Focal Adhesion Kinase (FAK) as a novel therapeutic target in HER2+ breast cancer

Focal Adhesion Kinase (FAK) is an intracellular kinase known to mediate integrin signalling following cell adhesion to the extracellular matrix. It is now emerging as a promising therapeutic target in many tumour types due to its overexpression in tumour cells and is associated with various cellular processes involved in cancer progression. Given that existing literature demonstrating that FAK plays a key role in the transduction of HER2 signalling in HER2+ cells and that the levels of FAK expression strongly correlated with HER2 overexpression in clinical samples, we explored the potential for improvement of current therapies for HER2+ breast cancer by combination treatment strategies with the small molecule FAK inhibitor, PF878. FAK activity was assessed in a panel of cell lines reflecting HER2- (MCF7, T47D) and HER2+ (BT474, MDA-361, SKBr3) disease by Western blotting. FAK activity was relatively increased in HER2+ versus HER2- cell lines with HER2+ cells demonstrating greatest sensitivity to PF878 with respect to suppression of FAK phosphorylation at Y397. The effects of PF878 on cell proliferation as a monotherapy and in combination with Herceptin were assessed using MTT and direct coulter cell counting and by Ki67 immuno-staining. Whilst PF878 did not affect the proliferation as a monotherapy, treatment of HER2+ cells with PF878 and Herceptin combined resulted in synergistic inhibitory action on cell proliferation with an associated suppression in AKT pathway activity. This combination treatment strategy produced the greatest effects in MDA-361 cells which were intrinsically insensitive to Herceptin-monotherapy. Inhibition of FAK activity also suppressed HER2+ cell migration in response to the (1) exogenous ligand Heregulin and (2) conditioned-media derived from fibroblasts (FCM), as assessed in Boyden Chamber migration assays. In this latter context, our data suggests that FAK may act through a STAT3-dependent mechanism to regulate fibroblast-stimulated migratory and invasive responses. Collectively, these data support a role for FAK in HER2+ breast cancer where its targeting has the potential to improve Herceptin response as well as suppress stromal-induced signalling that can contribute to disease progression and spread

Computer-aided identification of novel anticancer compounds with a possible dual HER1/HER2 inhibition mechanism

HER1 and HER2 are frequently overexpressed in human tumors where they drive cellular proliferation. For this reason they are considered important targets in anticancer therapy with dual HER1/HER2 inhibitors being recently approved and marketed. In this paper we report the identification of a series of compounds with anticancer activity by a combined virtual screening approach on the kinase domains of HER1 and HER2. 6 hit compounds that present a sub- or low-micromolar activity in two cell-based assays, were initially identified and a subsequent design cycle led to the synthesis of a compound with nanomolar activity in the cell-based assays

Nestin regulates prostate cancer cell invasion by influencing the localisation and functions of FAK and integrins

Nestin, an intermediate filament protein and marker of undifferentiated cells, is expressed in several cancers. Nestin is important for neuronal survival and is a regulator of myogenesis but its function in malignancy is ambiguous. We show that nestin downregulation leads to a redistribution of phosphorylated focal adhesion kinase (pFAK, also known as PTK2) to focal adhesions and alterations in focal adhesion turnover. Nestin downregulation also leads to an increase in the protein levels of integrin α5β1 at the cell membrane, activation of integrin β1 and an increase in integrin clustering. These effects have striking consequences for cell invasion, as nestin downregulation leads to a significant increase in pFAK- and integrin-dependent matrix degradation and cell invasion. Our results indicate that nestin regulates the localisation and functions of FAK and integrin. Because nestin has been shown to be prevalent in a number of specific cancers, our observations have broad ramifications for the roles of nestin in malignant transformation

Targeting focal adhesion kinase in ER+/HER2+ breast cancer improves trastuzumab response

The HER2 transmembrane receptor is a well-characterised predictive marker for trastuzumab benefit and may be associated with decreased benefit from endocrine therapy use. Despite the clinical effectiveness of anti-HER2 agents in such cases, resistance represents a significant limiting factor. Focal adhesion kinase (FAK) plays an important role in HER2 signalling, mediating downstream Akt activation in addition to HER2 cross talk with other growth factor receptors. In this study, we investigated the therapeutic potential of FAK in oestrogen receptor-positive (ERC)/HER2C breast cancer using the novel FAK-specific inhibitor PF4554878 (‘PF878’). The activation of the FAK/HER2 signalling pathway was assessed in ERC/HER2K(MCF7 and T47D) and ERC/HER2C(BT-474 and MDAMB361) breast cancer cells in the presence or absence of PF878 and PF878Gtrastuzumab. The effects of PF878 on cell growth as a monotherapy and in combination with trastuzumab were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Coulter counting with isobologram analysis to determine synergy/additive effects. FAK activation (at Y861 but not at Y397) was highest in ERC/HER2C cells, which also demonstrated the greatest sensitivity to PF878. As a monotherapy, PF878 prevented heregulin-induced MDA361 cell migration, but had no significant effect on cell growth. The treatment of ERC/HER2C cells with PF878 and trastuzumab in combination resulted in the synergistic inhibition of cell proliferation. Underlying this was an abrogation of Akt activity and increased poly(ADP-ribose) polymerase cleavage, effects that were greatest in trastuzumab-refractory MDA361 cells. Collectively, these data support a role for FAK in ERC/HER2C breast cancer, where its targeting has the potential to improve trastuzumab response. This is particularly important in the context of ERC/HER2C, trastuzumab-refractory disease, where FAK inhibition may present an important strategy to restore trastuzumab sensitivity

Targeting focal adhesion kinase in ER+/HER2+ breast cancer improves trastuzumab response

The HER2 transmembrane receptor is a well-characterised predictive marker for trastuzumab benefit and may be associated with decreased benefit from endocrine therapy use. Despite the clinical effectiveness of anti-HER2 agents in such cases, resistance represents a significant limiting factor. Focal adhesion kinase (FAK) plays an important role in HER2 signalling, mediating downstream Akt activation in addition to HER2 cross talk with other growth factor receptors. In this study, we investigated the therapeutic potential of FAK in oestrogen receptor-positive (ERC)/HER2C breast cancer using the novel FAK-specific inhibitor PF4554878 (‘PF878’). The activation of the FAK/HER2 signalling pathway was assessed in ERC/HER2K(MCF7 and T47D) and ERC/HER2C(BT-474 and MDAMB361) breast cancer cells in the presence or absence of PF878 and PF878Gtrastuzumab. The effects of PF878 on cell growth as a monotherapy and in combination with trastuzumab were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Coulter counting with isobologram analysis to determine synergy/additive effects. FAK activation (at Y861 but not at Y397) was highest in ERC/HER2C cells, which also demonstrated the greatest sensitivity to PF878. As a monotherapy, PF878 prevented heregulin-induced MDA361 cell migration, but had no significant effect on cell growth. The treatment of ERC/HER2C cells with PF878 and trastuzumab in combination resulted in the synergistic inhibition of cell proliferation. Underlying this was an abrogation of Akt activity and increased poly(ADP-ribose) polymerase cleavage, effects that were greatest in trastuzumab-refractory MDA361 cells. Collectively, these data support a role for FAK in ERC/HER2C breast cancer, where its targeting has the potential to improve trastuzumab response. This is particularly important in the context of ERC/HER2C, trastuzumab-refractory disease, where FAK inhibition may present an important strategy to restore trastuzumab sensitivity