Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 92-100).

Man, Cheuk Him. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 219-263). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xvi / List of Publications --- p.xvii / Chapter Ch.l --- Introduction --- p.1 / Chapter 1.1 --- Aim of study --- p.1 / Chapter 1.2 --- Literature review --- p.3 / Chapter 1.2.1 --- Nasopharyngeal carcinoma (NPC) --- p.3 / Chapter 1.2.1.1 --- Structure and function of nasopharynx --- p.3 / Chapter 1.2.1.2 --- Histopathology of NPC --- p.3 / Chapter 1.2.1.3 --- Epidemiology of NPC --- p.4 / Chapter 1.2.2 --- Etiology of NPC --- p.6 / Chapter 1.2.2.1 --- Genetic factors --- p.6 / Chapter 1.2.2.2 --- Environment factors --- p.13 / Chapter 1.2.2.3 --- Epstein-Barr virus (EBV) infection --- p.14 / Chapter 1.2.3 --- Therapeutic treatment of NPC --- p.24 / Chapter 1.2.3.1 --- Radiotherapy (RT) --- p.24 / Chapter 1.2.3.2 --- Chemotherapy --- p.25 / Chapter 1.2.4 --- Notch signaling pathway --- p.26 / Chapter 1.2.4.1 --- Notch receptors and their ligands --- p.26 / Chapter 1.2.4.2 --- Activation of Notch signaling pathway --- p.29 / Chapter 1.2.4.3 --- Regulators of Notch signaling pathway --- p.32 / Chapter 1.2.4.4 --- Effectors of Notch signaling pathway --- p.32 / Chapter 1.2.5 --- Role of Notch signaling pathway in tumorigenesis --- p.33 / Chapter 1.2.5.1 --- Cell proliferation --- p.34 / Chapter 1.2.5.2 --- Cell survival --- p.35 / Chapter 1.2.5.3 --- Angiogenesis --- p.36 / Chapter 1.2.5.4 --- Cell invasion and metastasis --- p.36 / Chapter 1.2.6 --- Notch and oncogenic virus --- p.37 / Chapter 1.2.7 --- Crosstalk between Notch and other signaling pathways --- p.38 / Chapter 1.2.7.1 --- NFkB signaling pathway --- p.38 / Chapter 1.2.7.2 --- Ras signaling pathway --- p.39 / Chapter 1.2.7.3 --- Wnt signaling pathway --- p.40 / Chapter 1.2.7.4 --- Akt signaling pathway --- p.40 / Chapter 1.2.7.5 --- ErbB2 signaling pathway --- p.41 / Chapter 1.2.8 --- Notch as therapeutic target for cancer --- p.41 / Chapter Ch.2 --- Materials and Methods --- p.45 / Chapter 2.1 --- "Cell lines, xenografts and primary tumors" --- p.45 / Chapter 2.1.1 --- Cell lines --- p.45 / Chapter 2.1.2 --- Xenografts --- p.46 / Chapter 2.1.3 --- Primary tumors --- p.48 / Chapter 2.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.2.1 --- Sample preparation for RT-PCR --- p.50 / Chapter 2.2.1.1 --- RNA extraction --- p.50 / Chapter 2.2.1.2 --- Quantitation of total RNA --- p.50 / Chapter 2.2.2 --- Conventional RT-PCR --- p.51 / Chapter 2.2.3 --- Quantitative RT-PCR --- p.51 / Chapter 2.3 --- Western immunoblot --- p.55 / Chapter 2.3.1 --- Protein extraction --- p.55 / Chapter 2.3.2 --- SDS-PAGE and immunoblotting --- p.55 / Chapter 2.4 --- Immunohistochemistry --- p.59 / Chapter 2.5 --- Cloning and plasmid DNA preparation --- p.62 / Chapter 2.5.1 --- Polymerase chain reaction (PCR) and purification of PCR products --- p.62 / Chapter 2.5.2 --- Restriction enzyme double digestion --- p.65 / Chapter 2.5.3 --- Ligation of plasmid and insert sequence --- p.65 / Chapter 2.5.4 --- Bacterial transformation --- p.66 / Chapter 2.5.5 --- Plasmid DNA extraction --- p.66 / Chapter 2.5.6 --- DNA sequencing --- p.67 / Chapter 2.6 --- Transient transfection of NPC cell lines --- p.67 / Chapter 2.7 --- Drug treatment on NPC cell lines --- p.69 / Chapter 2.8 --- Cell proliferation assays --- p.71 / Chapter 2.8.1 --- WST-1 assay --- p.71 / Chapter 2.8.2 --- BrdU assay --- p.71 / Chapter 2.9 --- Flow cytometry analysis --- p.72 / Chapter 2.9.1 --- Sample preparation --- p.72 / Chapter 2.9.2 --- Cell cycle analysis by propidium iodide staining --- p.73 / Chapter 2.9.3 --- Apoptosis analysis by AnnexinV-PI staining --- p.73 / Chapter 2.10 --- Apoptosis analysis by Caspase-3 activity assay --- p.74 / Chapter 2.11 --- RBP-Jk reporter assay --- p.75 / Chapter 2.12 --- NFKB1 reporter assay --- p.77 / Chapter 2.13 --- Dual luciferase reporter assay --- p.77 / Chapter 2.14 --- Expression array --- p.78 / Chapter 2.15 --- Statistical analysis --- p.79 / Chapter Ch.3 --- Characterization of Notch Signaling Molecules in NPC --- p.80 / Chapter 3.1 --- Introduction --- p.80 / Chapter 3.2 --- Results --- p.81 / Chapter 3.2.1 --- "Expression of Notch ligands, receptors, effectors and regulators in NPC cell lines and xenografts" --- p.81 / Chapter 3.2.2 --- "Expression of Notch ligands, receptors, regulators and effectors in NPC primary tumors" --- p.104 / Chapter 3.3 --- Discussion --- p.111 / Chapter 3.3.1 --- Overexpression of Jagl and D114 in NPC --- p.112 / Chapter 3.3.2 --- Overexpression of Notch receptors in NPC --- p.114 / Chapter 3.3.3 --- "Downregulation of Negative regulator, Numb, in NPC" --- p.116 / Chapter 3.3.4 --- Overexpression of Notch effectors in NPC --- p.117 / Chapter 3.4 --- Summary --- p.119 / Chapter Ch.4 --- Mechanisms of Activation of Notch Signaling Pathway in NPC --- p.120 / Chapter 4.1 --- Introduction --- p.120 / Chapter 4.2 --- Results --- p.122 / Chapter 4.2.1 --- EBV mediated Notch activation --- p.122 / Chapter 4.2.1.1 --- No effect of EBERs and EBNA1 on the expression of Notch Components --- p.122 / Chapter 4.2.1.2 --- LMP1 induces expression of Notch components --- p.129 / Chapter 4.2.1.3 --- LMP2A induces expression of Notch components --- p.133 / Chapter 4.2.2 --- Effect of CXCR4 on Notch signaling pathway in C666-1 --- p.137 / Chapter 4.3 --- Discussion --- p.139 / Chapter 4.3.1 --- EBV-mediated induction of Notch components --- p.139 / Chapter 4.3.2 --- Regulation of Notch expression by CXCR4 signaling pathway --- p.142 / Chapter 4.4 --- Summary --- p.145 / Chapter Ch.5 --- Investigation of the Oncogenic Role of Notch3 --- p.146 / Chapter 5.1 --- Introduction --- p.146 / Chapter 5.2 --- Results --- p.148 / Chapter 5.2.1 --- Effect of knockdown Notch 1 by siRNA on the growth of C666-1 --- p.148 / Chapter 5.2.2 --- Effect of knockdown Notch3 by siRNA on the growth of C666-1 --- p.151 / Chapter 5.2.2.1 --- Effect of knockdown Notch3 by siRNA on the RBP-Jk promoter activity of C666-1 --- p.153 / Chapter 5.2.2.2 --- Effect of knockdown Notch3 by siRNA on the proliferation of C666-1 --- p.155 / Chapter 5.2.2.3 --- Effect of knockdown Notch3 by siRNA on cell cycle progression of C666-1 --- p.158 / Chapter 5.2.2.4 --- Effect of knockdown Notch3 by siRNA on resistant to apoptosis in C666-1 --- p.160 / Chapter 5.2.3 --- Investigation of the anti-proliferation effect of therapeutic agents targeting Notch signaling pathway in NPC cells --- p.168 / Chapter 5.2.3.1 --- "Effect of DAPT on the proliferation of HEK293T, C666-1 and HK-1" --- p.168 / Chapter 5.2.3.2 --- Effect of AMD3100 on Notch signaling pathway and proliferation of NPC cells --- p.172 / Chapter 5.2.4 --- Study of downstream targets of Notch3 in NPC cells --- p.178 / Chapter 5.3 --- Discussion --- p.200 / Chapter 5.3.1 --- Oncogenic role of Notch3 in C666-1 --- p.200 / Chapter 5.3.2 --- Potential therapeutic approach in treating NPC via Notch inhibition --- p.206 / Chapter 5.3.2.1 --- "Gamma secretase inhibitor, DAPT" --- p.206 / Chapter 5.3.2.2 --- "CXCR4 antagonist, AMD3100" --- p.207 / Chapter 5.4 --- Summary --- p.209 / Chapter Ch.6 --- General Discussion --- p.210 / Chapter Ch.7 --- Conclusion --- p.217 / Reference --- p.219 / Appendices --- p.263 / Appendix 1 Summary of immunohistochemical staining results on 23 primary NPC samples --- p.264 / Appendix 2 Summary of 581 selected genes from the expression array --- p.265

A galectina-3, um membro da família das proteínas de ligação a glicanas, tem sido objeto de intensa pesquisa nos últimos anos devido ao seu importante papel na biologia tumoral, como a proliferação, transformação, apoptose, angiogênese, adesão, invasão e metástase tumoral. As diferentes funções de galectina-3 nas células tumorais resultam das suas diversas localizações inter- e subcelulares que lhe permite interagir com diferentes proteínas. Esta tese teve como objetivo identificar um papel específico de galectina-3 na regulação da via de sinalização Notch, que cada vez mais tem sido associada com a progressão tumoral e angiogênese. Inicialmente, demonstramos que galectina-3 interage com o receptor Notch-1 e modula diferencialmente a ativação da via pelos ligantes DLL4 e Jagged1. A galectin-3 regulou a expressão dos ligantes de Notch assim como o receptor Notch-1 e extracelularmente recuperou a ativação de Notch na ausência de galectina-3 endógena. Em câncer gástrico humano, a galectina-3 encontrou-se positivamente correlacionada com a expressão de Jagged1, enquanto que a galectina-1, um outro membro da família das galectinas, foi positivamente correlacionado com DLL4. De seguida estudou-se o papel biológico da regulação da via Notch pela galectina-3 na angiogênese. Demonstramos que nas células endoteliais, galectina-3 liga e aumenta a meia vida de Jagged1 promovendo a ativação preferencial da Jagged1/Notch em vez de DLL4/Notch de uma forma independente de VEGF. Verificamos que condições de hipóxia alteraram a expressão de galectina-3 assim como o status de glicosilação das células endoteliais de forma a promover a ativação de Jagged1/Notch e o aumento de angiogênese. A superexpressão de Jagged1 num modelo de carcinoma de pulmão de Lewis, acelerou o crescimento tumoral in vivo que foi inibido em camundongos Lgals3-/-. Por fim, avaliou-se o papel de galectina-3 na resistência das células tumorais a quimioterápicos. Observamos que a expressão de sialil-Tn, um produto biossintético da ST6GalNAc-I, diminuiu in vitro como in vivo a presença e os sítios de ligação de galectina-3 na superfície da células levando à sua acumulação no meio intracelular. Extracelularmente, galectina-3 não levou à indução de morte celular, no entanto contribuiu para a morte induzida por quimioterápicos. As células expressando sialil-Tn encontraram-se protegidas. Em amostras de tumor gástrico, os sítios de ligação de galectina-3 encontraram-se negativamente correlacionados com a expressão de sialil-Tn. Este conhecimento possui implicações diretas no desenvolvimento de estratégias visando o controle do crescimento tumoral e angiogênese e abre novas perspectivas no combate à resistência tumoral à terapia / Galectin-3, a member of a family of glycan binding proteins has been the subject of an intense research over the past few years due to its important role in cancer biology, such as cancer cell growth, transformation, apoptosis, angiogenesis, adhesion, invasion and metastasis. The different roles of galectin-3 on cancer cells behavior appears to have originated from its diverse inter- and subcellular localizations where it interacts with several different binding partners. The aim of this thesis was to pinpoint a specific role for galectin-3 in regulating Notch signaling pathway in cancer. Notch signaling has emerged as an important pathway in carcinogenesis, and activated Notch-1 signaling has being associated with cancer progression and angiogenesis. Initially, we found that galectin-3 was able to interact with Notch-1 receptor and to differentially modulate Notch signaling activation by DLL4 and Jagged1 ligands. Galectin-3 was found to regulate the expression of the Notch ligands and Notch-1 receptor and its extracellular form was able to rescue Notch activation in the absence of endogenous galectin-3. In human gastric cancer, galectin-3 was positively correlated with the expression of Jagged1 whereas galectin1, another member of the galectin family, was positively correlated with DLL4. Furthermore, we studied the biological role of Notch regulation by galectin-3 in angiogenesis. We showed that, in endothelial cells, galectin-3 binds to and increases Jagged1 protein half-life promoting Jagged1/Notch over DLL4/Notch signaling in a VEGF independent way. Hypoxic conditions changed galectin-3 expression and the glycosylation status of endothelial cells, acting in concert to promote Jagged1/Notch activation and sprouting angiogenesis. Jagged1 overexpression in Lewis lung carcinoma accelerated tumor growth in vivo that was prevented in Lgals3-/- mice. Finally, we evaluated the role of galectin-3 in cancer cell resistance to therapy. We found that the expression of sialyl-Tn, a biosynthetic product of ST6GalNAc-I, was able to decrease cell surface galectin-3 and galectin-3-binding sites both in vitro and in vivo leading to an intracellular accumulation of this protein. Exogenously added galectin-3 was found to have no effect on cancer cell death but contributed to chemotherapy-induced apoptosis. Sialyl-Tn expressing cells were protected. In human gastric cancer samples, galectin-3 binding sites were negatively correlated with the expression of sialyl-Tn. This knowledge has direct implications for the development of strategies aimed at controlling tumor growth and angiogenesis and open novel perspectives to overcome tumor resistance to therapy

A galectina-3, um membro da família das proteínas de ligação a glicanas, tem sido objeto de intensa pesquisa nos últimos anos devido ao seu importante papel na biologia tumoral, como a proliferação, transformação, apoptose, angiogênese, adesão, invasão e metástase tumoral. As diferentes funções de galectina-3 nas células tumorais resultam das suas diversas localizações inter- e subcelulares que lhe permite interagir com diferentes proteínas. Esta tese teve como objetivo identificar um papel específico de galectina-3 na regulação da via de sinalização Notch, que cada vez mais tem sido associada com a progressão tumoral e angiogênese. Inicialmente, demonstramos que galectina-3 interage com o receptor Notch-1 e modula diferencialmente a ativação da via pelos ligantes DLL4 e Jagged1. A galectin-3 regulou a expressão dos ligantes de Notch assim como o receptor Notch-1 e extracelularmente recuperou a ativação de Notch na ausência de galectina-3 endógena. Em câncer gástrico humano, a galectina-3 encontrou-se positivamente correlacionada com a expressão de Jagged1, enquanto que a galectina-1, um outro membro da família das galectinas, foi positivamente correlacionado com DLL4. De seguida estudou-se o papel biológico da regulação da via Notch pela galectina-3 na angiogênese. Demonstramos que nas células endoteliais, galectina-3 liga e aumenta a meia vida de Jagged1 promovendo a ativação preferencial da Jagged1/Notch em vez de DLL4/Notch de uma forma independente de VEGF. Verificamos que condições de hipóxia alteraram a expressão de galectina-3 assim como o status de glicosilação das células endoteliais de forma a promover a ativação de Jagged1/Notch e o aumento de angiogênese. A superexpressão de Jagged1 num modelo de carcinoma de pulmão de Lewis, acelerou o crescimento tumoral in vivo que foi inibido em camundongos Lgals3-/-. Por fim, avaliou-se o papel de galectina-3 na resistência das células tumorais a quimioterápicos. Observamos que a expressão de sialil-Tn, um produto biossintético da ST6GalNAc-I, diminuiu in vitro como in vivo a presença e os sítios de ligação de galectina-3 na superfície da células levando à sua acumulação no meio intracelular. Extracelularmente, galectina-3 não levou à indução de morte celular, no entanto contribuiu para a morte induzida por quimioterápicos. As células expressando sialil-Tn encontraram-se protegidas. Em amostras de tumor gástrico, os sítios de ligação de galectina-3 encontraram-se negativamente correlacionados com a expressão de sialil-Tn. Este conhecimento possui implicações diretas no desenvolvimento de estratégias visando o controle do crescimento tumoral e angiogênese e abre novas perspectivas no combate à resistência tumoral à terapia / Galectin-3, a member of a family of glycan binding proteins has been the subject of an intense research over the past few years due to its important role in cancer biology, such as cancer cell growth, transformation, apoptosis, angiogenesis, adhesion, invasion and metastasis. The different roles of galectin-3 on cancer cells behavior appears to have originated from its diverse inter- and subcellular localizations where it interacts with several different binding partners. The aim of this thesis was to pinpoint a specific role for galectin-3 in regulating Notch signaling pathway in cancer. Notch signaling has emerged as an important pathway in carcinogenesis, and activated Notch-1 signaling has being associated with cancer progression and angiogenesis. Initially, we found that galectin-3 was able to interact with Notch-1 receptor and to differentially modulate Notch signaling activation by DLL4 and Jagged1 ligands. Galectin-3 was found to regulate the expression of the Notch ligands and Notch-1 receptor and its extracellular form was able to rescue Notch activation in the absence of endogenous galectin-3. In human gastric cancer, galectin-3 was positively correlated with the expression of Jagged1 whereas galectin1, another member of the galectin family, was positively correlated with DLL4. Furthermore, we studied the biological role of Notch regulation by galectin-3 in angiogenesis. We showed that, in endothelial cells, galectin-3 binds to and increases Jagged1 protein half-life promoting Jagged1/Notch over DLL4/Notch signaling in a VEGF independent way. Hypoxic conditions changed galectin-3 expression and the glycosylation status of endothelial cells, acting in concert to promote Jagged1/Notch activation and sprouting angiogenesis. Jagged1 overexpression in Lewis lung carcinoma accelerated tumor growth in vivo that was prevented in Lgals3-/- mice. Finally, we evaluated the role of galectin-3 in cancer cell resistance to therapy. We found that the expression of sialyl-Tn, a biosynthetic product of ST6GalNAc-I, was able to decrease cell surface galectin-3 and galectin-3-binding sites both in vitro and in vivo leading to an intracellular accumulation of this protein. Exogenously added galectin-3 was found to have no effect on cancer cell death but contributed to chemotherapy-induced apoptosis. Sialyl-Tn expressing cells were protected. In human gastric cancer samples, galectin-3 binding sites were negatively correlated with the expression of sialyl-Tn. This knowledge has direct implications for the development of strategies aimed at controlling tumor growth and angiogenesis and open novel perspectives to overcome tumor resistance to therapy