeleton and nucleus organisation whereas in HA1ER FRA1 regulates a large group of transcription- and signalling-associated genes. Additionally it could be shown that in both cell lines FRA1 regulates genes in-volved in epigenetic processes as well as circadian rhythms which are known to be important aspects in oncogenic transformation. I verified 37 different putative target genes of FRA1 using qRT-PCR (Taqman) and partially also ChIP-analysis. Of these 37genes, 5 were fully validated as directly regu-lated targets of FRA1: FRA1, AEBP1, YWHAZ, NPAS2 and TCFL5. They imply functionalities connected to proliferation and differentiation (AEBP1, FRA1, TCFL5) as well as apoptosis (YWHAZ) cell cycle control and circadian rhythm (NPAS2, AEBP1), feedbacks into the signalling (YWHAZ, AEBP1) and metabolism (NPAS2, AEBP1). Summarised the work of this thesis contributes to the decipherment of the direct and indirect targets of the according transcription factors and strengthens the argument of a general and massive shift of the transcriptional network during oncogenic trans-formation of cells. The importance of all three transcription factors on the survival of genes could be proved via proliferation assays. Additionally the functionality of their according targets could be integrated into processes connected to oncogenic trans-formation.

In the present study, we designed a new chemical template that contains an oxindole moiety as potential dual-pathway inhibitors of the Raf/MEK/ERK and PI3K/Akt signaling pathways. The design hypothesis is to evaluate whether the oxindole ring system will approximately orient functional groups in a similar manner to the thiazolidinedione moiety, and thus maintain biological activity as dual-pathway inhibitors of the Raf/MEK/ERK and PI3K/Akt signaling pathways. Furthermore, the oxindole ring will provide the flexibility to allow the introduction of various substituents on the oxindole moiety, thereby facilitating comprehensive SAR studies to further explore the biological activity.

Master of Science
Biochemistry and Molecular Biophysics Interdepartmental Program
Anna Zolkiewska
Mitogen-activated protein kinase (MAPK) signaling plays an important role in the proliferation, survival, and therapy resistance of breast cancer cells. Two important protein kinases involved in the MAPK pathway are MEK and ERK. The MEK/ERK signaling cascade can be stimulated by activation of the epidermal growth factor receptor (EGFR) upon binding of EGF-like ligands, which are released from cells by ADAM proteases. EGFR is frequently overexpressed in triple-negative breast cancer (TNBC), a particularly aggressive form of breast cancer. Our analysis of clinical data revealed that high expression of ADAM12, but not other ADAMs, in TNBC is associated with poor patient survival. Thus, we hypothesized that ADAM12 plays a critical role in the progression of TNBC, possibly by stimulating MEK/ERK activity in an EGFR-dependent manner. To test this hypothesis, ADAM12 was knocked-down (KD) in SUM159PT TNBC cells, which express high levels of the endogenous ADAM12 protein. An antibody array assay indicated a significant decrease in the activation of the MAPK pathway in SUM159PT cells after ADAM12 KD. The decrease in MAPK activity was further confirmed by Western blotting using phospho-MEK and phospho-ERK specific antibodies. Additionally, conditioned media from ADAM12-deficient SUM159PT cells failed to support the survival of MCF10A cells, suggesting that ADAM12 KD reduced the release of pro-survival growth factors from SUM159PT cells. Based upon this data, we propose that ADAM12 is a novel regulator of the MAPK pathway and a potential therapeutic target in breast cancer.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
A via de sinalização da cinase regulada por fatores extracelulares, da família das proteínas cinases ativadas por mitógenos (MAPK/ERK) é importante tanto para a sobrevivência como para a progressão da diferenciação de oligodendrócitos. Neste trabalho, a via da MAPK/ERK foi avaliada na oligodendroglia in vitro com a utilização de inibidores da MEK. A morfologia celular, assim como a distribuição de proteínas foram analisadas em diferentes estágios de maturação da oligodendroglia. Culturas primárias de oligodendrócitos foram tratadas com os inibidores da MEK PD98059 ou U0126, aos 5 ou 11dias in vitro (div), por 30min, 24 ou 48h. A oligodendroglia foi distinguida com marcadores estágio-específicos: A2B5, 23nucleotídeo cíclico 3 fosfodiesterase (CNPase) e proteína básica de mielina (MBP), e classificada de acordo com sua morfologia em diferentes estágios de desenvolvimento. O tratamento aumentou significativamente o número de células com morfologia mais imatura e diminuiu o número de células maduras. Além disso, aumentou o número de células redondas e sem prolongamentos as quais não puderam ser classificadas em nenhum dos estágios de desenvolvimento da oligodendroglia. Os efeitos mais evidentes foram observados logo após o menor tempo de tratamento. Células redondas eram positivas para CNPase e MBP, porém não foram marcadas com A2B5 ou com NG2, indicando que seriam células maduras incapazes de estender ou manter seus prolongamentos. De fato, estas mudanças foram acompanhadas por alterações na distribuição de proteínas de oligodendrócitos como a MBP e a CNPase, assim como alterações em proteínas de citoesqueleto, como actina, tubulina e na cinase de adesão focal (FAK). A MBP foi observada nas células tratadas em um padrão de distribuição desorganizado e disperso, oposto ao padrão contínuo que é observado nas células das culturas controle. Além disso, o tratamento causou uma desorganização na distribuição da CNPase, actina e tubulina. Nas células das culturas controle, estas proteínas apresentam um padrão organizado compondo as estruturas de citoqueleto semelhantes a nervuras. Após um pequeno período de tratamento (30min), actina e tubulina apresentaram o mesmo padrão de marcação puntiforme que a CNPase apresentou. O tratamento também reduziu os pontos de adesão focal demonstrados pela FAK. Com o decorrer do tratamento, após 24 e 48h, actina e tubulina aparentavam estar se reorganizando em um padrão filamentar. Estes resultados indicam um efeito importante da via da MAPK/ERK na ramificação e alongamento dos prolongamentos dos oligodendrócitos, com possíveis consequências para a formação da bainha de mielina.
The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is important for both long-term survival and timing of the progression of oligodendrocyte differentiation. In this work, the MAPK/ERK signaling in oligodendroglia was studied in vitro by using MEK inhibitors. Cell morphology and distribution of proteins were analyzed in different stages of maturation. Primary cultures of oligodendroglia were treated with the MEK inhibitors PD98059 or U0126, at 5 or 11div for 30min, 24 or 48h. Oligodendroglial cells were distinguished by using stage specific markers: NG2 proteoglycan, A2B5, 23nucleotide-cyclic 3phosphodiesterase (CNPase) and myelin basic protein (MBP), and classified according to their morphology into different developmental stages. Treatment significantly increased the number of cells with more immature morphologies and decreased the number of mature cells. Furthermore, it increased the number of rounded cells that could not be classified into any of the oligodendroglial developmental stages. The strongest effects were usually observed shortly after treatment. Rounded cells were CNPase/MBP positive and they were not stained by anti-NG2 or A2B5, indicating that they were mature cells unable either to extend and/or to maintain their processes. In fact, these changes were accompanied by alterations in the distribution of the oligodendroglial proteins MBP and CNPase, and alterations in cytoskeleton proteins, as actin, tubulin and the focal adhesion kinase (FAK). MBP was observed in a continuous distribution in cell body and processes in control cultures. Furthermore, in treated cultures a disorganized pattern of distribution of CNPase, actin and tubulin was observed. In control cultures, these proteins compose the cytoskeleton vein-like structures. By the other side, after a short time of MEK inhibition (30min), actin and tubulin showed the same punctual pattern observed in CNPase distribution. Treatment also caused a reduction of focal adhesion sites showed by FAK. As treatment progressed, after 24 and 48h, actin and tubulin seemed to be rearranged into a filament-like pattern. These data showed an effect of the MAPK/ERK pathway on oligodendroglial branching, with possible consequences for the formation of the myelin sheath.

In vielen humanen Neoplasien findet sich eine erhöhte Aktivität des Raf-Mek-Erk-Signaltransduktionsweges. Zunächst wurde davon ausgegangen, dass diese erhöhte Aktivität hauptsächlich durch die Ras-Onkogene hervorgerufen wird. Doch mittlerweile konnten auch Mutationen der Raf Gene in humanen Neoplasien nachgewiesen werden. Gegen Raf und Mek konnten eine Anzahl von Enzyminhibitoren entwickelt werden. Der Nachteil vieler dieser Inhibitoren ist, dass sie nicht zwischen den einzelnen Kinaseisoformen unterscheiden können. In dieser Arbeit ist es nun das erste Mal gelungen, jede Komponente des Raf-Mek-Erk-Signaltransduktionsweges einzeln mittels RNA Interferenz effizient zu inhibieren. Dabei konnte die Rolle der verschiedenen Isoformen in der Proliferation, Morphologie und Genex-pression von transformierten Zellen definiert werden. In den NIH3T3-pEJ Zellen konnte A-Raf erstmals eine antiapoptotische Rolle zugewiesen werden. Diese Hemmung der Apoptose läuft möglicherweise über einen Mek2-abhängigen Weg und ist an die Mitochondrien gekoppelt. Für die beiden Mek Kinasen konnten unter-schiedliche Funktionen in der Signalweiterleitung gezeigt werden. Mek2 spielt die Hauptrolle in der Aktivierung der beiden Substratkinasen Erk1 und Erk2. Der Verlust der Mek1 Isoform wird dagegen möglicherweise durch eine erhöhte Expression von Mek2 kompensiert und wirkt sich nicht so stark auf die Phosphorylierung von Erk1/2 aus. Durch die Verwendung von Erk1 und Erk2 spezifischen siRNAs konnte eine Trennung zwischen der Proliferationsre-gulation und der Kontrolle der morphologischen Transformation herausgearbeitet werden. Durch die Verwendung von Mikroarrays ist es gelungen, beiden Phänotypen ein Genexpres-sionsprofil zuzuordnen. Neben Unterschieden zwischen den verschiedenen Kinaseisoformen konnten neue, potentielle Feedbacks beschrieben werden.
In many human neoplasia an increased activity of the RAF/MEK/ERK- signaling pathway is found. First it was assumed that this raised activity is caused primarily by the RAS onco-genes. However, meanwhile mutations in the RAF genes could be also proved in human neo-plasia. A number of enzyme inhibitors have been developed against the RAF and MEK pro-teins. The disadvantage of many of these inhibitors is that they cannot distinguish between the different kinase isoforms. In this work it has succeeded the first time to inhibit every compo-nent of the RAF/MEK/ERK- signaling pathway individually by means of interference RNA. Beside this, the role of the different isoforms in the proliferation, morphology and genetic profile of transformed cells could be defined. For the first time A-Raf could be assigned an anti-apoptotic role in NIH3T3-pEJ cells. This inhibition of the apoptosis possibly runs through a Mek2-dependent way and is coupled to the mitochondria. For both Mek kinases different functions could be shown in the downstream signaling. Mek2 plays the leading role in the activation of both downstream kinases Erk1 and Erk2. The loss of the Mek1 isoform expression is possibly compensated through an increased expression of Mek2 and does not affect the phosphorylation of Erk1 / 2 so strongly. A discri-mination between the regulation of proliferation and the control of the morphological trans-formation could be worked out by the use of Erk1 and Erk2 specific siRNAs. By the use of micorarray an expression profile of both phenotypes has assigned. Beside differences between the different kinases new, potential feedback pathways could be described.

Neutrophils form a first line of defence against infections. These short-lived, terminally differentiated cells perform many important functions, including chemotaxis, degranulation, reactive oxygen species (ROS) release and cytokine production. Whilst neutrophils are essential for host immunity, their inappropriate recruitment, activation and/or removal can contribute to excessive inflammation and host damage, as exemplified in autoimmune diseases such as rheumatoid arthritis. It is therefore essential that neutrophil function is tightly regulated. Neutrophils are activated by a range of stimuli, including immune complexes. Neutrophil functions are tightly regulated by intracellular signalling events that are induced by the ligation of cell surface receptors, for example, the binding of immune complexes to Fc receptors. Phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (Erk) are key signalling intermediates that act downstream of many cell surface receptors. They are involved in the regulation of numerous biological processes in the neutrophil. Using pharmacological interventions, I analysed PI3K signalling in immune complex-stimulated human neutrophils and uncovered a previously uncharacterised, noncanonical signalling pathway, PI3K-Cdc42-Pak-Mek-Erk. This represents an unusual situation where Pak acts as the MAP3K downstream of Cdc42 in a PI3K-dependent fashion. By performing a range of functional experiments, I showed that this unconventional signalling pathway promotes apoptosis in human neutrophils by regulating the ratio between anti- and pro-apoptotic members of the Bcl-2 family proteins. No other immune complex-induced, PI3K-dependent neutrophil function tested depended on PI3K-Cdc42-Pak-Mek-Erk signalling. Mouse knock-outs of all components of this signalling pathway have been described. Immune complex-induced apoptosis was also PI3K-dependent in mouse neutrophils, but experiments performed with inhibitors showed that, in contrast to human neutrophils, this was not dependent on PI3K-Cdc42-Pak-Mek-Erk signalling. The myeloid leukaemia cell line, PLB-985 is amenable to knock-down and can be differentiated to become neutrophil-like. These cells are not notably activated by immune complexes, perhaps because they do not express the major Fcγ receptor, CD16. Since retroviral expression of CD16 in PLB985 cells did not improve their response to activation by immune complexes, I was not able to confirm my observations with human neutrophils genetically. Collectively, I showed that a novel, pro-apoptotic signalling pathway operates downstream of Fcγ receptors in the human neutrophil. The fact that this signalling pathway appears to regulate apoptosis specifically suggests uncoupling pro- and anti-inflammatory effects induced by immune complexes might be possible. This may be helpful in the design of improved therapies of autoimmune diseases such as rheumatoid arthritis, in which immune complex-driven neutrophilic inflammation contributes to disease pathogenesis and where neutrophil apoptosis is disturbed.

El cáncer de páncreas es una de las enfermedades más letales, siendo la cuarta causa de muerte por cáncer en países occidentales. Con una supervivencia global a los 5 años de alrededor del 5%, el tratamiento estándar actual consiste en quimioterapia, y la amplia mayoría de terapias dirigidas que se han probado hasta ahora no han tenido éxito. Por ello, es necesario el desarrollo de nuevas terapias contra componentes clave de esta enfermedad, así como que combatan los mecanismos de resistencia. Alrededor del 90% de los cánceres de páncreas contienen mutaciones en K-Ras, clave para el desarrollo de esta enfermedad. K-Ras es una GTPasa que transduce señales iniciadas en receptores tirosina quinasa (RTKs) y controla proliferación celular y supervivencia, entre otras funciones. A pesar del desarrollo de inhibidores específicos de K-Ras durante las últimas décadas, ninguno ha conseguido demostrar eficacia en la clínica. Como consecuencia, los esfuerzos se han dirigido a inhibir a las proteínas reguladas por K-Ras, como las Raf o las Mek. Sin embargo, a pesar de que los inhibidores de B-Raf y Mek1/2 han demostrado eficacia clínica en melanoma, en cáncer de páncreas no se ha observado tal eficacia. En esta tesis, probamos la eficacia de la inhibición de Mek1/2 en modelos de cáncer de páncreas, en combinación con quimioterapia, utilizando líneas celulares y xenoinjertos derivados de pacientes con cáncer páncreas (PC-PDXs). Sin embargo, demostramos que este efecto se ve limitado por la aparición de clones resistentes, que resultan como consecuencia del alto grado de heterogeneidad de estos tumores. En este trabajo también se muestra que, cuando las células de cáncer de páncreas adquieren resistencia a la inhibición de Mek1/2, aumentan la expresión del factor de transcripción Slug. Este factor de transcripción induce resistencia, con un mecanismo de acción que consiste en desacoplar la división celular del control de la ruta de señalización Raf-Mek-Erk. Además, la expresión de Slug correlaciona con resistencia en células de cáncer de páncreas y melanoma. Asimismo, también mostramos que Slug controla la capacidad metastásica de las células de resistentes y es un indicador de mal pronóstico en pacientes con cáncer de páncreas y melanoma.
Pancreatic cancer is one of most lethal human diseases, being the fourth leading cause of cancer-related deaths in western countries. With a 5-year overall survival of around 5%, the development of novel therapies is needed. The current standard treatment for this disease consists of chemotherapy, and many targeted therapies have failed to improve patients’ survival so far. For this reason, the development of novel therapies targeting key components of this cancer as well as the study of resistance becomes necessary. 90% of pancreatic cancers are mutated in the GTPase K-Ras, required for the development of this disease. K-Ras is a GTPase that transduces signals from tyrosine kinase receptors (RTKs) and controls cell proliferation and survival. Despite the initial approaches for pharmacologically inhibiting K-Ras started more than twenty years ago, none of the inhibitors has succeed in the clinics so far. As K-Ras is still considered ‘undruggable’, the efforts have been focused on targeting its downstream effectors, as the Raf and Mek proteins. Despite B-Raf and Mek1/2 inhibitors have improved patients’ survival in melanoma, they have failed to succeed in pancreatic cancer patients. In this work, we assess the efficacy of Mek1/2 inhibition against pancreatic cancer. Using cell lines and pancreatic cancer-derived xenografts (PC-PDXs), we gauged the efficacy of the Mek1/2 inhibitor MEK162 in addition to the standard chemotherapy and we show that it impairs growth in vitro and in vivo. However, we also demonstrate that the effectiveness of Mek1/2 inhibition is limited by the emergence of resistant clonal populations, that result from the high degree of tumor heterogeneity and they compromise the effectiveness of this treatment. In this thesis, we also demonstrate that, when pancreatic cancer cells acquire resistance to Mek1/2 inhibition they increase the expression of the EMT zinc finger transcription factor Slug. This transcription factor regulates resistance to Mek1/2 inhibitors in pancreatic cancer cells by uncoupling the regulation of cell division by the Mek1/2-Erk1/2 pathway, and it correlates with resistance in a panel of pancreatic cancer and melanoma cell lines. Likewise, Slug is responsible for the enhanced metastatic ability of resistant cells, and it consistently correlates with poor survival in pancreatic cancer and melanoma patients.

Extracellular signal-regulated protein kinase 5 (ERK5) is a member of the mitogen activated protein (MAP) kinase family which is specifically activated by mitogen/extracellular signal regulated kinase kinase-5 (MEK5). Over recent years, abnormal MEK5/ERK5 signalling has been shown to be important in prostate carcinogenesis with increased levels of ERK5 immunoreactivity being associated with Gleason sum score (p<0.0001), bone metastases (p=0.0044) and locally advanced disease at diagnosis (p=0.0023). In addition PC3 cells over-expressing ERK5 displayed enhanced proliferation, migration and invasion. Taken together, these data suggest MEK5/ERK5 pathway to be biological important in prostate cancer and a potential target in invasive prostate cancer. Using siRNA to target ERK5 expression, I found that reduced ERK5 expression significantly inhibited cellular proliferation, motility and invasion in prostate cancer PC3 cells when compared to the controls, (p<0.005). Our group has previously reported upregulated ERK5 expression in primary human prostate cancer specimens. In this study, I was able to validate these results and demonstrate moderate-strong levels of cytoplasmic staining in 63% cases of PIN/PIA. High levels of cytoplasmic (55%) and nucleur (73%) immunoreactivity was also shown in a range of metastatic prostate tumours (n=11). A number of similarities and interactions between ERK5 and ERK1/2 have recently been identified and there is suggestion that ERK5 may in fact regulate some of the cellular functions originally attributed to ERK1/2. Potential ‘cross-talk’ between ERK5 and ERK1/2 signalling was investigated using siRNA for each individual isoform of ERK1/2. ERK1 knockdown resulted in increased ERK5 activation in addition to prolonged ERK2 phosphorylation. Proliferation studies were also performed in PC3 cells, the results of which support published data that ERK1 acts as a negative regulator and ERK2 as a positive regulator of cell proliferation. ERK5 has been shown to regulate the activity of several transcription factors and recent evidence suggests that ERK5 may be heat shock protein (HSP) 90 dependent. To further investigate the ERK5 signalling network and its interacting proteins, I performed mass spectrometry-based quantitative proteomics using SILAC labelled cells. Results from this study support the theory that HSP90 does associate with ERK5 however contrary to published data my results show that it is not involved in ERK5 activation. Our results validate the importance of the MEK5-ERK5 signalling pathway as a potential target for therapy in prostate cancer and highlight a novel functional and biochemical relationship between ERK1 and HSP 90 with ERK5 signalling.

碩士
國立臺灣大學
臨床牙醫學研究所
103
Aim : Basic fibroblast growth factor (bFGF) owns multiple biological functions in various tissues, and plays important roles in cell proliferation and differentiation. Human apical papilla cells have been reported to show characteristics of stem cells and are known as stem cells from apical papilla (SCAP). The purpose of this study is to investigate the effects of bFGF on apical papilla cells and the roles of MEK/ERK signaling. We hypothesize that bFGF regulates cell proliferation and differentiation through MEK/ERK. Materials and Methods : Primary-cultured human apical papilla cells were treated under different concentration with or without U0126 (an inhibitor of MEK/ERK). Cell proliferation was measured by MTT assay. The expressions of cell cycle-related and differentiation-related genes and proteins were examined by reverse transcription polymerase chain reaction (RT-PCR) and western blot, respectively. Phosphorylation of signaling molecules was examined by western blot. ALP activity was determined by ALP staining. FGF receptors (FGFRs) were detected by immunofluorescent (IF). Results : In human apical papilla cells, treatment of bFGF (10~500 ng/ml) enhanced the proliferation and the expression of cell cycle-related genes and proteins including cyclin B1, cdc2, and cdc25c. In osteogenic/ dentinogenic differentiation, bFGF promoted the expression of Runx2 and osteocalcin, but ALP activity was suppressed by treatment of bFGF for 5 days. FGFR1, 2, 3 and 4 were expressed abundantly in apical papilla cells. Using U0126 solely decreased the inherent proliferative ability in apical papilla cells, and combined with bFGF inhibited the bFGF-induced enhancement of proliferation and expression of cell cycle-related genes and proteins. The increase of osteocalcin and TIMP1 induced by bFGF was repressed by U0126, while Runx2 and ALP were not changed. Besides, ALP activity attenuated by bFGF could not be reversed by U0126. Conclusion : The effect of bFGF on apical papilla cells is complicated and might be divergent depending on the duration of treatment. MEK/ERK pathway plays important roles in miscellaneous cell functions. These results would be useful for clinical therapies in the future, including apexogenesis and pulp-dentin complex regeneration.

碩士
長榮大學
醫學研究所
97
The uncontrolled hyperglycemia of maternal diabetes has been found to negatively affect pregnancy and can result renal structure malformations. Histologically, cell apoptosis was significantly increased in fetal kidneys of diabetic group. Glial cell line-derived neurotrophic factor (GDNF) expression was down-regulated at peritubular region of early developing fetal kidney of diabetic group. GDNF binds to GDNF receptor (GFRα-1) and Ret. Phosphorylation of the tyrosine kinase receptor Ret, induced a signal cascade, lead to gene transcription. Advanced glycation end-products (AGE) were found in hyperglycemic condition. The aim of this study was to examine whether AGEs directly affect GDNF function in rat renal tubular epithelial cell line (NRK-52E). Diabetes mellitus mice induced by Streptozotocin as an animal model. Gene expressions of embryonic kidneys were quantified by Q-PCR. NRK-52E was used as target. AGE was prepared as originally described previously. NRK-52E apoptosis was determined by flow cytometer. Expression RAGE, GFRα-1, EGR-1, phosphorylated MEK and ERK of NRK-52E cells were quantified by Q-PCR and Western blot. The results showed that AGE up-regulated RAGE and EGR-1 expression on NRK-52E cells. RAGE and EGR-1 expression were increased at mRNA level in a time-dependent manner and peaked at 24 hrs and 48hr respectively. GDNF bind to GFRα-1 can induce NRK-52E cells apoptosis. AGE co-incubation with GDNF promoted NRK-52E cells apoptosis. This effect was via MEK/ERK pathway and could be significantly inhibited by ERK inhibitor. In conclusion, AGE might be promote diabetic embryopathy by up-regulating NRK-52E cells RAGE expression and enhancing NRK-52E cells apoptosis via MEK/ERK pathway.

碩士
國立陽明大學
微生物及免疫學研究所
101
Enterovirus 71 (EV71) belongs to the enterovirus genus within the Picornaviridae family. EV71 infection is generally mild or asymptomatic, and manifests most frequently as hand, foot, and mouth disease (HFMD). However, in some cases, the infection of EV71 may cause severe neurological disease, pulmonary edema and death, especially in children under 5 years old. At present, the cellular factors involved in the life cycle of EV71 are largely unknown. In this study, we construct an EV71 replicon to produce the recombinant virus and generate anti-sera against viral non-structural proteins, for the purpose of screening novel cellular factors involved in the life cycle of EV71. The EV71 replicon was constructed by placing a firefly luciferase gene in the capsid protein-coding region. To establish a packaging cell line expressing viral capsid proteins, we used a lentiviral vector containing the gene to infect 293T cell and RD cell. The EV71 replicon RNA was then transfected into the packaging cell line to generate recombinant virues. Using the recombinant virus system, we have identified Phosphatidylinositol-4 kinases (PI4KB) and Valosin-containing protein (VCP), which have been known previously to be essential for viral replication, and some other novel candidates which may participate in EV71 replication. In addition, we observed that EV71 infection induced an early activation of a tyrosine kinase receptor, insulin-like growth factor receptor (IGF1R), and the phosphatidylinositol 3-kinase (PI3K)/Akt and the Raf/MEK/ERK signaling pathways. Inhibition of IGF1R, PI3K, or ERK with AG1024, LY294002, or U0126, respectively severely impaired EV71 replication, suggesting that these signaling pathways may be important to EV71 replication. On the other hand, dephosphorylation of mTOR and 70S6K was observed at early stage of viral infection, and mTOR inhibitors significantly enhanced EV71 replication. These results suggest that mTOR may play a negative role in EV71 replication. In conclusion, we have identified several novel cellular factors which probably participate in EV71 replication. Our preliminary evidence also suggests that IGF1R, PI3K/Akt and Raf/MEK/ERK signaling pathways may play important role in the life cycle of EV71. Further studies to determine the underlying mechanisms of these molecules and the signaling pathways are warranted.

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates a plethora of functions including cell growth, development, and differentiation. TGF-β plays a major role in the inhibition of normal cell growth and proliferation. Quite dichotomously, at a late stage of cancer progression cells become refractory to the cytostatic actions of TGF-β, and in turn start to proliferate, invade, and metastasize beyond their tissue of origin. Acquisition of metastatic phenotypes by cancer cells is critically dependent upon their ability to undergo epithelial-mesenchymal transition (EMT). Pathophysiologic ally, EMT is a critical step in mediating the acquisition of metastatic phenotypes by localized carcinomas. TGF-β is a potent inducer of these two lethal aspects of cancer mainly EMT and metastasis. However, it is not very clear how TGF-β suppresses growth in normal epithelial cells and promotes metastatic processes like EMT in their malignant counterparts. TGF-β is known to mediate its function by binding to its cognate serine/threonine kinase receptor and activate downstream signaling mediated by the canonical Smad dependent as well as non-canonical Smad independent pathways. Through the activation of these pathways, TGF-β regulates the expression of several target genes either positively or negatively in a cell type and context dependent manner. TGF-β induced both canonical and non-canonical signaling has been implicated in mediating its pro-tumorigenic functions. However, there is no clear understanding of the mechanism (s) responsible for the differential responses of various cell types to TGF-β. Therefore, more studies are warranted to understand how TGF-β regulates differential gene expression to induce EMT and metastasis in cancer. This understanding will enable the development of novel targeted therapies, thereby improving the clinical course of cancer patients. Over the past few years, several independent transcriptome analyses revealed cell type dependent differential regulation of genes in response to TGF-β. A previous transcriptome analysis data from our laboratory identified several novel genes regulated by TGF-β in normal and transformed cell types including S100A2. S100A2 belongs to the S100 family of EF hand calcium binding proteins, which are heavily implicated in the progression of cancer. Differential expression of S100A2 has been reported in several cancers like breast, lung, gastric, ovarian, prostrate, endometrial, and pancreatic cancer. Over expression of S100A2 in lung and pancreatic cancer has been correlated with poor prognosis and recently, S100A2 has been reported to induce metastasis in the xenograft murine model of non-small cell lung cancer (NSCLC). However, the exact role of S100A2 in tumorigenesis remains elusive. Therefore, in view of the S100A2 and TGF-β involvement in the complex process of metastasis and tumor development, the present study is designed to understand the mechanism of S100A2 gene regulation by TGF-β and its function (s) with respect to tumor progression. The following sections highlight the mechanistic aspects of S100A2 regulation and function. • Characterization of S100A2 regulation by TGF-β induced signaling pathways In order to investigate the mechanism of S100A2 gene regulation by TGF-β, normal and transformed cell types of different tissue origins were treated with TGF-β and the levels of S100A2 transcript and protein were examined. The S100A2 mRNA expression increased as early as 1h and persisted until 24h, post TGF-β treatment. Maximal stimulation was observed at 6h after TGF-β treatment in immortalized HaCaT cell line. Similar regulation of S100A2 mRNA was observed in Hep3B and MDAMB-231 cells in response to TGF-β. In addition, increase in S100A2 mRNA and protein expression by TGF-β was completely blocked in the presence of actinomycin D, suggesting S100A2 as a transcriptional target of TGF-β in epithelial cells. To delineate the transcriptional regulation of S100A2 expression by TGF-β, we characterized 2.1kb 5’ flanking region of S100A2 gene. Treatment of cells with TGF-β induced S100A2 promoter activity in all the above-mentioned cell lines. In silico analysis revealed potential transcription factor binding sites, including one Smad binding Element (SBE), three each of AP-1 and p53 binding sites amongst others in the 2.1kb region of S100A2 promoter. Characterization of the promoter revealed activator protein-1(AP-1) element at -1161 to -1151 as most critical for TGF-β1 response. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) confirmed the functional binding of AP-1 complex, predominantly JunB, to S100A2 promoter in response to TGF-β1. JunB over expression markedly stimulated S100A2 promoter which was blocked by dominant-negative JunB and MEK1 inhibitor, PD98059 suggesting involvement of TGF-β induced MEK/ERK signaling in this regulation. Intriguingly, despite the presence of a putative SBE, S100A2 regulation by TGF-β1 was found to be independent of SBE element. This was confirmed by absence of Smad protein complex binding on the SBE element. Further, promoter deletion analysis and blocking Smad3 signaling had no effect on the induction of S100A2 mRNA by TGF-β1. Most importantly, our study demonstrates a unique synergistic regulation of S100A2 promoter by p53 and TGF-β induced MEK/ERK signaling, without direct involvement of Smads. Collectively, our findings demonstrate S100A2 gene as a direct transcriptional target of TGF-β in epithelial cells. TGF-β mediated regulation of S100A2 is dependent on the AP-1 binding to its consensus site in the promoter under the influence of MEK/ERK signaling. Further, our study demonstrates a novel interplay between TGF-β induced MEK/ERK and p53 signaling in the synergism of S100A2 promoter. Functional significance of S100A2 over expression in cancer S100A2 is an over expressed gene in several cancer including basal like breast tumors, endometrial cancer, gastric cancer, ovarian cancer, pancreatic cancer, and non-small cell lung cancer (NSCLC). As mentioned earlier, over expression of S100A2 is correlated with increased metastasis and poor prognosis of the patients in the early stage NSCLC. Few members of S100 proteins like S100A4 actions in the process of EMT and metastasis has just begun to unravel. However, the role of S100A2 in the progression of cancer is not clear. Hence, the present study has been designed to investigate the significance of S100A2 over expression in the progression of cancer. Towards understanding the functional role of S100A2 in cancer, neomycin resistant A549 clones (lung adenocarcinoma cell line) stably over expressing S100A2 were generated in our laboratory. In the present study, we utilized these S100A2 over expressing A549 clones in different functional assays like cell proliferation, cell invasion, anchorage independent growth potential and xenograft mouse model to gain insights into S100A2 role in cancer. In a monolayer culture, S100A2 over expression resulted in an altered morphology of cells from polarized cuboidal to spindle shaped mesenchymal cells, suggestive of EMT. In addition, in anchorage independent growth assay S100A2 over expressing clones resulted in increased number of colonies with a distinct “spread out” and loose appearance. This morphological feature of colonies in soft agar has been reported to be suggestive of increased metastatic potential of the cells. Further, S100A2 clones invaded the Matrigel more efficiently than the control cells in the Matrigel invasion assay. Effect of S100A2 in mediating EMT was biochemically confirmed by looking at the mRNA and protein expression of EMT markers. We observed an increase in the expression of mesenchymal markers like vimentin, N-cadherin, alpha-smooth muscle actin and a concomitant decrease in the expression of epithelial markers like E-cadherin and occluding. In addition, the transcription factor Snail, which is a negative regulator of E-cadherin expression, was induced in S100A2 clones compared to the control. S100A2 preferentially increased the activation of phospho-AKT in the clones compared to the vector clones. To corroborate these observations in vivo, these cells were injected subcutaneously in the flanks of immunocompromised mice and studied the phenotype of resultant tumors. Subcutaneous injection of S100A2 clones formed significantly large tumors which histologically appeared to have undergone EMT as seen by the hematoxylin and eosin staining. S100A2 induced EMT in vivo was observed by the altered mRNA expression of EMT markers as seen by RT-PCR and protein expression of few mesenchymal markers by immunohistochemistry. Increased AKT phosphorylation was also observed in the resected tumors over expressing S100A2 suggesting a role for AKT in S100A2 mediated invasive phenotype. To substantiate that these effects were specific to S100A2, we depleted the expression of S100A2 in Hep3B cell line using shRNA. In good correlation, knockdown of S100A2 reversed the EMT phenotype by repressing the expression of mesenchymal markers as well as significantly reduced phospho-AKT levels. Collectively, our data demonstrate the pro-tumorigenic role of S100A2 in cancer. • Mechanism of S100A2 mediated pro-tumorigenic actions Our data revealed that over expression of S100A2 regulates expression of genes involved in matrix modulation like MMP-9, MMP-1, MMP-7, TGM2, uPA, and EMT modulators like vimentin, Snail and E-cadherin, respectively. Interestingly, these gene targets are also shown to be regulated by TGF-β/Smad3 signaling. In addition, the role of TGF-β and Smad3 in mediating EMT has been well documented. Our observation on the involvement of S100A2 in the enhancement of EMT prompted us to evaluate a possible interaction of S100A2 with Smad3. By GST-pull down and co-immunoprecipitation experiments, we demonstrate a physical interaction between S100A2 and Smad3 proteins. In the presence of high calcium and TGF-β, S100A2-Smad3 interaction was further enhanced. Moreover, S100A2 positively induced Smad3 dependent transcriptional activation. Supporting this, knockdown of S100A2 expression abolished TGF-β/Smad3 mediated gene induction, which mainly included direct Smad3 targets like p21, PAI-1 and vimentin. Most importantly, loss of S100A2 significantly reduced TGF-β induced cancer cell migration and invasion. In summary, our study has for the first time delineated the mechanism of S100A2 gene regulation in the context of TGF-β and p53. Further, we established a pro-tumorigenic role of S100A2 through induction of EMT. Moreover, this study highlights a novel interaction of S100A2 with Smad3, which may have important implications in the progression of cancer. In our study, PI3/AKT signaling emerged as the important signaling pathway altered upon S100A2 over expression. This substantiates the well-documented role of AKT signaling in mediating EMT and increased survival of cancer cells. Finally, our study has identified S100A2 as a potential therapeutic target at least in lung cancer for combating EMT induced by activated TGF-β/Smad3 signaling.

Aberrant DNA methylation has been frequently observed in human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, representing approximately 5% of malignant solid tumour in the paediatric population. However, the specific mechanisms and targets are still poorly understood. We showed the up-regulation of DNA methyltransferase (DNMT) family members in 14 RMS primary tumour biopsies and 4 RMS cell lines in comparison to normal skeletal muscle (NSM). Our study focused on DNMT3B gene, which exhibited particularly high levels in RMS samples, in order to establish its individual role in this malignancy. RNA interference-mediated DNMT3B knock-down decreased cell proliferation, by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the p21 and p27 checkpoint regulators. DNMT3B depleted cells also showed a decreased migratory capacity and clonogenic potential in comparison to mocked controls. Interestingly, DNMT3B silencing was able to reactivate the skeletal muscle differentiation program in embryonal RMS (ERMS) cells, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, leading to cell cycle arrest and myogenic terminal differentiation, this supporting the methyltransferase as a down-stream molecule of the MEK/ERK oncogenic pathway. Moreover, we showed that DNMT3B is a target of specific miR-29 family members in RMS cells and that the restoration of miRNA expression levels, by miRNA mimic transfection, lead to decreased cell proliferation and migration and to G1 cell cycle arrest. Finally, DNMT3B silencing radiosensitizes ERMS cells by altering DNA damage response signalling. Taken together, our data shed further light on RMS development, underlying a pivotal role of DNMT3B gene in myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel and promising strategy against RMS tumour able to ameliorate traditional therapies in order to improve the survival rate for patients with this soft tissue sarcoma.

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011
O cancro e, actualmente, a segunda causa de morte no mundo ocidental, sendo o cancro colorectal, a terceira forma de cancro mais commumente diagnosticada, tanto no homem, como na mulher. Mais ainda, o cancro colorectal e a terceiraprincipal causa de morte relacionada com o cancro.A transformacao do tecido colonico normal e o desenvolvimento do cancro colorectal e considerado um processo faseado, com acumulacao de diferentes alteracoes geneticas e epigeneticas, que levam a transformacao do epitelio normal em adenoma e, mais tarde, em carcinoma. Adenomas ou polipos sao neoplasmas que, embora apresentem vantagem proliferativa inerente e reducao de diferenciacao, progridem para formas malignas em apenas 10% dos casos. Um dos principais eventos moleculares que pode conduzir ao desenvolvimento e progressao do cancro e a perda de estabilidade genomica que, no caso do cancro colorectal, esta associada a instabilidade de microssatelites e a instabilidade cromossomica. A instabilidade de microssatelites e caracterizada pela presenca de mutações em genes do sistema de reparacao de mismatchesno DNA, as quais podem resultar na inserção ou deleccao de repeticoes em microssatelites. Os microssatelites, por sua vez, sao pequenas repeticoes de sequencias de nucleotidos, contendo cerca de uma duzia de repeticoes de 4 a 6 sequencias nucleotidicas. As mutacoes geradas em cancros com instabilidade de microssatelites sao, inicialmente, aleatorias, afectando qualquer repeticao de microssatelite. Alguns clones podem, no entanto, adquirir mutacoes em genes chave, conferindo a celulavantagem proliferativa. A instabilidade cromossomica e o tipo de instabilidade genómica mais comum, ocorrendo em cerca de 80 a 85% dos tumores colorectais esporadicos, que não apresentam deficiencias no sistema de reparacao de mismatches noDNA. Tumores com instabilidade cromossomica estao associados a uma elevada frequencia de aneuploidias e desequilibrios alelicos, apresentando em geral, um prognostico pior do que tumores com deficiencias no sistema de reparacao demismatchesnoDNA. Nos neoplasmas colorectais e, ainda, comum a instabilidade epigenetica, a qual resulta da metilacao aberrante de genes supressores de tumores. Dentro dos tumores esporaricos com instabilidade de microssatelites, a instabilidade epigenetica manifesta-se atraves da metilacao do promotor de genes envolvidos na reparacao do DNA, nomeadamente do gene MLH1. Varias vias de sinalizacao que regulam o ciclo celular e a apoptose encontram-se desreguladas na transformacao, desenvolvimento e progressao tumoral, levando a um aumento descontrolado da proliferacao celular e a quase completa insensibilidade a estímulos pro-apoptoticos. Destacam-se a sobreactivacao das vias de sinalizacao das MAPKs, do NF-B e do PI3/AKT, que promovem a proliferacao celular, bem como a mutacao do p53, que inibe a apoptose numa elevada percentagem de carcinomas. A desregulacao da expressao de microRNAs (miRNAs) tem sido associada ao desenvolvimento de varios tipos de cancro, incluindo o cancro colorectal, atraves da alteração dos niveis de producao dos varios mediadores envolvidos nestas vias de sinalizacao celular. Os miRNAs sao pequenas moleculas de RNA nao codificante que regulam negativamente a expressao genica, a nivel pos-transcricional, atraves da repressao da traducao do mRNA alvo, ou da clivagem e degradacao do mesmo, estando este processo dependente do grau de complementaridade entre o miRNA maduro e o mRNA alvo. A accao dos miRNAs esta envolvida na regulacao de inumeros processos biologicos, nomeadamente na proliferação celular e na apoptose. Curiosamente, observou-se que o miRNA-143 (miR-143) apresenta expressao reduzida no cancro colorectal, assim como em outros tipos de cancro, sendo que um dos seus principais alvos biologicos e a ERK5. A accao do miR-143 na inibicao do crescimento tumoral, com modulacao da expressao de ERK5, foi ja demonstrada pelo nosso grupo, tanto in vitro, como in vivo. Nao existe, actualmente, qualquer informacao disponivel na literatura relativa a producao da proteina ERK5, ou da sua proteina activadora directa, a MEK5, no cancro colorectal. No entanto, o aumentonos niveis destas duas cinases foi ja observado em outros tipos de cancro, estando associado ao aumento de proliferacao, progressao tumoral e resistencia a terapeutica,para alem de se correlacionar com um pior prognostico da doenca. Um dos objectivos do presente trabalho foi analisar a producaodas proteina ERK5 e MEK5 durante o desenvolvimento e progressao do cancro colorectal, por forma a determinar se esta via de sinalizacao pode constituir um novo alvo terapeutico no cancro colorectal. Foi estudado um conjunto bem definido de 197 amostras humanas, incluindo tecido normal, adenomas e carcinomas, com e sem instabilidade de microsatelites. Os resultados demonstram, inequivocamente, que as cinases ERK5 e MEK5 se encontram aumentadas no cancro colorectal e que, possivelmente, a sinalizacao via ERK5 e um evento importante na iniciacao da transformacao tumoral. De facto, verificou-se um aumento significativo de ERK5 e MEK5 em adenomas, bem como em carcinomas, de forma independente da integridade do sistema de reparacao de mismatchesno DNA, relativamente ao tecido colonico normal. Sugere-se, assim, que a ERK5 podera representar um alvo terapeutico novo e relevante no cancro colorectal, possivelmente atraves da reducao da sua producao induzida pelo miR-143. O estudo de mediadores de outras vias de sinalizacao, importantes no desenvolvimento tumoral, como o NF-B e o AKT, revelou que estao alteradosdurante o desenvolvimento do cancro colorectal, tanto nos polipos, como nos carcinomas com e sem instabilidade de microsatelites. Confirmou-se, ainda, que a perda de funcao da proteina p53 esta envolvida na transicao de adenoma para carcinoma e que a frequencia de ocorrencia de mutacoes no p53 esta relacionada com a funcionalidade do sistema de reparacao de mismatches no DNA. Tendo em conta o conhecimento actual do potencial anti-proliferativo, pro-apoptotico e quimiosensibilizador do miR-143, possivelmente atraves da repressao da via de sinalização da ERK5, outro objectivo do presente trabalho consistiu no desenvolvimento de uma estrategia eficiente de entrega do miR-143, tendo em vista o seu uso como ferramenta terapeutica no tratamento do cancro colorectal. O maior obstaculo na aplicacao terapêutica dos miRNAs esta relacionado com o seu direcionamento para as celulas alvoin vivo, bem como com a sua estabilidade, uma vez que os miRNAs, nao modificados e desprotegidos, são instaveis em circulacao. O sistema de entrega em desenvolvimento neste estudo baseia-se na conjugacao do miR-143 com o cetuximab, ja utilizado no cancro colorectal como terapia dirigida. O cetuximab e um anticorpo monoclonal que se liga ao receptor EGFR, frequentemente aumentado no cancro colorectal, promovendo a sua internalizacao e degradacao. O tratamento com este anticorpo melhora significativamente a sobrevivencia dos doentes e a progressao livre de doenca, sendo, no entanto, a sua eficacia dependenteda ausencia de mutacoes no KRAS. Sabendo que o miR-143 apresenta efectores especificos a jusante na via do EGFR, KRAS e ERK5, como principais alvos, o potencial terapeutico do miR-143 e elevado e poderá aumentar a eficacia do cetuximab. O objectivo ultimo e tirar partido da especificidade do cetuximab para direccionar o miR-143 para as celulas com producao aumentada de EGFR. A estrategia de entrega desenvolvida baseou-se na conjugacao do cetuximab com fluoresceína 5(6)-isotiocianato (FITC). De seguida, o anticorpo conjugado foi complexado com um anticorpo anti-FITC, conjugado com protamina (anti-FITC-protamina), numa proporção superior ao numero de moleculas de FITC conjugadas com o cetuximab, para garantir que todos os residuos de FITC no cetuximab estejam ocupados e, desta forma, maximizar a carga de miRNA em cada molecula de cetuximab. Por fim, o miR-143 sera marcado com o fluoróforo Cy3 e carregado nas protaminas. A protamina e uma proteina carregada positivamente, com afinidade para acidos nucleicos, que apresenta um grande potencial para a entrega de pequenos RNAs em celulas de cancro, ja demonstrado in vitro e in vivo. O progresso conseguido no desenvolvimento desta ferramenta permitiu demonstrar que o cetuximab conjugado com FITC manteve a capacidade de ligacao ao EGFR, para alem de que o complexo terapeutico constituido por cetuximab-FITC e anti-FITC-protamina foi entregue em celulas de cancro colorectal com niveis elevados de EGFR. Uma vez que a eficiencia desta ferramenta depende, em grande medida, da ligacao e internalizacao do complexo nas celulas alvo, os resultados obtidos ate agora reforcam o potencial desta estrategia de entrega, para alem de estimularem estudos futuros com vista a sua utilizacao terapeutica.
Colorectal cancer (CRC) is a major health problem worldwide, with high incidence, representing a major cause of cancer related deaths. The development of CRC is considered a stepwise process, with the accumulation of genetic and epigenetic alterations, which contribute to transformation of normal colonic epithelium into adenoma, and eventually to carcinoma. The overexpression of ERK5 and MEK5 have been reported in many types of human cancer and correlated with increased cell survival, proliferation, chemoresistance and poor disease prognosis. Curiously, no information is available on ERK5 or MEK5 expression profiles in CRC. However, ERK5 expression is directly regulated by microRNA-143 (miR-143), typically downregulated in CRC. The aim of the present work was to evaluate the expression levels of ERK5 and MEK5 in normal colon, colon adenomas, and colon carcinomas. In addition, we aimed to develop an efficient delivery strategy for future use of miR-143 in CRC therapy, using cetuximab as a delivery vehicle. Our results provide the first human data demonstrating the aberrant overexpression of ERK5 and MEK5 in adenomas and, to a lesser extent, carcinomas. This suggests ERK5 as a potential target for the development of novel cancer therapies. Further, we confirmed a significant deregulation of NF-B and AKT signaling pathways, thought to be involved in cancer development and progression. p53, a key player in adenoma-carcinoma transition was also deregulated. Finally, we built an effective delivery complex, delivered to target cells with high affinity, highlighting the potential of this strategy for cancer cell delivery of miRNA therapeutics.

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2019
Colorectal cancer remains one of the leading causes of cancer morbidity and mortality in the world. Despite significant advances in early detection and multimodal care, drug resistance, recurrence, and metastasis persist as major challenges in the management of the disease, calling for the identification of alternative targets for therapeutic intervention. Interestingly, in human colorectal adenomas and adenocarcinomas, there is ample evidence that aberrant MEK5-ERK5 expression and/or activation correlates with tumor-node-metastasis stage. As such, the studies presented in this thesis were conducted with the purpose of defining the contribution of ERK5-mediated signaling to the regulation of hallmark traits associated with disease progression, namely tumor cell resistance to 5-fluorouracil (5-FU), the cornerstone of systemic colorectal cancer treatment; and malignant stem cell-like pools, major players in therapy-refractory disease. Using colorectal cancer cell lines as model, we showed that exposure to 5-FU impairs endogenous MEK5-ERK5 signaling, whereas ectopic expression of a constitutively active MEK5 increases resistance to this drug. Conversely, blocking ERK5 in combination with 5-FU results in impaired tumor cell survival and growth in vitro and in subcutaneous xenografts. Further studies established that ERK5 inhibition promotes 5-FU–induced apoptosis in a TP53-wild-type but not a TP53-null background, implying a p53-dependent axis mediating 5-FU sensitization. In parallel, we demonstrated that MEK5-ERK5 phosphorylation levels are increased in three-dimensional sphere cultures enriched for neoplastic stem-like cells. Further, targeting ERK5 suppresses the rates of tumorsphere formation and the expression/activity of representative markers of immature cancer cell fractions, while sensitizing to 5-FU–based chemotherapy. Moreover, downregulation of NF-κB–mediated IL-8 expression might be a crucial event for the impact of ERK5 inhibition on malignant stem-like phenotypes. Finally, analysis of publicly available databases revealed that increased ERK5 expression correlates with shorter overall survival in colorectal cancer patients, reinforcing the clinical relevance of the MEK5-ERK5 axis. Overall, our findings indicate that upregulated MEK5-ERK5 signaling in colorectal carcinoma cells contributes to a shift to an undifferentiated state, whilst providing a route for cancer (stem) cells to escape cytotoxic insults inflicted by classical chemotherapy, therefore encouraging future investigations on the translational potential of ERK5-targeted agents for antineoplastic treatment and chemosensitization.
Fundação para a Ciência e a Tecnologia (FCT), projeto SAICTPAC/0019/2015; Programa Operacional Factores de Competitividade (COMPETE 2020), Programa Operacional Regional de Lisboa, projeto LISBOA-01-0145-FEDER-016405

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2013
Colon cancer (CC) remains highly ranked in incidence and mortality worldwide. 5-Fluorouracil (5-FU) has been the mainstay for the treatment of CC for several decades. Nevertheless, chemoresistance remains a significant drawback to its clinical success. Therefore, a better understanding of the molecular mechanisms by which tumor cells circumvent 5-FU-induced cytotoxicity may lead to the identification of new therapeutic targets to improve CC treatment. Aberrant MEK5/ERK5 signaling has been reported in several types of human cancer, being associated with increased cell proliferation, survival, and chemoresistance. In CC, MEK5 overactivation was correlated with disease stage progression. Moreover, recent data from our group demonstrated that MEK5 and ERK5 expression is increased in human colon adenomas and adenocarcinomas, suggesting that MEK5/ERK5 signaling overactivation may contribute to CC initiation and progression. In the present study, using HCT116 and SW620 cells, we produced two stable CC models with differential activation of the MEK5/ERK5 pathway to investigate its role in cell proliferation and response to 5-FU. Our results demonstrate that MEK5 constitutive activation increased CC cell proliferation and KRAS expression, in both HCT116 and SW620 models. In turn, in the HCT116 model, MEK5/ERK5 signaling inhibition, using a dominant-negative MEK5, increased p53 and p21 expression, as well as cell death following 5-FU exposure, which was further associated with increased caspase-3/7 activation and apoptosis. Conversely, constitutive activation of MEK5/ERK5 signaling increased 5-FU chemoresistance, reducing 5-FU-induced cytotoxicity and apoptosis. Interestingly, 5-FU exposure markedly decreased the levels of endogenous MEK5 and ERK5 expression and activation. Finally, our results show that MEK5/ERK5 activation may modulate cell proliferation and sensitivity to 5-FU by downregulating the expression of the tumor-suppressors miR-143/-145/-34a. Collectively, these data unravel an important interplay between the MEK5/ERK5 pathway and the mechanisms of 5-FU-induced cytotoxicity, suggesting that ERK5-targeted inhibition may provide a promising therapeutic approach for CC treatment, warranting further investigation.
O cancro do cólon (CC) persiste como um dos cancros com maior incidência e mortalidade a nível mundial. De acordo com as estimativas atuais, o cancro do cólon e do reto apresenta-se como a terceira e segunda forma de cancro mais frequente, respetivamente em homens e mulheres. Para além disso, o cancro do cólon e do reto representa atualmente 8 % das mortes relacionadas com cancro, encontrando-se classificado como a quarta forma de cancro com maior mortalidade no mundo. Por fim, prevê-se que o número de novos casos diagnosticados de cancro do cólon e do reto aumente cerca de 30 % até 2020. Apesar dos avanços no tratamento desta neoplasia, os doentes metastáticos apresentam uma taxa de sobrevida a 5 anos limitada a 12 %. A intervenção cirúrgica visando a resseção do tumor primário e dos nódulos linfáticos regionais constitui a base da terapêutica do CC. Após cirurgia, o recurso à quimioterapia para a irradicação de células tumorais residuais irá depender do estadio da doença. Há várias décadas que o tratamento adjuvante do CC tem por base o 5-fluorouracilo (5-FU). No entanto, embora a combinação com novos fármacos antitumorais tenha resultado numa melhoria das taxas de resposta aos esquemas terapêuticos baseados no 5-FU, a resistência a este agente quimioterapêutico mantém-se como uma das principais limitações ao seu sucesso clínico. São bem conhecidos os mecanismos de ação através dos quais o 5-FU induz citotoxicidade, impossibilitando a replicação e reparação do DNA e inibindo a maturação do RNA. Continuam, no entanto, por esclarecer, as vias de sinalização para a inibição do crescimento celular e indução de apoptose moduladas pelo 5-FU, bem como as vias associadas ao desenvolvimento de resistência pelas células tumorais. Este conhecimento poderá revelar-se fundamental para a identificação de doentes com maior probabilidade de resposta a tratamentos com base neste agente quimioterapêutico. Para além disso, deverá contribuir para o desenvolvimento de novas estratégias terapêuticas que visem melhorar a eficácia clínica do 5-FU e, consequentemente, o panorama geral do tratamento do CC. O desenvolvimento do cancro é um processo faseado, através do qual as células cancerígenas adquirem progressivamente capacidade de proliferação anormal, resistência à apoptose, invasão de tecidos adjacentes e metastização. De entre as diferentes vias de sinalização implicadas neste processo, a família das MAPKs apresenta-se como uma das mais importantes. Em particular, a sinalização mediada por ERK5 e pela sua proteína ativadora direta MEK5, apresenta características únicas e desempenha um papel fundamental na transdução de sinais extracelulares, culminando na fosforilação e ativação transcricional de proteínas alvo. Desta forma, a via de sinalização MEK5/ERK5 participa na regulação de processos biológicos cruciais, como a proliferação, a diferenciação e a morte celular. O aumento dos níveis de expressão e/ou ativação de MEK5 e ERK5, encontra-se associado a um aumento da agressividade de diversos cancros, tendo sido já demonstrado como um fator de prognóstico desfavorável no cancro da mama e da próstata. No CC a sobre-ativação de MEK5 foi já correlacionada com o estadio da doença. Para além disso, demonstrámos recentemente que as proteínas MEK5 e ERK5 se encontram sobre-expressas em adenomas e adenocarcinomas do cólon, reforçando a noção de que a via de sinalização mediada por estas cinases poderá contribuir para a iniciação e progressão da doença. Mais ainda, a sobre-ativação da sinalização via MEK5/ERK5 mostrou estar associada à resistência aos mecanismos de indução de apoptose despoletados pela ação citotóxica de diversos agentes antitumorais. Em particular, demonstrámos anteriormente que o silenciamento de ERK5 induz apoptose e sensibiliza células de CC para o 5-FU. Os estudos apresentados nesta tese tiveram como principal objetivo a avaliação do papel da sinalização MEK5/ERK5 na modulação da proliferação celular e sensibilidade de células de CC ao tratamento com 5-FU. Para este propósito, utilizando células HCT116 e SW620, foram desenvolvidos dois modelos de CC com sobre-expressão estável de MEK5 constitutivamente ativa ou dominante negativa. Os nossos resultados demonstraram que a ativação constitutiva de MEK5 induz a proliferação celular, aumentando a expressão de KRAS e reduzindo a expressão de p53, tanto no modelo em HCT116 como no modelo em SW620. Por outro lado, em HCT116, a inativação da sinalização via ERK5 resultou na expressão aumentada de p53 e p21. Tendo em conta que a atividade antitumoral do 5-FU depende dos mecanismos de inibição do ciclo celular e indução de apoptose, regulados por p53, estes resultados sugeriram, desde logo, que a via MEK5/ERK5 poderia modular a resposta ao 5-FU. De acordo com esta hipótese, a inativação da via MEK5/ERK5 mostrou sensibilizar células HCT116 para o efeito citotóxico do 5-FU. Em paralelo, esta sensibilização mostrou estar associada a um aumento da atividade das caspases-3 e -7 e, em conformidade, dos níveis de apopotose. Em contrapartida, a ativação constitutiva da via de sinalização MEK5/ERK5 levou a uma diminuição da resposta de células HCT116 ao 5-FU, aumentando a viabilidade celular e reduzindo a apoptose, após exposição a este agente quimioterapêutico. Foi igualmente possível verificar que a exposição ao 5-FU reduziu os níveis endógenos de expressão e ativação de MEK5 e ERK5. Estes resultados indicam a existência de interação entre a sinalização mediada pela via MEK5/ERK5 e os mecanismos de citotoxicidade induzidos pelo 5-FU, cujo balanço poderá ser crucial para a determinação da resposta a este agente antitumoral. A ativação constitutiva de MEK5 mostrou também estar associada à resistência à oxaliplatina, outro agente quimioterapêutico frequentemente utilizado, em combinação com o 5-FU, no tratamento do CC. Os microRNAs (miRNAs) constituem uma classe endógena de pequenos RNAs não codificantes, cujo papel biológico se centra no silenciamento pós-transcricional de genes alvo. A expressão aberrante de miRNAs foi já descrita em vários tipos de cancro, incluindo no CC e encontra-se associada, não só ao processo de carcinogénese, mas também à alteração da sensibilidade celular a agentes quimioterapêuticos. A compreensão dos mecanismos que levam à desregulação dos níveis de expressão dos miRNAs reveste-se, por isso, de particular importância. Assim sendo, avaliámos o efeito da ativação diferencial de MEK5 nos níveis de expressão dos miRNAs supressores de tumor miR-143, miR-145 e miR-34a. O estado de ativação de MEK5/ERK5 mostrou estar inversamente relacionado com a expressão de miR-143/-143/-34a. Estes resultados sugerem que os mecanismos pelos quais a ativação desta via induz a proliferação e reduz a sensibilidade à sinalização apoptótica induzida pelo 5-FU em células de CC poderá, em parte, ser consequência da inibição da expressão destes miRNAs. Em suma, os nossos resultados indicam que a ativação aberrante da via MEK5/ERK5 poderá contribuir para a agressividade e a resistência à quimioterapia, sugerindo que a sua inibição específica poderá constituir uma abordagem terapêutica promissora no tratamento do CC. De futuro, será interessante avaliar os efeitos antitumorais da ação combinada do 5-FU com inibidores químicos seletivos para esta via de sinalização. Para além disso, será também aliciante determinar o potencial da re-introdução dos miR-143/-145, que têm como alvo direto a ERK5, como potencial ferramenta terapêutica para contrabalançar a resistência induzida pela sinalização através desta cinase.
This work was supported by grants PTDC/SAU-ORG/119842/2010 and PEstOE/SAU/UI4013/2011 from FCT (to C.M.P.R.)

Une dérégulation de la voie de signalisation Ras/Raf/MEK/ERK1/2 est observée dans plus de 30% des cancers et des mutations activatrices de RAS sont observées dans 30% à 50% des adénomes colorectaux. À la suite d’une analyse extensive de biopsies de tumeurs colorectales humaines par micromatrices tissulaires (TMA), nous avons observé que 44% des tissus cancéreux exprimaient MEK1/2 phosphorylés, contre 10% des tissus normaux. L'analyse des TMA a également révélé que 79% des tumeurs arboraient un marquage nucléaire de MEK1/2 phosphorylés, contre 4 % pour les tissus normaux. Bien que la voie MEK/ERK1/2 soit fréquemment activée dans les cancers, le rôle précis des isoformes de MEK1 et de MEK2 n'a jamais été clairement établie. De même, l'impact de cette localisation nucléaire aberrante de phospho-MEK1/2, dans l'initiation et la progression des cancers colorectaux, est inconnu. Lors d'un premier projet, nous avons démontré, que l’expression de MEK1 ou MEK2 activé est suffisante pour transformer in vitro des cellules intestinales épithéliales de rat (IEC-6). L'expression des mutants actifs de MEK1 ou MEK2 est suffisante pour induire une dérégulation de la prolifération cellulaire et engendrer la formation d'adénocarcinomes invasifs dans un modèle de greffe orthotopique du côlon chez la souris. Nous avons également démontré que l'inhibition de MEK2 par shRNA supprime complètement la prolifération des lignées humaines de cancer du côlon, alors que la suppression de MEK1 a peu d'effet sur la capacité de prolifération. Le deuxième projet, nous a permis d'observer que l'expression d'un mutant nucléaire de MEK1 dans les cellules IEC-6 transforme drastiquement les cellules. Une augmentation de prolifération, une résistance à l'anoikose, un dérèglement du cycle cellulaire, de l'instabilité chromosomique (CIN), de la tétra/aneuploïdie sont observés. La caractérisation des mécanismes responsables de cette localisation aberrante de MEK1/2 phosphorylés, a permis d'identifier la protéine Sef, un régulateur de la localisation cytoplasmique de MEK/ERK1/2. Nous avons démontré que l'expression d'une forme oncogénique de Ras (H-RasV12) inhibe l'expression de Sef, engendrant alors une accumulation nucléaire de MEK1/2 activés. Plus encore, la réexpression de Sef restaure la localisation cytoplasmique de MEK1/2 et renverse les propriétés tumorigéniques ainsi que l'aneuploïdie induite par Ras activé. Un troisième projet, visant la caractérisation des mécanismes associés à la CIN et à l'aneuploïde engendrés par l'activation aberrante de la voie de Ras-ERK1/2, a permis d'observer que l'hyperactivation de ERK1/2 induit des anomalies mitotiques menant à la binucléation. Une localisation erronée et une surexpression de la kinase Aurora A, de même que des protéines de passage du complexe chromosomique (CPC), Aurora B, Survivine et INCENP, sont observées. L'inhibition partielle de l'activation de ERK1/2 par de faible dose de PD184352, un inhibiteur de MEK1/2, est suffisante pour renverser la surexpression de ces régulateurs mitotiques, de même que corriger les anomalies de la mitose et réduire la tétra/aneuploïdie engendrée par Ras oncogénique. Ainsi, nous avons démontré, pour la première fois, que la voie des MAP kinases ERK1/2 est impliquée dans la CIN, la tétraploïdie et l'aneuploïdie. Nos résultats suggèrent que la perte de Sef est un événement oncogénique précoce, qui contribue à la localisation nucléaire aberrante de MEK1/2 qui est observée dans les tumeurs colorectales. Cette localisation anormale de MEK1/2 est associée à l'initiation de la transformation, la progression tumorale et la CIN, via l'activité soutenue de ERK1/2. Ces informations sont capitales et démontrent l’importance de la voie de signalisation Ras/Raf/MEK/ERK1/2 dans le processus de tumorigénèse colorectale.
The Ras-dependent Raf/MEK/ERK1/2 signaling pathway is frequently hyperactivated in human cancer as a result of receptor tyrosine kinase overexpression or gain-of-function mutations in RAS or RAF genes. More specificaly, activating mutation in RAS genes are found in ~ 30-50% of colorectal adenomas and phosphorylation of ERK1/2 is frequently observed in human colorectal cancer cells and tumor specimens. In a large TMA analysis, we found that MEK1/MEK2 are aberrantly activated in 44% of human colorectal cancers. In addition, our analysis revealed that 79% of colorectal cancers exhibit aberrant phospho-MEK1/2 staining in the nucleus, as compared to 4% of normal tissue. How dysregulation and mislocalization of MEK1/2 contribute to tumor initiation and progression is not well understood. In order to determine the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer, wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We found that the expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect. In a second project, we have investigated the impact of the nuclear mislocalization of phosphorylated MEK1/2 observed in colorectal tumors. We show that oncogenic activation of Ras is sufficient to induce the nuclear accumulation of phosphorylated MEK1/2 and ERK1/2 in intestinal epithelial cells. To evaluate the biological impact of the mislocalization of MEK1/2, we have forced the localization of MEK1 in the nucleus of epithelial cells. We found that sustained nuclear MEK1 signaling leads to hyperactivation of ERK1/2 and to enhanced cell proliferation. Nuclear localization of MEK1 also leads to tetraploidization, chromosomal instability (CIN) and tumorigenesis. Importantly, we show that oncogenic Ras downregulates the spatial regulator Sef, concomitant to nuclear accumulation of activated MEK1/2. Moreover, re-expression of Sef is sufficient to restore the normal localization of MEK1/2 and to revert the cell cycle defects and tumorigenesis induced by oncogenic Ras. Another project was initiated to characterize the tetraploidy and CIN observed upon hyperactivation of the Ras-ERK1/2 pathway. Aneuploidy and CIN are observed in the majority of colorectal cancers and are associated with a poorer prognosis. We show that hyperactivation of ERK1/2 by oncogenic Ras or sustained nuclear MEK-ERK1/2 signaling induces mitotic defects that lead to tetraploidy, aneuploidy and CIN. We also found that dysregulation of Ras-ERK1/2 signaling alters the expression and localization of Aurora A and the Chromosomal passenger complex proteins. In conclusion, we show for the first time that the MEK/ERK1/2 signaling pathway is implicated in aneuploidy and CIN. Our results suggest that sustained nuclear ERK1/2 signaling may contribute to the initiation and progression of colorectal cancer by rapidly inducing aneuploidy and CIN. We suggest that loss of Sef is an early oncogenic event that contributes to genetic instability and tumor progression by sustaining nuclear ERK1/2 signaling. These observations are significant and highlight the importance of the Ras-ERK1/2 signaling pathway in colorectal tumorigenesis.

Hepatitis B virus (HBV) infection is one of the major causes of chronic and cancerous liver disease. Elimination of HBV from chronically infected patients by recombinant interferon α (IFNα) monotherapy shows that the mechanisms of the innate immunity play an important role in suppressing viral infection. However, the mechanisms of recognition of the HBV genome and its escape from the mechanisms of natural immunity are still little known. One of the principal factors enabling the virus to escape from cellular restriction mechanisms is the HBx viral protein. HBx is a 154 amino acid pleiotropic multifunctional protein affecting transcription, signal transduction, cell cycle, protein degradation, apoptosis, and chromosomal stability in the host cell. Previous results from our laboratory have shown that activation of the MEK1/2-ERK signaling pathway in plasmacytoid dendritic cells leads to inhibition of IFNα production. The aim of my work was to determine whether HBx activates the MEK1/2-ERK pathway and thus inhibits IFN type I production also in hepatocytes. For this purpose, I monitored HBx production in the Huh7 hepatoma cell line by transfecting the bicistronic plasmid pHBx- IRES-EGFP and Western blotting. Using the same method, I monitored activation of the MEK1/2-ERK signaling pathway by ERK...