Here I describe a new function of Focal Adhesion Kinase (FAK) in driving anti-tumour immune evasion. The kinase activity of FAK in squamous cancer cells drives the recruitment of regulatory T-cells (Tregs) by transcriptionally regulating chemokine/cytokine and ligand-receptor networks, including the transcription of CCL5 and TGFβ, which are required for enhanced Treg recruitment. In turn, these changes inhibit antigen-primed cytotoxic CD8+ T-cell activity in the tumour microenvironment, permitting survival and growth of FAK-expressing tumours. I show that immune evasion requires FAK’s catalytic activity, and a small molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, drives depletion of Tregs and permits CD8+ T-cell-mediated tumour clearance. It is therefore likely that FAK inhibitors may trigger immune-mediated tumour regression, providing previously unrecognized therapeutic benefit.

Tumour angiogenesis is a hallmark of cancer. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in endothelial cells (ECs) survival, proliferation and migration. FAK has several tyrosine phosphorylation sites thought to be involved in FAK function but the requirement of phosphorylation of these residues in vivo is unknown. We have generated mice where endogenous FAK is deleted simultaneously with the expression of nonphosphorylatable FAK-Y397F or FAK-Y861F mutated or wild type forms of FAK in adult endothelium in order to test this. My data show that EC-FAK-Y397FKI mice present with decreased tumour angiogenesis (in sygeneic B16F0, CMT19T and LLC) but impaired B16F0 and CMT19T tumour growth only, with increased tumour hypoxia. FAK-Y397F tumour endothelium is not perfusion, leakage or vascular maturation defective. This mutation affects VEGF-, PlGF- and bFGF-driven angiogenesis in vivo and VEGF+Ang2 administration is able to partially rescue this phenotype ex vivo. In contrast, endothelial FAK-Y861F mutation leads to an initial delay in B16F0 tumour angiogenesis, that subsequently resolves, and does not affect B16F0 tumour growth. LLC and CMT19T tumour growth and angiogenesis are not affected by the endothelial FAK-Y861F mutation; neither are tumour blood vessel perfusion, leakage, vascular maturation or tumour hypoxia. VEGF-, PlGFand bFGF-driven angiogenesis in vivo and ex vivo was not affected by the endothelial FAK-Y861F mutation, whereas increased in vivo angiogenesis was triggered by Ang2 administration. Lastly, to understand whether cytokine profiles that might affect angiocrine signalling are affected differentially in FAK-Y397F vs FAK-Y861F endothelial cells, I show that CCL1 and CCL2 are increased in FAK-Y397F but IL- 13, IL-1F3, CCL4, IL-1F1, CCL2 and others are increased in FAK-Y861F endothelial cells. Overall my data indicates that endothelial-specific FAK mutations on two phosphorylation sites has different effects on tumour angiogenesis, tumour growth, growth factor stimulated angiogenesis in vivo and ex-vivo and cytokine production.

Integrin recycling has been generally assumed to be important for cell migration but the trafficking pathways and the molecules regulating integrin trafficking remain poorly characterized. Furthermore, little is known about the activation status of endocytosed integrins and how it affects the recycling of these receptors. It is likely that FA-engaged integrins will follow different trafficking pathways than bulk integrins and here I sought to study the endocytic fate of this particular integrin pool using the MT-induced FA disassembly assay. I found that integrins previously resident at FAs travel through different Rab compartments after FA disassembly and that their return to the plasma membrane is Rab11- and Src-dependent. Strikingly, I unveiled new functions for FAK and Src family kinases in this process by showing that these kinases are critical to keep integrins active during endocytic trafficking. This finding is unprecedented since it was not known whether endocytosed integrins were kept active during their trafficking. Interestingly, reassembly of FAs from endocytosed integrin occurred preferentially at the leading edge of migrating cells suggesting that integrins are trafficked in a polarized fashion. Furthermore, the recycling of integrins from the Rab11-positive compartment to the plasma membrane is a long-range transport implying the existence of a MT motor committed to this task. Consistently, I identified that a kinesin-II motor, Kif3AC, is engaged in this process. My work establishes a FAK- and Src family kinases-based mechanism for integrin "adhesion memory" during endocytic trafficking and identifies a direct link between FA disassembly and reassembly through an endocytic recycling pathway involving Rab5 and Rab11 and a kinesin-II family member.

Breast cancer most commonly metastasizes to the bone, where it perpetuates the vicious cycle leading to osteolytic lesions. This occurs when secreted factors from breast cancer cells disrupt bone homeostasis by deregulation of osteoblast bone formation, and enhance osteoclast bone degradation thereby releasing bone matrix bound growth factors leading to further tumor growth. Although the use of osteoclast targeting agents, such as bisphosphonates and RANK-L inhibitors, are common practice for the treatment of bone metastasis, they have not been shown to increase patient survival. We therefore sought to investigate the role of focal adhesion kinase (FAK), a potential therapeutic target, in the treatment of breast cancer mediated osteolysis. FAK is a non-receptor tyrosine kinase known to directly regulate tumor progression and metastasis; it is also expressed in all of the cell types involved in breast cancer mediated osteolysis. Thus, we hypothesized that the inhibition of FAK would restore normal bone homeostasis, as well as mediate direct anti-tumor activity. FAK depletion resulted in the decrease of expression of several osteolytic factors secreted by breast cancer cells. However, the use of FAK depleted breast cancer conditioned media did not prevent breast cancer mediated osteoclastogenesis in an osteoblast/osteoclast coculture. In monoculture however, using the FAK inhibitor PF-271, we have shown that FAK inhibition leads to increased apoptosis of mature osteoclasts, and their decreased ability to degrade mineralized bone matrix, perhaps in part due to reduced expression of lytic factors such as tartrate resistant acid phosphatase and cathepsin K. Further, FAK inhibition in osteoblast monoculture led to a decrease in their ability to express the maturation factor alkaline phosphatase, and also inhibited their ability to induce mineralization. This inhibition may be due in part to the specific effects of FAK inhibition using PF-271, which may result in decreased levels of p53 in treated osteoblasts. These results suggests that the pharmacological inhibition of FAK can effect all three cell types involved in the vicious cycle of bone metastasis, and as such could be a beneficial therapeutic for patients with bone metastasis resulting in prevention of bone degradation along with direct inhibition of tumor growth. However, it may require further evaluation in animal models to determine if observed effects on osteoblast activity in vitro also occurs in vivo with possible detrimental effects on restoration of damaged bone.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 83-95).

Invasion and metastasis are the major causes of treatment failure and high mortality rate in hepatocellular carcinoma (HCC) patients. Cell motility is crucial to tumor invasion and metastasis, requiring the ability of tumor cells to interact with extracellular matrix, which is regulated by integrins and integrin-associated molecules at the focal adhesions. Recent studies have demonstrated the role of β1 integrin (CD29) overexpression in HCC and its correlation with cancer cell invasiveness and metastastic potential, as well as its protective role against cancer cells against chemotherapeutic drug-induced apoptosis, yet the mechanism is not fully known. Focal adhesion proteins serve as binding platforms for additional cytoskeletal and signaling molecules in the CD29 signaling pathway. Recently, Actopaxin has been demonstrated to form complex with numerous molecules at the focal adhesions, including ILK, which interacts with the cytoplasmic tail of CD29. Through these interactions, Actopaxin has been shown to regulate different cellular events, including cell survival, spreading and cell migration. In this study, the role of Actopaxin in HCC was investigated. In particular, its role in the regulation of tumor invasion and metastasis of HCC cells was demonstrated. This study showed that Actopaxin expression was overexpressed in HCC specimens when compared with the adjacent non-tumorous liver, and that its overexpression positively correlated with tumor size, stage and metastasis in HCC specimens. Actopaxin expression was also correlated with the metastatic potential in HCC cell-lines. Functional studies established that overexpression of Actopaxin conferred invasive phenotypes in primary, non-metastatic HCC cells, whereas down-regulation of Actopaxin could revert the invasive phenotypes and metastatic potential of metastatic HCC cells in vitro and in vivo. Suppression of Actopaxin expression was associated with reduced expression of ILK, PINCH, Paxillin and cdc42, whereas expressions of E-cadherin, β-catenin and GSK3β were induced, indicative of a less invasive and invasive phenotype. Conversely, overexpression of Actopaxin in primary, non-metastasis HCC cells accordingly up-regulated the expression of ILK, PINCH, Paxillin and cdc42, and down-regulation of of E-cadherin, β-catenin and GSK3β, suggestive of an enhanced invasive phenotype. The expression of Actopaxin was found to be correlated with CD29 level, indicating that Actopaxin is a CD29-associated protein and involved in CD29-regulated signaling. Finally, Actopaxin down-regulation enhanced chemosensitivity of of HCC cells towards chemotherapeutic treatment. Treatment with Oxaliplatin was enhanced in Actopaxin-deficient HCC cells, which showed a stronger inhibitory effect on cell proliferation and cell cycle progression, accompanied with induction on apoptosis. The enhanced chemosensitivity effect was a collective result of suppression of Survivin protein, β-catenin and mTOR pathways; and up-regulation of p53. To conclude, this study demonstrated for the first time that Actopaxin is involved in HCC invasion, metastasis and chemosensitization, providing the basis to further investigate the potential role of this protein or its downstream effectors as a therapeutic target for inhibiting the development of metastasis and enhancing chemotherapy efficacy to combat HCC, and perhaps other invasive cancers. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

H κινάση εστιών προσκόλλησης (FAK) συμμετέχει στη μεταγωγή μηνυμάτων κατά την κυτταροφαγία. Στα αιμοκύτταρα των εντόμων υπάρχει σε διαφορετικές ποσότητες κατά την ανάπτυξη. Ενεργοποιείται με φωσφορυλίωση στην Tyr-397. Kατά την κυτταροφαγία του βακτηρίου Ε. coli ενεργοποιείται άμεσα. Ανάμεσα στα μόρια που σχηματίζουν σύμπλοκο με την FAK είναι και οι πρωτείνες pinch, Src και οι ΜΑΡΚ. Κάποια από τα παραπάνω σχηματίζουν σύμπλοκο κατά την κυτταροφαγία ενώ για άλλα το σύμπλοκο προυπάρχει. Ο κυτταροσκελετός ακτίνης και τουμπουλίνης χρειάζονται για την κυτταροφαγία, όπως χρειάζεται και η έκκριση. Τέλος διαπιστώθηκε πως κατά την κυτταροφαγία του βακτηρίου Ε. coli και του πεπτιδίου RGD, συμμετέχουν οι πρωτείνες Src, Ras, Rho και JNK. / Focal adhesion kinase (FAK) participates in signal transduction at phagocytosis. Insect hemocytes have FAK in different mounts during development. FAK becomes activated after phosphorylation at Tyr-397. During phagocytosis of E. coli, FAK becomes immediately activated. Pinch, Src and MAPK are some of the molecules which are in complex with FAK. Some of these molecules are in complex with FAK during phagocytosis, but some others there are in complex at any time. Phagocytosis, in order to happen, needs actin and tubulin cytoskeleton. Secretion is also needed for this purpose. Finally, we found that the proteins Src, Rho, Ras and JKN participate in phagocytosis of the RGD peptide and in phagocytosis of the microbe Escherichia coli.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that localises to focal adhesions. FAK is crucial for many cellular processes that are disturbed in malignancy, including proliferation, cell cycle, cell survival, adhesion, and migration. Mouse models have shown that FAK is involved in tumour formation and progression. Other studies demonstrated a functional correlation between FAK expression, tumour progression and malignancy in human cancer, making FAK a potentially important therapeutic target. Several FAK inhibitors have been developed most of which target the FAK kinase function. However, FAK may predominantly act as a scaffolding molecule rather than as a kinase, therefore, disruption of FAK’s interaction with protein binding partners could be a good strategy to inhibit some cancer processes. The identification and characterisation of novel FAK interactions may help to uncover important molecular mechanisms that, in turn, regulate key cellular processes involved in tumour formation and/or progression. Disruption of their function, or inhibition of their binding to FAK, will define their roles and identify whether they are good anti-cancer targets. In this thesis work, I set out to identify novel binding partners of FAK, and study the role of a sub-set of these in tumour biology by impairing them in squamous cell carcinoma cancer cells in vitro. To do this I employed protein microarray and phage display methodologies using FAKΔ375 and FAK-FERM recombinant proteins as bait, respectively. I identified a number of novel proteins that interact directly with FAK. Then I set out to characterise some of these proteins. The first of these, Axl, is a protein receptor tyrosine kinase that has previously been linked with tumour progression and metastasis in number of human cancers. I confirmed the interaction between FAK and Axl in SCC cells and showed that the FAK-Axl interaction is predominantly a scaffolding function of FAK, which seems to be unregulated, at least by any of the major phosphorylation events characterised for FAK. I also found that Axl controls cell spreading, cell polarisation and invasive migration in this cancer cell lines. The second protein I characterise is the autophagy protein Ambra1. I found that Ambra1 is required for selective targeting of active Src to the autophagy pathway – a process that SCC cancer cells use when they are under adhesion stress, such as when FAK is deleted. Thus, Axl and Ambra1 are potentially important proteins in SCC biology. They bind to FAK and function at cell adhesions to promote cancer-associated cellular processes. Analysis of FAK binding proteins may be a useful strategy to discover proteins that function in various aspects of cancer cell behaviour.

Focal adhesion kinase (FAK) controls the assembly of focal adhesion sites and transduces signals from several membrane receptors. Controlled activation and localization of FAK functionally links cell adhesion, migration and survival. FAK is overexpressed in many cancer types, promoting tumor invasiveness and metastasis. The molecular mechanisms allowing FAK to fulfil numerous different functions and act as versatile ‘nanomachines’ are poorly understood. We have previously revealed that ligand-induced dimerization along with intramolecular interactions control FAK activation and localization where the C-terminal focal adhesion targeting (FAT) domain is strictly involved. In this study, we combine NMR with X-ray crystallography, as well as biophysical and computational methods to understand the molecular mechanisms that link the large-scale dynamics and intramolecular and intermolecular interactions of FAT into FAK’s capacity to integrate various stimuli into a site-specific function. Our results reveal FAT-mediated dynamical interplays between binding of known and newly discovered FAT ligands, and multimerization and autoactivation of FAK. Additionally, we investigate the impact of neuronal alternative splicing on FAT dynamics and interactions. Collectively, our results elucidate FAT’s role in allosterically controlling various FAK functions, and might inspire allosteric protein-protein interaction inhibitors against FAK-dependent cancer cell proliferation.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23800号 / 医博第4846号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 髙折 晃史, 教授 辻川 明孝 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM