The role of IGF-I signalling in the pathogenesis of breast cancer : therapeutic potential of a protease resistant IGF binding protein 4 (IGFBP4)

摘要

The insulin-like growth factor (IGF) pathway promotes breast cancer and disease progression through cellular processes including proliferation, differentiation, invasion, migration and angiogenesis. Due to its involvement in breast cancer, targeting this pathway may prove beneficial in treating this disease. This thesis investigates the importance of the IGF-IR in breast cancer progression, the characterisation of preclinical mouse models of breast cancer using classification and prognostic markers of the human disease, and preclinical evaluation of a novel strategy to target the IGF pathway in breast cancer.To investigate the role of the insulin-like growth factor I receptor (IGF-IR) in breast cancer progression, the expression of total and phosphorylated IGF-IR was evaluated in two breast cancer patient cohorts. A correlation between IGF-IR expression in breast tumours and clinical parameters was identified using a tissue microarray study and reverse phase protein arrays containing 184 and 712 breast cancer patients, respectively. These studies identified a potential role for IGF-IR in breast cancer diagnosis and as a marker of prognosis. Patients with higher grade tumours had decreased IGF-IR expression, while an increase in IGF-IR expression was associated with patients with lymph node positive breast cancer. Breast cancers positive for activated IGF-IR were associated with decreased survival and increased disease relapse. In addition IGF-IR expression was associated with specific subtypes of the disease including Her2+ tumours and triple negative tumours. These results identify the IGF-IR as a potential new prognostic marker in breast cancer.Syngeneic mouse models offer clinically relevant systems in which to study the molecular mechanisms involved in breast cancer growth and metastasis. Mammary tumour cells originating from a spontaneous mammary tumour (SMT1) and its matched lung metastatic lesions (SMT1L) were isolated and shown to express established biomarkers of breast cancer along with members of the IGF family. SMT1 and SMT1L cells were capable of establishing lung metastases in an experimental metastasis model. The novel E0771.LMB model of metastatic breast cancer was also characterised and found to have increased metastatic capabilities in vitro in comparison to parental E0771 which were weakly/nonmetastatic. Several other syngeneic models of breast cancer, 67NR, 4T1.2, EMT 6.5, E0771 and E0771.LMB, were profiled and found to be similar to human breast cancer subtypes using immunohistochemistry, with tumours exhibiting features of triple negative/basal-like tumours and expressing the IGF-IR.A novel therapy to inhibit IGF pathway activation was developed. Insulin-like Growth Factor Binding Protein 4 (IGFBP4) binds IGF-I, regulating its bioavailability, inhibiting IGF-mediated stimulation of proliferation and/or angiogenesis. A protease, Pregnancy Associated Plasma Protein A (PAPP-A) is IGFBP4-specific and following proteolytic cleavage, causes the release of biologically active IGF-I. A modified IGFBP4 resistant to cleavage by PAPP-A but which retained IGF-I binding was expressed in Human Embryonic Kidney (HEK) 293 cells to allow production and purification of recombinant protease resistant IGFBP4 (dBP4). Functional assays were performed to determine biological activity of dBP4 including Biacore binding analysis and PAPP-A cleavage assays.In vitro and in vivo angiogenesis assays established an anti-angiogenic effect of dBP4. The 4T1.2 orthotopic model of metastatic breast cancer (4T1.2luc) was chosen as a model system within which to evaluate the anti-tumour efficacy of purified dBP4. 4T1.2luc tumour bearing mice were treated with dBP4 and its effects on tumour growth monitored with the IVIS in vivo imaging system. dBP4 treated mice had significantly lower lung metastatic burden in comparison to PBS treated controls. The in vivo anti-angiogenic effect of dBP4 on 4T1.2luc tumours was demonstrated following staining of 4T1.2luc tumours for the endothelial cell marker CD31.Overall, the IGF-IR has been identified as an important receptor in breast cancer progression and a potential therapeutic target. Characterisation of syngeneic mouse models of breast cancer in relation to human breast cancer subtypes enhances their preclinical applications. A protease resistant IGFBP4, dBP4, capable of inhibiting the actions of IGF-I has shown in vitro and in vivo anti-tumour effects, and may have value as a therapeutic for breast cancer.