Pyruvate kinase M2 (PKM2) has been studied for decades on its role in cancer metabolism. Recently, PKM2 is highlighted again for its new function: promoting gene transcription by acting as a protein kinase. Moreover, the PKM2 levels in patient circulation have been used as a diagnostic marker for many types of cancers. However, it remains unclear whether PKM2 in blood circulation has any physiological or pathological function. In my dissertation, I demonstrate that PKM2 released from cancer cells facilitates tumor growth by promoting tumor angiogenesis. Our experiments show that PKM2 promotes endothelial cell proliferation, migration and survival. Only the dimeric PKM2, not the tetrameric PKM2 possesses the activity in angiogenesis promotion. Our results further indicate that PKM2 regulates angiogenesis by integrin αvβ3 activation and integrin redistribution. I also found that PKM2 enhances drug resistance of cancer cells expressing integrin αvβ3.

In the Xenopus embryo, cell rearrangements during early development require the dynamic modulation of adhesion. Cells primarily use the integrin family of transmembrane receptors for attachment to and interpretation of the extracellular environment. While acting as adhesion receptors, integrins also have bidirectional signalling properties essential for driving cellular movements. The regulation of integrin activity is thought to stem from cytoplasmic assemblies of constitutively expressed molecules. PINCH (Particularly Interesting New cysteine-histidine rich protein), an adapter protein, is part of an IPP complex that has emerged as a key signalling scaffold indispensable for integrin function in vitro. As such, I tested the hypothesis that PINCH regulates integrin function in the Xenopus embryo. Xenopus PINCH was successfully cloned using RT-PCR. The predicted amino acid sequence of PINCH shares a 98% similarity with mammalian orthologs, and comprises of five highly conserved LIM domains. PINCH mRNA and protein are ubiquitously expressed throughout embryogenesis. In situ hybridization indicates that PINCH mRNA is expressed in the blastocoel roof and the pre-involution mesoderm. The localization and temporal expression of PINCH suggests a role in mediating cell adhesive events during gastrulation. A functional approach was used to examine the role of PINCH during gastrulation. I used site-directed mutagenesis to generate non-functional LIM1 (LIM1mut) and LIM4 (LIM4mut) domains that have been proposed to bind ILK and Grb4 respectively. Over-expression of PINCH leads to a delay in blastopore closures, while the expression of both LIM1mut and LIM4mut relieve this inhibition at lower concentrations. Further analysis indicates that PINCH, LIM1mut, and LIM4mut inhibit FN matrix assembly independent of integrin adhesion. Contradictory to in vitro studies, co-immunoprecipitation analysis indicates that endogenous PINCH does not bind ILK, confirming an integrin-independent role during gastrulation. Furthermore, in the embryo PINCH is found at cell boundaries but does not appear to directly modulate cadherin adhesion. As such this thesis provides evidence that PINCH regulates cell intercalation movements independent of integrin and cadherin receptors and raises the possibility that the LIM4 domain is involved in PINCH regulation of cell adhesion during early development.

Tip growth, the mechanism by which hyphae, pollen tubes, root hairs, and algal rhizoids extend, is a complex and dynamic process that is characterised by localised extension at the extreme apex of the cell and morphological polarity. Its complexity suggests that high degree of regulation is needed to ensure that the characteristics of a particular cell type are maintained during growth. Regulation is likely to come about through bidirectional interplay between the cell wall and cytoplasm, although the mechanisms by which such cross-talk might occur are unknown. Results of this thesis present immunocytochemical data that indicate the presence of, and a close association between β4 integrin subunit-like proteins and proteins containing phosphorylated tyrosine residues in the oomycete Achlya bisexualis. When hyphae were plasmolysed, these proteins were present in wall-membrane attachment sites where there was also F-actin. A combination of immunoblots, ELISA, and a coupled enzyme assay suggest that phosphorylation may occur by both autophosphorylation and through the possible action of a tyrosine kinase. Tyrphostins, which are inhibitors of tyrosine kinases, abolished the anti-phosphotyrosine staining, inhibited the kinase activity, slowed tip growth and affected the organisation of the actin cytoskeleton, in a dose-dependent manner. In addition, results show A. bisexualis contains proteins epitopically similar to the rod domain of animal talin. However, these proteins do not co-localise with F-actin, and mainly locate at the sub-apical region in hyphae. For comparative purposes, Saccharomyces cerevisiae was also used to investigate the presence of β4 integrin subunit-like proteins and tyrosine phosphorylation. Immunoblotting showed that S. cereviaise contains a protein, which is found in the microsomal pellet fraction, that cross reacts with anti-β4 integrin subunit antibody. Furthermore, there are a number of proteins containing phosphotyrosine residues. Immunocytochemistry shows that this anti-β4 integrin staining is at the cortical site but anti-phosphotyrosine residues are distributed throughout cells. On the basis of an ELISA and a coupled enzyme assay, it is suggested that a soluble fraction of S. cerevisiae contains tyrosine kinase activity. This activity is strongly inhibited by tyrphostins.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.

A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.

Laminin binding integrins (α6β1 and α3β1) are persistently but differentially expressed throughout prostate cancer progression and metastasis. Prostate cancer primarily invades through laminin rich nerve for extracapsular escape during cancer metastasis. An intense expression of the pro-metastatic α6 integrin was observed during perineural invasion with a heterogeneous distribution of the integrin on the cancer cell membrane as well as intracellularly. Bone and soft tissue metastasis of human prostate cancer demonstrated a similar pattern where 75-80% of the cancers had significant intracellular staining. This was correlated with an mRNA overexpression of various intracellular trafficking regulators. Using a prostate cancer cell culture model of DU145 cells, the α6 integrin was found to be constitutively internalized in cancer cells at a rate of 3.25 min-1, which was 3 fold greater than internalization rate of α3 integrin, classically considered a "non-circulating" receptor. α6 and α3 integrins function coordinately to regulate cell migration during development, wound healing. Their orchestrated redistribution during these processes is well-known, but the mechanism remains elusive. Current study identifies intracellular trafficking of these integrins as a key mechanism of their coordination. Depletion of α3 integrin in prostate cancer cells significantly increased internalization of α6 integrin up to 1.7-fold and increased localization of α6 integrin at cell-cell membrane locations. There was a concomitant 1.8-fold increase in cell migration significantly dependent on α6 integrin. Depletion of α6 integrin expression however, had no effects on the internalization of α3 integrin indicating that the identified coordination was unidirectional. α6 integrin trafficking drives cancer invasion, but its selective regulators are unknown. Here, Rab11FIP5 was identified as a selective regulator of α6 integrin recycling to cell membrane. Interestingly, α6 integrin was found to be primarily recycled to the cell-cell membranes where it colocalized with Rab11 and Rab11FIP5. Depletion of Rab11FIP5 reduced such membrane expression of α6 integrin, inhibited cell-cell cohesion in 3D culture and significantly reduced cell migration. The localization of α6 and α3 integrin at these locations have been implicated in cell adhesion. Based on current study α6 recycling by Rab11FIP5 might be key to such function. Another Rab11 effector protein Rab11FIP1 was identified as a regulator of both α3 and α6 integrin trafficking. Depletion of Rab11FIP1 reduced membrane expression of α3 integrin by significantly increasing its internalization and reducing the recycling. There was a major effect on α6 integrin internalization, which increased to an extent similar to that observed on α3 integrin depletion. Rab11FIP1 regulated α6 integrin recycling, in a pathway found to be independent of Rab11FIP5. Taken together, current research defined Rab11FIPs as regulators of α6 and α3 integrins. A unidirectional coordination between α6 and α3 integrin was identified such that loss of α3 integrin, representative of high grade prostate cancer, amplifies integrin α6 integrin internalization and a resultant migratory phenotype.

Cell migration is an essential physiological process required for embryogenesis, tissue repair and immune surveillance. Directional cell migration requires coordinated regulation of multiple integrin-mediated cellular processes, including dynamic regulation of the actin cytoskeleton, precise control of membrane protrusion events and the constant recycling of adhesion receptors. While it is clear that regulation of the small guanosine triphosphatases (GTPases) Rac1, Arf6 and RhoA is critical for these processes, the integrin-dependent mechanisms responsible for cyclic activation and dynamic coordination of GTPase signalling are only partially understood. Here, analysis of three published mass spectrometry (MS) studies cataloguing integrin-dependent adhesion complexes identified filamin-A and IQGAP1 as potential candidates linking β1 integrin to the regulation of Rac1 activity. Using immunoprecipitation, MS analysis, immunocytochemistry and RNAi, filamin-A and IQGAP1 were found to be recruited to integrin activation sites, where they constrained Rac1 activity via the recruitment of the GTPase-activating protein RacGAP1. The functional relevance of Rac1 deactivation, through a RacGAP1 and IQGAP1-mediated mechanism, is to permit efficient membrane protrusion and directional cell migration. Subsequently, IQGAP1 was identified as a molecule co-ordinating Rac1 and Arf6 activities downstream of β1 integrin engagement, via the recruitment of the GTPase activity modulators RacGAP1, srGAP2 and HERC1. This lead us to propose a model whereby IQGAP1, through the recruitment of multiple small GTPase activity modulators, co-ordinates the two small GTPases Rac1 and Arf6, to efficiently regulate directional cell migration. Dyregulated cell migration due to integrin over-activation is associated with tumour invasion. Increased recycling of α5β1 integrin, resulting from expression of mutant p53 or inhibition of αVβ3 integrin function, leads to random cell migration on 2D substrates and promotes tumour invasion via activation of the pro-invasive kinase Akt. Here, the RacGAP1- IQGAP1 complex was identified as a key component of this pathway. In particular, RacGAP1 was found to be phosphorylated by Akt2 on T249, a phosphorylation event that promoted RacGAP1 recruitment to IQGAP1, at the cell front, and triggered cell invasion by inducing a Rac1/RhoA activity switch. These findings demonstrated that Akt activation, downstream of α5β1 integrin recycling, promotes fibronectin-mediated cell invasion by activating a novel RacGAP1/IQGAP1/Rac1/RhoA pathway. Taken together, we identified a novel signalling nexus, downstream of integrin activation and/or recycling that co-ordinate the small GTPases Rac1, Arf6 and RhoA during cell migration and invasion.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21013号 / 医博第4359号 / 新制||医||1028(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 瀬原 淳子, 教授 小川 修 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Abstract Integrin-linked Kinase (ILK) is an integrin-associated protein kinase, which regulates growth factor-signalling pathways and cell-ECM adhesion events. Abrogated ILK expression or activity has been implicated in contributing to oncogenic transformation. We examined the role played by ILK in growth factor-stimulated and integrin signalling events in five human oesophageal squamous cell carcinoma cell lines (HOSCCs), known to overexpress the EGF receptor. Western blot analysis revealed the presence of ILK (59kDa) in all the moderately differentiated HOSCC lines. ILK1 was confirmed as being the predominant isoform. Densitometrically analysed Western blots showed that, per unit of protein, ILK is expressed uniformly across the cell lines under standard culture conditions. Following EGF (10 ng/ml) and TGFβ1 (1 ng/ml) treatment, ILK expression increased in all five HOSCCs. Indirect immunofluorescence microscopy showed the majority of ILK to localise at a cytoplasmic/nuclear level, with a proportion of ILK localising at the membrane, which resembled the distribution pattern of the β3 integrin subunit. This membranal distribution most likely follows that of the adhesion plaques although lesser, and variable, amounts were also identified throughout the cytoplasm. The functionality of the ILK1 kinase domain was demonstrated using myelin basic protein (MBP)-based kinase assays. EGF and TGFβ1 treatment produced an increase in ILK activity in the WHCO3 cell line of 3.5 fold, but a decrease in activity in the other cell lines, which are suggested to involve the actions of PTEN. The identification of nuclear ILK was surprising, and the mechanism for nuclear ILK localisation was suggested to involve a caveolae-associated protein, caveolin-1. Cell adhesion assays revealed that KP-392-mediated inhibition of ILK resulted in a nonsignificant reduction in cell adhesion to collagen and fibronectin. These data provide distinctive evidence for the influence of growth factors on ILK expression, but a duality in the effect on ILK activity. This apparent dichotomy is noteworthy and may be of particular relevance in HOSCC. It is further suggested that KP-392-induced ILK inhibition destabilises the αβ integrin heterodimer and that PI3K acts upstream of ILK-mediated cell adhesion events in HOSCCs. This suggests that ILK mediates integrin associated processes in human oesophageal SCC cell lines.