Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 3, 2010). Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains v, 42 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2009. / Includes bibliographical references. Also available online through Digital Dissertations.

Inhibitor of Differentiation (ID) proteins are a family of four (ID1-4) bHLH transcription factors that lack the DNA binding domain. They act by forming dimers with other transcriptional regulators and inhibiting their interaction with DNA. They play a crucial role during embryonic development and later in the adulthood, their expression is mostly restricted to a few populations of stem cells. In the last decades, many authors have described their re-activation and participation in tumor development, angiogenesis and EMT although the results are still controversial. In the first chapter of this research work, the role of ID genes as prognostic markers in breast cancer was evaluated. We studied the mRNA expression of the four ID genes and four markers of EMT by qRT-PCR in a clinical series of 307 primary breast carcinomas previously stratified in immunophenotypes. In addition, the expression of all these genes was measured in breast cancer cell lines and mammospheres. Overexpression of at least one ID gene was found in 48.9% of the studied samples. ID1 and ID4 were overexpressed mostly in TNBL and HER2-enriched subtypes, whereas ID2 and ID3 were overexpressed more frequently in luminal tumors. High ID1 and ID4 was associated with larger tumor size, histological grade 3, presence of necrosis and vascular invasion, and poorer outcome. Multivariate analysis revealed that ID4 and vascular invasion were independent factors for DFS. Regarding EMT markers, high levels of SNAI1 were associated with the overexpression of the four ID genes. Additionally, ID1 overexpression was positively related to TWIST1, and the overexpression of ID2 and ID3 was more frequently paired with tumors that conserve CHD1 expression. In vitro studies showed high expression of the four ID genes in all cell lines. However, when mammospheres were formed, mRNA levels of ID genes decreased, in contrast to SNAI1 and TWIST1, which mostly increased. In the second chapter of this thesis, we aimed to (a) describe the mechanisms of action of a small molecule pan-ID antagonist, (b) define its main targets and (c) investigate potential pathways of acquire resistance. Treatment with AGX51 led to Id protein loss, increase in ROS accumulation, cell cycle arrest, and cell death in all tumor cell lines tested. Here, we used an antioxidant compound in different cell lines to demonstrate that ROS are the main responsible of cell death following treatment with AGX51. A model of cultured quiescent cells not expressing ID proteins served to show that the main target of AGX51 are these proteins. Experiments with AGX-derivatives also supported these results. Finally, three mutagenizing agents were used in order to generate mutations that confer resistance to treatment with AGX51. Treatment with ENU gave rise to two clones apparently resistant to AGX51 effects. Based on our in vitro and clinicopathological studies, we conclude that ID1 and ID4 may act as biomarkers of worse prognosis in patients with breast cancer, and seem to be involved in the initiation of EMT mechanism. Therefore, they are potential targets for the development of novel drugs. In line with this, AGX51 arises as a potent anti-ID compound that has anticancer effects.

Das Bone Morphogenetic Protein 2 (BMP2) gehört zur TGF-beta Superfamilie und findet seinen Fokus in der osteogenen Aktivierung und in der Anwendung bei der Frakturheilung. Es wird angenommen, dass weitere, bisher unbekannte Verbindungen existieren, die die BMP2-Signalübertragung und die osteogene Aktivität verbessern und somit zu einer verbesserten klinischen Wirksamkeit von BMP2 führen. Für den Stickstoffoxid (NO)-Signalweg ist bereits bekannt, dass im endothelialen Kontext eine Verbindung zum BMP2-Signalweg existiert. Ziel dieser Arbeit war es daher, eine Verbindung zwischen dem NO- und BMP2-Signalweg bezüglich der Regulierung des BMP2-abhängigen Signalwegs und der Osteoinduktion aufzuzeigen. Dies erfolgte durch Anwendung von Inhibitoren (LNAME, ODQ und LY83583) und Aktivatoren (L-Arginin, Deta NONOate, SNAP und YC-1) des NO-Signalwegs, in Kombination mit BMP2. Eine mögliche Verbindung zwischen dem BMP2- und NO-Signalweg, über eine Protein Kinase A (PKA) Brücke, wurde durch die Anwendung des PKA Inhibitors H89 untersucht. Zusammenfassend zeigen diese Ergebnisse, dass der NO-Stoffwechselweg den BMP2-vermittelten Signalweg und die osteoinduktive Aktivität modulieren kann, wobei PKA beide Signalwege im Rahmen der BMP Signalübertragung verbindet, jedoch nicht zu einer BMP2-vermittelten Osteoinduktion führt. / Bone Morphogenetic Protein 2 (BMP2) is a TGF-beta superfamily member, with a major focus on osteogenic activity and application in fracture healing. In order to improve efficiency of BMP2 in the clinic, it is assumed that additional, yet unknown compounds can improve BMP2 signalling and osteogenic activity. The Nitric Oxide (NO) pathway has previously shown to be connected with the BMP2 pathway in an endothelial context. Therefore, it was the aim of this study to unravel connections between the NO and BMP2 pathway in regulating BMP2 mediated signalling and osteoinduction. This was carried out through the application of inhibitors (LNAME, ODQ and LY83583) and activators (L-Arginine, Deta NONOate, SNAP and YC-1) of the NO pathway in combination with BMP2. A proposed connection between BMP2 and NO pathways via a Protein Kinase A (PKA) bridge was investigated by application of H89 inhibitor. In summary, these results show that the NO pathway can modulate BMP2 mediated signalling and osteoinductive activity. The PKA bridge connects NO and BMP2 only for the process of BMP2 signalling, but not for BMP2 mediated osteoinduction.

Vascular remodelling due to excessive proliferation and apoptosis resistance of pulmonary arterial smooth muscle (PASMCs) and endothelial cells (ECs) has been attributed to the pathogenesis of pulmonary arterial hypertension (PAH). It is an incurable cardiovascular disorder, which leads to right heart failure and death, if left untreated. Heterozygous germline mutations in the bone morphogenetic protein receptor type II (BMPR2) have been linked with the majority (~75%) of the familial form of the disease (HPAH). Mutations in the BMPR2 gene impinge upon the BMP signalling which perturbs the balance between BMP and TGF-β pathways leading to the clinical course of the disease. Current therapies were discovered prior to the knowledge that PAH has substantial genetic components. Hence, this study aims to identify novel therapeutic intervention and provide novel insights into how the dysfunctional BMPRII signalling contributes to the pathogenesis of PAH. This work demonstrates that cryptolepines and FDA approved drugs (doxorubicin, taxol, digitoxin and podophyllotoxin) inhibit the excessive proliferation and induce apoptosis in BMPR2 mutant PASMCs by modulating the BMP and TGF-β pathways. Moreover, established drug PTC124 has also been tested but has failed to promote translational readthrough. I have also shown that dysregulated apoptosis of PASMCs and HPAECs is mediated through the BMPRII-ALK1-BclxL axis. Finally, the siRNA screen targeting approximately 1000 genes has identified novel proteins including PPP1CA, IGF-1R, MPP1, MCM5 and SRC each capable of modulating the BMPRII signalling. Taken together, this study for the very first time has identified novel compounds with pro-BMP and anti-TGFβ activities which may provide therapeutic intervention prior to or after the onset of PAH. / Commonwealth Scholarship Commission in the UK