Search

Aberrant activation of the PI3K pathway is one of the commonest oncogenic events in human cancer. AKT is a key mediator of PI3K oncogenic function, and thus has been intensely pursued as a therapeutic target. Multiple AKT inhibitors, broadly classified as either ATP-competitive or allosteric, are currently in various stages of clinical development. Herein, we review the evidence for AKT dependence in human tumours and focus on its therapeutic targeting by the two drug classes. We highlight the future prospects for the development and implementation of more effective context-specific AKT inhibitors aided by our increasing knowledge of both its regulation and some previously unrecognised non-canonical functions.

Background AKT, a critical effector of the phosphoinositide 3-kinase (PI3K) signalling cascade, is an intensely pursued therapeutic target in oncology. Two distinct classes of AKT inhibitors have been in clinical development, ATP-competitive and allosteric. Class-specific differences in drug activity are likely the result of differential structural and conformational requirements governing efficient target binding, which ultimately determine isoform-specific potency, selectivity profiles and activity against clinically relevant AKT mutant variants. Methods We have carried out a systematic evaluation of clinical AKT inhibitors using in vitro pharmacology, molecular profiling and biochemical assays together with structural modelling to better understand the context of drug-specific and drug-class-specific cell-killing activity. Results Our data demonstrate clear differences between ATP-competitive and allosteric AKT inhibitors, including differential effects on non-catalytic activity as measured by a novel functional readout. Surprisingly, we found that some mutations can cause drug resistance in an isoform-selective manner despite high structural conservation across AKT isoforms. Finally, we have derived drug-class-specific phosphoproteomic signatures and used them to identify effective drug combinations. Conclusions These findings illustrate the utility of individual AKT inhibitors, both as drugs and as chemical probes, and the benefit of AKT inhibitor pharmacological diversity in providing a repertoire of context-specific therapeutic options.

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, 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.

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 (ER+)/HER2+ breast cancer using the novel FAK-specific inhibitor PF4554878 (‘PF878’). The activation of the FAK/HER2 signalling pathway was assessed in ER+/HER2− (MCF7 and T47D) and ER+/HER2+ (BT-474 and MDAMB361) breast cancer cells in the presence or absence of PF878 and PF878±trastuzumab. 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 ER+/HER2+ 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 ER+/HER2+ 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 ER+/HER2+ breast cancer, where its targeting has the potential to improve trastuzumab response. This is particularly important in the context of ER+/HER2+, trastuzumab-refractory disease, where FAK inhibition may present an important strategy to restore trastuzumab sensitivity.

Focal Adhesion Kinase (FAK) is an intracellular kinase known to mediate integrin signalling following cell adhesion to the extracellular matrix. FAK is also implicated in invasive signalling pathways downstream of growth factor receptors including the HER2. Cross talk between tumor and stroma can result in the activation of growth factor signalling pathways and thus may play a significant role in promoting tumor progression and spread. In this study, we have investigated the metastatic responses of HER2+ breast cancer cells to fibroblasts and whether FAK is involved in these events. Conditioned medium derived from human lung fibroblast MRC-5 cells was used to stimulate Her2+ SKBr3 breast cancer cells in the presence or absence of FAK inhibitor, PF271, or after FAK gene knockdown, prior to determining changes in migration (Boyden Chamber assay), 3d matrix invasion, growth and FAK-mediated signalling. To compare the role of FAK with HER2 in these cells, the HER2 targeted agent, trastuzumab was additionally employed. Whilst fibroblast-conditioned medium (FCM) did not affect the growth of SKBr3 cells or their HER2- negative, ER+ counterparts MCF7 cells, FCM significantly stimulated SKBr3 cell migration (∼10 fold vs control) and promoted invasive dissociation of 3d SKBr3 cultures. FCM treatment of SKBr3 cells resulted in rapid (within 5 minutes) activation of MAPK and AKT and increased phosphorylation of the HER4 receptor and FAK (Y861). FCM also promoted an increase in STAT3 (Y704) activity with an accompanying re-distribution to sites of membrane ruffling and to the nucleus. Pharmacological inhibition of FAK, or FAK siRNA, in SKBr3 cells attenuated FCM-mediated FAK activity and suppressed STAT3 activation and subcellular re-distribution and increased the expression of E-Cadherin. Correspondingly, FCM treatment of PF271 or FAKsiRNA- treated SKBr3 cells poorly elicited migratory and invasive responses. Importantly, suppression of HER2 using trastuzumab did not significantly antagonise these FCM responses. Taken together, these results suggest that FAK plays a role in the signalling stimulated by fibroblast- derived stimulators of migration and invasion through a mechanism involving STAT3. These highlight the importance of understanding tumor-stromal interactions and support the potential of FAK as a novel therapeutic target in HER2+ breast cancer particularly in the context of tumor stroma interactions where it may suppress stromal-induced tumor signalling that can contribute to progression and spread. Citation Format: Glorianne Lazaro, Chris Smith, Stephen Hiscox. Focal adhesion kinase (FAK) mediates fibroblast-induced HER2+ breast cancer cell migration and invasion through a mechanism involving Stat3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1165. doi:10.1158/1538-7445.AM2014-1165