京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13062号 / 論医博第2120号 / 新制||医||1018(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 長船 健二, 教授 川口 義弥, 教授 松田 道行 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The goal of this study is to determine whether SKI-606, a Src/abl inhibitor, potentiates chk1 inhibitor UCN-01 to induce apoptosis in multiple myeloma cells, and what mechanism maybe involved. We found that the co-exposure of human myeloma cells (e.g., U266, RPMI8226, MM.1S and its dexamethasone-resistant counterparts MM.1R) to minimally toxic concentration of SKI-606 (e.g., 1-2 ƒÝM) and UCN-01 (e.g., 100-150 nM) resulted in dramatic increase in mitochondrial damage and apoptosis. In our previous reports, it has been well demonstrated that activation of Ras/Raf/MEK/ERK pathway represents a critical cytoprotective response in cells exposed to UCN-01. Moreover, Src is required to activate this pathway by growth factors and cytokines. To this end, we examined whether the Src inhibitor enhances UCN-01 lethality through interruption of Ras/Raf/MEK/ERK signaling cascade. Firstly, it was found that co-adminstration of SKI-606 markedly diminished ERK phosphorylation/activation induced by UCN-01, accompanied by an increase in cdc2 activation. Furthermore, myeloma cells with ecpotic expression of either active mutant Ras (Q61L) or constitutive active MEK1 were significantly resistant to combined treatment with SKI-606 and UCN-01, indicating Src inhibition acts upstream of Ras/Raf/MEK to potentiate UCN-01 lethality. Conversely, stable expression of dominant-negative mutant Ras (S17N) markedly sensitized myeloma cells to this combination regimen. Lastly, ectopic expression of kinase inactive (K297R) or dominant-negative (K296R/Y528F) mutant Src blocked ERK activation by UCN-01 and thereby sensitized myeloma cells to UCN-01. Together, these findings indicate that Src inhibitors act through a Ras/Raf/MEK-dependent mechanism to prevent ERK activation in UCN-01-treated cells, resulting in the synergistic induction of cell death.

iv Abstract Crosstalk of integrin and mTOR signalling is an essential process that monitors cellular interaction with extracellular matrix and transmits these inputs to cell growth signalling. Although adhesion status of the cell monitored by integrin signalling is clearly important for regulation of cellular growth, a little is known about the crosstalk of integrin and mTOR signalling. In this study, we employed two different approaches to describe and elucidate character of this crosstalk. p130Cas is an adaptor protein phosphorylated by Src kinase and focal adhesion kinase upon integrin ligand binding and implicated in cell adhesion, motility and survival in both Src-transformed and untransformed cells. Recently, p130Cas was also described in cellular pathology, mainly by its ability to stimulate cell invasion and metastasis. In this study, we described that p130Cas affects mTOR signalling in Src-transformed cells. Substrate domain of p130Cas was found to be indispensable for this effect and is also responsible for serum-induced activation of mTOR signalling. In addition, we prepared cell lines overexpressing various Rheb protein versions and characterized them in context of mTOR signalling, integrin signalling and cell cycle progression. Interestingly, a cell line overexpressing constitutively active...

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Les arrestines sont connues pour leurs rôles dans la désensibilisation et l'endocytose des récepteurs couplés aux protéines G (RCPGs). Au fil des ans, plusieurs partenaires de ces protéines adaptatrices ont été identifiés, notamment diverses molécules impliquées dans la signalisation, incluant les kinases ERK, JNK, Akt, Raf et Src. Ainsi, les arrestines interagissent avec plusieurs kinases, mais seulement avec deux phosphatases, PPA2 et MKP7. Récemment, notre laboratoire a démontré une nouvelle interaction entre les arrestines non-visuelles et la phosphatase Shp2. En effet, des essais in vitro et in cellulo ont montré une interaction directe entre les deux protéines peuvent interagir ensembles et ce directement. Or, nous en sommes venus à nous demander si les arrestines peuvent être phosphorylées sur leurs résidus tyrosine et si cette modification pourrait être régulée par des partenaires connus, soient Src et Shp2. Il a déjà été montré que l'arrestine 2 peut être phosphorylée sur un résidu tyrosine qui lui est unique et que cette modification expliquerait peut-être les différences de modulation entre les arrestines non-visuelles. Par contre, il n'existe encore aucune preuve que l'arrestine 3 puisse aussi être phosphorylée. D'abord, nous démontrons pour la première fois que l'arrestine 3, tout comme l'arrestine 2, est phosphorylée sur des résidus tyrosines et que cette modification peut être amenée par l'activité de Src. Ensuite, un double mutant ponctuel de l'arrestine 3 a été construit afin de cibler les tyrosines régulées. Il semble que les tyrosines 380 et 404 de l'arrestine 3, soient d'importants sites de phosphorylation. Ce nouveau mutant de l'arrestine 3 représente un excellent outil afin de déterminer le rôle de la phosphorylation des tyrosines de l'arrestine 3. Aussi, puisque ces deux tyrosines sont absentes de la séquence de l'arrestine 2, leur phosphorylation pourrait être à la base des différences fonctionnelles entre les arrestines 2 et 3. De plus, nous faisons la démonstration, en conditions de surexpression en cellules, que l'activité de Shp2 peut mener à la déphosphorylation des arrestines non-visuelles. Dès lors, nos études montrent d'abord que, tout comme son homologue, l'arrestine 3 peut être phosphorylée sur ses tyrosines. De plus, cette phosphorylation est non seulement régulée par la phosphatase Shp2, mais représente également un nouveau mécanisme potentiel de régulation des multiples fonctions des arrestines.

Src signaling plays an important role in prostate cancer (PrCa) progression. It has previously been shown that Src interacts with androgen receptor (AR) and enhances AR transactivation. Although it has been shown that Src promotes AR activity, the underlying pathway has not been defined. To help characterize the Src-AR pathway, the cellular localizations of Src, p-Src, AR, pAR, and Prostate Specific Antigen (PSA, an AR target gene) were analyzed in androgen-dependent (AD) LNCaP cells and in androgen-independent (AI) castration-resistant C4-2B cells. Using sub-cellular fractionation, the data showed that treatment of AD cells with synthetic androgen R1881 increased p-Src, AR, pAR, and PSA in the nucleus, while the levels of c-Src remained unchanged. Treatment of AI cells with R1881 increased pSrc and AR in the nucleus, while the levels of c-Src and PSA remained unchanged. When using immunofluorescence microscopy, R1881 did not appear to increase the nuclear levels of p-Src or c-Src, so perhaps this technique is not as sensitive or quantitative as subcellular fractionation immunoblots. The presence of PSA in the nucleus was unexpected given its well proven role as a secreted protein. Nuclear PSA was observed upon androgen stimulation in AD and AI cells, and in the nucleus of AI cells upon androgen deprivation. Given PSA's ability to induce cell division and decrease apoptosis when transfected into cells, its presence in the nucleus may imply that PSA acts there to help induce tumorigenesis. The effect of Src on AR activity was further studied by transfection of a dominant negative src (SrcK298M) in AD and AI cells. Transfection with SrcK298M did not affect PSA expression in LNCaP cells, but strongly inhibited PSA levels in AI cells. Integrin signaling through Src was investigated in PrCa by ligand binding assay in AD and AI cells. The data showed that alpha v beta 3 integrin (but not alpha v beta 6) upon attachment to fibronectin or TGF-beta-latency associated peptide (TGF- beta-LAP) increases p-Src levels in AD and AI cells, while the levels of c-Src, PSA, and AKT remain unchanged. Thus, alpha v beta 3 integrin facilitates Src signaling, but the activation does not appear to affect AR transactivation. In conclusion, these data show that Src is required for AR activity and, consequently, PSA expression in AI prostate cancer cells, but not in AD cells. These data also suggest that the nuclear co-localization of p-Src, AR and PSA might allow macromolecular interactions, which can further enhance AR transactivation and promote disease progression. With respect to the switch in tumor progression from an AD to AI state, the data indicate that the integrin-Src pathway does not include AKT or PSA (and not AR by deduction), so perhaps other non-AR pathways help facilitate tumor growth at the AI state.

Histone Deacetylase Inhibitors (HDACi) are a new class of chemotherapeutics which have shown promise in pre-clinical and clinical settings. HDACi have been shown to act by re-programming gene expression, with the transcription of some genes such as p21WAF1 being activated, while others like SRC and BCL2L1 are repressed. The mechanism behind HDACi gene expression changes remains unknown; although it has been shown to involve a direct interaction with gene promoters. Using a quantitative qRT-PCR approach, the effect of various HDACi on the transcription of p21WAF1, SRC and BCL2L1 was examined. TSA and apicidin led to an up regulation of p21WAF1 mRNA levels while c-Src and Bcl-xL mRNA levels were downregulated. Short c-Src mRNA transcripts were unaffected following TSA and apicidin treatments, despite the full length transcripts being repressed. Repression of full length c-Src and Bcl-xL mRNA transcripts was not seen following treatment with MS-275 and MGCD0103, although p21WAF1 mRNA expression was induced. ChIP experiments revealed that following HDACi treatment, histone acetylation levels and RNA Polymerase II occupancy increased in the promoter regions of both the SRC and BCL2L1 genes. RNA Polymerase II occupancy lasted less than 15 minutes in the 3’ regions of the gene following treatment with apicidin and TSA, but was more long-term following MS-275 and MGCD0103 treatment. The protein phosphatase inhibitor Calyculin A completely blocked HDACi mediated repression of c-Src and Bcl-xL mRNA, suggesting a role for protein phosphatases in the mechanism behind HDACi. It is therefore hypothesized that HDACi work through at least two different mechanisms. Whether or not an HDACi leads to gene repression depends on its ability to disrupt an HDAC/protein phosphatase complex and not on their HDAC specificities. The disruption of the complex leads to the release of an active protein phosphatase. The released phosphatase can then presumably act on various factors changing a gene from an active to paused state, possibly through promoter proximal pausing. HDACi unable to disrupt this complex are unable to induce gene repression. Collectively, these studies highlight not only the complexity of HDACi mediated effects within the cell, but also present a new explanation behind HDACi mediated gene repression.

Signal Transducer and Activator of Transcription 3, Stat3, has been associated with cytokine-induced proliferation, anti-apoptosis and neoplastic transformation, while constitutively active Stat3 has been found in many human tumors. Stat3 activation by the Src oncoprotein leads to specific gene regulation and the Stat3 mediated signaling pathway is one of the critical signaling pathways involved in Src oncogenesis. Our laboratory demonstrated that engagement of cadherins, which are a class of cell-cell adhesion molecules, can activate Stat3, even in the absence of direct cell to cell contact. Interestingly, a significant increase in total Rac1 and Cdc42 protein levels triggered by cadherin engagement, and an increase in Rac1 and Cdc42 activity, which led to Stat3 activation by a mechanism involving gp130, a common subunit of the IL-6 family of cytokines, was also observed. To investigate the role of gp130 in vSrc transformation, we knocked down gp130 in vSrc transformed cells and found a decrease in the levels of phosphorylated Stat3, the rate of cell migration, rate of cell proliferation and the anchorage-independent growth. It was also previously demonstrated that vSrc had a negative effect on the function of cadherins. Surprisingly, however, despite the fact that vSrc may reduce cadherin adhesion, previous results in our lab showed that cell density still caused a significant increase in Stat3 activity in vSrc transformed cells. Moreover, Stat3 downregulation induced apoptosis in transformed cells which was more pronounced at high cell densities. This may reflect an increased need for Stat3 activity at high densities, possibly to overcome apoptosis, which raised the question of the actual role of cadherins in the density-mediated activation of Stat3 in such cells. The expression of cadherin-11, a type II cell adhesion molecule, is associated with invasive breast cancer and many studies suggest that it may play a significant role in facilitating tumor cell invasion and the formation of metastatic tumors. Since Src and its family members participate in many aspects of tumor progression and metastasis, it was interesting to see if Src needed cadherin-11 for neoplastic transformation. To this effect, when we knocked down cadherin-11 in Balb/c3T3 cells, we observed a significant reduction in levels of phosphorylated Stat3-ptyr705 which was also observed when vSrc was expressed in them. Moreover, expressing vSrc in cells in which cadherin-11 was knocked down also decreased the anchorage –independent growth and increased apoptosis indicating that cadherin-11 is needed for transformation and survival respectively, in vSrc transformed cells. Our results thus demonstrate that cadherin-11 may be a good target for the selective elimination of cells expressing Src and presumably other oncogenes as well. Stat3 activation by cadherins is so potent and important that tumor cell death can be enhanced by cadherin inhibition. In our experiments, the inhibition of cadherin-11 induced apoptosis in Src expressing cells, while the normal cells were not affected. Elucidation of the functions and regulation of cadherin-11 may enhance our understanding of the roles of cadherins in invasive cancer and may provide future targets for therapy. Through our work, the cadherin/IL-6 pathway may emerge as a crucial Stat3 activator in vSrc expressing cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2011-11-27 18:54:05.777

The adaptor protein p130Cas (CAS, BCAR1) represents a nodal signaling platform for integrin and growth factor receptor signaling, and influences normal development and tissue homeostasis. Its altered expression drives many pathological conditions including tumor growth, metastasis and drug resistance in many cancer types. How p130Cas contributes to many of these pathologies is still poorly understood. Therefore, the overall aim of my PhD work was to provide new insights to p130Cas signaling and its regulation. The SH3 domain is indispensable for p130Cas signaling, but the ligand binding characteristics of the p130Cas SH3 domain, and the structural determinants of its regulation were not well understood. To be able to study various aspects of p130Cas signaling we identified an atypical binding motif in p130Cas SH3 domain by establishing collaborations with Dr Veverka (Structural biology) and Dr Lepšík (Computational biochemistry; Academy of Sciences, CZ) which gave new insight into this binding interface. Through these collaborations I generated chimeras of p130Cas SH3 domain with its ligands for structural NMR analysis and learned how to visualize and analyze structures. Furthermore, my work expanded our knowledge of p130Cas SH3 ligand binding regulation and led to a novel model of Src-p130Cas- FAK...

Src is a 60 kDa tyrosine kinase that is expressed in most of animal tissues. Src has three splice variants, C-src, which is ubiquitously expressed, and N1- and N2-src, which are neuronal specific splice variants. The srcs are differentially spliced at their SH3 domains, therefore the hypothesis is that this splicing allows them to have different binding partners and perform different roles in neurons. The aim of this project is to identify new interactions for the three src splice variants in neurons and their possible functional roles. The SH3 domains, kinase active truncated proteins ( 80) and kinase dead mutant full length versions of the three splice variants of src were cloned from a rat brain cDNA library into bacterial expression vectors. GST-pull downs from nerve terminal lysates showed that different src splice variants had different binding partners. These partners were identified by mass spectrometry and confirmed by western blotting. C-src binding partners included dynamin, synapsin, and synaptojanin, while N2-src binding partners included synaptophysin, Munc18-1, and NSF. The interaction between N2-src and Munc 18-1 was characterized further; however a number of in vitro interaction assays and kinase assays showed that Munc 18-1 may not be a direct binding partner for N2-src or substrate. N1-src displayed a stimulation-dependent interaction with dynamin I. This was shown to be phosphorylation-dependent in contrast to C-src binding. The major phosphorylation sites on dynamin I, S774 and S778, were not involved in the regulation of N1-src binding. The binding site for N1-src on dynamin I was different to C-src, with extensive mutagenesis studies suggesting that the interaction site is at the tail of the dynamin I xa splice variant, which has an additional two phosphorylation sites.