Transforming growth factor-£] (TGF-£])-activated kinase 1(TAK1), a member of the MAPKKK family, was identified as a protein kinase that involved in c-Jun N-terminal kinase(JNK)/p38 MAPKs and NF-£eB signaling pathways. This kinase also participated in inflammatory and innate immune responses as well as the aggressiveness in several types of cancer. Association with its specific activator protein, TAK1 binding protein1 (TAB1), triggers TAK1 kinase activity. Recent evidences suggest that the interaction between chemokines and their cognate receptors involved in cancer metastasis. Our previous results demonstrated that CC chemokine receptor 7 (CCR7), the receptor for the two major chemokines CCL19 and CCL21, is up-regulated in breast cancer cells and may play an important role in the trafficking and homing of breast cancer cells to lymph nodes. In addition, CCR7 expression is positively correlated with lymphatic metastasis in the primary tumors of patients with breast cancer. Here, we show that TAK1 is constitutively activated in MDA-MB-231 breast cancer cells and suppression of TAK1 signaling by using TAK1 shRNA or TAB1 shRNA significantly reduces expression of CCR7 in these cells. In addition, the TAK1 inhibitor 5Z-7-oxozeaenol, a resorcylic lactone of fungal origin, also inhibited TAK1 kinase activity and CCR7 expression. Promoter deletion assay identified a responsive element localized between -500/-223 bp from transcription start site is critical for TAK1 to activate CCR7 transcription. Furthermore, Chromatin immunoprecipitation assay(ChIP assay)identified two transcription factors, c-Jun and NF£eB participate in TAK1 regulating CCR7 expression. In addition, our study in breast tumor tissues indicated that CCR7 was significantly associated with p-TAK1 expression as found in breast cancer cell lines.

The metastatic spread of tumor cells is the major lethal aspect of cancer, and lymphatic metastasis is one of the most important routes. Recent studies indicated that cyclooxygenase-2 (COX-2) expression is frequently associated with lymph node metastasis and over-expression of COX-2 can enhance lymphatic invasion of cancer cells. The interaction of chemokines and their cognate receptors also plays a critical role in cancer metastasis. Previous results of our laboratory demonstrated that CCR7 is a downstream target for COX-2 and COX-2 up-regulated CCR7 expression via the EP2 and EP4 receptor. We also found that protein kinase A (PKA) and AKT kinase are involved in COX-2-induced CCR7. In this study, we provided further evidences that COX-2 directly stimulates CCR7 expression via promoter activation. Promoter deletion and mutation assay indicated that COX-2 stimulated CCR7 promoter via the Sp1 binding site located at the -61/-52 bp region upstream of the transcription start site. Increase of Sp1 binding to CCR7 promoter by COX-2 was confirmed by chromatin immunoprecipitation (ChIP) assay. Furthermore, knockdown of Sp1 expression resulted in inhibition of PGE2-induced CCR7, and over-expression of Sp1 potently up-regulated CCR7 in MCF-7 cells. In vitro kinase assay indicated that AKT could directly phosphorylate Sp1 at S42, T679 and S698 sites. And the phosphorylation of Sp1 by AKT led to enhanced protein stability and DNA binding affinity of Sp1. The results of immunohistochemistry indicated that CCR7 expression was significantly associated with Sp1 and phosphor-AKT. Taken together, COX-2 may act via the EP receptor/PKA/AKT/Sp1 signaling pathway to stimulate CCR7 expression in breast cancer cells to promote lymphatic spread.

Notre laboratoire a démontré que le récepteur de chimiokine CCR7 joue un rôle majeur dans la migration des monocytes en réponse à ses ligands naturels, les chimiokines CCL19 et CCL21. Plusieurs études ont permis de mettre en évidence que les monocytes en réponse à des infections, des traumatismes ou des maladies neurologiques, migrent vers les tissus en inflammation du cerveau par un mécanisme jusqu’à présent inconnu. D’un autre côté, l’activation de certains récepteurs TLR (de l’anglais « Toll-like receptors ») par des composants bactériens est caractéristique aux infections bactériennes et tient un rôle primordial dans la réponse immunitaire. L’objectif principal de ce projet de maîtrise est de déterminer si l’activation des TLR bactériens module l’expression de CCR7 chez les monocytes, les rendant ainsi aptes à transmigrer à travers la barrière hémato-encéphalique (BHE). Pour ce faire, nous avons d’abord développé un modèle in vitro de la BHE à partir de co- culture d’astrocytes humains et de cellules endothéliales microvasculaires humaines. Les résultats de résistance électrique transendothéliale (de l’anglais « Transendothelial Electrical Resistance » (TEER)) démontrent que ce modèle est fonctionnel et possède des jonctions serrées rendant la BHE imperméable et plus près de la réalité. Des essais de transmigration de monocytes démontrent que le modèle de la BHE construite limite le passage des monocytes à travers la barrière. Ces résultats laissent supposer que la BHE limite donc le passage des monocytes à travers le SNC. Cette imperméabilité aux cellules, propre à la BHE in vivo, rend le modèle idéal à des essais chimiotaxiques de transmigration. Par la suite, nos travaux ont permis de déterminer si l’activation de certains TLR par des composants provenant de bactéries Gram + et Gram – sont capables de moduler l’expression iv et la fonctionnalité de CCR7 chez les monocytes. Des PCR quantitatives effectuées avec la lignée MONO-MAC-1 ont démontré que l’activation des TLR1/2 et TLR4 promeut l’expression de l’ARNm codant pour CCR7. Des essais de cytométrie en flux ont démontré que l’activation de TLR1/2 et TLR9 chez la lignée MONO-MAC-1 ainsi que l’activation de TLR4 chez les monocytes sanguins augmentait le pourcentage de cellules exprimant CCR7 à leur surface. Des essais de migration ont démontré que le récepteur CCR7 était fonctionnel puisque la lignée MONO-MAC-1 et les monocytes sanguins traités avec l’agoniste de TLR4 migraient en plus grand nombre en réponse aux agonistes de CCR7, les chimiokines CCL19 et CCL21. Nos travaux ont aussi démontré que le récepteur CCR7 exprimé par les MONO- MAC-1 et monocytes sanguins avait une plus grande affinité envers CCL19 que CCL21. L’activation de TLR4 promeut la migration CCR7 dépendante des MONO-MAC-1 et des monocytes sanguins à travers le modèle in vitro d’une barrière hémato-encéphalique en réponse à CCL19. Nos résultats suggèrent une implication importante des récepteurs TLR4 dans la migration CCR7 dépendante des monocytes à travers la BHE et donc qu’une infection bactérienne ou que la présence de LPS dans le sang peut influencer la migration des monocytes au cerveau exacerbant ainsi des conditions neurologiques pathologiques. Davantage d’études portant sur les rôles des TLR dans la migration cellulaire pourraient permettre de développer de nouvelles thérapies.

Sialylation is catalyzed by sialyltransferases (STs) that adding sialic acids to the terminal positions of oligosaccharide of glycoproteins and glycolipids. This process is frequently enhanced in cancer and is associated with increased cancer metastasis. Recent studies demonstrated that over-expression of ST3Gal-I promotes mammary tumorigenesis. In our experiments, we also find overexpression of £\-2,3-ST in breast cancer cells. We previously synthesized a lithocholic acid-based ST inhibitor AL10 and demonstrated its anti-metastatic effect in vitro and in vivo. Our results showed that AL10 is an effective sialyltransferase inhibitor and exerts anti-metastatic effect in vivo via suppression of sialylation of beta1 integrin and CXCR4. Breast cancer cells expressing high level of chemokine receptors CXCR4 and CCR7 are prone to exhibit lymphatic metastasis because their cognate ligands CCL19, CCL21 and SDF-1 are continuously expressed by lymphatic endothelial cells. In this study, we demonstrate that AL10 can inhibit invasion, proliferation and induce anoikis of £\-2,3-ST-overexpressing MDA-MB231 human breast cancer cells. Our results indicate that inhibition of CCL19-induced invasion and CCL19-reduced anoikis by AL10 are associated with reduced sialylation of CCR7 and attenuated activation of the downstream signaling mediator ERK and p38. In addition, AL10 can inhibit proliferation by reducing activation of AKT via CCR7 sialylation independent pathway and p-38 via CCR7 sialylation dependent pathway which results in ubiquitin-dependent cyclin D1 degradation. Taken together, we conclude that sialylation of CCR7 is critical for CCL19-stimulated proliferation, invasion and anti-anoikis in breast cancer cells.

Les maladies cardiovasculaires étant la première cause de mortalité au monde depuis une quinzaine d’années, de nombreuses recherches ont vu le jour sur plusieurs récepteurs impliqués dans ces maladies. C’est le cas du récepteur « Liver X Receptor », un récepteur nucléaire responsable du maintien de l’homéostasie du cholestérol et du glucose ainsi que du contrôle de l’inflammation. Les travaux réalisés dans le cadre de cette maîtrise avaient pour objectifs de mieux comprendre les éléments immunologiques et moléculaires contrôlant la migration des monocytes humains. Tout d’abord, nous avons déterminé le rôle de la prostaglandine E[indice inférieur 2], une cytokine produite massivement en inflammation, en combinaison avec l’activation du récepteur « Liver X Receptor », dans le contrôle de l’expression du récepteur de chimiokine CCR7 chez les monocytes. Ce récepteur est responsable de la migration des leucocytes vers les ganglions lymphatiques sous l’action chimiotactique des chimiokines CCL19 et CCL21. Nous avons démontré que la prostaglandine E[indice inférieur 2] en combinaison avec l’activation du récepteur « Liver X Receptor » induit la transcription de l’ARNm de CCR7 chez les monocytes et leur migration subséquente vers les chimiokines CCL19 et CCL21. Cependant, cette augmentation de la capacité migratoire n’est pas corrélée avec une augmentation de l’expression protéique de CCR7. Nous avons également observé que la prostaglandine E[indice inférieur 2] cause une diminution importante de l’expression de ABCG1, l’un des gènes cibles du récepteur « Liver X Receptor » impliqué dans le relargage du cholestérol. L’importance des récepteurs EP[indice inférieur 2] et EP[indice inférieur 4] a été démontré dans l’expression de l’ARNm et la fonctionnalité de CCR7. La prostaglandine E[indice inférieur 2] semble lier préférentiellement le récepteur EP[indice inférieur 4] plutôt que le récepteur EP[indice inférieur 2]. De plus, l’importance de la voie de signalisation induite par PI3K dans la modulation de la capacité migratoire a également été démontrée. Ces travaux ont permis de mettre en lumière les principaux acteurs moléculaires impliqué dans la migration des monocytes suite à l’activation du récepteur « Liver X Receptor » en présence de la prostaglandine E[indice inférieur 2].

The expression of CCR7 was evaluated in different cancer cell lines by using flow cytometry, western blot, Immunofluorescence and immunohistochemistry. We showed for the selected cell lines that the expression is maintained in cells grown as spheroids, and xenoplanted in mice. Furthermore, we showed the expression of CCR7 correlates with stage of the disease in patient derived head and neck cancer tissue. We also showed that expression of CCR7 in cancer cell lines correlates with migratory aptitude towards CCL21 in a scratch assay, Boyden chamber assay and spheroid invasion assay. We then showed that the expression of CCR7 is elevated under serum starvation and under hypoxia in cancer cell lines grown as monolayers and as spheroids; and that there is a correlation between hypoxia and CCR7 expression in spheroids, xenografted cells and clinical cancer tissue. However, we found that in cell line OSC-19, the increase in the expression of CCR7 did not correlate to increased migration. Our investigations following this observation showed that whilst hypoxia increases the expression of CCR7, it concurrently causes a decrease in reactive oxygen species (ROS) which strongly abrogates migratory aptitude in OSC-19, resulting in an overall loss of migration in OSC-19 cells. In addition, we characterised OSC-19 as a suitable model to evaluate small molecule CCR7 antagonists using a number of different assays. In particular, we showed that ICT13069 antagonised response of this cell line across a number of drivers of malignancy such as migration, invasion in 2D and 3D models.

Lymphocyte migration is crucial for adaptive immunity. CCR7 and its ligands mediate the migration and positioning of T cells in lymph nodes but the underlying mechanism is complex. The research in this thesis investigated CCR7-mediated T cell migration using a microfluidics-based approach. A microfluidic method suitable for quantitative migration analysis of genetically modified lymphocyte transfectants was developed. Using this method, I demonstrated chemotaxis of Jurkat transfectants expressing wild-type or C-terminal mutated CCR7 to a CCL19 gradient, and characterized the difference in transfectant migration mediated by wild-type and mutant CCR7. The fluorescent tag allows identification of CCR7-expressing transfectants in cell migration analysis, and microscopy assessment of CCR7 dynamics in migrating cells. Furthermore, my results also showed interesting migratory behaviours of CCR7 Jurkat transfectants in a specific co-existing CCL19 and CCL21 fields. This developed method will be broadly useful for studying cell migration signalling.

The expression of CCR7 was evaluated in different cancer cell lines by using flow cytometry, western blot, Immunofluorescence and immunohistochemistry. We showed for the selected cell lines that the expression is maintained in cells grown as spheroids, and xenoplanted in mice. Furthermore, we showed the expression of CCR7 correlates with stage of the disease in patient derived head and neck cancer tissue. We also showed that expression of CCR7 in cancer cell lines correlates with migratory aptitude towards CCL21 in a scratch assay, Boyden chamber assay and spheroid invasion assay. We then showed that the expression of CCR7 is elevated under serum starvation and under hypoxia in cancer cell lines grown as monolayers and as spheroids; and that there is a correlation between hypoxia and CCR7 expression in spheroids, xenografted cells and clinical cancer tissue. However, we found that in cell line OSC-19, the increase in the expression of CCR7 did not correlate to increased migration. Our investigations following this observation showed that whilst hypoxia increases the expression of CCR7, it concurrently causes a decrease in reactive oxygen species (ROS) which strongly abrogates migratory aptitude in OSC-19, resulting in an overall loss of migration in OSC-19 cells. In addition, we characterised OSC-19 as a suitable model to evaluate small molecule CCR7 antagonists using a number of different assays. In particular, we showed that ICT13069 antagonised response of this cell line across a number of drivers of malignancy such as migration, invasion in 2D and 3D models. / Zarqa University / The full text was made available at the end of the embargo, 3rd December 2019

University of Tobruk, and the Ministry of Higher Education of Libya / The full text will be available at the end of the embargo: 25th Apr 2024

Yes / The CCR7 chemokine axis is comprised of chemokine ligand 21 (CCL21) and chemokine ligand 19 (CCL19) acting on chemokine receptor 7 (CCR7). This axis plays two important but apparently opposing roles in cancer. On the one hand, this axis is significantly engaged in the trafficking of a number of effecter cells involved in mounting an immune response to a growing tumour. This suggests therapeutic strategies which involve potentiation of this axis can be used to combat the spread of cancer. On the other hand, the CCR7 axis plays a significant role in controlling the migration of tumour cells towards the lymphatic system and metastasis and can thus contribute to the expansion of cancer. This implies that therapeutic strategies which involve decreasing signaling through the CCR7 axis would have a beneficial effect in preventing dissemination of cancer. This dichotomy has partly been the reason why this axis has not yet been exploited, as other chemokine axes have, as a therapeutic target in cancer. Recent report of a crystal structure for CCR7 provides opportunities to exploit this axis in developing new cancer therapies. However, it remains unclear which of these two strategies, potentiation or antagonism of the CCR7 axis, is more appropriate for cancer therapy. This review brings together the evidence supporting both roles of the CCR7 axis in cancer and examines the future potential of each of the two different therapeutic approaches involving the CCR7 axis in cancer.