Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, with two predominant histologic subtypes: embryonal and alveolar. These histologies display distinct clinical courses, and despite refinements in dose and duration of multimodality therapy, the 5-year overall survival of patients diagnosed with metastatic RMS remains <30%. Thus, there is an urgent need to define novel targets for therapeutic intervention. Interrogation of cancer cell signal transduction pathways that regulate the pathogenic behaviours of tumor cells has been successful in defining targets in numerous tumor types. These have ultimately yielded clinically-relevant drugs that have improved the disease-free and overall survival of patients diagnosed with cancer. Work contained in this thesis describes the interrogation of several potential targets for inhibition in RMS. Interruption of RMS cell proliferation, survival and apoptosis is examined through disruption of the protein kinase integrin-linked kinase (ILK) and the nuclear receptor estrogen-receptor β. ILK, in particular, is demonstrated to have dual competing functions through the regulation of c-jun amino-terminal kinase (JNK) signaling: an oncogene in alveolar, and a tumor suppressor in embryonal RMS. These findings are recapitulated in other tumor cell lines, indicating that expression levels of JNK1 correlate with ILK function in a broad spectrum of tumor types. Furthermore, interruption of rhabdomyosarcoma cell migration as a surrogate marker of metastasis is examined through disruption of the stromal-cell derived factor 1α/chemokine (CXC)receptor 4 signaling network, as well as through cooperative interactions between ILK and the mammalian target of rapamycin. Finally, we demonstrate that the insulin-like growth factor pathway is a potential target for therapeutic inhibition, which also distinguishes tumors of embryonal and alveolar histology. These studies provide a rationale for the development of novel agents, as well as the use of established drugs targeting these pathways in rhabdomyosarcoma.

The pathological process behind abdominal aortic aneurysm (AAA) formation is poorly understood and difficult to study. The aim of the thesis was to study the pathophysiology of AAA formation with positron emission tomography (PET) technology, a molecular imaging technique, allowing in vivo studies of pathophysiological changes. In study I 18F-FDG, a glucose analogue, was tested. It had previously been reported as a useful tracer studying inflammation in AAAs. These studies included, however, foremost large, symptomatic, and inflammatory AAAs. In the present study on five small and seven large asymptomatic AAAs, no increase in 18F-FDG uptake could be revealed in vivo. In study II 11C-PK11195, a macrophage tracer, and 11C-D-deprenyl, an unspecific inflammatory tracer, previously never tested on asymptomatic AAAs, were tested in vivo on five and 10 AAA-patients respectively, without signs of increased levels of inflammatory activity in the aorta. In study III several tracers were screened in vitro through autoradiography on AAA tissue. [18F]fluciclatide, targeting the integrin αVβ3 receptor upregulated in angiogenesis, was the only tracer with an increased uptake. In study IV [18F]fluciclatide-autoradiography was performed on AAA tissue from five patients and non-aneurysmal aortic tissue obtained from five age and sex matched organ donors. The study showed a 56% increased specific uptake in AAA, although not significant (P=0.136). Immunohistochemical revealed inflammatory cell foci in close relation to the vessels. In conclusion, PET has potential to elucidate the pathophysiology of AAA formation. For the large group of small asymptomatic AAAs, 18F-FDG is not suitable, as the chronic inflammation in asymptomatic AAA is not sufficiently metabolically active. Furthermore, 11C-PK11195 and 11C-D-deprenyl were unable to show the chronic inflammation seen in asymptomatic AAA. The interesting finding with uptake of [18F]fluciclatide showed that angiogenesis may be imaged in large asymptomatic AAAs in vitro, through the integrin αVβ3 receptor. Thus, it is likely that future studies of the role of angiogenesis in AAA formation in vivo, in small AAAs, could use this target site. The development of an integrin αVβ3 receptor tracer, preferably with higher affinity, is in progress for further in vitro and in vivo studies.

Οι φυλλοειδείς όγκοι του μαστού είναι σπάνια διφασικά νεοπλάσματα, με ποικίλου βαθμό κίνδυνο για τοπική υποτροπή και μετάσταση, που αποτελούνται από επιθηλιακό και στρωματικό στοιχείο. Η επιθηλιακή προς μεσεγχυματική μετατροπή, κρίσιμης σημασίας γεγονός κατά την εμβρυογένεση, έχει φανεί ότι συμμετέχει στην παθογένεια της ίνωσης διαφόρων οργάνων καθώς και στη διηθητική και μεταστατική ικανότητα πολλών τύπων καρκινωμάτων. Ο σκοπός της παρούσας μελέτης είναι η διερεύνηση του κατά πόσον το φαινόμενο ΕΜΤ συμμετέχει στην παθογένεια των φυλλοειδών όγκων. Υλικό-Μέθοδος: Η έφραση της Ε-καντχερίνης, της β-κατενίνης και της ILK μελετήθηκε ανοσοϊστοχημικά σε δείγματα μονιμοποιημένα σε φορμόλη και εγκλεισμένα σε παραφίνη από 71 εξαιρεθέντες φυλλοειδείς όγκους, 44 καλοήθεις (62%), 20 ενδιάμεσης κακοήθειας (28%) και 7 κακοήθεις (10%). Διερευνήθηκαν συσχετίσεις με κλινικοπαθολογικές παραμέτρους όπως τον ιστολογικό τύπο των όγκων (καλοήθεις, οριακής κακοήθειας και κακοήθεις). Αποτελέσματα: Στο μη νεοπλασματικό επιθήλιο παρατηρήθηκε μεμβρανικής εντόπισης ανοσοθετικότητα για την Ε-καντχερίνη και τη β-κατενίνη και αρνητική ανοσοχρώση για την ILK. Αντίθετα, στο επιθηλιακό στοιχείο των όγκων παρατηρήθηκε μείωση της μεμβρανικής εντόπισης ανοσοθετικότητας για την Ε-καντχερίνη και τη β-κατενίνη. Η απώλεια της μεμβρανικής Ε-καντχερίνης ήταν μεγαλύτερη στους οριακής κακοήθειας και κακοήθεις όγκους (Kruskal-Wallis test p=0.000). Πυρηνικής και κυτταροπλασματικής εντόπισης ανοσοθετικότητα για την Ε-καντχερίνη και τη β-κατενίνη, καθώς και πυρηνικής και κυτταροπλασματικής εντόπισης ανοσοθετικότητα για την ILK ανιχνεύθηκαν τόσο στο επιθηλιακό όσο και στο στρωματικό στοιχείο των όγκων. Στους οριακής κακοήθειας και κακοήθεις όγκους παρατηρήσαμε στατιστικώς σημαντικά μεγαλύτερη πυρηνική/κυτταροπλασματική ανοσοθετικότητα Ε-καντχερίνης ( Kruskal-Wallis test p=0.013/p=0.006 αντίστοιχα για επιθηλιακά κύτταρα και p=0.017 για στρωματικά κύτταρα) και μεγαλύτερη πυρηνική/κυτταροπλασματική ανοσοθετικότητα για την ILK (Kruskal-Wallis test p=0.031 /p=0.012 αντίστοιχα για στρωματικά κύτταρα). Συμπέρασμα: Τα ευρήματα αυτά υποδηλώνουν ότι μία διεργασία τύπου ΕΜΤ μπορεί να εμπλέκεται στην παθογένεια των φυλλοειδών όγκων συμβάλλοντας στην δημιουργία όγκων με πιο επιθετικό φαινότυπo. / Phyllodes breast tumors consist of epithelial and mesenchymal components. Epithelial-mesenchymal transition (EMT) has been implicated in carcinogenesis. The aim of this study was to investigate whether EMT is involved in the pathogenesis of phyllodes breast tumors. Methods: E-cadherin, β-catenin and integrin-linked kinase (ILK) expression was evaluated immunohistochemically in 71 FFPE human phyllodes breast tumors (44/71 (62%) benign, 20/71 (28%) bordeline and 7/71 (10%) malignant). Correlations with tumor histopathology (benign, borderline, malignant) were investigated. Results: Non-neoplastic breast epithelium showed membranous E-cadherin and β-catenin expression and negative ILK immunoreactivity. Decreased membranous expression of E-cadherin and β-catenin in tumor epithelial component was observed. Loss of membranous E-cadherin was higher in borderline/malignant tumors (Kruskal-Wallis test p=0.000). Nuclear/cytoplasmic immunoreactivity of E-cadherin and β-catenin, as well as nuclear/cytoplasmic expression of ILK was detected in both epithelial and stromal tumor cells. We observed significantly higher nuclear/cytoplasmic immunoreactivity of E-cadherin (Kruskal-Wallis test p=0.013/p=0.006 respectively for epithelial cells and p=0.017 for stromal cells) and nuclear/cytoplasmic stromal expression of ILK (Kruskal-Wallis test p=0.031 /p=0.012 respectively) in borderline/malignant tumors. Conclusion: These results suggest that an-EMT-like process may be implicated in phyllodes breast tumors pathogenesis contributing to a more malignant phenotype.

Molecular dynamics (MD) simulations were used to investigate the structural basis for the functions of three proteins: Fc(gamma) receptor III (CD16), von Willebrand factor (VWF), and integrin. CD16, a heavily glycosylated protein expressed on human immune cells, plays a crucial role in immune defense by linking antibody-antigen complexes with cellular effector functions. Glycosylation of CD16 decreases its affinity for IgG. MD simulations were run for CD16-IgG Fc complexes with or without an N-glycan on CD16. The two simulated complexes show different conformations. Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) approach was used to calculate the binding free energy of the CD16-IgG Fc complexes. The calculated binding free energy helped to identify critical residues. VWF, a multimeric multidomain glycoprotein, initiates platelet adhesion at the sites of vascular injury. A specific VWF metalloprotease, A Disintegrin And Metalloprotease with ThromboSpondin motifs member 13 (ADAMTS-13), cleaves the Tyr1605-Met1606 bond in the VWF A2 domain to generate the full spectrum of plasma VWF species. Shear stress or denaturants assist VWF cleavage by ADAMTS-13 due to the unfolding of A2. MD was used to simulate the unfolding processes of A2 under force or high temperature. The beta-strands of A2 were pulled out sequentially by force, during which the cleavage site changed in steps from the fully buried state to the fully exposed state. Thermal unfolding follows a very different pathway. Integrins are adhesion molecules mediating cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Experiments suggest that integrins can undergo a large-scale change from a bent to an extended conformation, associating with a transition from low to high affinity states, i.e., integrin activation. Steered MD was utilized to simulate the bent-to-extended conformational transition in time of aVb3 integrin. The integrin was observed to change smoothly from the bent to the extended conformation. One major energy barrier was overcome, corresponding to the disruption of the interactions at Hybrid/EGF4/bTD interfaces. A partially extended conformation tends to bend back while a fully extended conformation is stabilized by the coordination of Asp457 with Ca2+ at alpha-genu. Unbending with separated legs overcomes more energy barriers.

Introduction : La thérapie ciblée des cancers des VADS nécessite la mise au point de nouveaux vecteurs spécifiques. Ces vecteurs servent à acheminer des substances thérapeutiques, mais aussi ils peuvent être couplés à des fluorophores afin de les utiliser dans la chirurgie guidée par l'imagerie de fluorescence proche infrarouge.Objectifs : L'objectif de notre travail est de tester de nouveaux vecteurs des cancers des VADS et d'étudier l'apport de l'imagerie de fluorescence proche infrarouge dans la chirurgie des cancers des VADS chez un modèle animal orthotopique que nous mettons au point.Matériel et méthodes : La lignée cellulaire des cancers des VADS CAL33 est caractérisée in vitro et in vivo. De nouveaux vecteurs qui ciblent un ou plusieurs récepteurs des cellules CAL33 comme l'intégrine alpha v beta 3, l'EGFR et la NRP1, sont testés in vitro. Parallèlement, un modèle animal orthotopique des cancers des VADS est développé par implantation de fragments tumoraux des cellules CAL33, au niveau de la cavité buccale de la souris nude. La résection des tumeurs orthotopiques est guidée par l'imagerie de fluorescence proche infrarouge, après injection systémique du peptide RAFT-c[RGD]4 couplé à un fluorophore. Ce peptide cible l'intégrine alpha v beta 3 et est préalablement testé in vivo sur les cellules CAL33.Résultats : Nos résultats préliminaires montrent que certaines molécules bispécifiques présentent une liaison accrue in vitro aux cellules CAL33. Par ailleurs, la chirurgie guidée par l'imagerie de fluorescence proche infrarouge ciblant l'intégrine alpha v beta 3, présente un impact positif sur la survie sans rechute dans notre modèle orthotopique, à travers la détection de reliquats tumoraux qui pourraient passer inaperçus si l'exérèse tumorale avait été réalisée exclusivement d'une façon macroscopique. Elle permet aussi de détecter les adénopathies métastatiques.Conclusion : L'imagerie de fluorescence proche infrarouge améliore la qualité de l'exérèse tumorale dans notre modèle orthotopqiue optimisé des cancers des VADS. Cette étape préclinique est indispensable avant de tester cette technique chez l'être humain. / Introduction: Targeted therapy of head and neck squamous cell carcinoma (HNSCC) requires the development of novel specific vectors that can deliver therapeutic molecules. These vectors could also be coupled to fluorophores to be used in near infrared fluorescence imaging-guided surgery.Objectives: The aim of our work is to test new targeted vectors of HNSCC and to study the role of the near infrared fluorescence imaging-guided surgery in HNSCC resection in a novel orthotopic animal model that we develop.Materials and Methods: The HNSCC cell line CAL33 is characterized in vitro and in vivo. Novel vectors that target one or more receptors of this cell line such as alpha v beta 3 integrin, EGFR and NRP1, are tested in vitro. Meanwhile, an orthotopic animal model of HNSCC is developed by implanting tumor fragments of CAL33 cells, in the oral cavity of nude mice. Surgical resection of orthotopic tumors is guided by the near infrared fluorescence imaging after systemic injection of RAFT-c[RGD]4 peptide coupled with a fluorophore. This peptide targets alpha v beta 3 integrin and is previously tested in vitro.Results: Our preliminary results show that bispecific vectors would present an increased binding to CAL33 cells in vitro. On the other hand, near infrared fluorescence imaging-guided surgery has a positive impact on the recurrence-free survival rate in our orthotopic model, by detecting fluorescent cancer foci that could remain unidentified if resection was performed exclusively under visual guidance. Our results show also that near infrared fluorescence imaging can also help to detect metastatic lymph nodes.Conclusion: Near-infrared fluorescence imaging-guided surgery improves the quality of tumor resection in our optimized orthotopic animal model of HNSCC. This preclinical stage is essential before testing this novel technique in humans.

Le glioblastome multiforme (GBM) est la tumeur cérébrale primaire la plus fréquente. Une dérégulation des voies de signalisation de l’EGFR et un fort potentiel invasif sont les caractéristiques principales du GBM. Malheureusement, les essais cliniques impliquant des thérapies anti-EGFR dans le traitement des GBM demeurent inefficaces. Nous avons précédemment montré que le récepteur de la fibronectine, l’intégrine α5β1, est associé avec un mauvais pronostic et une résistance des patients au temodal. Les intégrines peuvent coopérer avec les récepteurs aux facteurs de croissance et ainsi amplifier leur potentiel oncogénique. Ici, nous avons cherché à déterminer le rôle de l’intégrine α5 dans la résistance aux thérapies anti-EGFR. Utilisant la lignée U87 de GBM, on a dans un premier temps confirmé que l’activation de l’intégrine sous l’influence de la fibronectine, potentialisait la signalisation de l’EGFR. La perte d’expression d’α5 sensibilise les cellules U87 aux anti-EGFR (cetuximab, gefitinib) dans des essais de clonogénicité en soft agar. L’expression d’ α5 favorise la résistance aux 2 drogues lors de la migration cellulaire. Pour aller plus loin, nous avons développé un nouveau test basé sur la quantification de l’évasion cellulaire à partir d’une sphère tumorale. La perte d’ α5 augmente la sensibilité des cellules U87 à 2 TKI réversibles spécifiques de l’EGFR, gefitinib et erlotinib, mais n’a pas d’effet sur l’efficacité du lapatinib, un TKI irréversible ciblant EGFR, ErbB2, ErbB3 et ErbB4. Grâce à la microscopie confocale, nous avons montré l’effet important du gefitinib sur l’endocytose de l’intégrine et de l’EGFR. Ces résultats suggèrent que l’expression d’ α5 favorise la résistance aux TKI par l’activation des voies de signalisation des récepteurs ErbB ou en contrôlant le trafic membranaire de l’EGFR. On a aussi montré que pour favoriser l’adhésion cellulaire, l’intégrine α5 stimulait la fibrillogénèse. Dans les cellules migrant à distance de la sphère, l’intégrine α5 est strictement engagée dans des adhésions cellule-substrat contenant la protéine FAK activée. Nos résultats soulignent le rôle central du couple fibronectine/ intégrine α5 dans l’invasivité du GBM et la résistance aux thérapies anti-EGFR. / Glioblastoma multiforme (GBM) is the most common primary brain tumor. Alteration of the EGFR pathway and high invasive potential are hallmarks of GBM. Unfortunately, trials using anti-EGFR therapies for the treatment of GBM reveal limited efficacy. We previously showed that overexpression of the fibronectin receptor, α5β1 integrin, is associated with a poor prognosis for patients and is responsible for chemoresistance to temodal. Integrins can cross-talk with growth factor receptors and amplified their oncogenic activity. Here, we sought to determine the potential role of α5 integrin in resistance to anti-EGFR therapy. Using U87 GBM cell line, we first confirmed that fibronectin-mediated integrin activation potentiated EGFR signaling. Loss of α5 integrin expression sensitized U87 cells to anti-EGFR drugs (cetuximab, gefitinib) in soft agar clonogenic assay. α5 expression can trigger resistance to both drugs on cell migration. To go further, we developed a new assay based on the quantification of cell evasion from tumor spheroids. α5 depletion increased U87 cell sensitivity to gefitinib and erlotinib, 2 EGFR-selective reversible TKI, but had not effect on lapatinib efficacy, an irreversible TKI that target EGFR, ErbB2, ErbB3 and ErbB4. Confocal microscopy revealed a strong impact of gefitinib on EGFR and integrin endocytosis. These results suggested that α5 expression may trigger resistance to TKI either by activating ErbB pathways or by controlling EGFR membrane trafficking. We also showed that to promote cell adhesion, α5 integrin stimulated fibronectin fibrillogenesis. As cells moved away from the spheroids, α5 became strictly engaged in cell-substratum adhesion sites where it recruited activated FAK. Our work highlights the pivotal role of fibronectin/α5β1 integrin in invasivity of GBM and resistance to anti-EGFR drugs.

L’hémostase primaire est un processus permettant la formation d’un clou plaquettaire qui sera stabilisé par un réseau de fibrine. Ce caillot est également consolidé grâce à des phases tardives de l’hémostase primaire résultant des fonctions plaquettaires ; il s’agit principalement de la rétraction qui diminue la taille du caillot afin de le stabiliser. Cette phase est déclenchée par une signalisation « outside-in », consécutive à l’activation de l’intégrine αIIbβ3 et à l’agrégation plaquettaire, et est dépendante d’une réorganisation du cytosquelette. Le premier objectif de ce travail a été d’étudier la signalisation impliquée dans la rétraction, et en particulier l’implication des protéines ROCK, MLCK, Rac-1 et de l’actine dans l’activité de la chaine légère de la myosine (MLC) . MLC est en effet une protéine clé de la réorganisation du cytosquelette. Nous avons mis en évidence une phosphorylation biphasique de MLC dont le deuxième pic, corrélé à la rétraction, est dépendant de Rac1 et de la polymérisation de l’actine. Cette étude a été appliquée à une pathologie, le syndrome de Lowe. Il s’agit d’une maladie génétique rare, également appelée OCRL (Oculo cérébro rénal de Lowe) en référence aux organes majoritairement touchés. Suite à l’observation d’événements hémorragiques per et postopératoires suggérant une instabilité du caillot et l’observation dans une étude précédente d’un temps d’occlusion allongé au PFA100®, nous avons mis en place une étude sur 15 patients et 15 témoins pour lesquels nous avons étudié les différentes phases de l’hémostase primaire. Outre une anomalie et un retard de maturation des mégacaryocytes, nous avons mis en évidence pour la première fois chez ces patients un défaut de la voie « outside-in » responsable d’une anomalie de l’étalement plaquettaire et de la rétraction du caillot. Ce défaut de rétraction, dû à un défaut d’activation de MLC, pourrait être en partie responsable des événements hémorragiques observés chez ces patients. / Primary hemostasis is a mechanism allowing platelet clot formation that is thereafter stabilized by a fibrin network. Fibrin clot is also consolidated following post occupancy events, mainly clot retraction that decrease clot size and thus strengthen it. This phase is triggered by « outside-in » signaling. It is consecutive to αIIbβ3 integrin activation and platelet aggregation, dependent on cytoskeleton organization. Our first objective was to investigate signaling events underlying retraction, and particularly the involvement of ROCK, MLCK, Rac-1, and actin in MLC (Myosin Light Chain) phosphorylation. Indeed, MLC, involved in cytoskeleton rearrangement, is a key protein of this mechanism. We described a MLC biphasic phosphorylation profile, which second peak was dependent of Rac1 and actin polymerization. In a second part, we studied clot retraction signaling in patients with the Lowe syndrome. It is a rare genetic disease, caused by absence of OCRL (oculo cerebro renal of Lowe) protein in reference to the majority of affected organs. The rationale of this study was a previous observation of hemorrhagic events during and after surgeries, suggesting clot instability. A thrombopathy was suggested by a closure time lengthening in the PFA-100 system. The study enrolled 15 patients and 15 controls. Besides a defect of megakaryocyte maturation, we described a defect of « outside-in » signaling responsible for spreading and clot retraction abnormality. This retraction defect, caused by a MLC activity defect, could be partly responsible for hemorrhagic events reported in these patients.

La néoangiogenèse tumorale est un élément pronostique de l’évolution de nombreux cancers. L’intégrine alphaVbeta3 ainsi que la métalloprotéase matricielle 9 (MMP-9), sont des marqueurs de ce processus. Leur ciblage offre la perspective d’une information diagnostique pour la détection précoce, l’évaluation de l’agressivité de pathologies et la sélection de patients répondeurs aux nouvelles thérapies anti-angiogéniques. Dans ce contexte, notre travail s’attèle à mettre au point les techniques nécessaires à la caractérisation de radiotraceurs. Des modèles de tumeurs richement néovascularisées ont été sélectionnés : le mélanome malin et le gliome malin. Nous nous sommes dans un premier temps intéressés à la détection de l’intégrine alphaVbeta3. Un traceur technétié, le 99mTc-DTPA-bis-c(RGDfK) a servi de support à la validation de nos techniques d’analyse. Cette méthodologie d’évaluation a ensuite été adaptée à des projets collaboratifs. L’étude du 18F-ribofuranose-RGD est réalisée avec le Centre de Recherche en Cancérologie de Toulouse (INSERM UMR 1037) et l’Institut des Sciences Moléculaires (CNRS UMR 5255). Un radioligand de la MMP-9, l’111In- DOTA-F3B, fait l’objet d’un partenariat avec l’ARNA (ARN : Régulations Naturelle et Artificielle, INSERM UMR 869) et l’Institut Lumière Matière (CNRS UMR 5306). Le composé technétié a démontré une bonne affinité et spécificité pour alphaVbeta3. In vivo, chez l’animal, les radioligands technétiés et fluorés ont permis l’identification de tumeurs alphaVbeta3 positives. L’111In-DOTA-F3B a, quant à lui, permis la visualisation de tumeurs chez l’animal et sur coupes tissulaires. Ces traceurs constituent une piste intéressante pour l’imagerie de la néoangiogenèse tumorale. / Tumor neoangiogenesis is a predictive element of the evolution of numerous cancers. AlphaVbeta3 integrin and matrix metalloprotease 9 (MMP-9) are markers of tumor neoangiogenesis. Their targeting appears of great interest either for early detection, aggressiveness staging of the disease or for selection of responders to new-targeted therapies. In this context, our objective is to develop methodologies needed for radiotracers characterization. Tracers have been investigated in different tumor models for which vascularization is very important: melanoma and glioma. First of all 99mTc-DTPA-bis-c(RGDfK) has been assessed in our laboratory and helped us to develop analytical methods. These methodologies were used in different partnership, the evaluation of 18F-ribofuranose-RGD targeting alphaVbeta3 with INSERM UMR 1037 and CNRS UMR 5255, and 111In-DOTA-F3B for molecular imaging of MMP-9 with INSERM UMR 869 and CNRS UMR 5306.The technetium peptide has demonstrated good affinity and specificity for alphaVbeta3. In vivo analysis in mice showed that both tracers were able to identify some alphaVbeta3-positive tumors. 111In-DOTA-F3B allowed us to detect hMMP-9 positive tumors in mice and in tumor tissue sections. In conclusion, these tracers still require to be investigated but represent promising tracers for tumor neoangiogenesis.

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis in cells. It has been implicated as a therapeutic target for various metabolic diseases like type II diabetes and obesity. However, its role in cancer is context-dependent and therefore warrants further studies to explore its possible use as a therapeutic target. AMPK can either promote or retard the growth of cancer cells depending on other cues and stresses in the milieu of the cancer cells. This study aims to understand AMPK signalling in response to extracellular cues of matrix deprivation and matrix stiffness that are important determinants of metastasis. 1) Calcium-Oxidant Signalling Network Regulates AMPK Activation upon Matrix Deprivation. Recent work from our lab, as well as others, has identified a novel role for the cellular energy sensor AMP-activated protein kinase in epithelial cancer cell survival under matrix deprivation. However, the molecular mechanisms that activate AMPK upon matrix-detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, while re-attachment to the matrix leads to its dephosphorylating and inactivation. Since matrix-detachment leads to loss of integrin signalling, we investigate whether integrin signalling negatively regulates AMPK activation. However, modulation of FAK or Src, the major downstream components of integrin signalling, fails to cause a corresponding change in AMPK signalling. Further investigations reveal that the upstream AMPK kinases, LKB1 and CaMKKβ, contribute to AMPK activation upon detachment. Additionally, we show LKB1 phosphorylation and cytosolic translocation upon matrix deprivation, which might also contribute to AMPK activation. In LKB1-deficient cells, we find AMPK activation to be predominantly dependent on Caskβ. We observe no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment is not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signalling and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that ER calcium release induced store-operated calcium entry (SOCE) contributes to intracellular calcium increase, leading to ROS production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. We find a significant increase in LKB1 as well as pACC levels in breast tumour tissues in comparison to normal tissues. Further, we observe a significant correlation between LKB1 and pACC levels in breast tumour tissues suggesting that LKB1-AMPK signaling pathway is active in vivo in breast cancers. Thus, the Ca2+/ROS triggered LKB1/CaMKK-AMPK signalling cascade may provide a quick, adaptable switch to promote survival of metastasising cancer cells. 2) Extracellular Matrix Stiffness Regulates Stemless through AMPK. Cancer cells experience changes in extracellular matrix stiffness during cancer progression. However, the signalling pathways utilised in sensing matrix stiffness are poorly understood. In this study, we identify AMPK pathway as a possible mechanosensory pathway in response to matrix stiffness. AMPK activity, as measured by downstream target phosphorylation, is found to be higher in soft matrix conditions. We additionally show that compared to stiff matrices, soft matrices increase stemless properties, as evidenced by the increased expression of stemless markers, which is dependent on AMPK activity. Thus, we elucidate a novel mechanotransduction pathway triggered by matrix stiffness that contributes to stemness of cancer cells by regulating AMPK activity. Taken together, our study identifies a novel calcium-oxidant signaling network in the rapid modulation of AMPK signaling in the context of matrix detachment. This pathway is especially relevant in the context of metastasising cancer cells that may not face energy stress in the blood stream but are matrix-deprived. Inhibition of AMPK might compromise the viability of these circulating cells thereby reducing the metastatic spread of cancer. Our study further suggests that varying stiffnesses experienced by cancer cells can modulate AMPK activity and this, in turn, regulates stem-like properties. Thus our study provides novel insights into various extracellular cues that regulate this kinase and contribute to cell survival and metastasis. This knowledge can be utilised in the stage-specific use of AMPK inhibitors in the treatment of breast cancer patients.

Breast cancer is one of the most prevalent cancers in the world. Each year, over 400,000 women die from breast cancer world wide and metastasis is the main cause of their mortality. Tumor cell invasion into the adjacent tissue is the first step in the multistep process of cancer metastasis and it involves multiple protein changes. The α6β4 integrin, a transmembrane heterodimeric laminin receptor is associated with poor prognosis in many tumor types, including breast cancer. Src family kinase (SFK) activity is elevated in many cancers and this activity also correlates with invasive tumor behavior. The α6β4 integrin can stimulate SFK activation and promote cancer invasion, however the mechanism by which it does so is not known. In the current study, I provide novel mechanistic insight into how the α6β4 integrin selectively activates the Src family kinase member Fyn in response to receptor engagement. Specifically, the tyrosine phosphatase SHP2 is recruited to α6β4 and its catalytic activity is stimulated through a specific interaction of its N-terminal SH2 domain with pY1494 in the β4 subunit. Importantly, both catalytic and non-catalytic functions of SHP2 are required for Fyn activation by α6β4. Fyn is recruited to the α6β4/SHP2 complex through an interaction with phospho-Y580 in the C-terminus of SHP2. In addition to activating Fyn, this interaction with Y580-SHP2 localizes Fyn to sites of receptor engagement, which is required for α6β4-dependent invasion. Moreover, the selective activation of Fyn, but not Src, requires the palmitoylation modification of Fyn on its N-terminus. Of clinical relevance, phospho-Y580-SHP2 and phospho-Y418-SFK could be used as potential biomarkers of invasive breast cancer because their expression are elevated in high-grade breast tumors.