Upon activation in epidermal stem cells, the proto-oncogene c-Myc triggers their exit from the stem cell compartment resulting in an increase in progenitor cell proliferation and an induction in terminal differentiation. Whether c-Myc plays a direct transcriptional role in epidermal stem cell differentiation was unknown. The exploration of c-Myc's transcriptional roles at the epidermal differentiation complex (EDC), a locus essential for skin maturation demonstrated that binding of c-Myc to the EDC can simultaneously recruit and displace specific sets of differentiation-specific transcriptional regulators to EDC genes. Among these factors, Sin3A acts as a transcriptional co-repressor and was initially discovered via its direct interaction with Mxi1 and Mxd1, which are antagonists of the Myc family network. As such, I concentrated on the role of Sin3A as a potential opposing factor to c-Myc activity in the epidermis. To analyse the role of Sin3A in regulating epidermal stem cell fate in vivo, I generated a number of transgenic mouse models. To determine whether Sin3A functions in hair follicle stem cells, I inducibly deleted Sin3A in the hair follicle bulge, where quiescent stem cells reside. However, lack of Sin3A in the hair bulge did not cause any aberrant phenotype and I concluded that Sin3A is dispensable for hair follicle homeostasis. I next analysed a mouse model in which Sin3A is inducibly deleted in the basal layer of the epidermis. Deletion of Sin3A resulted in a severe disruption of epidermal homeostasis-namely due to increases in proliferation and differentiation. Further investigation demonstrated that this phenotype is driven by enhanced genomic recruitment of c-Myc to the epidermal differentiation complex and reactivation of c-Myc target genes involved in cellular proliferation. I found that Sin3A causes de-acetylation of the c-Myc protein to directly repress c-Myc’s transcriptional activity and is antagonistic to c-Myc in the interfollicular epidermis. I hypothesised that simultaneous deletion of Sin3A and c-Myc might return the skin to normality. Indeed, when Sin3A and Myc are concurrently deleted, proliferation and differentiation levels returned to normal. These results demonstrate how levels of Sin3A and c-Myc must be carefully balanced for epidermal homeostasis to be maintained. Decreased expression of Sin3A has been linked to tumour susceptibility in other tissues for example in non-small cell lung carcinoma making Sin3A a candidate tumour suppressor gene. I therefore considered that loss of Sin3A may lead to increased susceptibility to skin cancer. To investigate this I performed pilot experiments using UVB irradiation of skin that has one copy of Sin3A deleted in the basal layer of the epidermis. Under normal conditions, these mice have no identifiable phenotype, but pilot experiments demonstrated that after short term and long term UVB irradiation, they exhibit increased epidermal thickness and proliferation relative to controls. This recapitulated the phenotype observed when Sin3A is inducibly deleted in the interfollicular epidermis and further demonstrates the role of SinA as an inhibitor of proliferation in this tissue. Overall, these results demonstrate that an interplay between the opposing functions of Sin3A and c-Myc are necessary to ensure that there is balanced homeostasis in the interfollicular epidermis.

Das kolorektale Karzinom ist eines der häufigsten beim Menschen vorkommenden Karzinome [2]. Diesem liegen unterschiedliche Mutationen zugrunde, die in knapp 100% der kolorektalen Karzinome zu einer Überexpression von MYC führen, welches als Transkriptionsfaktor maßgeblich den Zellzyklus, Proliferation und Vaskularisierung beeinflusst [10,16]. Damit stellt MYC ein potenzielles Therapieziel in der Behandlung des Kolorektalen Karzinoms dar. Zusätzlich konnte in den letzten Jahren ein Onkoprotein namens CIP2A identifiziert werden, welches nach Depletion mit einem Verlust von MYC Protein einhergeht [69]. Zusätzlich ist CIP2A ein unabhängiger prognostischer Faktor im Kolorektalen Karzinom [70]. Diese Arbeit konnte zeigen, dass CIP2A-depletierte Zellen einen deutlichen Wachstumsnachteil gegenüber unbehandelten Zellen zeigen. Dieser Unterschied kann nicht durch eine gesteigerte Apoptose, sondern vielmehr durch einen verlängerten Zellzyklus erklärt werden. Weiterhin konnte eine neue Zelllinie mit DOX-induzierbarer shCIP2A hergestellt werden, die für weitere Experimente genutzt werden kann. Entgegen der Wirkweise im Zervixkarzinom [69], konnte im kolorektalen Karzinom kein Einfluss auf die Stabilität von MYC Protein durch CIP2A nachgewiesen werden. Auch konnte der Verlust von MYC nach CIP2A Knockdown nicht durch gleichzeitige Inhibierung des Abbaus, durch Okadasäure, MG132 oder in den FBWX7-defizienten Zellen, verhindert werden. Stattdessen resultiert die Herunterregulation von CIP2A in einem leichten Rückgang der MYC-mRNA Menge und einem deutlichen Verlust an MYC-Protein. In Zellen mit verschiedenen Konstrukten der MYC Transkripte kann dieser Verlust an MYC Protein auf eine translationelle Regulation in der 5’UTR zurückgeführt werden, was eine bisher nicht beschriebene Wirkweise von CIP2A darstellt. Da CIP2A in normalen Zellen praktisch nicht exprimiert ist [78], könnte dies ein mögliches Ziel in der Tumortherapie darstellen. Dieses gilt es in weiteren Experimenten noch genauer zu untersuchen. / Colorectal Cancer is one of the most common type of cancer in human beings [2]. These are based on different mutations, which, in nearly 100%, lead to overexpression of MYC. As an transcription factor, MYC influences cell cyclus, proliferation and vascularization [10,16]. So MYC appears to be a good target in the therapy of colorectal cancer. Additionally a oncoprotein called CIP2A could be identified in the last years, which depletion leads also to a loss of MYC protein [69]. CIP2A was also found to be a independent prognostic factor in colorectal cancer [70]. In this work it could be demonstrated, that CIP2A-depleted cells show disadvantage in cell growth compared to the untreated cells. This difference could not be explained through an increased cell death, but an extended cell cycle. Additionally, a new cell line with DOX-inducible shRNA against CIP2A was established, which can be used for further experiments. Contrary to the mode of action which was found in cells of cervix carcinoma [69] we could not see an influence of CIP2A on the stability of MYC. Furthermore, the loss of MYC protein after knockdown of CIP2A could not be prevented by simultaneous inhibition of MYC degradation by okadaic acid, MG132 or in FBWX7-deficient cells. Instead knockdown of CIP2A lead to little decrease of MYC-mRNA and a clear loss of MYC protein. In cells with different constructs of MYC mRNA the loss of MYC protein can be attributed to a regulation in the 5’UTR. Because CIP2A is rarely expressed in normal tissue [78] it seems to be a possible target in the treatment of colorectal cancer. This should be further evaluated in future experiments.

The transcription factor MYC is deregulated in over 70% of all human tumors and, in its oncogenic form, plays a major role in the cancer metabolic reprogramming, promoting the uptake of nutrients in order to sustain the biosynthetic needs of cancer cells. The research presented in this work aimed to understand if MYC itself is regulated by nutrient availability, focusing on the two major fuels of cancer cells: glucose and glutamine. Initial observations showed that endogenous MYC protein levels strongly depend on the availability of glutamine, but not of glucose. Subsequent analysis highlighted that the mechanism which accounts for the glutamine-mediated regulation of MYC is dependent on the 3´-untranslated region (3´-UTR) of MYC. Enhanced glutamine utilization by tumors has been shown to be directly linked to MYC oncogenic activity and MYC-dependent apoptosis has been observed under glutamine starvation. Such effect has been described in experimental systems which are mainly based on the use of MYC transgenes that do not contain the 3´-UTR. It was observed in the present study that cells are able to survive under glutamine starvation, which leads to cell cycle arrest and not apoptosis, as previously reported. However, enforced expression of a MYC transgene, which lacks the 3´-UTR, strongly increases the percentage of apoptotic cells upon starvation. Evaluation of glutamine-derived metabolites allowed to identify adenosine nucleotides as the specific stimulus responsible for the glutamine-mediated regulation of MYC, in a 3´-UTR-dependent way. Finally, glutamine-dependent MYC-mediated effects on RNA Polymerase II (RNAPII) function were evaluated, since MYC is involved in different steps of global transcriptional regulation. A global loss of RNAPII recruitment at the transcriptional start site results upon glutamine withdrawal. Such effect is overcome by enforced MYC expression under the same condition. This study shows that the 3´UTR of MYC acts as metabolic sensor and that MYC globally regulates the RNAPII function according to the availability of glutamine. The observations presented in this work underline the importance of considering stress-induced mechanisms impinging on the 3´UTR of MYC. / In über 70% aller Krebserkrankungen ist der Transkriptionsfaktor MYC dereguliert. Dabei spielt onkogenes MYC unter anderem eine wichtige Rolle bei der Umprogrammierung metabolischer Prozesse indem es z.B. die Aufnahme von Nährstoffen wie Glutamin oder Glukose fördert, um den veränderten Bedürfnissen an den Stoffwechsel der Krebszellen Rechnung zu tragen. Die im Rahmen dieser Arbeit erzielten Ergebnisse zeigen, dass auch das MYC-Protein selbst durch die Verfügbarkeit von Nährstoffen in der Zelle reguliert werden kann. Erste Beobachtungen zeigten, dass die endogenen MYC Proteinlevel stark von der Verfügbarkeit von Glutamin, jedoch nicht von Glucose, abhängen. Weiterführende Experimente ergaben außerdem, dass der Mechanismus, der der Glutamin vermittelten Regulation von MYC zugrunde liegt, abhängig von der 3´-untranslatierten Region (3´-UTR) der MYC-mRNA ist. Es konnte bereits gezeigt werden, dass in Tumoren die verstärkte Nutzung von Glutamin in direktem Zusammenhang mit der onkogenen Aktivität von MYC steht und Zellen unter Glutaminentzug MYC-abhängig Apoptose einleiten. Diese Effekte wurden in experimentellen Systemen beschrieben, die auf einer Überexpression eines MYCTransgenes basierten, welches keine 3´-UTR enthält. In dieser Arbeit konnte jedoch beobachtet werden, dass Zellen, die ohne Glutamin kultiviert wurden, in der Lage waren zu überleben, da entgegen den Resultaten vorausgegangener Studien, ein Arrest des Zellzyklus und nicht Apoptose eingeleitet wurde. Die verstärkte Expression eines MYCTransgenes ohne 3´-UTR, erhöhte jedoch auch unter diesen Bedingungen die Anzahl apoptotischer Zellen. Weiterhin war es möglich Adenosin, für dessen Biosynthese Glutamin notwendig ist, als Stimulus zu identifizieren, der für die 3´-UTR abhängige Regulation von MYC verantwortlich ist. Da MYC in verschiedene Schritte der globalen Regulation der Transkription eingebunden ist, wurden abschließend die durch MYC vermittelten Glutaminabhängigen Effekte auf die RNA-Polymerase II (RNAPII) untersucht. Dabei zeigte sich, dass es nach Glutaminentzug zu einem globalen Verlust der Rekrutierung von RNAPII zu den Transkriptionsstartstellen kommt, was durch eine verstärkte MYC-Expression wieder aufgehoben werden kann. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass die 3´-UTR von MYC als metabolischer Sensor fungiert und dass MYC in Abhängigkeit der Verfügbarkeit von Glutamin global die RNAPII Funktion reguliert. Diese Studie hebt weiterhin die Bedeutung der 3´-UTR von MYC für die Vermittlung stressinduzierter Feedback-Mechanismen hervor.

Yes / Breast cancer is a leading cause of cancer-related deaths. Anemia is common in breast cancer patients and can be treated with blood transfusions or with recombinant erythropoietin (EPO) to stimulate red blood cell production. Clinical studies have indicated decreased survival in some groups of cancer patients treated with EPO. Numerous tumor cells express the EPO receptor (EPOR), posing a risk that EPO treatment would enhance tumor growth, but the mechanisms involved in breast tumor progression are poorly understood. Here, we have examined the functional role of the EPO-EPOR axis in preclinical models of breast cancer. EPO induced the activation of PI3K/AKT and MAPK pathways in human breast cancer cell lines. EPOR knockdown abrogated human tumor cell growth, induced apoptosis through Bim, reduced invasiveness, and caused downregulation of MYC expression. EPO-induced MYC expression is mediated through the PI3K/AKT and MAPK pathways, and overexpression of MYC partially rescued loss of cell proliferation caused by EPOR downregulation. In a xenotransplantation model, designed to simulate recombinant EPO therapy in breast cancer patients, knockdown of EPOR markedly reduced tumor growth. Thus, our experiments in vitro and in vivo demonstrate that functional EPOR signaling is essential for the tumor-promoting effects of EPO and underline the importance of the EPO-EPOR axis in breast tumor progression. / Cancer Research UK - C10141/A9977 (TRL, MET, KKC). European Commission FP7 (EpoCan) 282551 (TRL, KBM); Invest NI RD0914223 (TRL, KBM).

Drosophila melanogaster is a valuable model for studying various aspects of human cancer, including proliferative capacity, invasiveness and metabolic adaptation typical of tumour cells to support cell growth. One of the major players in this process is Myc, which can promote tumorigenesis by triggering a metabolic reprogramming that allows cells to produce macromolecules, by modulating glycolytic flux, glutaminolysis, lipidogenesis, and autophagy. The process by which hyperproliferative cells undergo metabolic reprogramming to sustain growth can be recapitulated in the epithelial cells from Drosophila imaginal discs, where different levels of Myc induce cell competition. This process is a mechanism for selection of cells expressing higher level of Myc that acquire a super-competitor condition, with the ability to non-autonomously kill the neighbouring slow-growing cells. The direct connection between Myc, glutamine metabolism and autophagy and their role in competitive events between cancerous cells and wild type cells have not been clearly explained; therefore, the main purpose of this project is to determine a plausible link between Myc and autophagy, by examining the dependency of Myc-induced autophagy on glutaminase and major regulators of autophagy, such as TOR, Atg1, Atg5 and ammonia, a by-product of glutamine catabolism, by dissecting these mechanisms both in normal epithelial clones and hyperproliferating RasV12 -expressing cells. Our results show that Myc promotes the transcription of glutamine-related genes and the production of ammonia, and that glutaminase is necessary for Myc-induced autophagy in epithelial cells of clones of the wing imaginal discs, with a mechanism independent from TOR and Atg1. Conversely, the effect of Myc on autophagy induction is mediated by Atg5. We then investigated the contribute of Myc in autophagy in RasV12-transformed cells, that upregulate Myc to sustain growth and hyperproliferation. Intriguingly, our data report that autophagy is increased non-autonomously in neighbouring wild type cells, and that this non-autonomous RasV12-driven autophagic flux depends on Myc activity. Moreover, downregulation of glutaminase in RasV12-expressing cells significantly reduces non-autonomous autophagy. Collectively, our results give new insights on how glutamine metabolism can contribute to Myc-induced autophagy and how this enhances cancerous cell fitness.

Pancreatic ductal adenocarcinoma (PDAC) is predominantly driven by mutations in KRAS and TP53. However, PDAC tumors display deregulated levels of MYC and are a paradigm example for MYC-driven and -addicted tumors. For many years MYC was described as a transcription factor that regulates a pleiotropic number of genes to drive proliferation. Recent work sheds a different light on MYC biology. First, changes in gene expression that come along with the activation of MYC are mild and MYC seems to act more as a factor that reduces stress and increases resilience towards challenges during transcription. Second, MYC is a strong driver of immune evasion in different entities. In this study we depleted MYC in murine PDAC cells and revealed the immune dependent regression of tumors in an orthotope transplant model, as well as the activation of the innate immune system using global expression analysis, immunoblotting and fCLIP. These experiments revealed that endogenous double-stranded RNA is binding as a viral mimicry to Toll-like receptor 3, causing activation of TBK1 and downstream activation of a proimmunogenic transcription program. The regression of tumors upon depletion of MYC is dependent on this pathway since the knockout of TBK1 prevents regression of tumors after depletion of MYC. We can summarize this study in three main findings: First, the dominant and most important function of MYC in tumors is not to drive proliferation but to promote immune evasion and prevent immune-dependent regression of tumors. Second, cells monitor defects or delay in splicing and RNA processing and activate the immune system to clear cells that face problems with co-transcriptional processing. Third, MYC suppresses the activation of the cell-intrinsic innate immune system and shields highly proliferating cells from the recognition by the immune system. To translate this into a therapeutically approach, we replaced the shRNA mediated depletion of MYC by treatment with cardiac glycosides. Upon treatment with cardiac glycosides tumor cells reduce uptake of nutrients, causing a downregulation of MYC translation, inhibition of proliferation, glycolysis and lactate secretion. Lactate is a major reason for immune evasion in solid tumors since it dampens, amongst others, cytotoxic T cells and promotes regulatory T cells. Treatment of mice with cardiac glycosides causes a complete and immune-dependent remission of PDAC tumors in vivo, pointing out that cardiac glycosides have strong proimmunogenic, anti-cancer effects. More detailed analyses will be needed to dissect the full mechanism how cardiac glycosides act on MYC translation and immune evasion in PDAC tumors. / Pankreaskarzinome entwickeln sich in den meisten Fällen durch die Mutation von KRAS und TP53. Nichtsdestotrotz weisen Pankreaskarzinome sehr hohe, deregulierte Level des MYC Proteins auf und sind exemplarisch für Tumore, deren Wachstum abhängig von MYC ist. Für lange Zeit wurde MYC als Transkriptionsfaktor beschrieben, der vor allem Gene aktiviert, die für die Proliferation von Zellen notwendig sind. Die jüngste Forschung wirft jedoch ein anderes Bild auf die Biologie von MYC. Zum einen sind die transkriptionellen Veränderung nach Aktivierung von MYC mild und vieles deutet darauf hin, dass die Funktion von MYC zum einen in der Reduktion von transkriptionellem Stress liegt. Zum anderen verhindert MYC die Erkennung von Tumorzellen durch das Immunsystem. In dieser Studie wurde MYC in murinen Pankreaskarzinomzellen depletiert und die immunabhängige Regression der Tumore in einem orthotopen Transplantationsmodel untersucht. Die Aktivierung der zell-intrinsischen Immunantwort wurde mittels globaler Expressionsanalyse, Immunoblots und fCLIP Experimenten untersucht. Diese Experimente haben gezeigt, dass endogene doppel-strängige RNAs als virales Mimikry an Toll-like Rezeptor 3 binden, worauf TBK1 phosphoryliert und ein pro-immunogenes Transkriptionsprogram aktiviert wird. Die Deletion von TBK1 konnte beweisen werden, dass die Regression der Tumore von diesem Signalweg abhängig ist. Die Ergebnisse der Studie lassen sich in drei zentrale Erkenntnisse zusammenfassen: Erstens ist die prä-dominante Funktion von MYC in Tumoren in vivo nicht die Proliferation der Zellen zu fördern, sondern zu verhindern, dass der Tumor vom Immunsystem erkannt und bekämpft wird. Zweitens überwachen Zellen das richtige Prozessieren von RNA und aktivieren bei Bedarf das Immunsystem um defekte Zellen zu entfernen. Drittens unterdrückt MYC die Aktivierung dieses zell-intrinsischen Signalweges und schirmt den Tumor dadurch vom Immunsystem ab. Um diese Ergebnisse in einen translationalen Ansatz zu überführen, haben wir die shRNA vermittelte Depletion von MYC durch die Behandlung mit Herzglykosiden ersetzt, die die Aufnahme von Nährstoffen reduzieren, Proliferation einschränken, die Translation des MYC Proteins stoppen und Glykolyse und Laktatsekretion herunterfahren. Laktat hat starke immunsuppressive Eigenschaften in soliden Tumoren, da es zytotoxische T Zellen erschöpft und regulatorische T Zellen aktiviert. Die Behandlung von Mäusen mit Herzglykosiden führte in Abhängigkeit vom Immunsystem zur kompletten Remission der Tumore, auch wenn noch weitere Forschung notwendig ist, um die exakten Zusammenhänge besser zu verstehen.

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Breast cancer (BC) is a complex disease with over 25,000 new diagnoses made in Canadian women every year. The disease can be caused by inactivation of the ataxia telangiectasia mutated (ATM) pathway, a major anti-tumor mechanism that protects against the abnormal cell division and growth that occurs in breast cancer, but how the pathway is inactivated has yet to be completely elucidated. BMI1 is an established oncogene that is overexpressed in BC and is associated with poor disease prognosis. BMI1 is a component of the polycomb repressive complex 1 (PRC1) that acts to repress transcription of the ARF/INK4A locus encoding two important tumor suppressor genes. We have recently shown a novel property of BMI1 in attenuation of ATM function independent of this locus. We thus hypothesize a role of BMI1 in promoting BC formation through inhibiting oncogene-induced ATM activation, allowing cancer-promoting genes to induce abnormal cellular growth. To examine this hypothesis, we transiently expressed oncogene c-Myc with or without BMI1 co-expression. As expected, ectopic c-Myc expression upregulated γH2AX, a demonstrated target of ATM; concurrent BMI1 expression reduced the γH2AX levels. Similar observations were also obtained using a BMI1 mutant deficient in promoting PRC1-mediated repression of the ARF/INK4A locus. These observations support the concept that BMI1 contributes to ATM inactivation during BC tumorigenesis through mechanisms independent of PRC1. To further examine this concept, we investigated the association of γH2AX and BMI1 in vivo. In MCF7 cell-produced xenograft tumors, the presence of γH2AX nuclear foci was clearly observed, indicative of ATM activation during BC tumorigenesis. In xenografts generated by MCF7 cells stably expressing BMI1, a trend of reduction in γH2AX nuclear foci was observed. To further model BMI1’s pathological relevance in c-Myc induced BC under a more physiological setting, we are developing transgenic mouse models (GEM) with breast-specific c-Myc expression with or without a breast-specific BMI1 knockout. The goal of these experiments is to recapitulate the above in vitro and in vivo observations. The expectation, should it be achieved, will significantly strengthen the connection between BMI1 and ATM during breast cancer tumorigenesis. / Thesis / Master of Science (MSc) / Breast cancer (BC) is a complex disease with over 25,000 new diagnoses made in Canadian women every year. Normally there are anti-tumor mechanisms in place to protect against the abnormal cell division and growth that is associated with breast cancer. We propose a novel function of protein BMI1 to explain how breast cancer cells override these protective pathways. BMI1 is known to contribute to BC through inhibiting production of key tumor suppressing proteins and has recently been shown to decrease activity of the ataxia telangiectasia mutated (ATM)- mediated tumor inhibiting pathway. We propose a novel role of BMI1 in promoting breast tumor formation through inhibiting the ATM-mediated anti-tumor barrier, allowing cancer-promoting genes (oncogenes) to induce abnormal cellular growth. BMI1 was shown to be able to reduce oncogene induced ATM activity, in an action independent of established mechanisms. Additionally in MCF7 BC tumors, the presence of BMI resulted in a trend of reduction in ATM activity. Continued work to develop a transgenic mouse model with a breast specific BMI1 knockout will help further our understanding of BMI1’s role in BC tumorigenesis.

La dérégulation de l'oncogène c-myc sensibilise les cellules à la mort programmée induite par le cisplatine en agissant sur la voie mitochondriale de l'apoptose. Les membres de la famille de protéines Bcl-2 sont essentiels à la régulation de ce sentier apoptotique. Parmi ceux-ci on retrouve une sous-famille de protéines pro-apoptotiques incluant Bid, Bad, Bim, Noxa et PUMA qui ont comme caractéristique de posséder un domaine BH3 unique. Suite à divers stress cellulaires, ces protéines BH3 interagissent avec les autres membres de la famille Bcl-2 via le domaine BH3 et favorisent ainsi la sortie du cytochrome c de la mitochondrie et l'initiation de la cascade des caspases. / L'influence de la dérégulation de l'oncogène c-myc sur l'expression, la phosphorylation et/ou localisation intracellulaire des protéines BH3 a donc été analysée. Ainsi, il semble que Bid et Noxa ne soient pas impliquées car ni leur expression ni leur localisation intracellulaire sont influencées par c-Myc. Par contre, c-Myc augmente légèrement l'expression de Bim et module sa localisation intracellulaire. Les résultats démontrent également que la phosphorylation de Bad est négativement régulée par c-Myc, mais que son expression et sa localisation en sont indépendantes. Finalement, l'expression de PUMA, tant au niveau de l'ARNm que de la protéine, est augmentée par c-Myc, mais cette protéine ne semble pas jouer de rôle fonctionnel dans cette apoptose. Ainsi, au moins trois protéines BH3 sont modulées par c-Myc mais aucune n'a pu être identifiée clairement comme étant impliquée dans l'apoptose dépendante de c-Myc suite à un traitement au cisplatine.