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: 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.