Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Overall survival is about 70% and in cases where current treatment fails, the disease recurs and most often is fatal. At the molecular level, MB can be divided into four defined subgroups: WNT, SHH, Group 3 and Group 4. Amplification of MYC family genes is common in MB and correlates with poor prognosis and tumor relapse. In this thesis we showed how MYCN initiates brain tumors when transduced in neural stem cells (NSCs). Prior to transduction, NSCs were isolated from different brain regions and at various time points. While overexpression of wild-type MYCN did not generate any tumors, orthotopic transplantation of MYCNT58A-expressing forebrain, brain stem and cerebellar NSCs induced diffuse malignant glioma, PNET-like tumors and MB, respectively. Interestingly, MYCNT58A-expressing cerebellar NSCs induced SHH-dependent MB from embryonic cells but SHH-independent MB from postnatal cells. We further showed that cerebellar NSCs transduced with both MYCNT58A and transcription factor SOX9 developed tumors faster and promoted distant migration into the forebrain. The function and regulation of SOX9 in MB cells is poorly understood. We identified SOX9 protein as target of FBW7 ubiquitin ligase and demonstrated the effects of SOX9 on MB cells migration, metastasis and drug resistance. We further blocked PI3K pathway to destabilize SOX9 which sensitized cells to cytostatic treatment. We used a (TetOFF) transgenic mouse model of MYCN-induced MB (GTML) and crossed it with a (TetON) transgene which allowed us to specifically target rare SOX9-positive cells in the tumor. In this system, MB develops spontaneously and SOX9-negative tumor cells can be killed off by doxycycline. The few remaining SOX9-positive cancer cells were able to promote distant MB recurrences. Such a pattern of relapse was recently shown for Group 3 and 4 human MB where about 90% of the recurrences were distant. In summary, this thesis demonstrates that MYCN can generate various types of brain tumors depending on the timing and location of its expression. It further defines the existence of a rare population of SOX9-expressing MB cells that are involved in causing distant MB recurrences. Finally, it describes how SOX9 is stabilized in MB cells and increases MB migration and therapy resistance.

The incidence of melanoma and non‐melanoma skin cancer is continuing to increase worldwide. Melanoma is the sixth most common cancer in the United States, making skin cancer a significant public health issue. Photo chemoprevention with natural products is an effective strategy for the control of cutaneous neoplastic. Polyphenols from fruits have been shown to protect the skin from the adverse effects of solar UVR, cancer, and the growth of cancer stem cells. In particular, blueberries are known for their high concentration of phenolic compounds that have the high antioxidant capacity, and their effectiveness in reducing UV damage and, therefore, skin cancer. In Matar's lab, we have shown that Polyphenol-Enriched Blueberry Preparation (PEBP), derived from biotransformation of blueberry juice through fermentation, is effective for targeting skin cancer stem cell proliferation in different skin cancer cell lines. We predicted that PEBP affects melanoma skin cancer stem cells (MCSCs) epigenetically by targeting miRNA pathways. We observed the effects of PEBP on sphere growth and cell motility in vitro. We performed RT2-qPCR analyses to determine PEBP influence on miRNA in B16F10 spheres. We transfected B16F10 cells with miR-200b and performed western blotting analyses. Our results demonstrated that PEBP reduced sphere growth and cell migration, and up regulated miR-200b expression in different biological settings. Inhibition of miR-200b increased Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression. Consequently, PEBP may influence MCSCs through miRNA pathways. Elucidating the mechanisms by which PEBP modulates CSCs biological behavior by controlling miRNAs will enhance our understanding of the molecular mechanisms in skin cancer chemoprevention and might result in their use as natural photo-protectants in skin cancer.

The overall objective of this research consists of two main parts: (1) provide evidence that autocrine production of IGF-I modulates tight junction permeability and (2) demonstrate the ability of IGFBPs to regulate IGF-I delivery across cell layers. To meet the first objective, parental and IGF-I secreting bovine mammary epithelial cells were tested for cell layer permeability, tight and adherens junction proteins, IGF-IR, and a downstream signaling components of IGF-IR. In comparison with parental cells, IGF-I secreting cells had high levels of IGF-IRs, but low levels of the junction components E-cadherin, b-catenin, and occludin. The differences in parental and IGF-I secreting cells was not due to extracellular stimuli since inclusion of IGF-I, IGFBP-3, or co-culture with SV40-IGF-I cells did not alter the barrier properties of parental cells, suggesting that intracrine signaling may alter cell connectivity. The second objective focused on exogenous rather than endogenous IGF-I and the role of IGFBPs and IGF-IRs in ligand transcytosis. Bovine aortic endothelial cells (BAECs) cultured on surfaces optimized to minimize paracellular transport were utilized to investigate the kinetics involved in the transport of insulin-like growth factor-I from the apical side of confluent monolayers to the basolateral side. Binding competitors were used to determine the role of the cell surface insulin-like growth factor-I receptor (IGF-IR) and cell surface insulin-like growth factor binding proteins (IGFBPs) in this transport process. Although IGFBPs initially retard delivery of IGF-I, using a computation model, this report shows that pulse durations of less than 6 hrs resulted in enhanced delivery of IGF-I in the presence of IGFBPs, above that for delivery in the absence of IGFBPs. In addition, the model was utilized to identify key parameters to target when developing engineered growth factors for the treatment of diseases. It is shown that the sorting factions and internalization rates are reasonable targets for the design of engineered growth factors. Since the sorting fractions are dictated by binding affinities in the acidic environment of the endosomes, it may be beneficial to design and analog of IGF-I that is more resistant to changes in pH, similar to those develop from epidermal growth factor. / Ph. D.

Tumor metastasis currently presents the greatest obstacle for effective cancer remediation. Metastatic growth necessitates both degradation of a specialized form of extracellular matrix (ECM) known as the basement membrane (BM) and the invasion of surrounding tissues thereafter. The thoracic air sacs of fruit flies (Drosophila melanogaster), which develop and operate in a fashion comparable to the human lung, provide a unique model for identifying and characterizing factors that contribute to its own development as well as tumoral invasion. We investigated the involvement of both Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) signaling and a BMassociated protein during the development of air sac primordia (ASPs), the precursors to Drosophila air sacs. We find that JAK/STAT signaling occurs in ASP tip cells and that misexpression of core pathway components via the GAL4/UAS system negatively impacts ASP development. Further, we identify Unpaired 2 (Upd2) as the primary activating ligand for JAK/STAT activity in the ASP. Knockdown of the BM-associated protein using GAL4 drivers associated with a fibroblast growth factor (FGF) receptor gene, breathless (btl), and segment polarity gene, patched (ptc), prevented larval development beyond the second larval instar (L2). Knockdown of the BM-associated protein in the wing also produced bristle defects, but its overexpression did not have an effect anywhere other than in the ASP, where the proportion of mutant phenotypes increased significantly (p < .0001) in response. Finally, we find that collagen IV localization was unaffected by knockdown of the BM-associated protein. Together, our data constitute a significant step forward in understanding the role of both this BM-associated protein and JAK/STAT signaling in the ASP and similar mammalian structures.

Theranostics is an emerging treatment approach that combines diagnostics with therapy in order to personalize treatment regimens for individual patients and decrease cancer mortality. Previously, nanoparticles entrapping conventional fluorescent dyes were developed for cancer theranostics, but fluorescent nanoparticles did not allow clinicians to significantly improve cancer treatments. The use of fluorescent dyes that are sensitive to solvent acidity (halo-fluorochromism) and polarity (solvato-fluorochromism) may overcome the limitations of fluorescent nanoparticles and improve cancer therapy by enabling researchers to detect chemical properties within the nanoparticle core environment. The model halo- and solvato-fluorochromic dye Nile blue was attached to the core of nanoscale drug delivery systems called polymer nanoassemblies (PNAs), which were created by tethering hydrophilic polymers and hydrophobic groups to a cationic polymer scaffold. The fluorescence of empty PNAs increased by 100% at pH 5.0 compared to pH 7.4, and the fluorescence of drug-loaded PNAs increased up to 300% compared to empty PNAs. A comparison of the fluorochromic properties between PNAs with various core properties indicated that both hydrophobic pendant groups and scaffold amines contributed to the fluorochromism of PNAs. The halo-fluorochromism of PNAs allowed investigators to minimize the detection of fluorescence signals in healthy organs such as the liver. Fluorescence imaging of halo-fluorochromic PNAs diffused into tissue mimics indicated that fluorescence of PNAs in tissues increased by 100% at pH 7.0 compared to pH 7.4. In addition, halo-fluorochromic PNAs identified the acidic perimeter surrounding metastatic tumors in orthotopic metastatic tumor models. Computational simulations of metastatic lesions verified that some halo-fluorochromic PNAs accumulate in the hypoxic/acidic regions of metastatic tumors following intravenous administration. These simulations also indicated that the accumulation of PNAs in the hypoxic regions of tumors doubles at 12 hours post-treatment compared to 1.8 hours post-treatment. The solvato-fluorochromism of PNAs enabled the fluorescence-based measurement of drug release from the nanoassembly core during dialysis-based drug release measurements. Solvato-fluorochromic methods indicated faster drug release rates than HPLC-based methods. Mechanistic modeling of drug release indicated that solvato-fluorochromic methods were unaffected by released drugs that interfered with HPLC-based methods. However, mechanistic modeling also indicated that drug rebinding and diffusion did not account for all of the differences between drug release rates determined by solvato-fluorochromic- and HPLC-based methods. Based on this evidence, it was hypothesized that solvato-fluorochromic drug release methods measure drug diffusion from near the scaffold of PNAs in a small region of the nanoassembly core, and that this process contributes to overall drug release but does not indicate apparent drug release rates for PNAs. In order to develop PNAs for potential clinical applications, ionizable amines were removed from the polymer scaffold to increase drug loading and sustain the release of model drugs carfilzomib and docetaxel. The removal of primary amines decreased drug diffusivity in the core of PNAs (D from 3.9*10-18 cm2/s to 0.1*10-19 cm2/s) and increased the drug release half-life (t1/2 from 4 to 26 hours). The controlled release of carfilzomib from PNAs reduced drug metabolism by 60% for up to one hour and sustained proteasome inhibition in cancer cells at 72 h post-treatment compared to free drug. Overall, this work provides insight into the design of theranostic nanoparticles with beneficial properties for improving cancer treatment.

Establishment and maintenance of the blood system relies on the cellular and spatial organization of bone marrow. In the BM niche, sinusoidal endothelial cells (SECs) are mainly located in the trabecular zone of the metaphysis area of long bones. SECs present a fenestrated structure characterized by high permeability, low shear rates, and oxygen pressure. The hypoxic environment surrounding SECs is needed for the movement and engraftment of hematopoietic cells, but it might also facilitate the homing of malignant cells. SECs’ adaptive response to hypoxia depends on the Hypoxia Inducible Factors (HIFs) promoting angiogenesis, hematopoiesis, and other processes. The upstream regulator of HIFs, the prolyl hydroxylase domain-2 (PHD2) is considered the key cellular oxygen sensor. Our group has shown the crucial regulatory effects that PHD2 has on different physiological and pathological settings. During steady state, PHD2 modulates proliferation and mobilization of hematopoietic progenitor cells (HSCPs), as well as bone metabolism. On the other hand, PHD2 is crucial for neutrophil motility affecting their extravasation during arthritis. We have also demonstrated that loss of PHD2 in different humans and mouse tumor cell lines, as well as in myeloid cells and T-lymphocytes where it impairs tumor development. Others have shown that heterozygous deletion of PHD2 in tumor ECs can reduce distant metastasis. Due to the intricate interplay between the different players of the PHD2/HIFs axis, as well as their effect on other signaling pathways, the positive or negative impact of the hypoxia pathways has been shown to be cell type and context dependent. In the present study, we analyzed the role of hypoxia pathway proteins, mainly PHD2, in ECs in the bone/BM niche, as well as its consequent influence on the environment during physiological and pathological scenarios. We have demonstrated that endothelial PHD2 has a profound intrinsic effect on vessel morphology and functionality, affecting the crucial communication between ECs and the bone/BM niche. Further, using different transgenic mouse lines, we have identified the BM endothelial cells (BM-ECs) PHD2/HIF-2α axis directly increasing leukocytosis via vascular cell adhesion protein 1 (VCAM-1) protein downregulation. Moreover, during steady state conditions we have discovered a novel regulatory effect that PHD2 exerts on VCAM-1 by increasing miR-126-3p transcription, a well-known inhibitor of VCAM-1 expression. Lastly, the PHD2/HIF-2/miR-126-3p/VCAM-1 axis not only influenced the myeloid cell intravasation towards circulation but also increased the extravasation and homing of metastatic breast carcinoma cells into the bone/BM tissue, increasing tumor burden throughout the bone. BM-ECs PHD2 offers a protective role against tumor homing cells in the bone/BM while serving as an important regulator in the communication between endothelium and BM niche. Concluding Remarks:  Loss of PHD2 in ECs generates profound changes in vessel function and in the hematopoietic compartment leading to increase myelopoiesis resulting in leukocytosis. The above-mentioned effect occurs in a HIF-2α-dependent manner.  PHD2/HIF-2α also transcribed in an increase of miR-126-3p expression leading to VCAM-1 downregulation. Process that resulted in increased leukocytes in circulation due to hematopoiesis dysregulation. The novel PHD2/HIF-2/miR-126-3p/VCAM-1 axis enhanced extravasation and homing of metastatic breast carcinoma cells into the bone/BM tissue, increasing tumor burden throughout the bone.:Introduction 8 The Endothelium 9 Endothelial barrier. 9 Leukocyte transendothelial migration. 11 Intracellular mechanism for activation of adhesion molecules. 12 Cellular migration in pathological conditions. 13 Tumor Dissemination: Metastasis. 14 Metastatic cascade. 15 The bone is a preferential site for malignant cells arrival. 16 Bone/BM physiology influences metastasis. 17 The endosteal niche. 18 The perivascular niche. 19 Hypoxia pathway proteins impact on metastasis. 20 Thesis Aims 23 Aim 1: Characterize the role of the hypoxia pathway proteins in bone vasculature and the impact on the niche. 24 Aim 2: Analyze the genetic changes that resulted from PHD2 deletion in BM-ECs and their impact on the cross-talk communication with the different BM-niches. 24 Aim 3: Investigate the impact of PHD2 and downstream regulators (HIF-1/2α) on vessel functionality. 25 Materials and Methods 26 Mice. 27 Histology: tissue processing and immunofluorescence staining 28 Bone tissue processing. 28 Staining of Bone cryosections. 28 Induced skin inflammatory model. 29 Staining of ears treated with PMA. 29 Vascular morphology quantification. 29 Microscopy 31 Antibodies used for immunofluorescence. 31 Bone analysis. 31 Bone µCT measurements and analysis 31 Tartrate-resistant acid phosphatase (TRAP) staining 32 Blood and BM analysis. 32 Sysmex. 32 Flow cytometry. 32 BM cell extraction from bones. 32 BM cells staining. 33 Meso Scale Discovery (MSD) 35 Evans Blue assay. 35 BM soup ELISA. 35 RNAseq of CD31+ EMCN+ BM-ECs 36 BM-EC cell sorting for RNAseq. 36 RNA extraction and qPCRs 36 Tumor model. 36 Tumor breast carcinoma cells. 36 Tumor homing model. 37 Statistical analyses. 37 Results 38 PHD2 Conditional Knockout from Endothelial Cell Compartment. 39 Further P2EC mice characterization. 41 Transgenic deletion of PHD2 showed slight developmental retardation. 41 Spleen size showed not to be affected by deletion endothelial PHD2. 41 P2EC mice displayed increased vessel leakiness. 42 Endothelial PHD2 deletion does not affect lung endothelial cells. 43 BM-ECs PHD2-HIF-2α axis modulates leukocytosis and vessel morphology. 44 HIF-2α modulates P2EC leukocytosis and thrombocytopenia. 44 BM-ECs PHD2 deficient mice hinder vessel morphology in a HIF-2α dependent manner. 44 Endothelial PHD2-deficient mice exhibit perturbed hematopoiesis. 45 P2EC mice early progenitor displayed reduced total cell number, but frequency remained unchanged. 46 P2EC mice favor differentiation of committed progenitors with a myeloid bias. 48 P2EC mice significantly reduced the numbers and frequency of megakaryocyte/erythroid progenitor’s linage. 48 PHD2-HIF-2α deletion restored normal hematopoiesis. 50 P2EC vascular functionality during pathological conditions. 53 Endothelial PHD2 modulates leukocyte migration during localized inflammation. 53 Endothelial PHD2 shapes bone/BM tumor homing. 55 Tumor homing in the bone: generation of an early metastatic model. 56 Early metastasis limitation. 58 Endothelial PHD2 modulates tumor colonization to the bone/BM. 59 Simultaneous deletion of PHD2 and HIF-1 in BM-ECs worsen tumor metastasis to bone. 61 Simultaneous deletion of PHD2 and HIF-2 in BM-ECs showed no differences in tumor homing. 62 Deep sequencing of PHD2 deficient BM-ECs. 63 BM EC from P2EC mice display enriched leukocyte migration gene signatures. 63 P2EC mice presented genetic dysfunction in the integrin-binding system. 64 P2EC steady-state VCAM-1 expression is HIF-2α dependent. 66 BM-ECs VCAM-1 + is regulated by PHD2 through HIF-2α. 66 PHD2-dependent downregulation of VCAM-1 does not affect VE-cadherin expression. 68 BM pro-inflammatory cytokines do not contribute to VCAM-1 lower expression. 69 During steady-state, loss of VCAM-1 increased frequency BM resident mature cells. 69 BM-ECs VCAM-1 deficient mice 71 VCAM1EC mice developed leukocytosis. 71 VCAM1EC does not exhibit significant changes in hematopoiesis. 73 P2EC vessel morphology is independent of downregulation of VCAM-1 74 VCAMEC mice showed increased tumor homing in the diaphysis. 75 PHD2-HIF-2 regulatory effect on VCAM-1 is modulated by mir-126-3p. 76 HIF-2α regulates mir-126 expression in PHD2 deficient BM-ECs. 77 BM-ECs PHD2 influence bone homeostasis. 78 Loss of BM-ECs PHD2 lead to increase Osteoclast numbers and activity. 79 Osteoclast differentiation and activity could be independent of OBs. 79 Loss of PHD2 in BM-ECs leads to osteoclastogenesis. 80 BM resident Tcell CD8+ could be Increasing Osteoclast Activation. 82 Discussion. 83 Mouse Model: Conditional Deletion of Endothelial PHD2. 85 Endothelial PHD2 Modulates Myelopoiesis. 86 BM-EC PHD2 regulates vessel morphology and functionality under steady-state independent of VCAM-1. 88 Endothelial VCAM-1 downregulation does not impaired neutrophil migration during inflammation. 88 BM-ECs PHD2 is a Gatekeeper of Tumor Homing in the Bone. 89 HIF-2α dependent Mir-126 activation leads to VCAM-1 downregulation 91 Endothelial PHD2 controls Osteoclastogenesis independent of BM RANKL. 94 References 96 List of Abbreviations 107 Summary 109 Zusammenfassung 111 Acknowledgements 113 Deklaration 114 Appendix 118 List of Figures. 118 List of tables 119 / Die Organisation und Aufrechterhaltung des Blutsystems hängt von der zellulären und räumlichen Organisation des Knochenmarks ab. In der BM-Nische befinden sich, hauptsächlich in der Trabekelzone des Metaphysenbereichs langer Knochen, die sinusoidale Endothelzellen (SECs). SECs weisen eine gefensterte Struktur auf, die von hoher Durchlässigkeit, geringer Scherrate und Sauerstoffdruck geprägt ist. Das hypoxische Milieu der SECs ist notwendig für Bewegung und Einwanderung der hämatopoetischen Zellen und könnte dies ebenso für bösartige Zellen begünstigen. Die Anpassung der SECs an Hypoxie hängt von den Hypoxia Inducible Factors (HIFs) ab. HIFs fördern die Angiogenese, die Hämatopoese und andere Prozesse. Die prolyl hydorxylase domain-2 (PHD2) ist der vorgeschaltete Regulator der HIFs und gilt als wichtigster zellulärer Sauerstoffsensor. Unsere Gruppe konnte zeigen welche zentralen regulatorischen Effekte die PHD2 auf verschiedene physiologische und pathophysiologische Mechanismen ausübt. Im Gleichgewichtszustand moduliert PHD2 Proliferation und Mobilisation der hämatopoetischen Vorläuferzellen (HSCPs) sowie den Knochenmetabolismus. Auf der einen Seite spielt PHD2 eine entscheidende Rolle bei der Motilität der Neutrophilen und beeinflusst daher die Extravasation bei Arthritis. Wir konnten außerdem zeigen, dass der Verlust von PHD2 in verschiedenen humanen und murinen Tumorzelllinien, sowie in myeloischen Zellen und T-Lymphozyten die Tumorentwicklung beeinträchtigt. In anderen Arbeiten wurde gezeigt, dass eine heterozygote PHD2-Deletion in Tumor-ECs eine Fernmetastasierung reduziert. In Anbetracht der komplizierten Wechselwirkung zwischen den verschiedenen Komponenten der PHD2/HIF-Signalkaskade sowie deren Effekte auf andere Signalkaskaden, übt sich in Abhängigkeit des Zelltyps und des Kontexts ein positiver oder negativer Einfluss auf die Hypoxie-Signalwege aus. In der vorliegenden Studie haben wir die Rolle von Proteinen des Hypoxiewegs, hauptsächlich PHD2, in den ECs der Knochen-/BM-Nische sowie deren daraus resultierenden Einfluss auf die Umwelt in physiologischen und pathologischen Szenarien analysiert. Wir konnten zeigen, dass das endotheliale PHD2 einen tiefgreifenden intrinsischen Effekt auf die Gefäßmorphologie und Funktionalität besitzt und damit entscheidend die Kommunikation zwischen ECs und der Knochen-/BM-Nische beeinflusst. Weiterhin konnte unter Nutzung verschiedener transgener Muslinien identifiziert werden, dass die Knochenmarksendothelzellen (BM-ECs) PHD2/HIF-2α-Achse direkt die Myelopoese, durch eine Herabsetzung der vascular cell adhesion protein 1 (VCAM-1) -Expression, steigert. Darüber hinaus haben wir einen neuartigen regulatorischen Effekt der PHD2 auf das VCAM-1 entdeckt. Hierbei wird die Expression des VCAM-1 Inhibitors miR-126-3p gesteigert. Des Weiteren beeinflusst die PHD2/HIF-2/miR-126-3p/VCAM-1 Achse nicht nur die Intravasation der Myloidzellen in Richtung des Kreislaufs, sondern auch eine Steigerung der Extravastion und Einwanderung von metastasierenden Brustkarzinomzellen in die Knochen/BM-Gewebe und steigert somit die Tumorlast in den Knochen. In Anbetracht klinischer Versuche der Krebsbehandlung mit PHD2-Inhibitoren, bietet BM-ECs PHD2 einen schützenden Effekt gegen Tumorzelleinwanderung in die Knochen/BM, während es gleichzeitig als ein wichtiger Regulator in der Kommunikation zwischen Endothelium und der BM-Nische dient. Abschließende Bemerkungen: Der Verlust von PHD2 in ECs führt zu tiefgreifenden Veränderungen in der Gefäßfunktion und im hämatopoetischen Kompartiment, was zu einer verstärkten Myelopoese und damit zu Leukozytose führt. Die oben erwähnte Wirkung tritt in einer HIF-2α-abhängigen Weise auf. PHD2/HIF-2α führte auch zu einem Anstieg der miR-126-3p-Expression, was zu einer Herunterregulierung von VCAM-1 führte. Dieser Prozess führte zu einer erhöhten Anzahl von Leukozyten im Blutkreislauf aufgrund einer Dysregulation der Hämatopoese. Die neuartige PHD2/HIF-2/miR-126-3p/VCAM-1-Achse förderte die Extravasation und Ansiedlung von metastatischen Brustkrebszellen im Knochen/BM-Gewebe, wodurch die Tumorlast im gesamten Knochen erhöht wurde.:Introduction 8 The Endothelium 9 Endothelial barrier. 9 Leukocyte transendothelial migration. 11 Intracellular mechanism for activation of adhesion molecules. 12 Cellular migration in pathological conditions. 13 Tumor Dissemination: Metastasis. 14 Metastatic cascade. 15 The bone is a preferential site for malignant cells arrival. 16 Bone/BM physiology influences metastasis. 17 The endosteal niche. 18 The perivascular niche. 19 Hypoxia pathway proteins impact on metastasis. 20 Thesis Aims 23 Aim 1: Characterize the role of the hypoxia pathway proteins in bone vasculature and the impact on the niche. 24 Aim 2: Analyze the genetic changes that resulted from PHD2 deletion in BM-ECs and their impact on the cross-talk communication with the different BM-niches. 24 Aim 3: Investigate the impact of PHD2 and downstream regulators (HIF-1/2α) on vessel functionality. 25 Materials and Methods 26 Mice. 27 Histology: tissue processing and immunofluorescence staining 28 Bone tissue processing. 28 Staining of Bone cryosections. 28 Induced skin inflammatory model. 29 Staining of ears treated with PMA. 29 Vascular morphology quantification. 29 Microscopy 31 Antibodies used for immunofluorescence. 31 Bone analysis. 31 Bone µCT measurements and analysis 31 Tartrate-resistant acid phosphatase (TRAP) staining 32 Blood and BM analysis. 32 Sysmex. 32 Flow cytometry. 32 BM cell extraction from bones. 32 BM cells staining. 33 Meso Scale Discovery (MSD) 35 Evans Blue assay. 35 BM soup ELISA. 35 RNAseq of CD31+ EMCN+ BM-ECs 36 BM-EC cell sorting for RNAseq. 36 RNA extraction and qPCRs 36 Tumor model. 36 Tumor breast carcinoma cells. 36 Tumor homing model. 37 Statistical analyses. 37 Results 38 PHD2 Conditional Knockout from Endothelial Cell Compartment. 39 Further P2EC mice characterization. 41 Transgenic deletion of PHD2 showed slight developmental retardation. 41 Spleen size showed not to be affected by deletion endothelial PHD2. 41 P2EC mice displayed increased vessel leakiness. 42 Endothelial PHD2 deletion does not affect lung endothelial cells. 43 BM-ECs PHD2-HIF-2α axis modulates leukocytosis and vessel morphology. 44 HIF-2α modulates P2EC leukocytosis and thrombocytopenia. 44 BM-ECs PHD2 deficient mice hinder vessel morphology in a HIF-2α dependent manner. 44 Endothelial PHD2-deficient mice exhibit perturbed hematopoiesis. 45 P2EC mice early progenitor displayed reduced total cell number, but frequency remained unchanged. 46 P2EC mice favor differentiation of committed progenitors with a myeloid bias. 48 P2EC mice significantly reduced the numbers and frequency of megakaryocyte/erythroid progenitor’s linage. 48 PHD2-HIF-2α deletion restored normal hematopoiesis. 50 P2EC vascular functionality during pathological conditions. 53 Endothelial PHD2 modulates leukocyte migration during localized inflammation. 53 Endothelial PHD2 shapes bone/BM tumor homing. 55 Tumor homing in the bone: generation of an early metastatic model. 56 Early metastasis limitation. 58 Endothelial PHD2 modulates tumor colonization to the bone/BM. 59 Simultaneous deletion of PHD2 and HIF-1 in BM-ECs worsen tumor metastasis to bone. 61 Simultaneous deletion of PHD2 and HIF-2 in BM-ECs showed no differences in tumor homing. 62 Deep sequencing of PHD2 deficient BM-ECs. 63 BM EC from P2EC mice display enriched leukocyte migration gene signatures. 63 P2EC mice presented genetic dysfunction in the integrin-binding system. 64 P2EC steady-state VCAM-1 expression is HIF-2α dependent. 66 BM-ECs VCAM-1 + is regulated by PHD2 through HIF-2α. 66 PHD2-dependent downregulation of VCAM-1 does not affect VE-cadherin expression. 68 BM pro-inflammatory cytokines do not contribute to VCAM-1 lower expression. 69 During steady-state, loss of VCAM-1 increased frequency BM resident mature cells. 69 BM-ECs VCAM-1 deficient mice 71 VCAM1EC mice developed leukocytosis. 71 VCAM1EC does not exhibit significant changes in hematopoiesis. 73 P2EC vessel morphology is independent of downregulation of VCAM-1 74 VCAMEC mice showed increased tumor homing in the diaphysis. 75 PHD2-HIF-2 regulatory effect on VCAM-1 is modulated by mir-126-3p. 76 HIF-2α regulates mir-126 expression in PHD2 deficient BM-ECs. 77 BM-ECs PHD2 influence bone homeostasis. 78 Loss of BM-ECs PHD2 lead to increase Osteoclast numbers and activity. 79 Osteoclast differentiation and activity could be independent of OBs. 79 Loss of PHD2 in BM-ECs leads to osteoclastogenesis. 80 BM resident Tcell CD8+ could be Increasing Osteoclast Activation. 82 Discussion. 83 Mouse Model: Conditional Deletion of Endothelial PHD2. 85 Endothelial PHD2 Modulates Myelopoiesis. 86 BM-EC PHD2 regulates vessel morphology and functionality under steady-state independent of VCAM-1. 88 Endothelial VCAM-1 downregulation does not impaired neutrophil migration during inflammation. 88 BM-ECs PHD2 is a Gatekeeper of Tumor Homing in the Bone. 89 HIF-2α dependent Mir-126 activation leads to VCAM-1 downregulation 91 Endothelial PHD2 controls Osteoclastogenesis independent of BM RANKL. 94 References 96 List of Abbreviations 107 Summary 109 Zusammenfassung 111 Acknowledgements 113 Deklaration 114 Appendix 118 List of Figures. 118 List of tables 119

No description available.

O câncer de mama é o tipo de câncer mais comumente detectado em mulheres de todo o mundo. Na maioria das pacientes, a causa de morte se deve, principalmente, à doença metastática que pode se desenvolver a partir do tumor primário. O processo metastático envolve uma complexa cascata de eventos, incluindo a quebra organizada dos componentes da matriz extracelular por metaloproteinases de matriz (MMPs). A atividade das MMPs é precisamente regulada por inibidores específicos, os inibidores teciduais das MMPs (TIMPs). Dado seu papel na progressão tumoral, níveis elevados de MMPs têm sido associados com prognóstico desfavorável para pacientes com câncer. Por outro lado, sendo os TIMPs proteínas multifuncionais, níveis elevados de TlMP-1 e de TIMP-2 correlacionam com agressividade do tumor e prognóstico ruim em diferentes tipos de câncer, incluindo o câncer de mama. O gene supressor de metástase RECK codifica uma glicoproteína de membrana capaz de inibir a invasão e a metástase tumoral através da regulação negativa da atividade de MMPs envolvidas em carcinogênese: MMP-2, MMP-9 e MMP-14 (MT1-MMP). A fim de analisar o papel das MMPs e de seus inibidores (TIMPs e RECK) na progressão tumoral do câncer de mama, o perfil de expressão destes genes foi detectado, através de ensaios de Real-Time PCR, em um painel de cinco linhagens celulares de carcinoma de mama humano com diferentes potenciais invasivos e metastáticos e em 72 amostras teciduais de tumores primários de mama e 30 amostras teciduais de borda normal adjacente ao tumor. O perfil de expressão protéica de RECK foi avaliado em 236 amostras de tumores primários de mama através de ensaios de Tissue Microarray. Além disso, a atividade proteolítica das MMPs foi detectada em ensaios de Zimografia. Os resultados obtidos indicam que a progressão do câncer de mama humano está relacionada com um aumento dos níveis de expressão das MMPs e de seus inibidores específicos. O aumento dos níveis de expressão dos TIMPs parece estar relacionado ao seu papel como proteína multifuncional que pode estar funcionando de maneira a promover, mais do que suprimir, a progressão tumoral. Níveis elevados da expressão protéica de RECK estão associados com pior prognóstico. No entanto, para pacientes em estádios clínicos avançados, altos níveis de expressão de RECK podem estar correlacionados com melhor prognóstico, dependendo do balanço MMP/inibidor. Os níveis de expressão das MMPs apresentaram correlação positiva em relação aos níveis de expressão de seus inibidores específicos, sugerindo a existência de fatores e vias de sinalização comuns envolvidas na regulação coordenada destes genes. Além disso, a síntese do inibidor pode estar relacionada a uma resposta celular ao aumento da expressão e atividade de proteases. O balanço transcricional enzima/inibidor favorece a enzima nas amostras tumorais e, de modo contrário, o inibidor específico nas amostras de borda normal, sugerindo o balanço como o principal fator na determinação da degradação da MEC em processos invasivos e metastáticos. Os resultados obtidos podem contribuir para um melhor entendimento da complexidade dos mecanismos envolvidos na metástase do câncer de mama. / Breast cancer is among the most common tumors affecting women. Like most solid tumors, metastatic disease rather than the primary tumor itself is responsible for death. The metastatic process involves a complex cascade of events, including the organized breakdown of the extracellular matrix by matrix metalloproteinases (MMPs). The activity of these proteases is tightly regulated by specific inhibitors, known as tissue inhibitors of MMPs (TIMPs). Consistent with their role in tumor progression, high levels of a number of MMPs have been shown to correlate with poor prognosis in human cancers. On the other hand, TIMPs are multifunctional molecules with high levels of TIMP-1 and TIMP-2 having been shown to predict adverse prognosis and correlate with tumor aggressiveness in several different human cancers, including breast cancer. The RECK metastasis suppressor gene encodes a membrane-associated MMP regulator protein that is able to suppress tumor invasion and metastasis by negatively regulating MMPs involved in carcinogenesis, namely: MMP-2, MMP-9 and MMP-14 (MT1-MMP). In order to analyse the role of these genes in breast cancer progression, the expression levels of MMPs and theirs inhibitors were detected by Real Time PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential and in 72 primary breast cancer and 30 adjacent normal tissue specimens. The RECK protein expression profile was also examined in 236 primary breast cancer tissue specimens by Tissue Microarray technology. The proteolytic activity of MMPs was examined by Zymography. The results suggest that high expression levels of MMPs and their inhibitors are correlated with breast cancer progression. High levels of TIMP transcript may be involved in tumor-promoting activity as a result of their multifunctional role. Increased levels of the RECK protein are correlated with poor prognosis for the patient. However, high levels of RECK would be expected to confer a favorable prognosis to patients with advanced disease. The expression levels of MMPs significantly correlated with the levels of TIMPs and may be explained by coordinate correlation of these molecules or, alternatively, the synthesis of an inhibitor may be a cellular reaction to the presence of the protease. The enzyme/inhibitor balance at the transcriptional level favors the enzyme in tumor tissue and the inhibitor in adjacent normal tissue. It is probably the parameter that will determine the matrix degradation at invasion and metastatic process. Our results are likely to contribute for better understanding of the complex mechanisms involved in breast cancer metastasis.

O câncer de mama é o tipo de câncer mais comumente detectado em mulheres de todo o mundo. Na maioria das pacientes, a causa de morte se deve, principalmente, à doença metastática que pode se desenvolver a partir do tumor primário. O processo metastático envolve uma complexa cascata de eventos, incluindo a quebra organizada dos componentes da matriz extracelular por metaloproteinases de matriz (MMPs). A atividade das MMPs é precisamente regulada por inibidores específicos, os inibidores teciduais das MMPs (TIMPs). Dado seu papel na progressão tumoral, níveis elevados de MMPs têm sido associados com prognóstico desfavorável para pacientes com câncer. Por outro lado, sendo os TIMPs proteínas multifuncionais, níveis elevados de TlMP-1 e de TIMP-2 correlacionam com agressividade do tumor e prognóstico ruim em diferentes tipos de câncer, incluindo o câncer de mama. O gene supressor de metástase RECK codifica uma glicoproteína de membrana capaz de inibir a invasão e a metástase tumoral através da regulação negativa da atividade de MMPs envolvidas em carcinogênese: MMP-2, MMP-9 e MMP-14 (MT1-MMP). A fim de analisar o papel das MMPs e de seus inibidores (TIMPs e RECK) na progressão tumoral do câncer de mama, o perfil de expressão destes genes foi detectado, através de ensaios de Real-Time PCR, em um painel de cinco linhagens celulares de carcinoma de mama humano com diferentes potenciais invasivos e metastáticos e em 72 amostras teciduais de tumores primários de mama e 30 amostras teciduais de borda normal adjacente ao tumor. O perfil de expressão protéica de RECK foi avaliado em 236 amostras de tumores primários de mama através de ensaios de Tissue Microarray. Além disso, a atividade proteolítica das MMPs foi detectada em ensaios de Zimografia. Os resultados obtidos indicam que a progressão do câncer de mama humano está relacionada com um aumento dos níveis de expressão das MMPs e de seus inibidores específicos. O aumento dos níveis de expressão dos TIMPs parece estar relacionado ao seu papel como proteína multifuncional que pode estar funcionando de maneira a promover, mais do que suprimir, a progressão tumoral. Níveis elevados da expressão protéica de RECK estão associados com pior prognóstico. No entanto, para pacientes em estádios clínicos avançados, altos níveis de expressão de RECK podem estar correlacionados com melhor prognóstico, dependendo do balanço MMP/inibidor. Os níveis de expressão das MMPs apresentaram correlação positiva em relação aos níveis de expressão de seus inibidores específicos, sugerindo a existência de fatores e vias de sinalização comuns envolvidas na regulação coordenada destes genes. Além disso, a síntese do inibidor pode estar relacionada a uma resposta celular ao aumento da expressão e atividade de proteases. O balanço transcricional enzima/inibidor favorece a enzima nas amostras tumorais e, de modo contrário, o inibidor específico nas amostras de borda normal, sugerindo o balanço como o principal fator na determinação da degradação da MEC em processos invasivos e metastáticos. Os resultados obtidos podem contribuir para um melhor entendimento da complexidade dos mecanismos envolvidos na metástase do câncer de mama. / Breast cancer is among the most common tumors affecting women. Like most solid tumors, metastatic disease rather than the primary tumor itself is responsible for death. The metastatic process involves a complex cascade of events, including the organized breakdown of the extracellular matrix by matrix metalloproteinases (MMPs). The activity of these proteases is tightly regulated by specific inhibitors, known as tissue inhibitors of MMPs (TIMPs). Consistent with their role in tumor progression, high levels of a number of MMPs have been shown to correlate with poor prognosis in human cancers. On the other hand, TIMPs are multifunctional molecules with high levels of TIMP-1 and TIMP-2 having been shown to predict adverse prognosis and correlate with tumor aggressiveness in several different human cancers, including breast cancer. The RECK metastasis suppressor gene encodes a membrane-associated MMP regulator protein that is able to suppress tumor invasion and metastasis by negatively regulating MMPs involved in carcinogenesis, namely: MMP-2, MMP-9 and MMP-14 (MT1-MMP). In order to analyse the role of these genes in breast cancer progression, the expression levels of MMPs and theirs inhibitors were detected by Real Time PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential and in 72 primary breast cancer and 30 adjacent normal tissue specimens. The RECK protein expression profile was also examined in 236 primary breast cancer tissue specimens by Tissue Microarray technology. The proteolytic activity of MMPs was examined by Zymography. The results suggest that high expression levels of MMPs and their inhibitors are correlated with breast cancer progression. High levels of TIMP transcript may be involved in tumor-promoting activity as a result of their multifunctional role. Increased levels of the RECK protein are correlated with poor prognosis for the patient. However, high levels of RECK would be expected to confer a favorable prognosis to patients with advanced disease. The expression levels of MMPs significantly correlated with the levels of TIMPs and may be explained by coordinate correlation of these molecules or, alternatively, the synthesis of an inhibitor may be a cellular reaction to the presence of the protease. The enzyme/inhibitor balance at the transcriptional level favors the enzyme in tumor tissue and the inhibitor in adjacent normal tissue. It is probably the parameter that will determine the matrix degradation at invasion and metastatic process. Our results are likely to contribute for better understanding of the complex mechanisms involved in breast cancer metastasis.