Targeting Focal Adhesion Kinase (FAK) in Triple Negative Breast Cancer (TNBC)

Abstract

Breast cancers are one of the most frequently diagnosed diseases in women and responsible for considerable mortality annually. Breast cancers are complex, with vast variations in terms of genetic, as well as epigenetic factors, which contribute to the ongoing challenges in developing efficacious treatments. The primary cause of mortality is due to metastasis and recurrence. Triple negative breast cancer (TNBC) is an aggressive subset of breast cancer which commonly metastasises to the lungs, liver, brain and bone, and lacks targeted therapeutics. Metastasis generally involves cancer cells penetrating the extracellular matrix (ECM), a major component of the cell microenvironment. The ECM is a dynamic and versatile part of the cell microenvironment which regularly interacts with cell membrane receptors, resulting in the regulation of cell proliferation, migration, invasion and metastasis. Focal adhesion kinase (FAK) is a cytoplasmic non-receptor protein tyrosine kinase that is up-regulated in many cancers including breast cancer. It is a signalling molecule regulated in tumour progression and metastasis and a key mediator in breast cancer cell survival. FAK can regulate the phosphorylation of downstream signalling pathways such as Akt and mitogen-activated protein kinase (MAPK). Moreover, FAK is also associated with cell metabolism, where, glucose consumption and lipogenesis can be upregulated due to FAK activity. Understanding the role ECM proteins have on the functionality of FAK and associated intracellular signalling pathways in TNBC, has the potential to improve therapeutic strategies. Moreover, targeting this essential signalling molecule is known to impact the metastasis and cell viability in breast cancer. By studying the interactions of TNBC cells with the surrounding ECM and involvement of key cellular signalling pathways such as FAK, will contribute to an improved understanding of the impact of these factors on breast cancer biology and potential therapeutic options. The aims of this project were to determine whether ECM components play a major role in FAK expression and activation in TNBC, and the impact of FAK inhibitors have in FAK activation, in the presence and absence of ECM components. In addition, the effect FAK inhibitors have on essential downstream signalling pathways, and vital cellular events such as cell cycle progression, oxidative stress and apoptosis in TNBC cells was assessed. The TNBC cell line, MDA-MB-231, grown with and without complete ECM Matrigel, and pure individual ECM proteins: Collagen IV, Fibronectin and Laminin were evaluated to determine the effect on FAK activation and expression. Collagen IV was selected for further exploration given the noticeable impact it had on FA related activities. As Collagen IV is a major constituent in the human mammary gland, its biological relevance to TNBC research was highlighted. FAK inhibitors with two distinct modes of action; Y15 (FAK inhibitor 14) and TAE226 were utilised to understand how FAK inhibition impacts FAK activation and expression in the presence of Collagen IV. Y15 is a small molecule FAK inhibitor that targets FAK specific 397 auto-phosphorylation sites, while TAE226 blocks the ATP binding site of FAK. The two inhibitors demonstrated differences in how they affect MDA-MB-231 cell metabolism as well as FA point formation. However, given that TAE226 has significant off-target effects, Y15 was selected to study specific FAK inhibition on FAK associated downstream pathways, cell cycle progression, oxidative stress and apoptosis. As FAK plays a vital role in cell attachment, FAK inhibition was investigated in cells treated both before and after cell attachment. A drastic reduction in FA point formation was observed when FAK inhibitors were added to attached cells. In contrast, this observation was not made for pre-adherent cells, with only minimal effects reported on FA point formation, in particular, in the presence of Collagen IV. Moreover, increased levels of apoptosis were observed following the FAK inhibition on the already attached cells, with less live cells present in comparison to the pre-adherent cells. Collectively, these results indicated that Y15 is more effective on adherent cells, resulting in changes to FA point formation and cell death. Nonetheless, the inhibitory concentrations of Y15 observed for the two conditions (i:e: Y15 treatment on pre and post attached cells) was very similar, both in the absence and presence of Collagen IV. This suggests that although a higher percentage of adherent cells had undergone cell death, the remaining live cells had an overall metabolic rate similar to that observed for pre-adherent cells, where the majority of the cell population was alive. The effects of FAK inhibition by Y15 also resulted in an upregulation of phosphorylation of Akt in the pre-adherent cells, which is an indication that MDA-MB-231 cells rely on the Akt pathway for survival. In addition, FAK inhibition using Y15 exhibited downregulation of reactive oxygen species (ROS) which is indicative of hindrance in breast cancer cell metabolism. Collectively, these findings suggest Collagen IV plays a pivotal role as an ECM component, in TNBC disease progression and the efficacy of FAK inhibition.