WWOX is a tumor suppressor and the down-regulation of WWOX has been demonstrated in prostate, lung, breast, gastric cancers. However, the role of WWOX in renal cell carcinoma (RCC) remains unknown. It has been demonstrated that WWOX addressed in mitochondria, golgi apparatus, rough ER, lysosome, plasma membrane and nuclear. The Subcellular localization of WWOX has been controversial. There are two parts in this study: (I) The expression of WWOX in RCC and the probability of WWOX to be a diagnostic and a prognostic marker. (II) The regulation of WWOX by phosphorylation. For the study of WWOX expression in RCC, we prepared polyclonal WWOX antibody and characterized the specificity of the antibody. We applied this specific antibody to 33 NT paring RCC tissue specimen for immunoblotting study and 138 cases of paraffin-embedded specimens for IHC, respectively. Our results demonstrated that hWOX1 was specifically down-regulated in clear cell type RCC (p=0.018). The percentage of down-regulation in patient specimen is 60.7 % and 90.7 % in immunoblotting and IHC study, respectively. And in clear cell and clear-granular combined type RCC, down-regulation of WWOX was significantly correlated with the survival rate of patients (p=0.0482). Therefore, WWOX could be used as a diagnostic and a prognostic marker in clear cell type RCC. Besides, we performed bioinformatics to predict the phosphorylation site of WWOX and investigated the effect of phosphorylation on WWOX subcellular localization. Our results demonstrated that hWOX1 was phosphorylated by PKC at Thr49 and Thr102 and the phosphorylation regulated the subcellular localization of WWOX.

The peptidyl prolyl cis/trans isomerase, PPIase, has been the focus of numerous studies in the field of cell cycle regulation since proline-directed phosphorylation is an essential signaling mechanism that might arrest cancer proliferation. Pin1 is the first phosphorylation-dependent PPIase enzyme to be discovered. The Pin1 regulatory mechanism, acting on other mitotic proteins in vivo and in vitro, remains largely unknown. For the study of Pin1 function, two types of assays were used to identity ligands for Pin1: (1) The Enzyme-Linked Enzyme Binding Assay (ELEBA) for the identification of WW domain ligands, (2) a catalytic assay to identified inhibitors of Pin1 catalytic activity. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain from chemical libraries. By using the ELEBA, a pSer-Pro peptidomimetic library of 315 ligands was screened, identifying three promising ligands cis-D2, O2, and M18. Competitive Kd values for cis-D2, O2, and M18 were determined to be 263 ± 6.4, 206 ± 3.4, and 130 ± 3.0μM, respectively. Furthermore, we screened the pSer-Pro peptidomimetic library using a Pin1 discontinuous-catalytic assay to identify inhibitors of Pin1. Ligands D20 and K7 were identified to decrease more than 90% of the Pin1 catalytic activity. To investigate the nature of the Pin1 interaction with c-Myc, we synthesized and characterized four peptides corresponding to the c-Myc sequence. These peptides were used in NMR isomerization studies of Pin1 by our collaborator Dr. Jeffry Peng (University of Notre Dame). Preliminary work shows that Pin1 binds and isomerizes the Ac–LLPpTPPLSPS–NH₂ peptide at the cMyc pThr58 position. Finally, we measured a secondary kinetic isotope effect (2º KIE) to study the Pin1 catalytic mechanism of proline isomerization. The ratio of kH/kD for unlabeled and [d₃]Ser-labeled substrate gave a SKIE value of 1.34 ± 0.01. The normal 2º KIE value indicates that carbonyl-serine hybridization is not changing from sp² to sp³. This result supports substrate analogue inhibitor studies, and previous solvent and SKIE results on Pin1, that suggest a twisted amide mechanism assisted by a transient hydrogen bond in the transition state. / Ph. D.

abstract: Cyanovirin-N (CVN) is a cyanobacterial lectin with potent anti-HIV activity, mediated by binding to the N-linked oligosaccharide moiety of the envelope protein gp120. CVN offers a scaffold to develop multivalent carbohydrate-binding proteins with tunable specificities and affinities. I present here biophysical calculations completed on a monomeric-stabilized mutant of cyanovirin-N, P51G-m4-CVN, in which domain A binding activity is abolished by four mutations; with comparisons made to CVN<super>mutDB</super>, in which domain B binding activity is abolished. Using Monte Carlo calculations and docking simulations, mutations in CVN<super>mutDB</super> were considered singularly, and the mutations E41A/G and T57A were found to impact the affinity towards dimannose the greatest. <super>15</super>N-labeled proteins were titrated with Man&#945;(1-2)Man&#945;, while following chemical shift perturbations in NMR spectra. The mutants, E41A/G and T57A, had a larger Kd than P51G-m4-CVN, matching the trends predicted by the calculations. We also observed that the N42A mutation affects the local fold of the binding pocket, thus removing all binding to dimannose. Characterization of the mutant N53S showed similar binding affinity to P51G-m4-CVN. Using biophysical calculations allows us to study future iterations of models to explore affinities and specificities. In order to further elucidate the role of multivalency, I report here a designed covalent dimer of CVN, Nested cyanovirin-N (Nested CVN), which has four binding sites. Nested CVN was found to have comparable binding affinity to gp120 and antiviral activity to wt CVN. These results demonstrate the ability to create a multivalent, covalent dimer that has comparable results to that of wt CVN. WW domains are small modules consisting of 32-40 amino acids that recognize proline-rich peptides and are found in many signaling pathways. We use WW domain sequences to explore protein folding by simulations using Zipping and Assembly Method. We identified five crucial contacts that enabled us to predict the folding of WW domain sequences based on those contacts. We then designed a folded WW domain peptide from an unfolded WW domain sequence by introducing native contacts at those critical positions. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2014

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18095号 / 理博第3973号 / 新制||理||1573(附属図書館) / 30953 / 京都大学大学院理学研究科化学専攻 / (主査)教授 三木 邦夫, 教授 杉山 弘, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Le cancer colorectal est la première pathologie cancéreuse de la sphère digestive, tant en terme de fréquence que de mortalité par an. Chaque année, 41 000 nouveaux cas sont diagnostiqués et 17 000 décès sont dus à ce cancer en France. Deux paramètres cliniques expliquent la mortalité de ce cancer; d'une part le fait qu'un patient sur deux est diagnostiqué au stade métastatique ou va présenter des lésions métastatiques durant l'histoire de sa pathologie, d'autre part le fait que les patients après traitement vont fréquemment présenter une récidive de leur pathologie. L'utilisation de régimes de chimiothérapies avant et après résection métastatique améliore la survie sans récidive à court terme, mais à 2 ans post chirurgie l'avantage apporté est perdu. Ainsi, la compréhension des mécanismes d'échappement à la chimiothérapie et régissant la croissance tumorale est d'intérêt pour tenter de limiter la récidive tumorale. L'objectif de ce travail de thèse a consisté en l'analyse de sous-populations obtenues sous pression de chimiothérapie au 5-Fluorouracile (5FU) dérivées de la lignée cancéreuse colique HT29, ainsi que les mécanismes moléculaires associés. Notre clone le plus chimiorésistant isolé, le modèle cellulaire 5F31, quitte le compartiment prolifératif sous traitement à fortes doses de 5FU, ceci étant associé à une perturbation de la voie de signalisation de la Src kinase c-Yes et de son partenaire, le co-activateur transcriptionnel YAP. Sous traitement, les cellules chimiorésistantes entrent en quiescence, le complexe protéique entre c-Yes et YAP est perdu et la quantité totale et nucléaire de YAP diminue de manière significative (Igoudjil, Touil, Corvaisier et al. 2014 Clinical Cancer Research). Dès lors, la suite des travaux a consisté en l'étude du rôle potentiel de YAP sur la balance quiescence/prolifération sous 5FU. L'inhibition pharmacologique ou l'inhibition transitoire de l'expression de YAP et de son paralogue, la protéine TAZ, dans plusieurs lignées cancéreuses coliques induit l'augmentation de la fraction de cellules quiescentes, associée au ralentissement significatif de la croissance tumorale. A l'inverse, la surexpression d'une forme constitutivement active de YAP demeurant nucléaire sous 5FU maintient les cellules 5F31 en prolifération et sensibilise les cellules à la chimiothérapie. Au niveau des effecteurs protéiques, l'induction de quiescence (par traitement à la chimiothérapie ou inhibition de YAP/TAZ) est associée à la perte d'expression de la Cycline E1 et du facteur de transcription c-Myc. A l'inverse, la surexpression du dominant constitutivement actif de YAP dans les cellules 5F31 conduit à l'expression soutenue de la Cycline E1 sous 5FU, expression nécessitant l'activation du facteur de transcription CREB. L'inhibition de la Cycline E1 permet d'induire la quiescence cellulaire, proposant cette protéine comme l'un des effecteurs des protéines YAP/TAZ dans la régulation entre la quiescence et la prolifération cellulaire (Corvaisier et al, Oncotarget, 2016). En conclusion, nos données montrent l'importance du rôle des protéines YAP/TAZ dans le maintien des cellules en prolifération via l'expression notamment de la Cycline E1. Nos résultats sur cohorte de patients atteints de métastases hépatiques de cancers colorectaux montrent que l'expression des co-activateurs YAP/TAZ est liée à un index prolifératif plus important, confortant nos données sur le rôle de ces protéines dans la croissance tumorale. De plus, l'expression élevée de YAP et TAZ est associée en analyses multivariées à une récidive plus précoce et à une survie globale plus faible. Ainsi, l'étude de l'expression et du niveau d'activation de ces acteurs serait un marqueur pronostic intéressant dans l'anticipation de la récidive métastatique ; ainsi que des cibles thérapeutiques intéressantes pour tenter de limiter la rechute tumorale. / Colorectal cancer is the most frequent and lethal cancerous pathology from the digestive system. Each year in France, 41 000 new cases are diagnosed and 17 000 patients die due to this pathology. This high mortality is mainly due to the rate of patients with liver metastatic lesions and the early relapse of those metastases after treatment. The use of chemotherapy prior to surgery induces a decrease of early relapse, however 2 years after resection this advantage is lost. Thus, understanding the mechanisms underlying escape to treatment is required to try to delay or prevent tumor recurrence.The aim of this doctoral work was to analyze clonal chemoresistant subpopulations derived from the colorectal cancer cell line HT29 after chronic exposure to 5-Fluorouracil (5FU) and molecular mechanisms associated with chemoresistance. The most chemoresistant clonal subpopulation, 5F31, stops its proliferation after treatment with high dose of 5FU, this behavior being associated with the modulation of the c-Yes/YAP axis. After treatment, 5F31 cells enter quiescence, interaction between c-Yes and YAP is lost and total and nuclear YAP protein expression reduces significantly (Igoudjil, Touil, Corvaisier et al. 2014, Clinical Cancer Research). The next step was to study functions of YAP protein in this chemotherapy- induced quiescence.Pharmacological or transient inhibition of YAP and its homolog TAZ, induces quiescence and reduces cellular growth in several colorectal cancer cell lines. On the other hand, overexpression of a constitutively active form of YAP in 5F31 cells forces cells to remain proliferative under 5FU treatment, enhancing 5F31 cell chemosensitivity to 5FU.Regarding proteic effectors, quiescence (either induced by 5FU or YAP/TAZ inhibition) is associated with loss of expression of the transcription factor c-Myc and Cyclin E1. In 5F31 cells expressing the active mutant form of YAP, Cyclin E1 expression is sustained after 5FU treatment through the activation of the transcription factor CREB. Cyclin E1 inhibition is sufficient to induce quiescence, therefore introducing this protein as one of the final effectors of YAP/TAZ co-activators in the regulation of the proliferation/quiescence switch in colorectal cancer cells (Corvaisier et al. 2016, Oncotarget).To conclude, our work reveals the importance of YAP/TAZ proteins for the maintenance of colorectal cancer cells proliferation through Cyclin E1 expression. Our work on liver metastases from patients with colorectal cancer shows that high expression of YAP/TAZ is connected to a higher proliferative index in metastatic lesions. Moreover, high YAP/TAZ expression is associated with shorter patient progression-free survival and shorter overall survival. Studying the expression and level of YAP/TAZ activation could be an interesting prognosis marker to anticipate metastatic relapse and potent druggable target to delay tumoral recurrence.

Pin1 is a peptidyl prolyl isomerase (PPIase) enzyme with two domains, the catalytic domain and the WW domain. Both domains specifically bind pSer/pThr–Pro motifs. Pin1 plays an important role in regulating the cell cycle, and it is involved in many diseases, such as cancer, HIV-1, Alzheimer's disease, asthma, hepatitis B, and rheumatoid arthritis. Pin1 is a very promising target for new drug development. Three stereoisomers: (R,S)-, (S,R)- and (S,S)-Ac–pSer–Ψ[(Z)CH=C]–Pip–2-(2-naphthyl)ethylamine were synthesized as inhibitors binding to the Pin1 catalytic domain. The (R,S)- and (S,R)-isomers were synthesized via a 13-step route, with overall yields of 2.0% and 1.4%, respectively. The newly formed stereogenic center in the piperidyl ring was introduced by a Luche reduction, followed by a stereoselective [2,3]-Still-Wittig rearrangement. The configuration of the stereocenter was determined by NOESY of a bicyclic derivative. The (Z)- to (E)-alkene ratio in the rearrangement was (5.5:1). The (S,S)-isomer was obtained as the epimerized by-product resulting from the (S,R)-isomer in the Na/NH3 deprotection step. The IC50 values for Pin1 inhibition were: 52, 85, and 141 μM, respectively. We concluded that in this Z-alkene isostere, the R-configuration would be preferred at both stereogenic centers, as mimics of L-Ser and L-Pip, to improve the affinity. Combinatorial chemistry is a powerful method to discover biologically active compounds, and solid-phase synthesis is most commonly used to synthesize combinatorial libraries. To identify ligands for the Pin1 WW domain, a library, R1CO–pSer–Pro–NHR2, was designed. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite function was synthesized in one step from commercially available Wang resin. The SPPR was applied in the preparation of a designed library through parallel synthesis. The library contained 357 members (17 Ã 21), and was screened by an enzyme-linked enzyme binding assay (ELEBA). The best hits were resynthesized, and the competitive dissociation constants, Kd-rel, were measured by ELEBA, with a Kd-rel value of 130 μM for the best ligand. The absolute dissociation constants will be measured by our collaborator, Prof. Jefferey Peng, University of Notre Dame, using NMR methods. Besides the identification of the Pin1 WW domain ligands, I created a practical method for solid-phase synthesis of phosphopeptides. / Ph. D.

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