This work sought to investigate the clinical phenotypes and molecular basis of cardio-facio-cutaneous syndrome (CFC), a germline disorder of the Ras-MAPK pathway, like Noonan syndrome (NS) and neurofibromatosis type I, caused by mutations in genes encoding proteins that act within this signal transduction pathway. CFC is most commonly due to mutation in BRAF, and less commonly MAP2K1, MAP2K2 or KRAS. A proportion of patients currently have no mutation identified. Mutations and clinical features of patients with a molecular diagnosis of CFC were investigated, which demonstrated a wide range of causative mutations, and some unclassified variants. Both known and novel clinical features of CFC were identified. A strong association between severe contractures and the p.(Tyr130Cys) mutation in MAP2K1 was found, which has not previously been reported. In contrast to the large number of patients with a confirmed molecular diagnosis, several with a highly suggestive clinical phenotype have been found to have no mutationin any of the known CFC genes. The molecular basis of these presentations was investigated by conventional Sanger sequencing of candidate genes. Fourteen patients with the p.(Ser2Gly) mutation in SHOC2 were identified, with clinical presentations consistent with CFC, NS or CS. Target enrichment and massively parallel sequencing of selected genes was undertaken in ten patients. Mutations in known genes were identified in four patients (including the positive control). Candidate causative variants in novel genes were suggested in two further patients, one of which was confirmed on Sanger sequencing. Whole exome sequencing of patient-parent trios was also undertaken to identify de novo variants. Three trios were analysed, and in one patient with a clinical diagnosis of CFC, a frameshift mutation in NF1 was identified, which was confirmed by Sanger sequencing to be present and de novo. The molecular effects of CFC-associated mutations in BRAF on Ras-MAPK pathway signalling were studied in cell culture systems, using Western blotting for ERK1/2 phosphorylation, in vitro kinase assays and luciferase assays, to assess activity of downstream targets of the Ras-MAPK pathway. Altered pathway activity was demonstrated for novel variants that had not previously been characterised at the molecular level, which was in keeping with the findings of the effects of previously studied mutations. The cardiac phenotype in animal models of CFC, CS and NS/CFC was explored using expression microarrays to identify potentially important genes and pathways in the pathogenesis of hypertrophic cardiomyopathy (a progressive but potentially treatable disease feature) in these conditions. A signature of increased expression of Myh7, the embryonic form of myosin, was identified in the heart of the mouse model of CFC due to a B-Raf mutation at four weeks postnatal age, but comparative analysis suggested significant differences in either the mechanisms causing cardiac phenotypes, or the timescales over which these may exert their effects, in the three models. In summary, the most significant findings of this work were that SHOC2 mutation is a frequent cause of a severe NCFC presentation, and massively parallel sequencing can be an effective means of molecular investigation of this group of disorders. Novel features of CFC syndrome that were identified include severe contractures in association with p.(Tyr130Cys) mutations in MAP2K1. The analysis of mouse models of the NCFCs was hampered by heterogeneity within the expression microarray results, and low levels of expression of the H-Ras mutant allele in the mouse model of Costellosyndrome.

No description available.

Les léiomyosarcomes (LMS) sont des tumeurs d’origine mésenchymateuse caractérisées par une différenciation musculaire lisse. La voie de signalisation PI3K/mTOR (qui contrôle la prolifération et la survie cellulaire) joue un rôle majeur dans le développement de ces tumeurs. De nos jours, cette voie est devenue une cible thérapeutique majeure en oncologie. Cette étude est la première qui évalue le bénéfice thérapeutique de l’inhibition de la voie PI3K/mTOR pour des patients atteint de LMS. Nous avons mis en évidence qu’une double inhibition de PI3K et mTOR est associée à une activité antitumorale supérieure à celle observée avec une inhibition de PI3K ou mTOR seule. Nous avons également montré que l’inhibition de la voie PI3K/mTOR est associée à une activation paradoxale de la voie MAPK et qu’un ciblage concomitant de cette voie est associé à une synergie antitumorale in vitro et in vivo. Afin de caractériser les mécanismes de résistance secondaire à l’inhibition de la voie PI3K/mTOR, nous avons développé in vitro et in vivo un modèle de résistance secondaire à l’inhibiteur double cible PI3K/mTOR. Nous avons notamment détecté une sous-population de cellules résistantes à l’inhibiteur et ayant des caractéristiques proches de celles des cellules souches. Nous avons mis en évidence que l’inhibition pharmacologique d’EZH2, une protéine cruciale du complexe Polycomb, permet de restaurer la sensibilité des modèles résistants. Ces résultats apportent de nouvelles perspectives thérapeutiques pour les patients atteints de LMS. / Leiomyosarcomas (LMS) are tumors of mesenchymal origin characterized by a smooth cell differentiation. The PI3K/mTOR pathway has been shown to play a crucial role in the tumorigenesis of LMS. Several agents targeting this pathway are under clinical development for the treatment of solid tumors and hematological malignancies. We report here the first study evaluating its potential therapeutic benefit for patients with LMS. We have demonstrated that dual inhibition of PI3K and mTOR is associated with more effective antitumor activity than agents targeting PI3K or mTOR only. We have also shown that PI3K and mTOR inhibition is associated with a paradoxal activation of the MAPK pathway and that combined treatment with MEK inhibitor resulted in synergistic antitumor activity in vitro and in vivo. Moreover, we developed in vitro and in vivo resistant model to dual PI3K/mTOR inhibitor. Interestingly, we have found that a cancer stem cell-like subpopulation may be involved in treatment resistance. We have shown that pharmacological inhibition of EZH2, a crucial protein of the Polycomb complex, is able to reverse dual PI3K/mTOR inhibitor resistance in vitro and in vivo. These results provide new therapeutic strategies for patients with LMS.

\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Introduction: Breast cancer is the second highest cause of cancer mortality in women worldwide. Hormonal therapy is considered one of the most effective therapies and is used against luminal-type malignancies. However, 40-50% of tumour cells can develop resistance, thereby limiting the success in breast cancer treatment. In this study, mechanisms of resistance were investigated using a novel multi-stable isotope labelled amino acids (SILAC) proteomics approach in phenotype-specific breast cancer cell lines resistant to endocrine treatment. Method: In vitro chemo-sensitivity (IC50) was determined for MCF7, T47D, MDA-MB-231, MDA-MB-468, MDA-MB-453, BT-20 and MCF-10A breast cell lines using four endocrine-based therapeutic agents (Tamoxifen, 4-Hydroxytamoxifen OHT, Raloxifene, Anastrozole) to select viable strains for resistance studies. MCF7 (luminal-type A) and MDA-MB-231 (triple negative breast cancer, TNBC) were selected and initially subject to OHT or raloxifene exposure with gradual increments for 10 months. WT cells were grown in the absence of drug in parallel as passage controls. Resistant cell lines were assessed by MTT and IF for comparison with parental cell lines. Resistant cell lines, along with the passage control and a SILAC control, were grown in “light” SILAC medium together with WT strains cultured in “heavy” SILAC medium. Proteins were extracted, concentrations determined and analysed by SDS PAGE for quality control. An aliquot of each “light” cell line (resistant, passage control or SILAC control) was combined with an equal amount of “heavy” WT, trypsin digested and analysed by nano-HPLC Orbitrap Fusion mass spectrometry (2D-LC MS/MS). Proteins were identified by database searching using MascotTM. Relative changes (resistant/WT ratio) in protein levels were determined and bioinformatics tools (STRING and UniProt) used to explore significantly changed pathways associated with resistance. Western blotting was used to verify selected target proteins. Results: Four consistently resistant sublines were generated MCF7 OHT Res (2.00-fold more resistant), MCF7 Ralx Res (2.00-fold), MDA-MB-231 OHT Res (1.90-fold change) and MDA-MB-231 Ralx Res (2.00-fold), in addition to two high passage controls. ER expression by IF was decreased in MCF7 OHT Res compared to the WT and MCF7 Ralx Res, whereas CD44 was increased. Proteomic analysis revealed 2247 and 2880 total proteins in MCF7 OHT Res and MCF7 Ralx Res whilst 3471 and 3495 total proteins were identified in MDA-MB-231 OHT Res and MDA-MB-231 Ralx Res, respectively. Bioinformatics tools identified significantly changed pathways included apoptosis, cytoskeleton, cell motility and redox cell homeostasis. Components of the MAPK-signalling cascade were consistently found to be upregulated in resistant cell lines. MAPK1 (ERK2), previously associated with tamoxifen resistance was increased in MDA-MB-231 Ralx Res cell lines by 4.45-fold and confirmed by Western blotting. Sorcin, which contributes to calcium homeostasis and is also linked to multidrug resistance was increased 4.11- and 2.35-fold in MCF7 OHT Res and Ralx Res sub cell lines, respectively. Some results, such as those for c-Jun, were inconsistent between proteomic analysis and Western blotting and require further investigation. Conclusion: The unique resistant cell lines generated here, as well the MCF7 OHT resistant line, provided novel data that give insights into the biological pathways involved in mechanisms of endocrine drug resistance in breast cancer. Proteomics analysis provided extensive data on common functionality and pathways across the resistant cell lines independent of phenotype or SERM. Overall, the results provided interesting targets for re-sensitising resistant breast cancer and the potential to investigate novel combination therapies in the future. / Al-Ahliyya Amman University scholaships

Phosphorylierung und Dephosphorylierung gehören zu den zentralen Regulationsmechanismen jeder Zelle. Während einer der bekanntesten Signalwege, der Mitogen-aktivierte Protein-Kinase(MAPK)-Signalweg bereits intensiv untersucht wurde, ist über die MAPK-Phosphatasen als wesentliche Inaktivatoren dieser Signalwegfamilie bisher nur wenig bekannt. Die MAPK-Signalwege sind in Tumoren häufig aktiv. Dies ließe sich durch aktivierende Mutationen der Kinasen oder ihrer übergeordneten Rezeptoren erklären. Ein anderer Ansatz geht von der stromalen Entzündungsreaktion aus, die viele solide Tumoren begleitet. Zytokine, die durch die Entzündungszellen gebildet werden, sind physiologische Aktivatoren der MAPK und eine fehlende Gegenregulation durch inaktivierende Phosphatasen würde ebenso zu hoher Aktivität der MAPK führen. In der vorliegenden Arbeit wurde ein solches Entzündungsgeschehen in Ovarialkarzinomzellinien simuliert und die Reaktion der Phosphatasen MKP-1 und MKP-3 auf proinflammatorische Zytokine untersucht. MKP-1 und MKP-3 reagierten mit erheblichen Unterschieden auf die Zugabe proinflammatorischer Zytokine, das Reaktionsmuster reichte von starker Aktivierung (MKP-1 in SKOV-3 und OVCAR-3) bis hin zu verringerter Aktivität der Phosphatasen (MKP-1 in OAW42 und CAOV-3). Zur Ergänzung der Zellkulturstudien wurde die Expression der Phosphatase MKP-1 in primären Ovarialkarzinomen, Zystadenomen sowie gesunden Ovarien immunhistochemisch untersucht. Der Proteinnachweis in insgesamt 101 Gewebeproben ergab eine signifikant geringere Expression der Phosphatase MKP-1 in Karzinomen im Vergleich zu normalem Ovarialepithel oder Zystadenomen. Innerhalb der Gruppe der Karzinome zeigte die MKP-1-Expression dennoch eine hohe Varianz, hierbei waren Malignome mit deutlicher MKP-1-Expression mit einer wesentlich schlechteren Prognose der Erkrankung verbunden. Die Phosphatase war in der multivariaten Analyse des rezidivfreien Überlebens ein unabhängiger Prognoseparameter (RR=4,03; 95%CI=1,72-9,48; p=0,001). Ein kürzeres rezidivfreies Überleben ist häufig mit der frühen Entwicklung von Chemoresistenzen verbunden. Ein möglicher Zusammenhang zwischen der Phosphatase MKP-1 und Chemoresistenz wurde in Zellkulturversuchen unter Verwendung von Cisplatin, einem wesentlichen Bestandteil der Standardchemotherapie bei Ovarialkarzinomen, untersucht. Die Expression der MKP-1 konnte durch Zugabe von Cisplatin deutlich induziert werden. Bemerkenswerterweise zeigten resistente Zellinien dabei eine frühe Reaktion, sensible Zellen reagierten deutlich verzögert. Diese frühe Induktion der MKP-1 könnte die therapeutisch induzierte Apoptose blockieren. Weitere Erkenntnisse über die daran beteiligten Signalwege sowie pharmakologische Inhibitoren der Phosphatasen sind daher vielversprechende Ansätze zur Optimierung der Chemotherapie. / Protein phosphorylation and dephosphorylation is a central regulatory system of cells. The mitogen-activated-protein-kinase(MAPK)-pathway as a typical example is one of the most investigated signalling pathways in cancers. In contrast, much less is known about MAPK-phosphatases, their physiological inactivators. MAPK pathways are frequently up-regulated in cancers. This might be explained by activating mutations of kinases or of up-stream receptors. Another view is based on the inflammatory stroma infiltrate that accompanies most solid carcinomas. Cytokines produced by inflammatory cells are physiological activators of MAPK pathways and missing balance of inactivating phosphatases would also result in up-regulated MAPK pathways. In this study, such an inflammatory situation was simulated in cell culture models and expression patterns of MAPK-phosphatases MKP-1 and MKP-3 were investigated after addition of proinflammatory cytokines. The expression of MKP-1 and MKP-3 after cytokine addition differed widely between the ovarian cancer cell lines investigated, ranging from strong induction in SKOV-3 and OVCAR-3 to down-regulation of phosphatases in OAW42 and CAOV-3. In addition to cell culture experiments, expression of MKP-1 was examined immunohistochemically in primary ovarian cancers, adenomas and normal ovaries (total of 101 samples). There was a lower expression of phosphatase MKP-1 in ovarian cancers compared to surface epithelium of normal ovaries and cystadenomas. However, MKP-1 expression in the group of carcinomas showed a high variation, including also a number of negative cases. Among all investigated cancer samples, those with a higher expression of MKP-1 were associated with poorer prognosis. Multivariate survival analysis revealed this phosphatase as an independant prognostic factor for progression-free survival (RR=4,03; 95%CI=1,72-9,48; p=0,001). Short progression-free survival is usually associated with early development of chemoresistance. Consequently, poor prognosis might result from different efficiencies of initial adjuvant chemotherapeutical treatments. Based on this presumption, the effects of cisplatin, a typically used drug against ovarian cancer, were investigated in cell culture. The phosphatase MKP-1 was highly inducable by cisplatin, remarkably as early reaction in cisplatin-resistant cell lines and with distinct delay in sensitive cells. This early induction of MKP-1 in resistant cells might block drug-induced apoptosis. Further studies about influencing pathways and pharmacological inhibitors of phosphatase MKP-1 might be promising efforts to optimize chemotherapy.

Carcinomas papilíferos (CP) estão relacionados à ativação da via de sinalização MAPK por diversos oncogenes. Estudos têm relacionado a incidência de CP com a disponibilidade de iodo, um dos mais importante reguladores da função tiroidiana. Recentemente, nosso grupo descreveu que a inibição da via de sinalização TGF<font face=\"symbol\">b pode estar vinculada ao desenvolvimento de CP. Nosso objetivo foi avaliar os efeitos do excesso de iodo na transformação de células tiroidianas. Utilizamos células PCCL3 com indução do oncogene RET/PTC3, tratadas com excesso de iodo. Observamos que o iodo reduziu a proliferação, sem alterar a morte das células e recuperou a expressão de genes específicos tiroidianos, evento geralmente vinculado à transformação maligna tiroidiana. O tratamento com excesso de iodo, ainda, reduziu a expressão de proteínas envolvidas na via MAPK e promoveu o restabelecimento da expressão da via TGF<font face=\"symbol\">b, uma importante via inibitória da proliferação de células tiroidianas. Concluímos que o excesso iodo tenha uma ação antioncogênica durante a transformação maligna tiroidiana. / Papillary Carcinomas (PC) are frequently related to MAPK signaling pathway, activation by several oncogenes. Recent studies had related PC incidence and iodine availability, one of the most important thyroid function regulators. Our group has recently showed that TGF<font face=\"symbol\">b pathway inhibition is directly related to PC development. Our aim was to evaluate iodine excess effects during thyroid cells transformation. We used the PCCL3 cells with RET/PTC3 oncogene induction, treated with iodine excess. We observed that iodine reduced the cell proliferation, not altering cell death and recovered thyroid specific genes expression, event frequently liked to thyroid cells malignant transformation. Iodine excess treatment also reduced MAPK proteins expression and reestablished TGF<font face=\"symbol\">b pathway components expression, an important inhibitory pathway of epithelial cells. We can conclude that iodine excess treatment plays an antioncogenic role during thyroid malignant transformation.

La résistance à l’hormonothérapie est un challenge majeur en clinique dans le choix du traitement chez les patientes atteintes de cancer du sein RE+ et l’activation des voies de signalisation PI3K/Akt/mTOR ou MAPK semble être impliquée dans la résistance à l’hormonothérapie. L’objectif de ce travail a été dédié à l’exploration de stratégies de réversion de la résistance à l’hormonothérapie et à l’identification de nouveaux biomarqueurs associés à cette résistance. Nous avons identifié dans des lignées résistantes à l’hormonothérapie ayant acquis l’activation endogène des voies PI3K/Akt/mTOR et MAPK, l’activation aberrante du système ErbB, pouvant être à la base de l’activation de ces deux voies de signalisation. L’inhibition d’au moins une des deux voies (par un inhibiteur de mTOR, un inhibiteur de PI3K et/ou un inhibiteur de MEK) a conduit dans la lignée sensible à une augmentation de la sensibilité au tamoxifène et au fulvestrant et dans les lignées résistantes à une restauration de la sensibilité à l’hormonothérapie. La réversion de la résistance au fulvestrant par la rapamycine a été démontrée non seulement au niveau de la prolifération cellulaire mais aussi au niveau de l’expression des gènes, explorés par une approche génomique. Par ailleurs, par une approche gènes candidats, nous avons identifié une signature de trois gènes (TACC1, NOV et PTTG1) présentant une valeur pronostique et associée à la résistance à l’hormonothérapie pouvant représenter un nouvel outil de diagnostic des patientes atteintes de cancer du sein hormono-dépendant. / Endocrine therapy resistance is one of the main challenges in the treatment of estrogen receptor positive breast cancer patients and the activation of PI3K/Akt/mTOR or MAPK signaling pathways seems to be implicated in the acquisition of the resistance. The objective of this work has been dedicated to the exploration of strategies to reverse the resistance to endocrine therapy and the identification of new biomarkers associated with this resistance. We have identified in cellular models resistant to hormone therapy, which have acquired the endogenous activation of PI3K/Akt/mTOR and MAPK pathways, the aberrant activation of the ErbB system which may be the cause of the activation of these pathways. The inhibition of at least one of these two pathways (by an mTOR inhibitor), a PI3K inhibitor) and/or a MEK inhibitor) was shown to increase sensitivity to tamoxifen and fulvestrant in the sensitive cells and to restore sensitivity to endocrine therapy in the resistant cells. The reversion of resistance to fulvestrant by rapamycin has been demonstrated not only at the cell proliferation level but also at the gene expression level explored by a genomic approach. In addition, using a candidate gene approach, we identified a signature of three genes (TACC1, NOV and PTTG1) with prognostic value and associated with resistance to endocrine therapy. These genes may represent a new diagnostic tool for patients treated with endocrine therapy.

Carcinomas papilíferos (CP) estão relacionados à ativação da via de sinalização MAPK por diversos oncogenes. Estudos têm relacionado a incidência de CP com a disponibilidade de iodo, um dos mais importante reguladores da função tiroidiana. Recentemente, nosso grupo descreveu que a inibição da via de sinalização TGF<font face=\"symbol\">b pode estar vinculada ao desenvolvimento de CP. Nosso objetivo foi avaliar os efeitos do excesso de iodo na transformação de células tiroidianas. Utilizamos células PCCL3 com indução do oncogene RET/PTC3, tratadas com excesso de iodo. Observamos que o iodo reduziu a proliferação, sem alterar a morte das células e recuperou a expressão de genes específicos tiroidianos, evento geralmente vinculado à transformação maligna tiroidiana. O tratamento com excesso de iodo, ainda, reduziu a expressão de proteínas envolvidas na via MAPK e promoveu o restabelecimento da expressão da via TGF<font face=\"symbol\">b, uma importante via inibitória da proliferação de células tiroidianas. Concluímos que o excesso iodo tenha uma ação antioncogênica durante a transformação maligna tiroidiana. / Papillary Carcinomas (PC) are frequently related to MAPK signaling pathway, activation by several oncogenes. Recent studies had related PC incidence and iodine availability, one of the most important thyroid function regulators. Our group has recently showed that TGF<font face=\"symbol\">b pathway inhibition is directly related to PC development. Our aim was to evaluate iodine excess effects during thyroid cells transformation. We used the PCCL3 cells with RET/PTC3 oncogene induction, treated with iodine excess. We observed that iodine reduced the cell proliferation, not altering cell death and recovered thyroid specific genes expression, event frequently liked to thyroid cells malignant transformation. Iodine excess treatment also reduced MAPK proteins expression and reestablished TGF<font face=\"symbol\">b pathway components expression, an important inhibitory pathway of epithelial cells. We can conclude that iodine excess treatment plays an antioncogenic role during thyroid malignant transformation.

<p>There is increasing evidence that prostate tumours are organized as a hierarchy with rare cancer stem cells (CSCs) implicated in initiating and maintaining the tumour. However, prospective prostate cancer stem cells (PCSCs) have not been thoroughly characterized from primary tissue specimens. Using the DU145 cell line, PCSCs have been propagated as non-adherent spheres <em>in vitro</em>. Approximately 1.25% of monolayer DU145 cells formed primary spheres while 26% of sphere cells formed subsequent spheres; a measure of PCSC self-renewal capacity. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins and CSC markers (CD44, CD24, integrin alpha2beta1). PCSCs initiate xenograft tumours with enhanced capacity compared to monolayer cells. While epidermal growth factor (EGF) promoted PCSC propagation, basic fibroblast growth factor (bFGF) inhibited these events. Activation of EGF receptor (EGFR) signalling, following EGF treatment or expression of constitutively-active EGFR (EGFRvIII), increased sphere formation. Conversely, attenuation of EGFR signalling inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signalling, the MEK-ERK pathway contributes to EGFR-facilitated PCSC propagation. Inhibition of ERK activation following MEK inhibitor treatment, expression of dominant-negative MEK1(K97M), or knockdown of ERK1 or ERK2 reduced PCSC propagation. Therefore, EGFR signalling promotes PCSC self-renewal by activating the MEK-ERK pathway.</p> <p>SOX2 is an essential transcription factor for stem cells, however, its role in PCSCs remains unclear. SOX2 protein is upregulated in PCSCs propagated as spheres, and its expression is regulated by EGFR signalling. EGFR activation, following EGF treatment or expression of constitutively-active EGFRvIII, increased SOX2 expression and PCSC self-renewal, while being attenuated by EGFR inhibitor treatment. Ectopic SOX2 expression enhanced EGF-induced PCSC self-renewal, while SOX2 knockdown renders PCSCs non-responsive to EGF-induced self-renewal and reduced their anchorage-independent growth. Furthermore, SOX2 expression is associated with the ability of PCSCs to form aggressive xenograft tumours. Collectively, SOX2 regulates EGFR-mediated PCSC self-renewal.</p> / Doctor of Philosophy (PhD)