Current approaches aimed at improving the efficacy of radiation as a cancer treatment modality involve the development and application of molecularly targeted radiosensitizers, a strategy that requires a thorough understanding of the fundamental processes comprising the cellular radioresponse. Recent data indicating that radiation modifies gene expression primarily through translational control rather than transcriptional events suggests that mRNA translation contributes to cell survival after irradiation. The overall goal of this project is to determine whether the regulatory/rate-limiting components of the translational machinery provide targets for tumor cell radiosensitization. The majority of translation in mammalian cells occurs in a cap-dependent manner and is highly dependent on eIF4E. As such, we investigated a regulatory role for eIF4E in cellular radiosensitivity. eIF4E knockdown enhanced the radiosensitivity of tumor but not normal cells. eIF4E knockdown inhibited the dispersal of radiation-induced γH2AX foci. Furthermore, radiation was found to increase the binding of >1000 unique mRNAs to eIF4E, many involved in DNA replication, recombination, and repair. S6 kinase 1 (S6K1), also an important regulatory component of the translational machinery, enhances the translation of specific mRNA subpopulations, independent from eIF4E, and mediates ribosome biogenesis. The role of S6K1 in determing cell survival after radiation was determined in several tumor cell lines and one normal cell line. S6K1 knockdown enhanced the radiosensitivity of all 3 tumor lines. In contrast S6K1 knockdown had no effect on the cellular radiosensitivity of the one normal line tested. The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of gene translation and has been suggested as a potential target for radiosensitization. Importantly, it plays a major role in regulating eIF4E availability as well as S6K1 activity. The radiosensitizing activities of the allosteric mTOR inhibitor rapamycin with that of the ATP competitive mTOR inhibitor PP242 were compared. Based on immunoblot analyses, whereas rapamycin only partially inhibited mTORC1 activity and had no effect on mTORC2, PP242 inhibited the activity of both mTOR containing complexes. In the two tumor cell lines evaluated, PP242 treatment 1h before irradiation increased radiosensitivity, whereas rapamycin had no effect. PP242 had no effect on the cellular radiosensitivity of a normal lung fibroblast line. PP242 exposure did not influence the initial level of γH2AX foci after irradiation, but did significantly delay the dispersal of radiation-induced γH2AX foci. Finally, PP242 administration to mice bearing U251 xenografts enhanced radiation-induced tumor growth delay. A next generation analog of PP242, INK128, is currently undergoing analysis in clinical trials. Given our data showing ATP-competitive mTOR inhibition is a strategy for tumor radiosensitization as well as the fact that radiotherapy is a primary treatment modality for locally advanced pancreatic ductal adenocarcinoma, the effects of INK128 on pancreatic cancer radiosensitivity were determined. In three pancreatic cancer cell lines addition of INK128 immediately after radiation resulted in radiosensitization. Consistent with the effects of PP242 on other cell lines, INK128 exposure did not influence the initial level of γH2AX foci after irradiation, but did significantly delay the dispersal of radiation-induced γH2AX foci. Furthermore, in pancreatic tumor xenografts INK128 inhibits mTOR activity as well as cap-complex formation in a time-dependent manner. Lastly, INK128 treatment significantly prolonged the radiation-induced tumor growth delay of pancreatic tumor xenografts. In summary, the data provided in this thesis have begun to characterize the role of the translational machinery in determining the cellular response to radiation.

Το αδενοκαρκίνωμα του προστάτη αποτελεί σημαντικό πρόβλημα υγείας σε παγκόσμια κλίμακα. Οι διαθέσιμες μέθοδοι κλινικής σταδιοποίησης φαίνεται να μην επαρκούν εφόσον 10% περίπου των ασθενών στους οποίους διαγιγνώσκεται εντοπισμένη νόσος και υποβάλλονται σε ριζική προστατεκτομή εμφανίζουν ήδη μετάσταση στους λεμφαδένες. Το αναπτυσσόμενο ενδιαφέρον για τη μοριακή σταδιοποίηση βασίζεται στα αποτελέσματα μελετών που χρησιμοποιούν δείκτες λεμφαγγείων και λεμφαγγειογενετικούς παράγοντες στην πρόβλεψη της κατάστασης των λεμφαδένων. Ερευνητικές μελέτες στο καρκίνωμα του μαστού, του τραχήλου και του στομάχου υποδεικνύουν τη στενή σχέση ανάμεσα στη λεμφαγγειακή πυκνότητα (LVD) και τις λεμφαδενικές μεταστάσεις. Ο διατηρημένος στόχος της ραπαμυκίνης στα θηλαστικά (mTOR) αποτελεί κινάση σερίνης/θρεονίνης του σηματοδοτικού μονοπατιού (PI3K)/AKT. Μεγάλος αριθμός μελετών υποδεικνύει το πιθανό ρόλο του m-TOR στην αγγειογένεση μέσω της επαγωγής του VEGF-A. Αποτελέσματα μελετών συνδέουν τη κινάση m-TOR με την αγγειογένεση μέσω της επαγωγής του αυξητικού παράγοντα VEGF-A. Η συμμετοχή του mTOR στη λεμφαγγειογένεση δεν είναι τόσο σαφής εφόσον φαίνεται να κατέχει σημαντικό ρόλο στην επαγόμενη λεμφαγγειογένεση από τον VEGF-C αλλά όχι από τον VEGF-A. Επιπλέον παραμένει ασαφές εάν η έκφραση του m-TOR συνδέεται με την παρουσία λεμφαδενικών μεταστάσεων. Παρόλο που ο COUP-TFII είναι απαραίτητος για τη λεμφαγγειογένεση, ελάχιστα είναι γνωστά για τη σχέση ανάμεσα στον μεταγραφικό παράγοντα το mTOR και τη δημιουργία λεμφαδενικών μεταστάσεων στο αδενοκαρκίνωμα του προστάτη. Σκοπός της παρούσας μελέτης ήταν ο προσδιορισμός της έκφρασης της ενεργοποιημένης κινάσης (p-mTOR) και η σύγκριση της με παραμέτρους που σχετίζονται με τα λεμφαγγεία (LVD, παρουσία καρκινικών εμβόλων κτλ) σε ασθενείς με αρνητικούς και θετικούς λεμφαδένες. Τα λεμφαγγεία καθορίσθηκαν από τους ειδικούς δείκτες D2-40 και LYVE-1. Επιπλέον εξετάσαμε την έκφραση του μεταγραφικού παράγοντα COUP-TFII και των αυξητικών παραγόντων VEGF-A και VEGF-C σε σχέση με την έκφραση του p-mTOR τη μέση λεμφαγγειακή πυκνότητα την παρουσία εμβόλων και την κατάσταση των λεμφαδένων. Για να επιτευχθεί ο στόχος της μελέτης χρησιμοποιήθηκαν ιστοί εγκλεισμένοι σε παραφίνη από 92 ασθενείς οι οποίοι υποβλήθηκαν σε ριζική προστατεκτομή. Εφαρμόσθηκε ανοσοϊστοχημική μέθοδος, χρησιμοποιώντας αντισώματα έναντι των p-mTOR, D2-40, CD-31, LYVE-1, VEGF-C, VEGF-A και COUP-TFII. Οι σχέσεις ανάμεσα στα αποτελέσματα εξετάσθηκαν με πρωτόκολλα στατιστικής ανάλυσης. Στην παρούσα μελέτη, η περιφερική του όγκου LVD η οποία καθορίσθηκε από το D2-40 ήταν αυξημένη στα περιστατικά με pN1. Η ένταση και η κατανομή της ανοσοϊστοχημικής χρώσης για το p-mTOR ήταν αυξημένη στα καρκινώματα με λεμφαδενικές μεταστάσεις σε σχέση με αυτά που παρουσίαζαν αρνητικούς λεμφαδένες. Επιπλέον, τα περιστατικά με εντονότερη κυτταροπλασματική έκφραση του p-mTOR παρουσίασαν αυξημένη LVD και μεγαλύτερο αριθμό διηθημένων λεμφαγγείων. Αντίστοιχα οι περιπτώσεις με pN1 παρουσίασαν εντονότερο ανοσοϊστοχημικό εντοπισμό των VEGF-C, VEGF-A και COUP-TFII που επίσης σχετίσθηκε με την παρουσία περισσότερων λεμφαγγειακών εμβόλων και με υψηλότερα επίπεδα έκφρασης του p-mTOR. Τέλος ο αριθμός των COUP-TFII θετικών αγγείων ήταν μεγαλύτερος στα περιστατικά με λεμφαδενικές μεταστάσεις και διηθημένα λεμφαγγεία. Τα ευρήματα μας υποδεικνύουν ότι τόσο το D2-40 όσο και το LYVE-1 αποτελούν ειδικούς δείκτες για τον εντοπισμό των λεμφαγγείων. Παρόλα αυτά η έκφραση του LYVE-1 περιοριζόταν σε έναν υποπληθυσμό λεμφαγγείων ελαττώνοντας την ευαισθησία του μορίου ως δείκτη. Η ενεργοποίηση του mTOR σηματοδοτικού μονοπατιού φαίνεται να διευκολύνει τη δημιουργία λεμφαδενικών μεταστάσεων πιθανώς επάγοντας την έκφραση των VEGF-C και VEGF-A. Η επαγωγή των αυξητικών παραγόντων έχει ως επακόλουθο την δημιουργία νέων λεμφαγγείων και πιθανόν την αύξηση της διαπερατότητας τους. Επιπλέον τα αποτελέσματα μας υποδηλώνουν ότι ο COUP-TFII ευοδώνει όχι μόνο τη νέο-λεμφαγγειογένεση αλλά και τη λεμφαγγειακή διήθηση. Τέλος μπορεί να υποτεθεί ότι τα νεοδημιουργηθέντα λεμφαγγεία είναι σημαντικότερα από τα ώριμα για τη εξάπλωση του προστατικού αδενοκαρκινώματος στους επιχώριους λεμφαδένες. / Adenocarcinoma of prostate is a major health problem worldwide. The available methods for clinical staging seem to be inefficient, since approximately 10% of patients who will undergo radical prostatectomy for clinically localized disease, harbor metastasis in the regional lymph nodes. Recent studies that utilize lymphangiogenic factors and lymphangiogenesis markers to predict the nodal status trigger the interest in molecular staging. Research studies in breast, cervical and gastric cancers suggest a high correlation between the lymphatic microvessel density (LVD) and nodal metastasis. The conserved mammalian target of rapamycin (mTOR) is a serine/threonine kinase of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. Many studies suggest a role of m-TOR in angiogenesis via the upregulation of VEGF-A. In lymphangiogenesis the participation of mTOR is more complicated as it seems to be essential for the VEGF-C induced lymphangiogenesis but not for lymphangiogenesis that is induced from VEGF-A. Furthermore it is unclear if the expression of m-TOR correlates with the presence of lymph node metastasis or not. Although studies indicate that the transcription factor COUP-TFII is indispensable for lymphangiogenesis, little is known about the relationship between the transcription factor, mTOR and lymph node metastasis in prostate adenocarcinoma. The aim of this study was to determine the expression status of the activated kinase p-mTOR and compare it with lymphatic parameters (LVD, lymphatic invasion etc.) in patients that were found to have positive lymph nodes and patients with negative lymph node status. The lymphatics were determined with the lymphatic-specific markers D2-40 and LYVE-1. Moreover, we examined the expression of the transcription factor COUP-TFII, the vascular endothelial growth factors A and C (VEGF-A, VEGF-C) in relation to each other, p-mTOR expression, mean LVD, lymphatic invasion and lymph node status. In order to accomplish our objective paraffin-embedded tissues obtained from 92 patients who underwent radical prostatectomy were immunostained with p-mTOR, D2-40, CD-31, LYVE-1, VEGF-C, VEGF-A and COUP-TFII antibodies. The associations between the results were analyzed with statistical analysis protocols. In the current study, peritumoral (but not intratumoral) mean LVD assessed by D2-40 was higher in pN1 tumors. Intensity of p-mTOR staining was higher in carcinomas with lymph node metastasis than in those with negative lymph nodes. Furthermore, higher p-mTOR cytoplasmic expression was strongly associated with LVD and lymphatic invasion. Intense VEGF-C, VEGF-A and COUP-TFII immunostaining was observed in N1 cases and correlated with the presence of lymphatic emboli and higher p-mTOR expression. Finally the number of COUP-TFII positive vessels was positively associated with lymph node metastasis and lymphatic invasion. Our findings suggest that both D2-40 and LYVE-1 are useful markers that may reveal lymphatic vasculature, the latter however showing lower expression, probably limited to a specific lymphatic vessel cellular sub-population. Activation of mTOR pathway may facilitate nodal metastasis, possibly due to increased lymphangiogenesis and lymphatic vessel invasion via increased VEGF-C and VEGF-A expression. Additionally our results indicate that COUP-TFII may facilitate not only neo-lymphangiogenesis but also promotes lymphatic invasion. Finally it seems that newly formed lymphatic vessels are more important than mature ones for lymphatic spread in prostate adenocarcinoma.

Equine pituitary pars intermedia dysfunction (PPID) typically occurs in horses older than 15 years of age and is characterized by hair coat abnormalities, muscle atrophy and decreased insulin sensitivity. The first objective of this research was to compare the rate of whole body protein metabolism and relative abundance of key factors in the signaling pathways associated with muscle protein synthesis and protein breakdown in response to feeding in Control and PPID horses. No differences (P > 0.05) were seen between the PPID and Control groups in whole-body protein metabolism or post-prandial activation of the muscle signaling pathways regulating skeletal muscle protein synthesis and breakdown. The second objective of this research was to determine if aged horses with PPID had reduced insulin sensitivity and alterations in the insulin-mediated signaling pathways in the skeletal muscle when compared to non-PPID, aged Control horses. Measures of insulin sensitivity and the activation of factors associated with protein synthesis and breakdown were similar between the PPID and Control groups (P > 0.05). Overall, insulin sensitivity and protein metabolism are similar between the PPID and Control groups. The studies suggest that abnormalities may exist as a function of advanced age rather than PPID status directly.

Le récepteur de l'inositol 1,4,5-trisphosphate (IP?R), un canal calcique, est un des principaux régulateurs de la mobilisation de Ca[indice supérieur 2+] intracellulaire. En regardant globalement les mécanismes de régulation des IP?Rs, on peut se rendre compte que cette régulation se fait bien souvent dans le contexte précis d'une ou de plusieurs voies de signalisation. Bien que de nombreux modulateurs de l'activité calcique des IP?Rs soient déjà connus, encore aujourd'hui beaucoup de questions sont en suspens. En fait, un nombre croissant de preuves et d'observations suggèrent que la régulation spécifique des mécanismes de signalisation calcique peut être accomplie par l'activation simultanée de plusieurs voies de signalisation ce qui permet de soutenir la diversité d’action du Ca[indice supérieur 2+]. Une de ces voies, la voie de croissance et de prolifération cellulaire PI3K-AKT-mTOR fut d’abord investiguée dans la première partie de cette thèse afin de comprendre l’importance de l’activité calcique des IP?R dans ces phénomènes d’importance majeure. Nous avons montré que mTOR interagit et phosphoryle l'IP?R-3 dans les cellules RINm5F puis déterminé que le niveau d’activité de mTOR affectait les relâches calciques médiées par l'IP?R-3 de ces cellules. Sachant que mTOR contrôle la prolifération et l'homéostasie cellulaire et que le Ca[indice supérieur 2+] joue aussi un rôle clé dans ces deux phénomènes, il est logique de croire que mTOR facilite les relâches calciques dépendantes des IP?Rs afin de fournir le Ca[indice supérieur 2+] nécessaire aux actions physiologiques de mTOR. La deuxième section de cette thèse a exploré l’interaction entre la voie dopaminergique et la voie calcique dépendante des IP?Rs. Nos résultats suggèrent que calcyon, une protéine liant le Ca [indice supérieur 2+] surexprimée dans plusieurs neuropathologies, est impliquée dans l'internalisation des récepteurs D2 par un mécanisme encore inconnu qui cause par le fait même une relâche calcique tardive dans les cellules PC-12. Nous avons montré que cette protéine affecte aussi l’activité calcique des IP?Rs. Nos résultats suggèrent que calcyon est la protéine reliant les voies de signalisation calcique et dopaminergique et que son implication est d’importance majeure afin d’assurer l’homéostasie calcique neuronale. L’ensemble de ces travaux montre toute l’étendue de la régulation calcique et de l’interaction entre les voies de signalisation.

Protein synthesis is frequently dysregulated in cancer cells; such conditions are known to favor aberrant cell growth and proliferation which lead to cancer. LARP1 is a novel target of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, a circuitry often hyperactivated in cancer which regulates cell growth and proliferation primarily through the regulation of protein synthesis. I aimed to determine if LARP1 plays a role in cancer progression by comparing its expression in normal versus cancer tissues. My results demonstrate that LARP1 expression is altered (lost or overexpressed) in various cancers and correlates with cancer patients survival. My systematic bioinformatics assessment, the results of my functional assays assessing the effect of LARP1 knockdown on cancer cells, together with my antibody validation do not only provide new insights for its role in cancer progression and mRNA translation, but also emphasizes the potential of LARP1 as a cancer therapeutic target.

Metformin är ett biguanidderivat som främst används vid diabetes mellitus typ 2. Det verkar genom ett flertal mekanismer och ökar bland annat insulinkänsligheten. Forskning har visat att det finns ett samband mellan metformin och överlevnad i cancer. Studier pågår för att undersöka metformins antineoplastiska effekt. Övervikt och diabetes ökar risken för endometriecancer och epidemiologiska studier har visat att metformin ger bättre total överlevnad vid endometriecancer. Genom artikelsökning via PubMed erhölls fem studier där metformins effekt på endometriecancervävnad undersöktes. Resultaten i dem varierar men majoriteten pekar på en antiproliferativ effekt av metformin, med avseende på Ki-67. Med vilken verkningsmekanism den effekten erhålls är tvetydligt, då vissa resultat tyder på inhiberad mTOR-signalering medan andra inte visar effekt på de variablerna. En minskning i cirkulerande tillväxtfaktorer insulin och IGF-1 ses, vilket kan ha en inhiberande effekt. Större studier krävs för att kunna avgöra vilken effekt metformin har på endometriecancer.

Lithium is given to millions of bipolar disorder or post-traumatic disorder patients. The recent studies also support a role for lithium in treating neurodegenerative disease such as Parkinson's disease and stroke. Lithium treatment leads to lithium nephropathy, which includes lithium-induced nephrogenic diabetic insipidus (NDI), lithium-induced renal cell proliferation leading to the formation of microcysts in the kidney, and lithium-induced renal fibrosis. However, there is still a gap in understanding the mechanisms and signaling pathways involved in regulating lithium-induced nephropathy. mTOR pathway activation and primary cilia are known to be associated with the abnormal renal cell proliferation and the formation of renal cysts in polycystic kidney disease, a renal disease model similar to our lithium model. The activation of hedgehog pathway is associated with the renal fibrosis observed in the unilateral ureteral obstruction and unilateral ischemia reperfusion injury models of chronic renal injury. Thus, I hypothesize that mTOR signaling pathway, primary cilia and hedgehog pathway may all contribute to lithium-induced nephropathy. To address the hypothesis that the mTOR signaling pathway may be responsible for lithium-induced renal collecting duct proliferation, mTOR pathway activation was assessed in lithium-treated mice and lithium-treated mouse inner medullary collecting duct (mIMCD3) cells. Lithium activated mTOR signaling pathway in renal collecting duct cells both in vivo and in vitro. Rapamycin, an inhibitor of mTOR, blocked lithium-induced renal cell proliferation in renal cortex and medulla in vivo and in renal collecting duct cells in vitro, supporting the hypothesis. However, rapamycin did not improve lithium-induced reduction of urine osmolality, suggesting mTOR signaling pathway may not contribute to lithium-induced NDI. To address the hypothesis that primary cilia may be necessary for lithium-induced mTOR activation and renal cell proliferation, primary cilia deficient cells were used to assess mTOR pathway activation and cell proliferation in response to lithium treatment. The absence of primary cilia abolished lithium-induced activation of mTOR pathway and cell proliferation, which supports the hypothesis. To address the hypothesis that lithium elongates primary cilia length, which is mediated by mTOR signaling pathway, primary cilia length alternation was assessed in the kidney and in mIMCD3 cells in response to lithium treatment. Lithium increased primary cilia length in renal collecting duct cells of cortex, outer medulla, and inner medulla kidney regions in vivo and in mIMCD3 cells in vitro. Rapamycin reversed lithium-induced elongation of primary cilia in renal cortical and outer medullary collecting duct cells in vivo, and blocked the increase of primary cilia length in mIMCD3 cells in vitro, which support the hypothesis. To address the hypothesis that lithium activates the hedgehog pathway in a Smoothened (smo, a key regulator of the hedgehog pathway)-dependent manner in renal collecting duct cells, mIMCD3 cells were treated with lithium or lithium/Smo inhibitor or lithium/Smo activator. Hedgehog signaling pathway is activated by lithium in mIMCD3 cells, which is partially Smo-dependent. However, the role of hedgehog signaling pathway in regulating lithium-induced fibrosis was not assessed in the study. Future studies are required to determine the role of the hedgehog pathway in the lithium model.

TRIM7 is a member of the TRIM (tripartite motif-containing) protein superfamily. This family has been implicated in many disorders such as genetic diseases, neurological diseases, and cancers. Little is known about the function of TRIM7 except that it interacts with glycogenin and may regulate glycogen biosynthesis. Recently, a yeast two-hybrid protein-protein interaction screen revealed the binding of TRIM7 to Sin1, a protein found in a complex with the mammalian target of rapamycin (mTOR) protein kinase. mTOR can form two complexes, mTORC1 and mTORC2, which are important for cell growth, differentiation, and survival. Sin1 is a core component of mTORC2 and is critical for mTORC2 stability and activity. It was confirmed by co-immunoprecipitation that TRIM7 associates with Sin1 and mTOR in cultured mammalian cells. Furthermore, it was demonstrated that TRIM7 is a phosphoprotein, although it was not directly targeted by mTOR in vitro. Similar to some other TRIM family proteins, it was demonstrated that TRIM7 has a ubiquitin E3 ligase function allowing it to autoubiquitinate both in vitro and in cells. The autoubiquitination of TRIM7 was dependent on its RING domain. Further characterization of TRIM7 indicated that it can both homo-oligomerise as well as hetero-oligomerise with other members of its sub-class of TRIM proteins and that it co-localises with them into discrete cytoplasmic loci. To determine the cellular function of TRIM7, a stable cell line expressing an shRNA directed against TRIM7 was generated. Successful knock down of TRIM7 was achieved and this led to an increase in the protein levels of components of the mTORC2 complex, including Sin1. This coincided with an increase in cell proliferation. In conclusion, this research identifies a novel role for TRIM7 as a ubiquitin ligase involved in regulating cell proliferation and provides a potential link between TRIM7 and the mTOR pathway, a major transducer of proliferative and cell survival signals.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13400号 / 論医博第2224号 / 新制||医||1051(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 藤田 恭之, 教授 松田 文彦, 教授 柳田 素子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13418号 / 論医博第2226号 / 新制||医||1052(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 羽賀 博典, 教授 長船 健二, 教授 伊藤 貴浩 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM