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Type: Theses
Title: Characterisation of proteaseresistant, non-matrix-binding variant of IGFBP-2 as a potential tumour growth inhibitor
Author: Soh, Cher-Lynn
Issue Date: 2016
School/Discipline: School of Biological Sciences
Abstract: The insulin-like growth factor (IGF) system includes two ligands (IGF-I and IGF-II), which bind and activate the type 1 IGF receptor (IGF-1R). IGF-II also binds with high affinity to the structurally similar insulin receptor isoform A (IR-A). Activation of these receptors elicits mitogenic responses for normal growth and development. IGF bioavailability is modulated by high-affinity binding proteins (IGFBPs). When in complex with IGFBPs the IGF are unable to interact with their receptors. IGF is released from the IGF:IGFBP complex via IGFBP interaction with extracellular matrix (ECM) or through proteolysis of IGFBPs, increasing bioavailability of IGF. This effectively increases IGF-1R and IR-A activation (in the case of IGF-II). In cancer, expression of IGF-I, IGF-II, and IGF-1R is upregulated, while compensatory signalling via IGF-II/IR-A (in the event of perturbed IGF/IGF-1R signalling) was documented. Additionally, tumours overexpress IGFBP specific proteases to accelerate IGF release. Collectively, mitogenic signalling via this system is upregulated, thereby promoting tumour growth, survival and metastasis. The main objective was to investigate the use of IGFBPs as potential anti-cancer agents. Here, IGFBP-2 was used to sequester IGF as it has similar affinities for both ligands. Two novel modified IGFBP-2 analogues were designed to block mitogenic signalling of IGF-1R and IR-A by impairing their IGF releasing abilities. The protease resistant (PR) mutant was produced by deleting residues 114-170 of the linker domain (which contains cleavage sites), while the protease resistant/non-matrix binding (PR/NMB) mutant was generated by further mutation of the PR mutant by Lys→Ala substitutions at positions 180, 181, 227, 234, and 237 (disrupting the ECM binding sites). The initial work was dedicated to in vitro characterisation of modified IGFBP-2. Truncation of the linker domain in PR and PR/NMB IGFBP-2 significantly improved resistance to tumour proteases: plasmin, MMP-1 and MMP-7. However, there is evidence for the presence of MMP cleavage sites outside the truncated linker domain. Furthermore, IGF binding did not alter rate of IGFBP-2 cleavage by the aforementioned proteases. Additionally, IGFBP-2 was cleaved by plasmin C-terminally to Arg56, Lys150, Arg156, Lys158, Arg188, and Arg287. Together these data provided a detailed understanding of the mechanisms of proteolytical degradation of IGFBP-2, which in a cancer setting may increase IGF bioavailability. Additionally, both IGFBP-2 mutants exhibited low affinity to ECM components including vitronectin, fibronectin and heparin, which are known to be involved in tumour progression. Lack of ECM binding discourages dissociation of the IGF:IGFBP-2 complex hence preventing IGF-mediated signalling. An in vitro proliferation assay demonstrated that these mutants significantly inhibited proliferation of HT29 cells despite the presence of exogenous IGF-I. Additionally, growth of breast cancer xenograft tumours treated with PR/NMB IGFBP-2 was significantly inhibited. Interestingly, sequestration of IGF by IGFBP-2 did not trigger a compensatory increase of IGF-I in the circulation, an event commonly accompanying treatment using anti-IGF-1R monoclonal antibodies to perturb IGF signalling. Furthermore, analyses of the tumours indicated that PR/NMB IGFBP-2 treatment disrupted vascularisation with a corresponding decrease in the expression of VEGFA, a major angiogenic factor. Through development of these IGFBP-2 mutants, the importance of IGFBP-2 proteolysis and matrix binding in the control of IGF action was confirmed. This research also provided an understanding of the mechanism by which mutant IGFBP-2 inhibits tumour growth. Ultimately, this data can be used to develop mutant IGFBP-2 as a therapeutic agent.
Advisor: Forbes, Briony Evelyn
Booker, Grant William
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2016.
Keywords: insulin-like growth factor
IGFBP
xenograft
breast cancer
vascularisation
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals
DOI: 10.4225/55/5af3da2cb6545
Appears in Collections:Research Theses

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