Acquired drug resistance and promotion of angiogenic responses in breast cancer
Introduction
Acquired resistance to anti-oestrogens in breast cancer is a major clinical problem, limiting the effectiveness of these agents and resulting in disease progression. In vitro, acquired resistance is associated with increased Src kinase activity and a gain in aggressive, invasive cell features, which may facilitate disease progression and spread in vivo. Recently, we have identified that, in contrast to endocrine-sensitive cells, antioestrogen-resistant breast cancer cells also overexpress several pro-angiogenic factors (VEGF, IL8); subsequently, conditioned medium from these cells is able to stimulate both ERK1/2 activity in, and growth of, human endothelial cell (HUVEC) cultures. Significantly, conditioned medium from endocrine-sensitive breast cancer cells engineered to express a constitutively active form of Src kinase also stimulate HUVEC signalling and proliferation.
Hypothesis:
Based on our preliminary data and recent literature, our hypothesis is that endocrine resistance in breast cancer cells promotes a pro-angiogenic phenotype through a mechanism involving Src kinase.
Aims of Project
Brief experimental plan:
1. Determine the ability of acquired endocrine resistant cells to modulate endothelial cell growth and tubule formation in vitro and in vivo. To determine whether an increased angiogenic capacity represents a unifying feature of anti-oestrogen resistance, HUVEC growth assays will be performed using conditioned medium from, and in co-culture with, breast cancer cells (MCF7, T47D) that have acquired resistance to a range of anti-oestrogens (tamoxifen, fulvestrant) along side additional models representative of de-novo endocrine resistance. Further functional in vitro assays will be performed to determine the ability of drug-resistant cells to induce HUVEC survival, migration, permeability and tubule formation. Establishment of the widely-used chick embryo chorioallantoic membrane (CAM) assay will allow measurement of in vivo capillary growth in response to both endocrine-sensitive and resistant breast tumour cells. To determine the relevance of pro-angiogenic factors (e.g. VEGF, IL8) previously identified by microarray analysis as overexpressed by resistant cells, on HUVEC cell function, the assays above will be performed in the presence of neutralising antibodies against these factors and/or by pre-treatment of tumour cells with siRNAs specific for these pro-angiogenic elements. Western blotting will identify expression of appropriate receptors on HUVEC cells, whether the expression/activity of these is modulated upon co-culture with tumour cells and the induction of signalling pathways controlling proliferation and migration in HUVEC cells following tumour cell co-culture/ conditioned medium stimulation as above.
2. Establish the role of Src, and its interplay with FAK, in the expression of angiogenic factors in endocrine-resistant breast cancer cells. Conditioned medium collected from breast cancer cells following their treatment with a Src inhibitor will be used to treat HUVEC cells in vitro and used in CAM assays. The expression of pro-angiogenic factors known to be regulated by Src (VEGF, IL8) will also be assessed in these cells alongside endocrine-sensitive MCF7 cells engineered to express constitutively-active Src. The contribution of the Src substrate, focal adhesion kinase (FAK) in this process, with particular reference to its activity at Y925 as reported recently, will be investigated by Western blotting; inhibition of Src kinase and/or siRNA knockdown of FAK will demonstrate whether FAK contributes to VEGF/IL8 production in a Src-dependent or independent manner
3. Identify whether pharmacological targeting of Src is suitable for anti-angiogenic therapy in drug-resistant breast disease. Given that some pro-angiogenic factors are induced by anti-oestrogen treatment and thus may promote angiogenic responses during the drug-responsive phase, we will examine the efficacy of novel drug combinations (e.g. Src inhibition plus anti-oestrogen) previously shown to effectively prevent other aggressive features (e.g. cell invasion) associated with acquired resistance, in suppressing this action in anti-oestrogen sensitive cells. Additionally, in cancer cells where drug-resistance already exists, combination therapy targeting Src alongside the dominant growth factor pathway will be examined for its potential to suppress their angiogenic activity.
