Prostate cancer is the most common invasive cancer diagnosed in men and a major and growing health problem in Western countries. Deregulation of different pathways has been implicated in progression of prostate cancer, namely nuclear factor kappa enhancer binding protein (NF-κB), transforming growth factor β (TGFβ), phosphoinositide 3ʹ-kinase/AKT (PI3K/AKT) and Src kinase pathways. However, the detailed mechanisms by which TGFβ activates these pathways to contribute in tumorigenesis and invasive behavior of prostate cancer cells have not been elucidated. We have demonstrated (paper I) that the E3 ligase activity of TRAF6 is crucial for recruitment of the regulatory subunit of PI3K, p85α, to TβRI and for TGFβ-induced Lys63-linked polyubiquitination of p85α. TRAF6 is required for the TGFβ-induced recruitment of AKT to the complex of PI3K and TβRI, where the polyubiquitination and activation of AKT occurs. When activated, AKT promotes TGFβ-induced cell migration which is dependent on p85 and PI3K activity, as well as on TRAF6, but not on TβRI kinase activity. Thus, TGFβ-induced activation of PI3K/AKT induces cell motility contributing to the progression of cancer. We have demonstrated (paper II) a pivotal role of TAK1 polyubiquitination in three different pathways, including TNFR, IL-1R, and TLR4 signaling. Lys63-linked polyubiquitination of TAK1 at Lys34 is essential for downstream signaling to NF-κB-mediated target gene expression in both cancer and immune cells. These findings are of importance for the understanding of the mechanism of activation of NF-κB in inflammation and may aid in the development of new therapeutic strategies to treat chronic inflammation and cancer. We have also shown (paper III) that TGFβ activates the tyrosine kinase Src via formation of a complex between TβRI and Src. The E3 ligase TRAF6 promotes the formation of the complex in a manner not dependent on its ubiquitin ligase activity, suggesting that TRAF6 acts as an adaptor. Moreover, the activation of Src is not dependent on the kinase activity of TβRI. On a functional level, Src activity was found to be necessary for TGFβ-induced chemotaxis. In conclusion, we have elucidated molecular mechanisms whereby TGFβ activates non-Smad pathways, i.e. PI3K and Src. Our findings shed light on the pro-tumorigenesis mechanisms of TGFβ. In addition, we have demonstrated how the activation of TAK1, an important component of the TGFβ non-Smad pathway, by TGFβ and other stimuli leads to the activation of NF-κB and thereby induction of inflammation which likely contributes to prostate cancer progression.

Energy balance refers to the relationship between energy intake and energy expenditure. Epidemiological studies have established a clear association between energy balance and cancer, however the underlying mechanisms are unclear. The objective of the current study was to evaluate the impact of caloric consumption on epithelial carcinogenesis and identify potential mechanisms of inhibition or enhancement. Using ICR female mice, we demonstrated that positive energy balance enhanced, while negative energy balance inhibited susceptibility to multistage carcinogenesis in mouse skin. We next evaluated diet-induced changes in the epidermal proliferative response. Calorie restriction (CR) significantly reduced epidermal hyperproliferation, in the presence and absence of tumor promotion, as compared to diet-induced obesity (DIO). Additional studies were conducted to determine the impact of dietary manipulation on TPA-induced growth factor signaling. CR reduced, while DIO increased insulin like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) activation, which subsequently modulated signaling downstream to Akt and mTOR. These diet-induced changes in growth factor signaling were confirmed under steady-state conditions in multiple epithelial tissues (i.e., skin, liver and dorsolateral prostate) in multiple mouse strains (FVB/N, C57BL/6 and ICR). Further analyses demonstrated that caloric consumption directly correlated with levels of cell cycle progression related proteins and inversely correlated with levels of cell cycle inhibitory proteins. Genetic reduction of circulating IGF-1, liver IGF-1 deficient (LID) mouse model, inhibited two-stage skin carcinogenesis, reduced epidermal hyperproliferation and attenuated IGF-1R and EGFR growth factor signaling during tumor promotion, similar to CR, suggesting a potential for IGF-1R and EGFR crosstalk. Further studies, demonstrated that IGF-1 induced EGFR activation in cultured mouse keratinocytes, possibly due to IGF-1R and EGFR heterodimerization or IGF-1 induced changes in EGFR mRNA expression. In vivo, CR reduced, while DIO increased IGF-1R and EGFR association during tumor promotion. Furthermore, CR attenuated EGFR ligand mRNA expression both in the presence and absence of TPA treatment. Collectively, these findings suggest that dietary energy balance modulates epithelial carcinogenesis, at least in part due to diet-induced changes in levels of circulating IGF-1, which then modulate IGF-1R and EGFR crosstalk and downstream signaling to cell cycle related proteins, subsequently altering epidermal hyperproliferation. / text

MicroRNAs are small noncoding RNAs that post-transcriptionally reduce protein output from most human mRNAs by mechanisms that are still obscure. This thesis provides insights into three aspects of microRNA biogenesis and function described below. MicroRNA precursors are excised from primary transcripts by the Microprocessor complex containing Drosha and DGCR8. Although most microRNAs are located in introns of protein-coding and noncoding genes, the mechanisms coordinating microprocessing and splicing are unclear. MiR-211 is a microRNA expressed from intron 6 of melastatin, a suspected melanoma tumor suppressor. We demonstrate that miR-211, and not melastatin, is responsible for the tumor suppressive function of this locus, that Drosha-mediated processing of the miR-211 precursor promotes splicing of melastatin exon 6-exon 7 junctions, and that perturbing 5' splice site recognition by the U1 snRNP reduces Drosha recruitment to intron 6 specifically and intronic microRNA levels globally. Thus we identify a novel physical and functional coupling between microprocessing and splicing. Typically, Agos stabilize mature microRNAs and as a complex stoichiometrically bind to complementary mRNAs. We demonstrate an alternative order of events in which Agos bind and repress pre-formed imperfect microRNA-mRNA duplexes in processing bodies of live cells, and cleave pre-formed perfect microRNA-mRNA duplexes in vitro. Our data support a novel catalytic model whereby Agos first deposit microRNAs onto mRNAs and dissociate, thus priming multiple microRNA-mRNA duplexes for concurrent repression by a single Ago. Despite key roles in development and pathogenesis, effectors and regulators of microRNA-mediated repression are still poorly characterized. An RNAi screen revealed that depletion of ribosomal proteins of either small or large ribosomal subunit dissociates microRNA-containing complexes from mRNAs repressed at translation initiation, increasing their polysome association, translation, and stability relative to untargeted mRNAs. Thus ribosomal proteins globally regulate microRNA function. Another RNAi screen revealed that Akt3 phosphorylates Ago2, which negatively regulates cleavage and positively regulates translational repression of microRNA-targeted mRNAs. Thus Ago2 phosphorylation is a molecular switch between its mRNA cleavage and translational repression activities. The following pages will place these novel insights into biological and disease-relevant context, will describe what was known prior to these studies, and will provide perspectives for future studies.

Pleckstrin homology (PH) domains are structurally conserved domains, which generally bind to phosphatidylinositol phosphate (PtdInsP) lipids. They are present in a variety of proteins, including those that are upregulated in cancer growth and metastasis, and represent a crucial component of intracellular signaling cascades and membrane translocation. Thus, they may be considered as attractive targets for cancer drug therapy. AKT (protein kinase B), a pleckstrin homology lipid binding domain and a serine/threonine kinase-containing protein, is a key component of the phophatidylinositol-3-kinase (PI3K)/AKT cell survival signaling pathway which is activated in a variety of cancers, including prostate, pancreatic, and skin cancers. In this study, I report the finding of a novel inhibitor of AKT; PH-427. I describe its effects on binding to the PH domain of AKT thus preventing its binding to PtdIns3-P at the plasma membrane and subsequent activation. In vivo testing of the drug led to reduction of tumor size and numbers in a mouse pancreatic cancer model. Additional testing of PH-427 on squamous cell carcinomas revealed that the drug is able to reduce tumor burden and multiplicity in vivo when topically applied. Thus, we demonstrate proof-of-principle in targeting PH domains as a viable cancer drug therapy option. The effects of PH-427 raised the intriguing possibility that targeting PH domains may have beneficial effects in other signaling pathways with PH domain-containing proteins. Guanine exchange factors (GEFs) contain a Dbl homology (DH) domain and a PH domain and have been shown to be involved in the process of metastasis. More specifically, RacGEFs activate Rac1 GTPase by facilitating the exchange of GDP to GTP. Over-expression of certain GEFs has been shown to contribute to increased malignancy in a variety of cancers. T-lymphoma invasion and metastasis-inducing protein-1 (Tiam1) is a highly conserved GEF and contains an N-terminal pleckstrin homology domain (nPH) and a DH/C-terminal PH domain (cPH). Tiam1 has been found to be over-expressed in several cancers, including breast, colon and prostate cancers. In this study, I describe the identification, development, experimental testing, and potential mechanism of action of novel small molecule inhibitors targeting the RacGEF Tiam1 to inhibit prostate cancer bone metastasis.

Η ηπατική καρκινογένεση είναι μια πολυσταδιακή διαδικασία που οδηγεί προοδευτικά στον κακοήθη μετασχηματισμό του ηπατικού κυττάρου, μέσω ποικίλων μοριακών μηχανισμών. Πρόσφατα αναδεικνύεται ολοένα και σε μεγαλύτερο βαθμό, η τεράστια σημασία που έχει για την ανάπτυξη και εξέλιξη του καρκίνου το μικροπεριβάλλον του όγκου, η αλληλεπίδραση δηλαδή των καρκινικών κυττάρων με την εξωκυττάρια ουσία. Ιδιαίτερο ενδιαφέρον παρουσιάζει η αλληλεπίδραση των καρκινικών κυττάρων με την εξωκυττάρια ουσία μέσω των ιντεγκρινών, η οποία φαίνεται ότι εμπλέκεται σε όλα τα στάδια καρκινογένεσης. Σημαντικό μόριο στην προαναφερθείσα διαδικασία αποτελεί η ILK (Integrin-Linked Kinase), μία κινάση σερίνης-θρεονίνης που παρεμβάλλεται στα σηματοδοτικά μονοπάτια που ξεκινούν από ιντεγκρίνες, αυξητικούς παράγοντες και συμμετέχει στη ρύθμιση κομβικών για το καρκινικό κύτταρο λειτουργιών όπως έλεγχο του κυτταρικού κύκλου, απώλεια των δομών συνοχής του κυττάρου, αναστολή της απόπτωσης και ενεργοποίηση της αγγειογένεσης. Διαπιστώθηκε πως το σηματοδοτικό μονοπάτι ILK/p-Akt διαδραματίζει κομβικό ρόλο τόσο στη βιολογία της κίρρωσης όσο και του ηπατοκυτταρικού καρκίνου και πιθανόν αποτελεί μοριακό σύνδεσμο μεταξύ των δύο καταστάσεων, επιβεβαιώνοντας τη διατυπωμένη θεωρία περί άξονα χρόνιας φλεγμονής-ίνωσης (κίρρωσης)-καρκίνου. Επίσης στην παθογένεια της κίρρωσης και του καρκίνου ήπατος, φαίνεται πως ρόλο έχει το φαινόμενο της επιθηλιακής προς μεσεγχυματική μετατροπή (EMT-epithelial to mesenchymal transition). Αυτό αποδεικνύεται από την απώλεια έκφρασης της E-καντχερίνης και τη μεταφορά της β-κατενίνης στον πυρήνα που οδηγούν σε απώλεια των κυτταρικών συνδέσεων από τη μεμβράνη καθώς και από την υπερέκφραση των μορίων ILK και p-Akt. Τέλος η υπερέκφραση survivin και κυκλίνης-D1 στην κίρρωση και στο ηπακυτταρικό καρκίνωμα καταδεικνύουν τη σημασία της αντιαποπτωτικής δραστηριότητας και του αυξημένου κυτταρικού πολλαπλασιασμού στις συγκεκριμένες νοσολογικές οντότητες. / The hepatic carcinogenesis is a many phased process that leads progressively to the malignant transformation of hepatic cell via various molecular mechanisms. Recently, the enormous importance of the tumor microenvironment for the growth and development of cancer, i.e. the interaction between cancer cells and the extacellular matrix, has been proven. Particular interest is presented in the interaction of tumor cells with the extracellular matrix via integrins, which appears to be involved in all the stages of carcinogenesis. An important molecule in the aforementioned process is the ILK (Integrin-Linked Kinase), a serine-threonine kinase that is implicated in the signal transduction pathways that begin from integrines, growth factors and participate in the regulation of nodal for the tumor cell functions, such as control of cell cycle, loss of cell adhesion structures, suppresion of apoptosis and activation of angiogenesis. It has been realized that the signal transduction pathway ILK /p-Akt plays a nodal role so much in the biology of cirrhosis as well as in hepatocellular cancer and probably constitutes a molecular connection between the two conditions, confirming the formulated theory of axis of chronic inflamation-chirrosis-cancer. Also in the pathogenicity of cirrhosis and liver cancer, it appears that the epithelial to mesenchymal transition (EMT) phenomenon plays a role. This is proven by the loss of expression of E-cadherin and the transport of b-catenin in the nucleus that leads to loss of cellular connections from the membrane as well as from the overexpression of molecules ILK and p-Akt. Finally, the overexpression of survivin and cyclin-D1 in cirrhosis and hepatocellular carcinoma show the importance of antiapoptotic activity and increased cellular proliferation in the particular disease entities.

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La relation entre l’obésité et le cancer, bien qu’établie par des études épidémiologiques, est peu connue. Pourtant, environ 25 % des cancers pourraient y être attribuables. Parmi les cancers reliés à l’obésité, les cancers du côlon, du sein chez les femmes ménopausées et de la prostate sont les plus fréquents. Des études sur modèles animaux ont suggéré une association positive entre une diète riche en gras et le développement du cancer mammaire et de la prostate. Nous avons étudié les mécanismes moléculaires par lesquels les acides gras influencent le devenir de lignées de cellules cancéreuses du sein et de la prostate. Ces travaux ont montré que les acides gras insaturés, dont l’oléate, induisent la prolifération cellulaire tandis que les acides gras saturés, dont le palmitate, diminuent la prolifération. Un traitement à l’oléate stimule la formation de gouttelettes lipidiques dans le cytoplasme des cellules de cancer du sein MDA-MB-231 et de la prostate DU145 alors qu’un traitement au palmitate entraîne l’apoptose. Le mécanisme d’action de l’oléate sur la prolifération a été étudié de façon plus approfondie. L’utilisation d’inhibiteurs pharmacologiques nous a permis de déterminer que l’effet prolifératif de l’oléate implique la voie PI3K/Akt, la voie ERK1/2 et l’activation d’un ou de plusieurs récepteur(s) couplé(s) aux protéines G (GPCR). L’oléate induit la phosphorylation rapide des protéines Akt et ERK1/2 dans les cellules de cancer du sein MDA-MB-231 et de la prostate DU145. Au cours des dernières années, deux GPCRs ont été identifiés comme étant activables par des acides gras à moyennes et à longues chaînes, GPR40 et GPR120. GPR40 étant exprimé dans plusieurs lignées cellulaires de cancer du sein et de la prostate contrairement à l’expression de GPR120 qui était inexistante dans la plupart des lignées, nous avons étudié l’implication de GPR40 dans l’effet prolifératif de l’oléate. Ces deux récepteurs n’étant pas exprimés dans les cellules épithéliales mammaires humaines en culture primaire, ces cellules ne répondent pas aux effets de l’oléate sur la prolifération et l’activation des voies de signalisation. L’activation des voies Akt et ERK1/2 par l’oléate dans les cellules MDA-MB-231 et DU145 est potentialisée par la surexpression du récepteur GPR40 et inhibée par l’utilisation d’un siRNA dirigé contre ce récepteur. Cependant, la prolifération induite par l’oléate ne semble pas affectée par la présence d’un siRNA dirigé contre GPR40. L’oléate étant un acide gras, il est capable d’entrer librement dans les cellules et une partie de ses effets sur la prolifération pourrait être attribuée à sa métabolisation. Un agoniste de GPR40, le GW9508, est en mesure d’activer GPR40 sans toutefois entrer dans les cellules ni activer le métabolisme de l’oléate. Le GW9508 stimule la phosphorylation des protéines Akt et ERK1/2 dans les cellules du cancer du sein MDA-MB-231 et de la prostate DU145, mais il n’est pas en mesure d’induire la prolifération cellulaire comme le fait l’oléate. Ces résultats nous permettent de mieux comprendre le mécanisme d’action de l’oléate sur les cellules de cancer du sein et de la prostate. L’oléate induit la signalisation de GPR40 qui est impliquée dans l’activation rapide des voies de signalisation Akt et ERK1/2. De son côté, l’effet prolifératif induit par l’oléate s’effectue par un mécanisme GPR40-indépendant, possiblement lié au métabolisme de l’oléate. / The relationship between obesity and cancer, although established by epidemiological studies, remains relatively unknown. However, about 25 % of cancers could be attributed to obesity. Among cancers that are affected by obesity, colon cancer, post-menopausal breast cancer and prostate cancer are the more frequent. Studies on animal models have suggested a positive association between high fat diets and de development of mammary and prostate cancer. We have studied the molecular mechanisms by which fatty acids influence breast and prostate cancer cells fate. This work has shown that unsaturated fatty acids, including oleate, can induce cellular proliferation while saturated fatty acids, including palmitate, reduce proliferation. An oleate treatment stimulates lipid droplets formation in the cytoplasm of breast cancer cells MDA-MB-231 and prostate cancer cells DU145 while a palmitate treatment induces apoptosis. The action mechanism of oleate on proliferation was studied more closely. Using pharmacological inhibitors, we determine that oleate-induced cell proliferation involves PI3K/Akt signaling pathway, ERK1/2 signaling pathway and the activation of one or many G protein coupled receptor(s) (GPCR). Oleate induces rapid Akt and ERK1/2 phosphorylation in breast cancer cells MDA-MB-231 and prostate cancer cells DU145. In the last few years, two GPCRs were identified as being activated by medium and long chain fatty acids, GPR40 and GPR120. GPR40 being expressed in many breast and prostate cancer cell lines while GPR120 expression was null in most cell lines tested, we studied the role of GPR40 in oleate-induced proliferation. Human epithelial mammary cells in primary culture did not express GPR40 nor GPR120 and failed to respond to oleate-induced cell proliferation or activation of signaling pathways. Akt and ERK1/2 signaling pathways activation by oleate in MDA-MB-231 and DU145 cells is potentiated by GPR40 over-expression and inhibited by the use of an siRNA directed against that receptor. However, oleate-induced cell proliferation does not seem to be affected by the presence of the siRNA directed against GPR40. Oleate being a fatty acid, it can enter cells freely by crossing the plasma membrane and part of its effects on proliferation could be attributed to its metabolism. A GPR40 agonist, GW9508, is able to activate GPR40 without entering the cells nor activating oleate’s metabolism. GW9508 stimulates Akt and ERK1/2 phosphorylation in breast cancer cells MDA-MB-231 and prostate cancer cells DU145, but does not induce cell proliferation as does oleate. These results help us to understand the action mechanism of oleate in breast and prostate cancer cells. Oleate induces GPR40 signalization which is involved in the rapid Akt and ERK1/2 signaling pathways activation. On the other hand, oleate-induced cell proliferation is carried out by a GPR40-independent mechanism, possibly linked to oleate’s metabolism.

Cocaine is interrelated with the opioid system on many levels, especially via the mu opioid receptor (MOR). Also, cocaine has been involved in modulating nitric oxide (NO) actions within the cell. The effect of cocaine was first assessed on the MOR, and then on transcription by the use of 1 µg/ mL actinomycin D inhibitor. Several signaling pathways that cocaine may exert its action in modulating the MOR up-regulation in protein expression were also explored. Two dosage regimens were used in cocaine treatment, single continuous treatment (SCT), and repeated intermittent treatment (RIT). Different pathway inhibitors were used on PC12 cells, as follows: the PLC-PKC inhibitors 5 µM U-73122 and 10 µM BIS-1 used to investigate the involvement of the PKC signaling pathways in MOR expression levels, the evaluation of MAPK pathway by the use of 50 µM U0126 inhibitor, and the 10 µM LY94002 inhibitor was used to investigate the PI3K/Akt pathway. Moreover, the effect of NO on these signaling pathways was investigated by the use of 20 mM nonselective L-NAME inhibitor and qualitatively by DAF-2 florescence. Western blot analysis indicated that cocaine up-regulated MOR protein expression. Also, RIT cocaine treatment increased MOR protein levels via transcription. All three signaling pathways, MAPK, Akt and PKC modulated cocaine-induced increase of MOR following SCT cocaine treatment (post-transcriptional). Both MAPK and Akt have been found to modulate the cocaine-induced transcription of MOR via the two dosage regimens of cocaine, SCT and RIT. Also, inhibition of both PLC and PKC did not prevent cocaine-induced increase in MOR transcription, according to RIT of cocaine. Furthermore, Akt and PKC appeared to modulate cocaine-induced NO production while MAPK did not. NO seemed to be involved with the PKC and Akt pathways in up-regulating MOR in RIT of cocaine directly by the Akt pathway, and indirectly by the PKC pathway. On the other hand, NO and MAPK modulated the MOR up-regulation expression simultaneously, but in an individual/parallel manner. Furthermore, signaling pathway activation levels were tested using L-NAME which concluded that NO modulated cocaine-induced increase in total Akt protein levels, but did not appear to have an effect on phosphorylated MAPK activation levels. In conclusion, different treatment regimens of cocaine activate different pathways; SCT of cocaine activated all three signaling pathways, however, RIT of cocaine activated only the MAPK and Akt pathways. / Saudi Bureau in Canada

Le neuroblastome (NB) représente 8% de tous les cancers pédiatriques et est caractérisé par sa grande hétérogénéité clinique. Afin d’évaluer son pronostic, plusieurs facteurs génétiques sont utilisés : amplification de MYCN, délétion 1p, gain 11q et gain 17q. Les buts de notre travail étaient d’abord de vérifier si l’hybridation in situ en fluorescence (FISH) permet une analyse complète de ces anomalies et ensuite, en utilisant une analyse globale du génome telle le polymorphisme nucléotidique simple (SNP), de vérifier la concordance avec les résultats de la FISH et le pronostic potentiel des anomalies du 14q, en particulier du gène AKT. Nous avons donc établi un panel de sondes pour la FISH qui a été appliqué sur 16 tumeurs non-fixées. Après isolation de l’ADN de 36 tumeurs, nous avons effectué une analyse génotypique par SNP utilisant les puces « Affymetrix Genome-Wide Human SNP Array 6.0 » contenant 945,826 sondes non polymorphiques et 906,000 sondes polymorphiques. Nos résultats ont démontré que la FISH permet l’évaluation complète des anomalies génétiques importantes du NB et que les anomalies déséquilibrées sont détectées très précisément par SNP. Les anomalies du 14q tendent à être associées avec des facteurs cliniques comme le grade et l’évolution, contrairement aux anomalies d’AKT. L’analyse du 14q a révélé trois gènes d’intérêt, MAX, BCL11B et GPHN, qui devraient être analysés sur un plus grand échantillon. Ainsi, l’étude par FISH semble adaptée pour détecter les anomalies génétiques classiques du NB, alors que celles retrouvées en 14q représentent de potentielles cibles thérapeutiques pour cette tumeur. / Neuroblastoma (NB) accounts for 8% of all childhood cancers and is characterized by its clinical heterogeneity. To evaluate its prognostic, many genetic markers are used: MYCN amplification, 1p deletion, 11q gain and 17q gain. Our goals were first to verify if fluorescence in situ hybridization (FISH) allows a complete analysis of these abnormalities and, second, using a global genomic analysis as single nucleotide polymorphism (SNP), to verify the concordance with FISH results and the prognostic potential of 14q abnormalities, especially these of AKT gene. We then established a FISH panel that has been applied on 16 unfixed tumors. After DNA isolation of 36 tumors, we made a genotypic analysis by SNP using « Affymetrix Genome-Wide Human SNP Array 6.0 » containing 945,826 nonpolymorphic probes and 906,000 polymorphic probes. Our results have demonstrated that FISH allows a complete evaluation of the NB’s important genetic abnormalities and that unbalanced abnormalities are detected very precisely by SNP. 14q abnormalities seem to be associated with clinical factors such as tumor grading and evolution, unlike AKT abnormalities. Analysis of 14q abnormalities revealed three genes of interest, MAX, BCL11B and GPHN, which should be analyzed on a larger sample. Thereby, FISH study seems appropriate to detect the NB’s classic genetic abnormalities, while those found in 14q represent potential therapeutic targets for this tumor.