La formation de métastases osseuses implique de nombreuses interactions entre les cellules de cancer du sein et le microenvironnement osseux. Les gradients d’hypoxie et l’activation de HIF (hypoxia inducible factor) 1alpha sont essentiels au maintien de l’homéostasie osseuse. Le rôle de la signalisation HIF dans les ostéoblastes lors du processus métastatique n’a pourtant jamais été exploré. Dans cette étude, nous montrons que les cellules ostéoprogénitrices (OPC), se situent dans des niches hypoxique, et que l’activation de la signalisation HIF dans ces cellules augmente la masse osseuse et favorise les métastases osseuses du cancer sein. L’effet de la signalisation HIF dans les OPC n’est pas limité au squelette, en effet celle-ci stimule aussi la croissance des tumeurs mammaires et la dissémination tumorale dans les poumons et d’autres organes distants. Nous avons mis en évidence que la signalisation HIF dans les OPC induit l’augmentation de la concentration plasmatique de la chimiokine C-X-C motif ligand 12 (CXCL12), qui entraine une augmentation systémique de la prolifération et de la dissémination tumorale, via l’activation de son récepteur CXCR4 sur les cellules cancéreuses. Ainsi, nos résultats mettent en évidence le rôle protumorigénique de l’hypoxie dans le lignage ostéoblastique, lors de la formation de métastases osseuse, mais également par une action systémique sur les tumeurs mammaires et les métastases dans les tissus mous. Nous démontrons également que des altérations de l’anabolisme osseux peuvent affecter la progression du cancer du sein, révélant un nouveau rôle du squelette au sein du macroenvironnement tumoral. / Bone metastasis involves dynamic interplay between tumor cells and thelocal stromal environment. In bones, local hypoxia and activation of the hypoxiainducible factor (HIF)-1alpha in osteoblasts are essential to maintain skeletalhomeostasis. However, the role of osteoblast-specific HIF signaling in cancermetastasis is unknown. Here we show that osteoprogenitor cells (OPC) are locatedin hypoxic niches in the bone marrow, and that activation of HIF signaling in thesecells increases bone mass and favors breast cancer metastasis to bone locally.Remarkably, HIF signaling in osteoblast lineage cells also promotes breast cancergrowth and dissemination remotely, in the lungs and in other tissues distant frombones. Mechanistically, we found that activation of HIF signaling in OPC increasesblood levels of the chemokine C-X-C motif ligand 12 (CXCL12), which leads to asystemic increase of breast cancer cell proliferation and dissemination, throughdirect activation of the CXCR4 receptor. Hence, our data reveal a previouslyunrecognized role of the hypoxic osteogenic niche in promoting tumorigenesisbeyond the local bone microenvironment. They also indicate that alterations inbone formation can affect breast cancer progression, and support the concept thatthe skeleton is an important regulator of the systemic tumor environment.

CXCR4 ist der spezifische Rezeptor für das Chemokin CXCL12 und ist überexprimiert in Vestibularisschwannomzellen. Das Ziel dieser Arbeit war es den Effekt des spezifischen Inhibitors AMD3100 auf die CXCR4 vermittelte Proliferation und Migration der Vestibularisschwannomzellen in verschiedenen Zellkuturmodellen zu analysieren. Die nachgewiesene Inhibition von CXCR4 deutet auf einen möglichen Einsatz von AMD3100 in der systemischen Therapie von NF-2 Patienten hin. / CXCR4 is the specific receptor for the chemokine CXCL12 and is hyperexpressed in vestibular schwannoma cells. The aim was to analyse the influence of the specific inhibitor AMD 3100 on the CXCR4 stimulated proliferation and migration of vestibular schwannoma cells in different cell culture models. The demonstrated inhibition of CXCR4 suggests the use of AMD3100 in the systemic therapy of NF-2 patients.

A growing body of work implicates chemokines and their receptors in the progression of various types of cancer, including breast cancer. However, as potent chemotactic factors for leukocytes, chemokines also have the potential to enhance anti-cancer immunity. Evidence suggests that the chemokine CXCL12 and its receptors may be important in a number of aspects of breast cancer progression and site-specific metastasis. Another chemokine, CXCL16, has been identified as a specific chemotactic factor for Type Ipolarised T lymphocytes, which are major effectors of cell-mediated immunity and hence efficacious anti-tumour immune responses. The aim of this study, therefore, was to further elucidate the roles of CXCL12 and CXCL16 in breast cancer development and metastasis. To achieve this, wild-type CXCL12 and CXCL16 and antagonists of CXCL12 and CXCL16 activity, CXCL12[subscript](P2G) and CXCL16[subscript](9-220) respectively, were overexpressed in the 4T1.2 mouse model of breast carcinoma. Overexpression of wild-type CXCL12 potently inhibited both primary tumour growth and metastasis in this model. This was attributed to the induction of an anti-tumour response dependent, in part, on T cells, interferon-g and the cytotoxic mediators perforin and TRAIL. This response was characterised by increased numbers of CD11c⁺ cells in the tumour-draining lymph nodes and enhanced cytolytic activity of lymph node-derived effector cells against tumour cells. Unexpectedly, CXCL12[subscript](P2G) inhibited metastasis of tumour cells to the lungs of tumour-bearing mice, without affecting primary tumour growth. Intravenous injection of tumour cells revealed that CXCL12[subscript](P2G) expression could block metastatic steps occurring post tumour cell escape from the primary tumour, though a role for CXCL12([subscript](P2G) at earlier metastatic steps could not be ruled out. Further work is needed to clarify the precise stages of metastasis at which CXCL12[subscript](P2G) exerts its effects. No obvious effects on primary breast tumour growth were observed when CXCL16 or CXCL16([subscript](9-220) were overexpressed in tumour cells. Interestingly, CXCL16[subscript](9-220) expression inhibited experimental metastasis but not spontaneous metastasis. The findings of this study begin to shed light on the roles of CXCL12 and CXCL16 in breast cancer progression and also highlight the potential therapeutic applications of CXCL12, CXCL16 and/or their antagonists in the treatment of breast cancer and breast cancer metastasis. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297662 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2007

A growing body of work implicates chemokines and their receptors in the progression of various types of cancer, including breast cancer. However, as potent chemotactic factors for leukocytes, chemokines also have the potential to enhance anti-cancer immunity. Evidence suggests that the chemokine CXCL12 and its receptors may be important in a number of aspects of breast cancer progression and site-specific metastasis. Another chemokine, CXCL16, has been identified as a specific chemotactic factor for Type Ipolarised T lymphocytes, which are major effectors of cell-mediated immunity and hence efficacious anti-tumour immune responses. The aim of this study, therefore, was to further elucidate the roles of CXCL12 and CXCL16 in breast cancer development and metastasis. To achieve this, wild-type CXCL12 and CXCL16 and antagonists of CXCL12 and CXCL16 activity, CXCL12[subscript](P2G) and CXCL16[subscript](9-220) respectively, were overexpressed in the 4T1.2 mouse model of breast carcinoma. Overexpression of wild-type CXCL12 potently inhibited both primary tumour growth and metastasis in this model. This was attributed to the induction of an anti-tumour response dependent, in part, on T cells, interferon-g and the cytotoxic mediators perforin and TRAIL. This response was characterised by increased numbers of CD11c⁺ cells in the tumour-draining lymph nodes and enhanced cytolytic activity of lymph node-derived effector cells against tumour cells. Unexpectedly, CXCL12[subscript](P2G) inhibited metastasis of tumour cells to the lungs of tumour-bearing mice, without affecting primary tumour growth. Intravenous injection of tumour cells revealed that CXCL12[subscript](P2G) expression could block metastatic steps occurring post tumour cell escape from the primary tumour, though a role for CXCL12([subscript](P2G) at earlier metastatic steps could not be ruled out. Further work is needed to clarify the precise stages of metastasis at which CXCL12[subscript](P2G) exerts its effects. No obvious effects on primary breast tumour growth were observed when CXCL16 or CXCL16([subscript](9-220) were overexpressed in tumour cells. Interestingly, CXCL16[subscript](9-220) expression inhibited experimental metastasis but not spontaneous metastasis. The findings of this study begin to shed light on the roles of CXCL12 and CXCL16 in breast cancer progression and also highlight the potential therapeutic applications of CXCL12, CXCL16 and/or their antagonists in the treatment of breast cancer and breast cancer metastasis. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297662 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2007

CAVALCANTE, Galyléia Menezes. Estudo da imunoexpressão dos sistemas CXCR4-CXC112/SDF-1, CCR7-CCl21 e KI-67 no carcinoma de células escamosas oral e sua associação com indicadores clínicopatológicos, metástase linfonodal e sobrevida. 2013. 57 f. Dissertação (Mestrado em Odontologia) - Universidade Federal do Ceará. Faculdade de Farmácia, Odontologia e Enfermagem, Fortaleza, 2013. / Submitted by denise santos (denise.santos@ufc.br) on 2014-07-14T13:15:32Z No. of bitstreams: 1 2013_dis_gmcavalcante.pdf: 1293725 bytes, checksum: fa485fabd530db7d0d4df8d64c549656 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2014-07-14T13:16:38Z (GMT) No. of bitstreams: 1 2013_dis_gmcavalcante.pdf: 1293725 bytes, checksum: fa485fabd530db7d0d4df8d64c549656 (MD5) / Made available in DSpace on 2014-07-14T13:16:38Z (GMT). No. of bitstreams: 1 2013_dis_gmcavalcante.pdf: 1293725 bytes, checksum: fa485fabd530db7d0d4df8d64c549656 (MD5) Previous issue date: 2013 / Chemokines are responsible for the directed migration of leukocyte chemotactic cytokines, coordinating cell movement during inflammation and the transport of hematopoietic cells. In addition to leukocytes, chemokine receptors are also found in neoplastic cells and tumors associated with stromal cells. Among chemokines, and the CXCR4/CXCL12 CCR7/CCL21 systems have been shown the involvement of lymph node metastases or distant metastases in different cancers. Thus, aim of this study was to evaluate the expression of CXCR4, CXCL12, CCR7, CCL21 and Ki-67 in oral squamous cell carcinoma (SCC) and to correlate these markers with clinicopathological indicators, lymph node metastasis and survival. We conducted a survey of reports and paraffin blocks of excisional biopsies of patients with SCC treated at the Hospital Haroldo Juaçaba (2001-2009). Data on anatomic location of the lesion, sex, age, patient survival, degree of histological differentiation of the tumor, tumor stage and presence or absence of lymph node metastasis, lymphovascular and perineural invasion, nuclear grade and depth of invasion were collected. For immunohistochemical analysis, followed by the technique of streptavidin-biotin-peroxidase using the anti-CXCR4, anti-CXCL12, anti-CCR7, anti-CCL21 and Ki-67 antibody. Histological sections were photomicrographed in 10 fields chosen randomly and measured for the number of labeled tumor cells and determined the percentage of each labeling antibody. The marking of CXCR4 was detected in the cytoplasm and nucleus, CXCL12, CCR7 and CCL21 were only cytoplasmic, their expression was observed in 18 (60%) 8 (22.66%) 16 (53.3%) and 3 (12%) cases, respectively. We found a significant positive association between lymphovascular invasion and immunostaining of CXCR4 (p = 0.007) and CCR7 (P = 0.01) and among these cases metastasis was present in 62.5% and 37.5%, respectively. When in combination with Ki67, we found a significant positive correlation between CXCR4 (p = 0.0086), CXCL12 (p = 0.036) and CCR7 (p = 0:04). Among patients CXCR4 + over 111 months, only 38.4% were alive (p = 0.845), whereas both patients CCR7 + (p = 0.398) as well as CXCR4 +, and CCR7 + (p = 0.441) after 62 months, everyone had already died. We conclude that these chemokines are associated with lymphovascular invasion and cell proliferation, perhaps favoring the development of metastasis and poor prognosis. / As quimiocinas são citocinas quimiotáticas responsáveis pela migração direcionada de leucócitos, coordenando o movimento celular durante a inflamação e o transporte de células hematopoiéticas. Além dos leucócitos, os receptores de quimiocinas também são encontrados em células neoplásicas e em tumores associados com células estromais. Dentre as quimiocinas, os sistemas CXCR4/CXCL12 e CCR7/CCL21 têm sido demonstrado no envolvimento de metástases linfonodais ou à distância em diferentes tipos de câncer. Dessa forma, foi objetivo desse trabalho avaliar a expressão de CXCR4, CXCL12, CCR7, CCL21 e Ki-67 em carcinoma de células escamosas orais (CEC) e correlacionar estes marcadores com indicadores clínicopatológicos, metástase linfonodal e sobrevida. Realizou-se um levantamento de laudos e blocos parafinados de biopsias excisionais de pacientes portadores de CEC tratados no Hospital Haroldo Juaçaba (2001 a 2009). Foram coletados dados sobre localização anatômica da lesão, sexo, idade, sobrevida do paciente, grau de diferenciação histopatológica do tumor, estadiamento tumoral e presença ou ausência de metástase linfonodal, invasão linfovascular e perineural, grau nuclear e profundidade de invasão. Para reação de imunohistoquímica, seguiu-se a técnica da estreptavidina-biotina-peroxidase, utilizando os anticorpos anti-CXCR4, anti-CXCL12, anti-CCR7, anti-CCL21 e Ki-67. As secções histológicas foram fotomicrografadas em 10 campos escolhidos aleatoriamente e quantificadas quanto ao número de células tumorais marcadas e determinado o percentual de marcação de cada anticorpo. A marcação de CXCR4 foi detectada em citoplasma e núcleo, CXCL12, CCR7 e CCL21 tiveram marcação apenas citoplasmática, sendo observada suas expressões em 18 (60%), 8 (22,66%), 16 (53,3%) e 3 (12%) casos, respectivamente. Encontrou-se uma associação significativa positiva entre a invasão linfovascular e a imunomarcação do CXCR4 (p=0.007) e CCR7 (p=0.01) e dentre esses casos a metástase esteve presente em 62,5% e 37,5%, respectivamente. Quando em associação com o Ki67, encontrou-se uma correlação positiva significante entre o CXCR4 (p=0.0086), CXCL12 (p=0.036) e CCR7 (p=0.04). Dentre os pacientes CXCR4+, ao longo de 111 meses, apenas 38,4% estavam vivos (p=0.845), ao passo que tanto para pacientes CCR7+ (p = 0.398), quanto CXCR4+ e CCR7+ (p = 0.441), após 62 meses, todos haviam ido a óbito. Conclui-se que essas quimiocinas estão associadas com a invasão linfovascular e proliferação celular, talvez favorecendo o desenvolvimento de metástases e um pior prognóstico.

CXCR4 is a chemokine receptor that has been linked to several disease related pathways including: HIV-1 proliferation, autoimmune disorders, inflammatory disease and cancer metastasis. The interaction of the C-X-C chemokine receptor type 4 (CXCR4) with C-X-C chemokine ligand 12 (CXCL12) plays a key role in triggering these disease related pathways. Various antagonists for these receptors have been synthesized and tested, but many are not useful clinically either because of toxicity or poor pharmacokinetics. Some of the most extensive CXCR4 antagonist libraries stem from a class of compounds, p-xylyl-enediamines, which all feature a benzene ring as the core of the compound. This work focuses on the design and synthesis of a new class of compounds that show potential as CXCR4 antagonists by using heterocyclic aromatic rings (2,6-pyridine, 2,5-furan, 2,5-pyrazine and 3,4-thiophene) as the core of the scaffold. After synthesis, these analogues were probed through a variety of assays and techniques by our collaborators in the Shim lab at Emory University including: preliminary binding assays, Matrigel invasion assays, carrageenan mouse paw edema tests, and in silico analysis. In silico analysis also probed 2,5-thiophene-based analogues previously synthesized. This work has produced the beginnings of a new library of CXCR4 antagonists and has identified fifteen hit compounds that are promising leads for further testing and modification.

No description available.

Multiple myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of plasma cells (PCs) within the bone marrow (BM). MM PC survival and expansion is dependent upon an adequate supply of oxygen and nutrients, and increased BM angiogenesis is a critical feature of MM progression. While MM PCs express and secrete a number of angiogenic factors, our current understanding of the precise mechanisms by which MM-induced angiogenesis occurs is incomplete. In this study, we collected specimens from patients with MM and the benign precursor condition MGUS, and demonstrated for the first time that circulating levels of the CXCL12 chemokine positively correlate with the degree of BM angiogenesis. Using conditioned media from a MM PC line, the contribution of MM PC-derived CXCL12 to angiogenesis was also examined and found to strongly induce vascular tube formation in vitro. In several other cell types, hypoxia has been shown to up-regulate CXCL12 expression. Studies investigating the hypoxic regulation of CXCL12 in MM PCs revealed that, while acute hypoxia is unable to stimulate CXCL12 expression, prolonged hypoxia significantly up-regulates CXCL12 mRNA and protein expression. To determine the mechanism(s) responsible for this, over-expression and RNA interference technology was employed to create genetically modified MM cells in which either HIF-1α or HIF-2α were overexpressed or knocked down. These studies showed that HIF-2α is the predominant mediator of the hypoxic induction of CXCL12 in MM PCs. The ability of HIF-2α to bind to the CXCL12 promoter was confirmed using EMSA and ChIP analyses. The role of CXCL12 in in vivo angiogenesis and the contribution of HIF-1α and HIF-2α were also examined. In these studies, transduced MM cells, in which HIF-1α, HIF-2α and CXCL12 were over-expressed or knocked down, were implanted into a vessel-poor, subcutaneous environment in immunocompromised mice. Tumour-induced angiogenesis was assessed after two weeks by measuring the haemoglobin content of excised implants. These studies confirmed that over-expression of CXCL12, HIF-1α and HIF-2α each stimulates a strong angiogenic response. Using the well-characterised CXCR4 antagonist, T140, CXCL12 was found to play a key role in the increased angiogenesis observed in response to HIF-1α and HIF-2α over-expression. These novel studies have shown that CXCL12 is an important mediator of angiogenesis in MM patients, and that aberrant CXCL12 expression by MM PCs is due, in part, to its hypoxic up-regulation mediated predominantly by HIF-2. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1365126 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

Multiple myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of plasma cells (PCs) within the bone marrow (BM). MM PC survival and expansion is dependent upon an adequate supply of oxygen and nutrients, and increased BM angiogenesis is a critical feature of MM progression. While MM PCs express and secrete a number of angiogenic factors, our current understanding of the precise mechanisms by which MM-induced angiogenesis occurs is incomplete. In this study, we collected specimens from patients with MM and the benign precursor condition MGUS, and demonstrated for the first time that circulating levels of the CXCL12 chemokine positively correlate with the degree of BM angiogenesis. Using conditioned media from a MM PC line, the contribution of MM PC-derived CXCL12 to angiogenesis was also examined and found to strongly induce vascular tube formation in vitro. In several other cell types, hypoxia has been shown to up-regulate CXCL12 expression. Studies investigating the hypoxic regulation of CXCL12 in MM PCs revealed that, while acute hypoxia is unable to stimulate CXCL12 expression, prolonged hypoxia significantly up-regulates CXCL12 mRNA and protein expression. To determine the mechanism(s) responsible for this, over-expression and RNA interference technology was employed to create genetically modified MM cells in which either HIF-1α or HIF-2α were overexpressed or knocked down. These studies showed that HIF-2α is the predominant mediator of the hypoxic induction of CXCL12 in MM PCs. The ability of HIF-2α to bind to the CXCL12 promoter was confirmed using EMSA and ChIP analyses. The role of CXCL12 in in vivo angiogenesis and the contribution of HIF-1α and HIF-2α were also examined. In these studies, transduced MM cells, in which HIF-1α, HIF-2α and CXCL12 were over-expressed or knocked down, were implanted into a vessel-poor, subcutaneous environment in immunocompromised mice. Tumour-induced angiogenesis was assessed after two weeks by measuring the haemoglobin content of excised implants. These studies confirmed that over-expression of CXCL12, HIF-1α and HIF-2α each stimulates a strong angiogenic response. Using the well-characterised CXCR4 antagonist, T140, CXCL12 was found to play a key role in the increased angiogenesis observed in response to HIF-1α and HIF-2α over-expression. These novel studies have shown that CXCL12 is an important mediator of angiogenesis in MM patients, and that aberrant CXCL12 expression by MM PCs is due, in part, to its hypoxic up-regulation mediated predominantly by HIF-2. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1365126 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

Multiple myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of plasma cells (PCs) within the bone marrow (BM). MM PC survival and expansion is dependent upon an adequate supply of oxygen and nutrients, and increased BM angiogenesis is a critical feature of MM progression. While MM PCs express and secrete a number of angiogenic factors, our current understanding of the precise mechanisms by which MM-induced angiogenesis occurs is incomplete. In this study, we collected specimens from patients with MM and the benign precursor condition MGUS, and demonstrated for the first time that circulating levels of the CXCL12 chemokine positively correlate with the degree of BM angiogenesis. Using conditioned media from a MM PC line, the contribution of MM PC-derived CXCL12 to angiogenesis was also examined and found to strongly induce vascular tube formation in vitro. In several other cell types, hypoxia has been shown to up-regulate CXCL12 expression. Studies investigating the hypoxic regulation of CXCL12 in MM PCs revealed that, while acute hypoxia is unable to stimulate CXCL12 expression, prolonged hypoxia significantly up-regulates CXCL12 mRNA and protein expression. To determine the mechanism(s) responsible for this, over-expression and RNA interference technology was employed to create genetically modified MM cells in which either HIF-1α or HIF-2α were overexpressed or knocked down. These studies showed that HIF-2α is the predominant mediator of the hypoxic induction of CXCL12 in MM PCs. The ability of HIF-2α to bind to the CXCL12 promoter was confirmed using EMSA and ChIP analyses. The role of CXCL12 in in vivo angiogenesis and the contribution of HIF-1α and HIF-2α were also examined. In these studies, transduced MM cells, in which HIF-1α, HIF-2α and CXCL12 were over-expressed or knocked down, were implanted into a vessel-poor, subcutaneous environment in immunocompromised mice. Tumour-induced angiogenesis was assessed after two weeks by measuring the haemoglobin content of excised implants. These studies confirmed that over-expression of CXCL12, HIF-1α and HIF-2α each stimulates a strong angiogenic response. Using the well-characterised CXCR4 antagonist, T140, CXCL12 was found to play a key role in the increased angiogenesis observed in response to HIF-1α and HIF-2α over-expression. These novel studies have shown that CXCL12 is an important mediator of angiogenesis in MM patients, and that aberrant CXCL12 expression by MM PCs is due, in part, to its hypoxic up-regulation mediated predominantly by HIF-2. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1365126 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009