Acquired resistance to endocrine therapy in breast cancer is a major clinical problem.Previous reports have demonstrated that cell models of acquired endocrine resistance have altered cell-matrix adhesion and a highly migratory phenotype, features which may impact on tumour spread in vivo. Focal adhesion kinase (FAK) is an intracellular kinase that regulates signalling pathways central to cell adhesion, migration and survival and its expression is frequently deregulated in breast cancer. In this study, we have used the novel FAK inhibitor PF573228 to address the role of FAK in the development of the adverse characteristics that accompany acquired endocrine resistance. Whilst total FAK expression was similar between endocrine-sensitive and endocrine-resistant MCF7 cells, FAK phosphorylation status (Y397 or Y861) was altered in resistance. PF573228 promoted a dose-dependent inhibition of FAK phosphorylation at Y397 but did not affect other FAK activation sites (pY407, pY576 and pY861). Endocrine-resistant cells were more sensitive to these inhibitory effects

Background: In the treatment of pre-menopausal women with oestrogen positive (ER+) breast cancer, tamoxifen represents a first line of adjuvant treatment with demonstrable benefits. Despite this, resistance is frequently acquired to tamoxifen with an associated poor prognosis. Breast cancer cell models have revealed the importance of growth factor signalling networks in sustaining growth of endocrine-resistant cancers and, more recently, their ability to promote a highly migratory and invasive phenotype, together with the expression of genes with pro-angiogenic ontology. The potential of endocrine resistant cells to elicit angiogenic responses, however, remains unknown. Materials and Methods: Real-time PCR was used to validate results from preliminary Affymetrix-based gene profiling of pro-angiogenic gene expression in endocrine-sensitive MCF7 cells and their tamoxifen-resistant (TamR) counterparts. The expression of pro-angiogenic factors in conditioned media (CM) from these cells was assessed by ELISA. The proliferative and migratory effects of MCF7 and TamR CM on vascular endothelial cells (HUVEC and HECV cells), was determined by MTS cell proliferation assay, wound closure assays and Matrigel tubule formation assays. Changes in endothelial cell migration following co-culture with endocrine-resistant cells were examined using Boyden-chamber chemotaxis assays. Growth factor signalling and migration pathway activation in endothelial cells in response to CM was determined by Western blotting. Results: TamR cells were found to express high levels of HIF-1α, IL-8 and VEGF-A at an mRNA level compared with expression in MCF7 cells. High levels of VEGF-A protein were also confirmed in the conditioned media from TamR cells versus their endocrine-sensitive counterparts. TamR conditioned media promoted endothelial cell proliferation, migration and the formation of tubules to a greater extent than that seen in MCF7 CM treated cells. TamR conditioned media was found to stimulate VEGFR2 phosphorylation and downstream activation of MAPK in endothelial cells compared to MCF7 CM. Pharmacological inhibition of VEGFR2 activity in endothelial cells suppressed TamR-induced endothelial cell proliferation and VEGFR phosphorylation. Further pharmacological manipulation of erbB receptors and intracellular kinases in TamR cells revealed an ERGF/Her2-Src kinase dependent mechanism of VEGF-A production in these cells. Discussion: These data suggest acquired tamoxifen resistance is accompanied by development of an erbB receptor/Src kinase-dependant pro-angiogenic phenotype which, if recapitulated in vivo, may promote tumour progression. Therapeutic targeting of erbB/Src axis may prove beneficial in such cases. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-01.

Background: Focal adhesion kinase (FAK) regulates signalling pathways central to cell adhesion, migration and survival and its expression is frequently deregulated in cancer, including breast cancer. Acquired resistance to endocrine therapy in breast cancer is a major clinical problem: relapses present as local and/or regional recurrences, frequently with metastases and the outlook for these patients is poor. Interestingly, cell models of acquired endocrine resistance have altered cell-matrix adhesion and a highly migratory phenotype, features which may impact on tumour spread in vivo. We have used the novel FAK inhibitor, PF573228 ('PF228'), to address the role of FAK in the development of these characteristics.Material and Methods: FAK expression and activity was determined in endocrine-sensitive MCF7 cells and their acquired tamoxifen-resistant ('TamR') and fulvestrant-resistant ('FasR') counterparts in response to PF228 (0-5µM) by Western blotting using phospho-specific antibodies. Changes in cell adhesion were measured by seeding cells, pre-treated with PF228, onto matrix (fibronectin and laminin)-coated plates. After 20 minutes adherent cells were quantified by MTT assay. Cell migration was assessed by seeding cells onto matrix-coated porous membranes ± PF228. After 24hrs, migratory cells were stained and counted. Changes in cell morphology and focal adhesion structure were determined using interference contrast microscopy and immunofluorescence staining respectively. The effects of FAK inhibition on cell proliferation was determined by MTT assay.Results: Whilst FAK expression was similar between MCF7, TamR and FasR cells, phosphorylation of FAK (Y397 and Y861) was elevated in the resistant cells. PF573 promoted a dose-dependent inhibition of phosphorylated FAK (Y397) but did not affect other FAK activation sites (Y407, Y576 and Y861). Endocrine-resistant cells were significantly more sensitive to these inhibitory effects versus MCF7 (mean IC50 for FAK Y397 inhibition: 0.26µM, 0.077µM and 0.011µM for MCF7, TamR and FasR cells respectively). Inhibition of FAK Y397 was associated with a reduction in TamR and FasR adhesion to, and migration over, matrix components; accompanying this was a loss of focal adhesion integrity and a change in appearance, with cells reverting from an EMT-like morphology to one akin to their endocrine-sensitive MCF7 counterparts. Whilst PF228 modestly inhibited proliferation of all three cell types, with resistant cells appearing most sensitive, treatment of endocrine-sensitive MCF7 cells with endocrine agent and PF228 combined resulted in greater suppression of proliferation versus single agent treatment.Conclusion: These data suggest a role for FAK in mediating the aggressive behaviour of endocrine-resistant breast cancer cells. Combining FAK inhibitors and endocrine agent may provide additional benefit and circumvent the development of these adverse characteristics. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3130.