Univ., Diss., 2010--Marburg.

Hannover, Universiẗat, Diss., 2002.

The major cause of therapy failure and death of cancer patients is metastasis. RECK is a newly identified gene which was isolated by screening for human fibroblast cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH3T3 cells. It can inhibit the release and activation of MMP-2 and MMP-9, and prevent cell invasion in vitro. In addition, RECK can repress tumor metastasis in experimental animal. Thus, RECK is a metastasis suppressor gene. However, RECK gene is a common target that is negatively regulated by oncogenic signals. Overexpression of c-myc protooncogene is frequently found in several types of human cancer and contributes to multiple steps of tumorigenesis. Ecto-expression of c-Myc in NIH3T3 cells inhibited RECK expression. Promoter activity assay suggested c-Myc repressed RECK at transcriptional level. By using DNA affinity precipitation assay and chromatin immunoprecipitation assay, we found that oncogenic c-Myc bound to the SP1 sites of RECK promoter in vitro and in vivo. It is possible that c-Myc could repress RECK expression via SP1. Our data suggest that c-Myc may inhibit the metastatic/angiogenic suppressor RECK to enhance cell invasiveness and restoration of RECK may be a novel strategy to inhibit c-Myc-mediated invasion.

Ulm, Univ., Diss., 2007.

Murine plasmacytoma models human cancers that involve deregulation of MYC. Overexpression and duplication of the immature colon carcinoma transcript 1 gene, Ict1, along with MYC deregulation may contribute to the aggressive mechanism for disease development in fast-onset mouse plasmacytomas. This study looks at Ict1 and c-MYC overexpression in mouse PreBmycER cells that serve as a cell culture model for MYC-dependent plasmacytomagenesis. An Ict1 inducible vector was transfected into the mouse PreBmycER cell line that contains inducible c-MYC. This allowed us to examine the effect of overexpression of ICT1 and c-MYC proteins simultaneously or each separately, on selected hallmark cancer cell traits such as increased proliferation, evasion of apoptosis and increased genomic instability. An increase in the number of cells in the S-phase was observed by 15 % and up to 20 % at 24 and 36 hours respectively, and cell doubling time shortened by almost 2 hours at 24 hours during peak ICT1 and c-MYC overexpression. Although, no noticeable change in apoptosis levels, or large scale genomic alterations were detected up to 96 hours post-ICT1 and c-MYC peak-overexpression, genomic instability was observed when MYC protein was overexpressed with or without ICT1 protein overexpression. Extrachromosomal elements increased in number and size during conditional MYC deregulation, and most of these elements (25 %) classified as Chromosome 11. These findings support Ict1 as a candidate gene that is selected for by MYC-deregulation during plasmacytomagenesis, and show promise that the experimental model of induced MYC and ICT1 overexpression in mouse PreB cells, deserves further investigation, specifically with in vivo studies. / October 2016

RESUME Le proto-oncogène c-myc code pour un facteur de transcription qui est impliqué dans de multiples processus cellulaires tels que la prolifération, la différenciation et l’apoptose. Une dérégulation de son expression suite à des altérations génétiques (mutation, translocation, amplification) est retrouvée dans plusieurs tumeurs telles que le lymphome de Burkitt, des plasmacytomes murins ainsi que des tumeurs non-lymphoïdes. c-myc est un gène dont l’expression est régulée à différents niveaux. Chez l’homme, le gène c-myc est transcrit à partir de quatre promoteurs alternatifs appelés respectivement P0, P1, P2 et P3. P1 et P2 sont les deux promoteurs les plus utilisés. Ensemble, ils permettent de former 90% des transcrits c-myc dans des cellules normales. Les promoteurs P0, P1 et P2 permettent la transcription de trois ARNms qui comportent deux codons d’initiation de la traduction (un CUG et un AUG). L’utilisation alternative de ces deux codons d’initiation est à l’origine de la synthèse de deux protéines (c-Myc 1 et c-Myc 2) ayant à la fois des fonctions identiques et distinctes. La grande taille des parties 5’ non-traduites ainsi que la présence dans celles-ci de phases ouvertes de lecture sont des éléments défavorables à la traduction de l’ORF codant pour les protéines Myc par un mécanisme classique d’initiation de la traduction. Notre laboratoire avait précisément montré que les protéines c-Myc sont synthétisées par un processus d’initiation interne de la traduction. Les ARNms dont l’initiation de la traduction s’effectue par entrée interne des ribosomes présentent une structure spécifique appelée IRES (Internal Ribosome Entry Site). Cette structure permet la fixation du ribosome directement à proximité du codon d’initiation. Dans le cas des ARNms c-myc, on retrouve une IRES se situant en amont des codons CUG et AUG qui permet la synthèse des protéines c-Myc1 et 2 respectivement. Un tel mécanisme permet la synthèse des protéines c-Myc dans des conditions où toute traduction dépendante de la coiffe est inhibée (mitose, apoptose). Au cours de mon travail, tout d’abord j’ai montré qu’une séquence de 40 nt dans les transcrits P2 permet à elle seule une initiation interne efficace de la traduction. Nous avons déterminé aussi que cette séquence, appelée B4, est active dans quatre types cellulaires différents avec une efficacité variable et qu’elle active la traduction indépendamment de l’ORF placée en aval. D’autre part, il a été déterminé que la séquence B4 recrute le complexe de préinitiation 43S, qui ensuite scanne le messager jusqu’aux codons initiateurs comme c’est le cas de l’IRES du rhinovirus. Une analyse plus détaillée de la séquence B4 a permis d’identifier trois plus petites séquences de plus ou moins 14 nt (Ti1, Boucle, Ti2), qui indépendamment l’une de l’autre permettent une entrée interne des ribosomes. Il a été déterminé que la présence du motif A-N6-AC dans la séquence de Ti2 était importante pour l’activité IRES de celle-ci. Cependant, ce même motif également présent dans la séquence Ti1 n’est pas essentiel à l’activité IRES de Ti1. Par la suite, nous avons démontré que l’IRES de c-myc nécessite pour son activité un évènement nucléaire. Nous avons donc entrepris la recherche de facteurs cellulaires impliqués dans l’activité de l’IRES de c-myc. Dans un premier temps, nous avons exclu le rôle de certaines protéines connues pour activer d’autres IRES dont le mécanisme de recrutement du complexe de préinitiation est similaire. Ainsi, nous avons montré, par des expériences de complémentation d’un RRL, que les protéines PTB et unr connues pour activer l’IRES du rhinovirus ne contribuent pas à l’activité de l’IRES de c-myc. De plus, la complémentation de RRL avec des extraits S10 ou nucléaires de cellules HeLa n’a pas permis d’identifier des protéines impliquées dans l’activité IRES de c-myc. D’autre part, des méthodes alternatives d’interaction d’ARN et de protéine comme le triple hybride ou la chromatographie d’affinité d’ARN n’a pas permis dans un premier temps de détecter une interaction entre un facteur non canonique et l’IRES de c-myc. Dès lors, l’existence de facteurs cellulaires impliqués dans l’activité de l’IRES de c-myc reste à déterminer.

Deregulated MYC expression contributes to cellular transformation as well as progression and maintenance of human tumours. Interestingly, in the absence of additional genetic alterations, potentially oncogenic levels of MYC sensitise cells to a variety of apoptotic stimuli. Hence, MYC-induced apoptosis has long been recognised as a major barrier against cancer development. However, it is largely unknown how cells discriminate physiological from supraphysiological levels of MYC in order to execute an appropriate biological response. The experiments described in this thesis demonstrate that induction of apoptosis in mammary epithelial cells depends on the repressive actions of MYC/MIZ1 complexes. Analysis of gene expression profiles and ChIP-sequencing experiments reveals that high levels of MYC are required to invade low-affinity binding sites and repress target genes of the serum response factor SRF. These genes are involved in cytoskeletal dynamics as well as cell adhesion processes and are likely needed to transmit survival signals to the AKT kinase. Restoration of SRF activity rescues MIZ1- dependent gene repression and increases AKT phosphorylation and downstream function. Collectively, these results indicate that association with MIZ1 leads to an expansion of MYC’s transcriptional response that allows sensing of oncogenic levels, which points towards a tumour-suppressive role for the MYC/MIZ1 complex in epithelial cells. / Eine Deregulation der MYC Expression trägt entscheidend zur malignen Transformation und Progression humaner Tumoren bei. In Abwesenheit von zusätzlichen genetischen Läsionen machen potentiell onkogene MYC Proteinmengen Zellen jedoch anfällig für eine Reihe Apoptoseauslösender Reize. Daher kann MYC-induzierte Apoptose als bedeutende tumorsuppressive Maßnahme und wichtige Barriere gegen die Entstehung von Krebs betrachtet werden. Mechanistisch unklar ist allerdings wie genau Zellen physiologische von supraphysiologischen MYC-Mengen unterscheiden um adäquat darauf reagieren zu können. Die Experimente in dieser Dissertation zeigen, dass die repressive Eigenschaft von MYC/MIZ1 Komplexen für die Induktion von Apoptose in Brustepithelzellen essentiell ist. Die Analyse von Genexpressions- und ChIP-Sequenzier-Experimenten verdeutlicht, dass hohe Level an MYC benötigt werden um niedrig-affine Bindestellen im Genom zu besetzen und Zielgene des SRF (serum response factor ) Transkriptionsfaktors zu reprimieren. Diese Gene haben eine wichtige Funktion in Prozessen wie Zytoskelettdynamik und Zelladhäsion und sind vermutlich daran beteiligt notwendige Überlebenssignale an die Kinase AKT weiterzuleiten. Eine Wiederherstellung der SRF Aktivität revertiert die MIZ1-abhängige Repression der Zielgene und führt zu einer vermehrten AKT Phosphorylierung und Funktion. Insgesamt deuten diese Resultate auf eine tumorsuppressive Rolle des MYC/MIZ1 Komplexes in epithelialen Zellen hin, da eine Veränderung der genregulatorischen Aktivität als Folge der Assoziation mit MIZ1 dazu beiträgen könnte onkogene Mengen an MYC zu erkennen.

Marburg, Univ., Diss., 2001

Many current efforts in cancer research focus on understanding the molecular mechanisms driving oncogenesis and to advance molecular diagnostics and targeted therapeutics. The MYC oncoprotein is estimated to be deregulated in over 50% of human cancers, and its deregulation is often associated with aggressive disease and poor patient outcomes. While the ability of MYC to promote cellular transformation is well established, a better understanding of the mechanisms promoting MYC-mediated tumorigenesis is essential. While MYC has been shown to undergo a number of post-translational modifications (PTMs), our current understanding of biological significance of these modifications is largly limited to two phosphorylation sites located in the N-terminal domain of the protein. Our work, therefore, aimed to further our understanding of how PTMs regulate MYC-dependent transformation. To this end we have identified and characterized three novel human cell line models of MYC-dependent transformation: MCF10A, SH-EP Tet21/N-Myc, and LF1/TERT/LT/ST cells. Using a combination of these novel models and classic systems, we have evaluated point mutants of MYC at key serine/threonine and lysine residues for their ability to influence MYC-dependent transformation. Using a six lysine to arginine substitution mutant, we have identified and chacterized six C-terminal lysines to be important for the negative regulation of MYC activity. We have additionally demonstrated for the first time that MYC can undergo SUMOylation at one of the lysines in this region. We further completed a functional and transcriptional characterization of MYC phosphorylation mutants. We have assigned biological significance to previously identified phosphorylation sites through the characterization of two mutants that have increased transformation potential over wild-type MYC. Expression array analysis identified gene expression changes both common to deregulated MYC and unique to the different gain-of-function phosphorylation mutants. Combined, this work has advanced our understanding of several of the mechanisms that may regulate MYC-induced transformation.

Myc is an oncogene that is commonly deregulated in human cancers. Mechanistic studies reveal that Myc is a transcription factor that interacts with a protein partner called Max. Heterodimerization and the formation of the Myc:Max complex enables Myc:Max to bind to the E-box and subsequently regulate the activation and repression of Myc target genes. Since regulation of its target genes are essential for Myc to drive transformation, the Myc and Max interaction has been targeted in mouse model studies to determine whether the oncogenic activity of Myc can be inhibited. Surprisingly, these studies reveal that targeting Myc is not only possible but a powerful way to suppress tumour growth. Since a better understanding of how Myc carries out its biological functions makes the possibility of targeting Myc a reality, it is essential to investigate and study the mechanisms of how Myc promotes tumourigenesis. In the first part of this thesis, we investigate the idea that the N-terminal end of Myc may be post-translationally modified and this modification may dictate Myc activities. Indeed we report here that the N-terminal end of Myc can be ubiquitylated as well as acetylated, and that the loss of these modifications results in a decrease in Myc activities. Furthermore, we characterize Mdm2 as a potential E3 ubiquitin ligase that may ubiquitylate the N-terminal end of Myc. In the second part of this thesis, we investigate CUL7 as a novel oncogene that may inhibit Myc-potentiated apoptosis and cooperate with Myc in transformation. Indeed, CUL7 is a novel p53 interacting protein that inhibits Myc potentiated apoptosis through the inhibition of p53. We have also characterized CUL7 to be overexpressed in primary human lung cancers, and a higher level of CUL7 expression associates with short-term survival of lung cancer patients. Through the better understanding of the enzymes that post-translationally modify the N-terminal end of Myc and proteins, such as CUL7, that can cooperate with Myc to drive tumourigenesis, we may begin to devise ways to target and control deregulated Myc in cancer cells.