<p>Neuroblastoma is a pediatric cancer arising from undifferentiated neural crest-derived precursor cells. Treatment strategies for neuroblastoma aim to promote neuroblast differentiation, however current therapies available are only modestly effective. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that act to promote neuroblast differentiation, though the precise factors released and their mechanism of action in neuroblastoma remains unclear. Here, we identify a novel component of the differentiating stroma secretome and harness stroma biology to inform the use of a combination therapy for neuroblastoma treatment.</p><p>HBEGF expression is decreased in neuroblastoma compared to benign disease, correlating to an increase in mortality. HBEGF protein is expressed only in stromal compartments of tumor specimens, with tissue from late-stage disease containing very little stroma or HBEGF. Addition of soluble HBEGF to neuroblastoma cell lines leads to increased neuroblast differentiation and decreased proliferation. Heparan sulfate proteoglycans (HSPGs) and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor (EGFR), leading to activation of the ERK1/2 and STAT3 pathways and upregulation of the inhibitor of DNA binding 1 transcription factor. </p><p>Expression of the type III TGF-β receptor (TβRIII), an HSPG, is epigenetically regulated in neuroblastoma cells via direct binding of the N-Myc transcription factor to Sp-1 sites on the TβRIII promoter. Analysis of patient microarray data demonstrate that other members of the differentiating stroma secretome, including HBEGF and EGFR, are positively correlated with TβRIII expression, suggesting that these proteins may be co-regulated. Treatment with inhibitors aimed at blocking N-Myc function, including inhibitors of histone deacetylases, DNA methyltransferases (DNMTs), and aurora kinase A (AurkA) can promote neuroblast differentiation and decrease proliferation. The combination of the DNMT inhibitor decitabine with the AurkA inhibitor MLN8237 enhances differentiation and reduces proliferation compared to either agent alone. Importantly, the combination of clinically achievable doses of these targeted agents dramatically reduces tumor growth in orthotopic xenograft models of neuroblastoma, identifying a novel combination therapy that may benefit children with this disease.</p><p>In conclusion, these studies dissect the tumor microenvironment to identify an important member of the differentiating stroma secretome, while also revealing a combination therapy for clinical development that has the potential to decrease adverse side effects and increase effectiveness of neuroblastoma treatment.</p> / Dissertation

L'acide lysophosphatique est un biolipide naturel actif capable de réguler diverses fonctions biologiques et d'agir en tant que facteur de croissance, via l'activation de six différents récepteurs de surfaces couplées aux protéines G (LPA1-6). Notre laboratoire a montré que le ciblage thérapeutique du récepteur LPA1 bloque de façon remarquable la dissémination métastatique des cellules de cancer du sein. Les mécanismes moléculaires et génétiques impliqués dans ce processus sont cependant encore inconnus. De plus, la plupart des cellules de mammifères co-expriment plusieurs formes de récepteurs du LPA. La réponse cellulaire est la résultante de l'activation de multiples voies de signalisation, parfois synergiques ou opposées, compromettant la validation chez le patient de l'efficacité des thérapies ciblant ces récepteurs. Au cours de cette thèse, nous avons dans un premier temps montré que HB-EGF est un marqueur spécifique de l'activité de LPA1. Le blocage pharmacologique de ce récepteur via des antagonistes des récepteurs LPA1-3 (Ki16425/Debio0719) ou l'invalidation de son expression par une technique d'ARN interférence entraine une inhibition de la surexpression en HB-EGF. Le ciblage thérapeutique de LPA1 via l'antagoniste Ki16425, dans notre modèle animal préclinique de xénogreffe de cancer de la prostate PC3, conduit également à une diminution de l'expression en ARNm de HB-EGF au niveau de la tumeur primaire et à une diminution des concentrations en HB-EGF humain circulants dans le sérum. Dans un deuxième temps, nous nous sommes intéressé au rôle des miRNAs, qui sont impliqués dans la régulation de l'expression de gènes. Grâce à l'analyse de 1488 patients atteins de cancers du sein référencés sur des bases de données publiques, nous avons pu établir une corrélation entre le gène LPA1 et le gène ZEB1. Nous avons également trouvé que le coefficient de corrélation entre ZEB1 et LPA1 était supérieur au niveau des tumeurs mammaires basales / Lysophosphatidic acid (LPA) is a natural bioactive lipid with growth factor-like functions due to activation of a series of six G protein-coupled receptors (LPA1-6). It has been demonstrated that blocking LPA1 activity in vivo inhibits breast cancer cell metastasis, however, activated genes involved in LPA-induced metastasis have not been defined yet. In addition most mammalian cells co-express multiple LPA receptors, resulting in the co-activation of multiple intracellular signaling pathways with potential redundant or opposite effects impairing the validation of target inhibition in patients because of missing LPA receptor-specific biomarkers. In the first part of this thesis I found that HB-EGF is a specific biomarker of LPA1 activity. HB-EGF upregulation was inhibited by LPA1-3 antagonists (Ki16425, Debio0719) and by stably silencing LPA1. Using a human xenograft prostate tumors mouse model with PC3 cells, we found that a five-day treatment with Ki16425 significantly decreased both HB-EGF mRNA expression at the primary tumor site and circulating human HB-EGF concentrations in serum. In the second part of experimental work, we focused our attention on miRNAs that are master gene regulators. We carried out correlation studies in 1488 human primary breast tumors from publically available databases and found ZEB1 as the most correlated gene with LPAR1. The coefficient of correlation between ZEB1 and LPAR1 was higher in human basal tumors than in non basal tumors. In three different basal cell lines LPA up-regulated ZEB1 through an LPA1/Phosphatidylinositol-3-Kinase (Pi3K)/AKT-dependent pathway. Based on microarray and real-time PCR analyses we found that LPA up-regulated the oncomiR miR-21 through an LPA1/Pi3K/AKT/ZEB1-dependent mechanism. MirVana miR-21 inhibitor, silencing LPA1 or silencing ZEB1 totally blocked in vitro LPA-induced cell migration and invasion, and in vivo tumor cell bone colonization. In all cases, basal breast cancer cell functions were rescued with mirVana miR-21 mimic. All together our results identify HB-EGF as a new and relevant biomarker with potentially high value in quantifying LPA1 activation state in patients receiving anti-LPA1 therapies

Indiana University-Purdue University Indianapolis (IUPUI) / FGF23 is the central regulator for phosphate homeostasis. Both FGF23 and phosphate dysregulation are highly related with the progression of chronic kidney disease (CKD), which is a global health problem. In previous studies, FGF23 was found to be produced in bone and targeting the kidneys to regulate phosphate reabsorption and excretion. In the FGF23 signaling axis, it binds a receptor complex (αKlotho and FGFRs) in the distal convoluted tubules (DCT) and causes its biological effects in the proximal tubules (PT). The mechanism of how the signals passing on from DCT to PT is not clear. In my research, experiments were focused on the FGF23 signaling pathway within the kidney to study the communication steps between tubular cells. HBEGF treatment was given to FGF23 signaling impaired mouse models resulting in significant change of genes regulated by FGF23, indicating that HBEGF was important in the FGF23 signaling axis. Then high quality rabbit anti-mouse HBEGF antibodies were made to better study HBEGF activity in vivo and in vitro. A new cell model was characterized to test FGF23 effects on HBEGF signaling using Western blots and immunofluorescence. Lastly, the location of HBEGF activity was examined in the kidney in vivo. Immunostaining suggested that HBEGF activated the mitogen activated protein kinase (MAPK) pathway. This mapping may provide important information for the molecular relationships between FGF23 and HBEGF.