In recent years, research in the area of cancer stem cells has spiked tremendously. Numerous investigators have found that several types of cancers contain a subpopulation of tumor cells that display many defining characteristics of normal tissue stem cells, including multipotent differentiation potential, long-term self-renewal capacity, and expression of molecular markers of stemness. Most importantly, these cancer stem cells (CSCs) have very high tumor initiating potential, a finding that has led to the development of the cancer stem cell model for tumor progression. This model suggests that tumors are organized in a developmental hierarchy (similar to healthy tissue), with long-term tumor progression being driven by self-renewing CSCs at the top of the hierarchy. The CSC model represents a significant shift in our understanding of tumor progression, and as such, it may be possible to expand our knowledge of other aspects of tumor biology by re-examining them in the context of the CSC model. My work focuses on investigating interactions between CSCs and the tumor vasculature. Previous work has demonstrated heterogeneity in the proangiogenic potential of cells in a tumor. Considering the possibility that angiogenesis may be driven by specific subsets of tumor cells, I investigated the contribution of the CSC fraction to tumor angiogenesis. Comparing tumors with low or high CSC fractions, I have found that CSCs contribute to tumor vascular development through promotion of endothelial cell activity and recruitment of bone marrow-derived proangiogenic cells, mediated in part by vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF1). Since some tissue stem cells are known to reside in a vascular niche, I investigated the possibility that CSCs may also be supported by blood vessels in the tumor microenvironment, and that consequently CSCs may be targeted by disruption of tumor vasculature with antiangiogenic therapy. By testing multiple antiangiogenic therapeutic strategies, I have found that antiangiogenic therapy sensitizes CSCs to the effects of cytotoxic chemotherapy. Taken together, my work demonstrates a bi-directional relationship in which CSCs promote tumor vascular development, and tumor vasculature supports and protects CSCs. This work has implications for our understanding of CSC biology, tumor angiogenesis and antiangiogenic therapy, and provides insight into strategies for targeting the critical CSC population.

Vascular endothelial growth factor receptor-2 (VEGFR2) is a key angiogenic factor, and angiogenesis is an important physiological process associated with neovascularization, growth, and metastasis of many different tumors. The mechanism of VEGFR2 gene expression was investigated in MiaPaCa-2, Panc-1, and AsPC-1 pancreatic cancer cells transfected with a series of VEGFR2 promoter deletion/mutated constructs, and the results indicated that the GC-rich –60 to –37 region of the promoter was essential for VEGFR2 expression in these cell lines. EMSA and ChIP assays showed that Sp proteins are expressed and bind to the proximal GC-rich region of the VEGFR2 promoter. RNA interference studies on Sp proteins demonstrated that Sp1, Sp3, and Sp4 all contributed to VEGFR2 gene/protein expression in pancreatic cancer cells. VEGFR2 gene expression was also investigated in ZR-75 and MCF-7 breast cancer cells. ZR-75 cells treated with 10 nM 17b-estradiol (E2) increased VEGFR2 mRNA levels/protein expression. The VEGFR2 promoter was induced by E2 in ZR-75 cells, and analysis of the VEGFR2 promoter identified the GC rich -60 to -37 region that was required for E2-mediated transactivation. EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed in ZR-75 cells and bind the proximal GC-rich region of the VEGFR2 promoter. RNA interference was used to determine the relative contributions of Sp proteins on hormonal regulation of VEGFR2 through ER/Sp complexes, and interestingly, in ZR-75 cells, hormone-induced activation of VEGFR2 involves ERa/Sp3 and ERa/Sp4 but not ERa/Sp1. In MCF-7 cells treated with 10 nM E2, VEGFR2 mRNA levels were decreased. Analysis of the VEGFR2 promoter revealed that the same GC-rich region important for E2-mediated upregulation in ZR-75 cells was responsible for E2-dependent downregulation of VEGFR2 gene expression in MCF-7 cells. EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed in MCF-7 cells and bind to the proximal GC-rich region of the VEGFR2 promoter. RNA interference studies showed that Sp1, Sp3, and Sp4 are involved in the E2-mediated downregulation of VEGFR2 in MCF-7 cells, and ERa/Sp protein-promoter interactions are accompanied by recruitment of the corepressor SMRT using the ChIP assay.

Η μελέτη των αναστολέων της αγγειογένεσης έχει γνωρίσει μεγάλη ανάπτυξη κατά την τελευταία δεκαετία, συγκεκριμένα από τότε που ανακαλύφθηκε συσχέτιση της αγγειογένεσης με πάνω από 70 νοσηρές καταστάσεις. Έχει δειχθεί ότι η παρστατίνη μπλοκάρει αποτελεσματικά την αγγειογένεση και έτσι μπορεί να αναπτυχθεί ως ένα πεπτίδιο με σημαντικά αποτελέσματα στην θεραπεία των νεοαγγειακών παθήσεων. Το πεπτίδιο 1-41 της παρστατίνης καταστέλλει την βασική μεμβράνη ή διεγείρει την αγγειογένεση με bFGF και VEGF αυξητικούς παράγοντες στο μοντέλο όρνιθας χοριοαλλαντοϊκής μεμβράνης (CAM) ή στο μοντέλο αγγειογένεσης με αορτικό δακτύλιο. Ο κύριος στόχος του προγράμματος αυτού είναι να διευκρινιστεί η μοριακής οδός μέσω της οποίας η παρστατίνη παίζει ρόλο στην αναστολή της αγγειογένεσης και να διερευνηθεί ο τρόπος με τον οποίο συγκεκριμένα πεπτίδια παρστατίνης (1-24 και 24-41) εμπλέκονται στην εξέλιξη της οδού αυτής. Για αυτό το σκοπό, μοντέλα αρουραίου και ποντικού χρησιμοποιούνται (στα ποντίκια μοντέλα απευθείας αγγειογένεσης - DIVAA - και στους αρουραίους μοντέλα δοκιμασίας αορτικών δακτυλίου) καθώς και ορισμένες κυτταρικές γραμμές των ενδοθηλιακών κυττάρων (ΜΤΤ και οι δοκιμασίες εκχύλισης πρωτεΐνης σε Ανθρώπινο Coron). Στο ένα μέρος του προγράμματος που περιελάμβανε απευθείας αγγειογένεση DIVAA σε μοντέλο μυός καθώς και στα άλλα μέρη του προγράμματος, επιβεβαιώθηκε ότι η παρστατίνη έχει ισχυρή αντί-αγγειογενετική δράση. Ενώ το πεπτίδιο 24-41 δεν έδειξε να προκαλεί αναστολή της αγγειογένεσης, αντίθετα προήγε την αγγειογένεση. Αυτό μας οδηγεί στο συμπέρασμα ότι το πεπτίδιο 24-41 δεν συμβάλει στην αναστολή της αγγειογένεσης. Το συμπέρασμα αυτό όμως θα πρέπει να επιβεβαιωθεί και με άλλα πειράματα στο εγγύς μέλλον. / Angiogenesis inhibitors have been under a great development in the last decade - precisely, since angiogenesis was associated with more than 70 disease conditions. Parstatin has been shown to effectively block angiogenesis and thus, it can be considered as an important molecule in the treatment of neovascular diseases. Parstatin 1-41 peptide inhibits basal membrane or induces angiogenesis with bFGF and VEGF growth factors in the chicken chorioallantoic membrane model (CAM) or in the angiogenesis aortic ring model. The main objective of this project is to clarify the molecular pathway through which parstatin plays a role in the inhibition of angiogenesis and to explore how two specific parstatin peptides (1-26 and 24-41) are involved in this development. For this, rat and mouse models were used (direct angiogenesis in mouse - DIVAA - and rat aortic ring assay in rats) and some cell lines of endothelial cells (MTT and protein extraction tests on Human Coron). A part of the program which included the direct angiogenesis DIVAA murine model and also other parts of the program, confirmed that parstatin has strong anti-angiogenic properties. Peptide 24-41 activity did not inhibit angiogenesis, but on the contrary it actually triggered the process. Hence, the submolecule 24-41 seems not to contribute to the inhibition of angiogenesis, but this has to be confirmed with more similar experiments in the future.

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Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Angiogenesis is the formation of new blood vessels from pre-existing vessels. It is highly regulated in a normal physiological system. Dysregulated angiogenesis is associated with a number of pathological states such as cancer and rheumatoid arthritis. Thus angiogenesis is proposed as a target for control of such diseases. The identification of novel genes involved in angiogenesis will improve the likelihood of the development of efficacious drugs. To identify genes essential to angiogenesis, we used an in vitro model of capillary tube formation and a PCR based subtraction hybridisation approach for isolation of regulated genes. We identified 125 different genes including 96 known and 29 novel genes and of these, 84 genes were confirmed to be regulated during the formation of capillary tubes. From the 125 isolated genes, one novel gene displayed characteristics suitable for further investigation. The gene, p73RhoGAP is a new member of the RhoGAP family. It is highly upregulated at 3 and 6 h in the tube formation process but only in an angiogenic milieu with little or no regulation seen under non-angiogenic conditions, and is restricted in its expression to vascular cells. Thus, we propose that p73RhoGAP is called VASGAP. Bioinformatics analysis of the protein sequence revealed the presence of a putative Rho GAP domain, indicating a potential role in the activation of RhoGTPases. Our study suggests that VASGAP displays RhoGAP activity for Rho but not Rac or Cdc42. RhoGTPase activity assays demonstrated that mutation of the conserved arginine at residue 82 (R82A) in the RhoGAP domain is crucial to the p73 effect on Rho. VASGAP localises to actin stress fibres. To characterise the function of VASGAP, knockdown of the protein was achieved by adenovirus delivery of VASGAP antisense into EC. Such VASGAP depleted cells showed enhanced actin stress fibres and basal permeability consistent with alteration of Rho activity. Cell migration, proliferation and capillary tube formation were inhibited, and enhanced apoptosis was evident. In an in vivo model of angiogenesis, antisense of VASGAP inhibited blood vessel invasion. Thus, the results suggest VASGAP is an important and novel modulator of angiogenesis and cell survival and displays many of the features worthy of a drug target. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1170778 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2005