Une faible biodisponibilité et une haute toxicité des médicaments anticancéreux, ajoutées à une résistance aux médicaments, constituent des obstacles majeurs pour le traitement du cancer. L'application des nanotechnologies pour la délivrance de médicaments est largement pressentie pour aborder ces problèmes. Premièrement, nous avons utilisé un peptide CRGDK comme ligand spécifique pour les cellules cancéreuses que nous avons conjugué au DSPE-PEG2000 pour préparer les nanomicelles encapsulant le médicament anticancéreux doxorubicine. Le peptide CRGDK conjugué aux nanomicelles provoque la liaison aux récepteurs à NRP-1, conduisant à l'absorption cellulaire spécifique et à l’amélioration de l'activité anticancéreuse in vitro. Les résultats in vivo ont également confirmé que les nanomicelles décorées de CRGDK pourraient efficacement pénétrer et s’accumuler dans les tumeurs profondes.Deuxièmement, nous avons a été consacrée à la mise au point de nanomicelles originales utilisant un dendrimère amphiphile (AmDM). Ces nanomicelles sont capables d’encapsuler efficacement la doxorubicine. Les taux de remplissage de ces nanomicelles sont extrêmement élevés. Ces nanomicelles montrent une efficacité supérieure à la doxorubicine libre et ceci sur divers types de cellules cancéreuses. De plus, ce mécanisme de pénétration cellulaire permet à ces nanomicelles de contourner le relargage du médicament médié par la pompe à efflux glycoprotéine, et ainsi surmonter la résistance à la doxorubicine. L’étude sur souris montre également un excellent effet anticancéreux associé à une diminution des effets toxiques de la doxorubicine. / Poor tumor penetration and high toxicity of anticancer drugs, together with the developed drug resistance constitute challenging hurdles for cancer therapy. The application of nanotechnology for anticancer drug delivery is expected to address these issues and bring new hope for cancer treatment. In the first part of my PhD thesis, we used a new tumor-penetrating peptide, CRGDK, to conjugate onto the surface of doxorubicin encapsulated DSPE-PEG2000 nanomicelles. The CRGDK peptide conjugated on the nanomicelles triggered specific binding to Nrp-1 receptors, leading to enhanced cellular uptake and anticancer activity in vitro. The in vivo results further confirmed that the CRGDK-decorated nanomicelles could efficiently accumulate and penetrate into deeper tumors. In the second part of my PhD thesis, we established an original nanomicellar drug delivery system based on an amphiphilic dendrimer (AmDM), which could generate supramolecular micelles to effectively encapsulate the anticancer drug doxorubicin (DOX) with high drug loading capacity (> 40%), thanks to the unique dendritic structure creating large void space for drug accommodation. The resulting AmDM/DOX nanomicelles are able to specifically accumulate at tumor sites via EPR effect and penetrate deeper into tumor tissues thanks to their small size. Most importantly, these nanomicelles exhibit significantly improved anticancer activity and reduced systemic toxicity, and are very effective even towards drug resistant cancers by virtue of their macropinocytotic cell uptake mechanism and their ability to bypass cell drug efflux pumps.

Energy production is a very important function for the cells to maintain homeostasis, survive and proliferate. Cellular energy can be produced either through oxidative phosphorylation (OXPHOS) in the presence of oxygen or glycolysis in its absence. Cancer cells, even in the presence of oxygen prefer to produce energy through glycolysis and this confers them a survival advantage. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. For that purpose the role of various transcription factors in determining the pathway of energy production has been investigated extensively and there is evidence to suggest that oncogenic transcription factors promote glycolysis whereas tumour suppressors demote it. In line with this notion, the master regulator of cellular response to hypoxia, the Hypoxia Inducible Factor 1 (HIF-1) has been shown to induce the expression of a variety of genes encoding enzymes involved in glucose metabolism as well as OXPHOS favouring energy production through glucose metabolism in hypoxic cells. The tumour suppressor p53 on the other hand inhibits glycolysis and stimulates OXPHOS. One of the pathways through which p53 exerts these effects, is by inducing the inhibitor of glycolysis TIGAR and the cytochrome c oxidase assembly factor SCO2 gene expression under DNA damage conditions. However, the regulation of the expression of these genes in hypoxic conditions has been only partially elucidated. We hypothesised that under hypoxic conditions, TIGAR and SCO2 gene expression might be differentially regulated in cells bearing mutated p53 and in these cells the involvement of HIF-1 could be crucial. Indeed under hypoxia mimicking conditions, the TIGAR and SCO2 protein and mRNA levels were found to be modulated differentially in p53 wild type and mutant cell lines. The bioinformatics analysis revealed the presence of hypoxia responsive elements (HREs) within the regulatory region of the promoters of TIGAR and SCO2 genes. Firefly reporter assays and chromatin immunoprecipitation (ChIP) assays have indicated that HIF-1 plays a crucial role in the regulation of TIGAR gene expression. The direct involvement of HIF-1 in the regulation of SCO2 gene expression requires further investigation. We and others have recently reported that PCAF is a common cofactor for p53 and HIF-1α, regulating the protein stability and transcription target selectivity of both transcription factors thereby orchestrating the balance between life and death in cancer cells. We hypothesised that PCAF plays a similar role in the regulation of cellular energy metabolism by differentially targeting HIF-1α and p53 to the promoter of TIGAR and SCO2 genes. In this study we present evidence to support the notion that PCAF plays an import role in the regulation of TIGAR and SCO2 gene expression under hypoxic mimicking conditions. This conclusion was supported by assessing the functional consequences of PCAFwt and PCAFΔHAT overexpression on the intracellular lactate production, cellular oxygen consumption, NAD+/NADH ratio and ROS generation in cells under hypoxia mimicking conditions.

The introduction of an increasing number of individualized molecular targeted therapies into clinical routine mirrors their importance in modern cancer prevention and treatment. Well-known examples for targeted agents are the monoclonal antibody trastuzumab and the selective estrogen receptor modulator tamoxifen. The identification of an unaltered gene in tumor tissue in colon cancer (KRAS) is a predictor for the patient’s response to targeted therapy with a monoclonal antibody (cetuximab). Targeted therapy for hereditary breast and ovarian cancer has become a reality with the approval of olaparib for platin-sensitive late relapsed BRCA-associated ovarian cancer in December 2014. This manuscript reviews the status quo of poly-ADP-ribose polymerase inhibitors (PARPi) in the therapy of breast and ovarian cancer as well as the struggle for carboplatin as a potential standard of care for triple-negative and, in particular, BRCA-associated breast cancer. Details of the mechanism of action with information on tumor development are provided, and an outlook for further relevant research is given. The efficacy of agents against molecular targets together with the identification of an increasing number of cancer-associated genes will open the floodgates to a new era of treatment decision-making based on molecular tumor profiles. Current clinical trials involving patients with BRCA-associated cancer explore the efficacy of the molecular targeted therapeutics platinum and PARPi.

No description available.

Zelluläre Seneszenz wird als terminaler Zellzyklusarrest definiert, der mit dem Altern und funktionellen Verlust von Geweben verknüpft ist. Eine Seneszenzreaktion wird ebenso durch Onkogene und zytotoxischen Stress verursacht. Die Ausführung des Seneszenzprogramms wird durch eine zeitlich hochdynamische Aktivität von Transkriptionsfaktoren (TF) bedingt. Interessanterweise kann die Zelllinienzugehörigkeit einer Zelle durch die Expression von linien-aberranten TF überschrieben werden. Die vorliegende Arbeit untersucht Chemotherapie-induzierte Seneszenz (TIS) in Bcl-2 überexprimierenden, deshalb vor Apoptose geschützten, murinen Eµ-Myc B-Zell Lymphomen in An- oder Abwesenheit der Seneszenz-essentiellen Histonmethyltransferase Suv39h1. Analysen auf Transkriptom- und auf Proteinebene ergeben dabei, dass in einer Seneszenz-spezifischen Weise die TF AP-1, PU.1 und C/EBPβ induziert werden, welche normalerweise für die Funktion und Entwicklung myeloischer Zelllinien bedeutend sind. Dementsprechend korreliert der Seneszenzzustand mit Transkripten, Oberflächenmarkern und einer enzymatischen Funktion der myeloischen Linie. Indem die identifizierten TFs heruntergeschaltet oder überexprimiert werden, wird ihre direkte Beteiligung an der Linienuntreue der TIS Lymphome demonstriert. TIS-Kapazität wird als für den Erfolg von Krebstherapie günstige Eigenschaft betrachtet, da sie zu einem Wachstumsblock führt. Nichtsdestotrotz können sich verweilende TIS Zellen krebsbiologisch auch nachteilig auswirken. Anhand von murinen und humanen, klinisch annotieren Transkriptomdatensätzen kann hier in beiden Spezies ein myeloisch verschobenes, Linienuntreue anzeigendes Genexpressionsprofil mit einer besseren Überlebensprognose korreliert werden. Die vorliegenden Befunde legen nahe, dass die Modulation von TF Aktivitäten in Seneszenz einen potentiellen therapeutischen Angriffspunkt darstellt, um den für den Therapieerfolg nützlichen Zweig des TIS Phänotyps zu befördern. / Cellular senescence is regarded as an irreversible cell cycle arrest associated with tissue aging and its functional decline. A senescence response is also evoked by oncogenic and cytotoxic stress. The execution of the senescence program relies on a highly dynamic sequence of transcription factor (TF) activities. Interestingly, cell lineage commitment can be overridden by the expression of lineage-aberrant TFs. This thesis examines chemotherapy-induced senescence (TIS) in Bcl-2 overexpressing, thus apoptosis-protected, murine Eμ-Myc B-cell lymphomas with or without the senescence-essential histone methyltransferase Suv39h1. Transcriptome as well as protein level analyses reveal senescence-specific induction of the TFs AP-1, PU.1 and C/EBPβ which are typically crucial for myeloid lineage commitment and function. Correspondingly, the senescent state associates with myeloid lineage transcripts, surface markers and enzymatic function, reminiscent of, but not equal to a transdifferentiation phenotype. By knocking down and overexpressing the identified TFs, we demonstrate their direct involvement in the lineage infidelity of TIS lymphomas. TIS-capacity is viewed as beneficial to cancer therapy outcome due to its block on proliferation. However, lingering TIS cells can also be detrimental due to the acquisition of latent stemness properties or tumor-protective remodeling of their microenvironment. By interrogating murine and human, clinical course-annotated transcriptome data sets, an association between a myeloid-skewed, lineage infidelity indicating gene expression profile and better tumor prognosis is established in both species. The presented findings suggest that modulation of the senescent TF activities could be therapeutically exploited to foster the cancer patient-beneficial branch of the TIS phenotype.

Error-free DNA replication is paramount to maintaining genomic integrity. Despite being highly regulated, the process of DNA replication is often challenged by various intrinsic and extrinsic sources of replication stress. Failure to maintain the DNA replication quality reduces genomic stability, cell survival and results in diseases, such as cancer. Thus, cells rely on the replication stress response that detects perturbations in DNA replication and pauses or arrests cellular replication. Similar to other intrinsic replication obstacles, oncogene expression also induces the replication stress response that acts as a barrier to cancer, thereby mystifying how cancer develops. Here, we demonstrate that oncogene expression, similar to other replication stress inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness unless counteracted by translesion synthesis (TLS). Moreover, we find that TLS subverts the replication stress response in a wide range of cancer cell lines indicating that TLS is a previously unappreciated and unique cancer vulnerability. Mechanistically, we reveal that upon replication stress, TLS restricts replication fork slowing, reversal, and fork degradation, while maintaining continuous replication. Furthermore, we demonstrate that a small molecule inhibitor targeting the TLS factor, REV1, not only disrupts DNA replication and cancer cell fitness, but also synergizes with other therapies that induce replication gaps. Thus, our study places TLS at the center of cancer cell fitness as a necessary adaptation to overcome replication stress.

Established biopharmaceuticals such as antibodies and derivatives thereof are relatively large. In cancer therapy, this creates a steep drug concentration gradient within tumors, leaving cells far from blood vessels effectively untreated. Continuous pseudo treatments should foster the development of drug resistance and might lead to eventual disease relapse. Drug concentration gradients can be operationalized as tissue penetration efficiencies, which are functions of molecular size. However, small particles are also subject to potent renal clearance, collapsing the therapeutic window beyond clinical applications. In this master’s thesis, spatial bispecificity was engineered into a single albumin binding domain (ABD). Resulting ABD derived affinity proteins (ADAPTs) are saved from urinary excretion by the grace of HSA, but in the more static microenvironment of tumors, following HSA dissociation, they are capable of tissue penetration efficiencies bestowed only upon smaller particles. To this end, phage display was used to raise ADAPTs against the cancer associated proteins human epidermal growth factor receptor 2 (HER2) and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), but also the inflammation marker C-reactive protein (CRP). Via Sanger sequencing, 9 variants were picked for protein production and characterization, among which two spatially bispecific binders were found. ADAPTs were also evaluated for aggregation tendencies, structural conformity to library design, and thermal stability. One ADAPT, binding HER2, passed all tests of initial characterizations. Deep sequencing was used to analyze selection output, from which many more binders should be screened in future experiments. / Etablerade bioläkemedel liksom antikroppar och deras derivat är relativt stora protein. Som cancerterapeutiska skapar de således branta koncentrationsgradienter utgående från tumörpenetrerande blodkärl. Detta riskerar att lämna vissa cancerceller utanför det terapeutiska fönstret. Det svaga selektionstryck som således verkar i tumörperiferin fostrar cancerceller till att utveckla resistens mot detsamma. Koncentrationsgradienten beror på proteinets vävnadspenetrarande förmåga, vilken är en funktion av proteinets storlek. Mindre proteiner borde därmed lättare ackumuleras i hela tumören och förebygga resistensutveckling. Problemet med små proteiner är deras mycket korta halveringstid i serum, en följd av relativt obehindrad filtrering ut i urinen via njurarna. I det här examensarbetet utvecklades rumsbispecifika bindare av cancerassocierade protein med hjälp av fagdisplayselektioner från ett proteinbibliotek baserat på en enda albuminbindande domän (ABD). Resulterande ABD deriverade affinitetsprotein (ADAPT) undkommer ovan nämnda filtrering tack vare sin naturligt starka interaktion med humant serumalbumin (HSA). I den mer långsamt flödande tumörmikromiljön tillåts ADAPTerna efter albumindissociation sedan utöva en bland bioläkemedel överlägsen vävnadspenetration. Tre parallella selektionsspår utfördes mot de cancerassocierade målproteinerna human epidermal growth factor receptor 2 (HER2) och carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) samt den utsöndrade inflammationsmarkören C-reaktivt protein (CRP). Via Sangersekvensering kunde flera kandidater identifieras. Bland 6 karakteriserade ADAPTer uppvisade samtliga hög HSA-affinitet, tre konstaterades interagera specifikt med sitt målprotein, och två verkade binda även rumsbispecifikt. ADAPTer utvärderades även för sin benägenhet att bilda aggregat, strukturell överensstämmelse med experimentell design, och värmestabilitet. Endast en bindare, mot HER2, klarade sig genom alla prövningar som proteinkarakteriseringen innebar utan underkänt. Även en högparallel sekvensering utav selektionsresultat utfördes, men utanför de tidsramar som tillät ytterligare karakterisering.

No description available.

The development of novel, tumor-selective and boron-rich compounds as potential agents for use in boron neutron capture therapy (BNCT) represents a very important field in cancer treatment by radiation therapy. Here, we report the design and synthesis of two promising compounds that combine meta-carborane, a water-soluble monosaccharide and a linking unit, namely glycine or ethylenediamine, for facile coupling with various tumor-selective biomolecules bearing a free amino or carboxylic acid group. In this work, coupling experiments with two selected biomolecules, a coumarin derivative and folic acid, were included. The task of every component in this approach was carefully chosen: the carborane moiety supplies ten boron atoms, which is a tenfold increase in boron content compared to the l-boronophenylalanine (l-BPA) presently used in BNCT; the sugar moiety compensates for the hydrophobic character of the carborane; the linking unit, depending on the chosen biomolecule, acts as the connection between the tumor-selective component and the boron-rich moiety; and the respective tumor-selective biomolecule provides the necessary selectivity. This approach makes it possible to develop a modular and feasible strategy for the synthesis of readily obtainable boron-rich agents with optimized properties for potential applications in BNCT.

The amount of boron accumulated in tumor tissue plays an important role regarding the success of the boron neutron capture therapy (BNCT). In this article, we report a modular system, combining readily available starting materials, like glycine, 1,3,5-triazine and the well-known 9-mercapto-1,7-dicarba-closo-dodecaborane(12), as well as alpha-d-galactopyranose for increased hydrophilicity, with a novel boron-rich tris-meta-carboranyl thiol.