Vascular endothelial growth factor is an autocrine survival factor for breast tumour cells under hypoxia.

Vascular endothelial growth factor (VEGF) is produced by most tumour types and stimulates the growth of new blood vessels in the tumour. The expansion of a solid tumour ultimately leads to the development of hypoxic regions, which increases VEGF production and further angiogenesis. In this study, we examined the role of VEGF in the survival of breast tumour cells under hypoxia. Murine 4T1 and human MDA-MB-231 tumour cells were cultured under normoxic and hypoxic growth conditions in the presence or absence of VEGF neutralising antibodies. Apoptosis was assessed in addition to changes in expression of the anti- and pro-apoptotic proteins, Bcl-2 and Bad, respectively. The effect of hypoxia on the novel VEGF receptor, NP1 (neuropilin-1) and the role of the PI3K (phosphatidylinositol-3-kinase) signalling pathway in response to VEGF were examined. VEGF blockade resulted in direct tumour cell apoptosis of both tumour cell lines under normoxia and hypoxia. While blocking VEGF resulted in a downregulation of hypoxia-induced Bcl-2 expression, there was a significant increase in the pro-apoptotic protein Bad relative to cells cultured under hypoxia alone. Both hypoxia and VEGF phosphorylated Akt. Neutralising antibodies to VEGF abrogated this effect, implicating the PI3K pathway in VEGF-mediated cell survival of mammary adenocarcinoma cells. This study demonstrates that VEGF acts as a survival factor not only for endothelial cells as previously thought, but also for some breast tumour cells, protecting them from apoptosis, particularly under hypoxic stress. The data presented provide an additional rationale for combining anti-VEGF strategies with conventional anti-cancer therapies such as chemotherapy and radiotherapy

Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF).

Vascular endothelial growth factor (VEGF) was originally identified as an endothelial cell specific growth factor stimulating angiogenesis and vascular permeability. Some family members, VEGF C and D, are specifically involved in lymphangiogenesis. It now appears that VEGF also has autocrine functions acting as a survival factor for tumour cells protecting them from stresses such as hypoxia, chemotherapy and radiotherapy. The mechanisms of action of VEGF are still being investigated with emerging insights into overlapping pathways and cross-talk between other receptors such as the neuropilins which were not previously associated with angiogenesis. VEGF plays an important role in embryonic development and angiogenesis during wound healing and menstrual cycle in the healthy adult. VEGF is also important in a number of both malignant and non-malignant pathologies. As it plays a limited role in normal human physiology, VEGF is an attractive therapeutic target in diseases where VEGF plays a key role. It was originally thought that in pathological conditions such as cancer, VEGF functioned solely as an angiogenic factor, stimulating new vessel formation and increasing vascular permeability. It has since emerged it plays a multifunctional role where it can also have autocrine pro-survival effects and contribute to tumour cell chemoresistance. In this review we discuss the established role of VEGF in angiogenesis and the underlying mechanisms. We discuss its role as a survival factor and mechanisms whereby angiogenesis inhibition improves efficacy of chemotherapy regimes. Finally, we discuss the therapeutic implications of targeting angiogenesis and VEGF receptors, particularly in cancer therapy

Polymeric prodrug combination to exploit the therapeutic potential of antimicrobial peptides against cancer cells.

Antimicrobial Peptides (AMPs) have unique anticancer properties, but their clinical application is currently limited by an inadequate margin of safety. A prodrug strategy associated with a combination therapy approach could address this limitation by increasing their therapeutic index and their efficacy. Accordingly, the first targeted anticancer polymeric prodrug candidates of AMPs, intended for combination therapy with another polymeric prodrug of an approved antineoplastic agent (doxorubicin), were synthesized as either a PEG-based dual-release prodrug or two individual pegylated prodrugs. The latter are based on a cathepsin B-labile peptide linker and an acid-sensitive acyl hydrazone bond for the AMP and doxorubicin prodrugs, respectively. Anticancer activities and toxicity differentials achieved with the free peptide and its polymer conjugates against ovarian, cancer and non-malignant, cells, indicate that protease-dependent reversible pegylation could be implemented to increase the therapeutic indices of AMPs in cancer therapy. The results obtained also show that this approach can be developed if the releasable PEG linker can be optimised to conciliate the attributes and restrictions of pegylation against proteases. In addition, combination of the polymeric prodrugs of the AMP and of doxorubicin provides additive antitumor effects which could be exploited to enhance the efficacy of the AMP candidate

Recombinant PAPP-A resistant insulin-like growth factor binding protein 4 (dBP4) inhibits angiogenesis and metastasis in a murine model of breast cancer.

BACKGROUND: The Insulin-like growth factor (IGF) pathway plays a role in tumour development and progression. In vivo, IGF1 activity is regulated by the IGF binding proteins (IGFBPs). IGFBP4 inhibits the activity of IGF1 but proteolytic cleavage by pregnancy-associated plasma protein-A (PAPP-A) releases active IGF1. A modified IGFBP4, dBP4, which was resistant to PAPP-A cleavage but retained IGF1 binding capacity, was engineered, expressed in Human Embryonic Kidney (HEK) 293 cells and purified. This study examined the effects of dBP4 on IGF1-induced cell migration, invasion and angiogenesis in vitro. The effect of intra-tumour injections of dBP4 on tumour angiogenesis and metastasis was examined using the 4T1.2luc orthotopic model of breast cancer. METHODS: PAPP-A resistance and IGF binding capacity of dBP4 were characterized by Western blot and surface plasmon resonance, respectively. 4T1.2luc are mouse mammary adenocarcinoma cells transfected with luciferase to allow in vivo imaging. The effect of dBP4 on IGF1-induced Akt activation in 4T1.2luc cells was assessed by Western blot. Cell migration and invasion assays were performed using 4T1.2luc cells. Angiokit™ assays and Matrigel® implants were used to assess the effects of dBP4 on angiogenesis in vitro and in vivo, respectively. An orthotopic breast cancer model - 4T1.2luc cells implanted in the mammary fat pad of BALB/c mice - was used to assess the effect of intra tumour injection of purified dBP4 on tumour angiogenesis and metastasis. Tumour growth and lung metastasis were examined by in vivo imaging and tumour angiogenesis was evaluated by CD31 immunohistochemistry. RESULTS: Our engineered, PAPP-A resistant IGFBP4 (dBP4) retained IGF1 binding capacity and inhibited IGF1 activation of Akt as well as IGF1-induced migration and invasion by 4T1.2 mammary adenocarcinoma cells. dBP4 inhibited IGF1-induced angiogenesis in vitro and in Matrigel implants in vivo. Direct intra-tumour injection of soluble dBP4 reduced angiogenesis in 4T1.2 luc mammary tumours tumour and reduced lung metastasis. CONCLUSION: A PAPP-A resistant IGFBP4, dBP4, inhibits angiogenesis and metastasis in 4T1.2 mammary fat pad tumours. This study highlights the therapeutic potential of dBP4 as an approach to block the tumour-promoting actions of IGF1.</p