No description available.

Bone metastases are a major cause of morbidity in patients of advanced breast cancer. Development of osteolytic bone metastasis depends on the interaction between malignant tumour cells and bone microenvironment. Receptor Activator of Nuclear Factor Kappa B (RANK) is a member of tumour necrosis factor (TNF) superfamily that is expressed by osteoclasts (the bone resorbing cells) and primary breast tumour cells. Previous studies demonstrated that RANK receptor and its ligand (RANKL) play an important role in bone remodelling, mammary gland development and immune system. RANKL was also found to serve as a chemotactic factor that facilitates breast tumour metastasis to bone. However, the role of the RANK receptor in breast cancer cell metastatic behaviour in bone is not fully understood. Therefore, the aim of this thesis was to explore the role of the RANK receptor in parental and bone-tropic breast cancer cell growth, motility and invasion, and assess these cells influence on breast cancer cell induced osteoclastogenesis. Functional studies in breast cancer cells showed that RANKL (100 - 300 ng/ ml) significantly enhanced parental human MDA-231 (MDA-231P) and mouse 4T1 breast cancer cell spreading within minutes. RANKL induced chemotactic cell migration of MDA-231P cells in vitro. I also found that RANKL significantly stimulated random and directional 2D and 3D cell migration of parental MDA-231P and bone-tropic (MDA-231BT) breast cancer cells in vitro. These effects were observed at concentrations (100 – 300 ng/ml) that were sufficient to induce osteoclast formation in the presence and absence of breast cancer cells in vitro. In contrast, high concentrations of RANKL (1000 ng/ ml) dramatically suppressed human MDA-231P breast cancer cell invasion in vitro. These data indicate that the RANK receptor in the breast cancer cell lines tested influences cancer cell spreading, migration and invasion in vitro. Thus, targeting RANK in tumour cells may be of value in the prevention of tumour burden associated with breast cancer bone metastasis. Mechanistic studies revealed that RANKL stimulated the phosphorylation of p38 kinase in human and mouse breast cancer cells. Interestingly, RANKL had no effect on NFᴋB, JNK and AKT pathways in parental human MDA-231 and mouse 4T1 breast cancer cells at concentrations up to 300 ng/ ml. These data implies that the RANK receptor modulates human and mouse breast cancer cell metastatic behaviour via p38 activation and independently of the NFᴋB and PI3K/AKT pathways. Silencing of the RANK receptor in the bone-tropic human breast cancer cells MDA- 231BT2 reduced directional cell migration without affecting cell viability and growth. Functional studies in osteoclast and breast cancer cell revealed that knockdown of RANK expression in both parental and bone-tropic human breast cancer cells significantly inhibited the ability of these cells to stimulate osteoclast formation. Although, I cannot exclude the possibility of the involvement of other signalling pathways downstream of the RANK receptor, these studies suggest that the RANK/P38 signalling in osteoclast and breast cancer cells contributes significantly to breast cancer cell behaviour in bone. Genetic analysis of the RANK gene in human parental and bone-tropic MDA-231 breast cancer cells showed a number of polymorphisms. One variant detected was found to be deleterious for the RANK protein. This variant changes the amino acid sequence from alanine to threonine (Ala ˃ Thr) and only appeared in the RANK gene in the parental human MDA-231P breast cancer cells. Moreover, of the four known RANK isoforms that were detected in the parental and bone-tropic breast cancer cells tested, two lacked the TRAF6 binding motifs associated with NFκB activation. All RANK isoforms detected on the bone-tropic MDA-231BT breast cancer cells expressed the P38 binding motifs. Altogether, these findings support the role of the RANK/P38 signalling pathway in breast cancer cell behaviour in bone. Epigenetic analysis in parental human MDA-231P breast cancer cells showed that continuous and long-term exposure to RANKL (10 and 100 ng/ ml) for up to 50 passages (approximately 120 days) did not induce epigenetic changes, particularly DNA methylation, in the RANK gene. However, I found DNA methylation changes in a set of genes that are known to be involved in cell development and regulation. The methylation status of the altered CpG loci either hypermethylated or hypomethylated are located at different parts in the CpG islands. Whole genome DNA methylation pattern of the bone-tropic breast cancer cells showed a number of genes that appeared in both bone-tropic variants are correlated with different biological function of the cells. I also found that long-term exposure of human MDA-231P to RANKL (100 ng/ ml) enhanced the ability of these cells to stimulate osteoclastogenesis in vitro. These data together indicate that long-term exposure to RANKL induces “boney” epigenetic changes in a set of genes that enhances breast cancer cell behaviour in bone. Overall, this thesis illustrated that the RANK receptor on human parental and bone-tropic breast cancer cells plays an important role in cell motility and ability of these cells to influence osteoclastogenesis and ultimately osteolysis. Therefore, agents that selectively target the RANK receptor may be of value in the treatment of both tumour burden and osteolytic bone disease associated with breast cancer. However, the role of the RANK receptor in bone metastasis will require further in vivo investigation.

Cancer is a global epidemic; over 1.5 million new cancer diagnoses and greater than 600,000 deaths due to cancer are estimated to occur in the United States within the year 2015 alone. Approximately two-thirds of patients with bone metastases will experience pain, pathological fractures, spinal cord or nerve root compression, paralysis, impaired mobility, bone marrow infiltration and hypercalcemia of malignancy. We induced bone metastases through inoculation of rat femurs with MDA-MB-231 human breast cancer cells in order to compare the effectiveness of various treatment modalities, disease progression and recovery through the use of imaging methods in current clinical practice. CTRA provides highly accurate monitoring of metastases progression and treatment through both Ibandronate and Paclitaxel therapies. Using computed tomography (QCT)-based analysis to calculate the load bearing capacity of bone infiltrated with metastatic breast carcinoma, fracture risk threshold was predicted using Computed Topography Rigidity Analysis (CTRA) with 100% sensitivity and 90% specificity. The results of this study further validate that there is an existing gap between clinical guidelines and physician’s recommendations. This inconsistency necessitates that the decision making process for the selection of surgical or non-surgical treatment must be narrowed by more advanced prognostic tools such as CTRA.

This study tests whether musculoskeletal markers are attributable to occupational categories. It is hypothesized that individuals over the age of 30 years with a lifetime occupation as a laborer will demonstrate a significantly different pattern of activity markers from individuals in the white collar classifications. A sample of n=69 from the Maxwell Museum's Documented Skeletal Collection are investigated. Upper and lower extremities were scored for MSM type (robusticity, stress lesions, and ossification exostoses) and severity (grades 0 - 3) following Hawkey and Merbs (1995) visual reference system. To evaluate methodological approaches to MSM scoring, ossification exostoses and stress lesions were also scored using the Mariotti et al. (2004) proposed methods. Upper limb muscle insertion sites on the humerus, radius, and ulna and lower limb insertion sites on the femur, fibula, patella, calcaneus, and tibia were studied. The Kruskal Wallis test was used to predict occupational class according to an individual's aggregate MSM z-score. The Mann-Whitney test was used for comparison of aggregate MSM z-scores between the two occupational categories and for comparison of aggregate MSM z-scores between males and females. The Spearman correlation was used for non-parametric correlation analysis of aggregate MSM z-scores and the occupational categories of white collar and labor. The data were analyzed using the statistical software program SPSS (version 17.0). Results of this study show that musculoskeletal markers cannot statistically predict, nor can they be used to distinguish between, occupational categories of white collar and labor. Comparison of MSM shows no significant difference in the overall patterns of enthesopathies between individuals who report an occupation of white collar or those who report an occupation of laborer as defined by the U.S. Office of Personnel. Comparison of MSM in this population shows no significant difference between males and females, regardless of occupational category, a finding which runs counter to many earlier studies. Using dichotomous data it is revealed that laborers develop MSM symmetrically, evidence of whole-body activity. Further, white collar MSM can be associated with sitting and elevating the arm. Laborer's MSM are associated with lifting, twisting, pushing, squatting, walking, running and standing. Recommendations on methodology are provided.

No description available.

ZNF217 est un oncogène codant pour un facteur de transcription de la famille Krüppel-like. Nos objectifs sont d'explorer le rôle de l'oncogène ZNF217 dans le développement de métastases du cancer du sein à tropisme osseux et la valeur pronostique ainsi que les fonctions d'une nouvelle isoforme de ZNF217. Nous avons identifié que de forts niveaux d'expression de l'ARNm de ZNF217 dans les tumeurs primitives du sein pourrait être un indicateur d'un développement ultérieur de métastases osseuses. Nous avons montré que ZNF217 est un nouvel activateur de la voie BMP et que l'inhibition de cette voie permet de reverser les propriétés métastatiques des cellules ZNF217 positives in vitro (migration, invasion et chimiotactisme vers des cellules osseuses). In vivo chez la souris, les cellules ZNF217-positives de cancer du sein développent très rapidement des métastases ostéolytiques. Dans notre deuxième axe de travail, nous avons prouvé l'existence de l'isoforme ZNF217-?E4 et montré qu'elle possède une valeur de mauvais pronostic dans le cancer du sein ER-a+. Les cellules surexprimant ZNF217-?E4 développent un phénotype encore plus agressif que les cellules possédant la forme WT (prolifération, résistance au paclitaxel), et de manière intéressante, ZNF217-?E4 semble jouer un rôle de régulateur de l'expression de ZNF217-WT. En conclusion, ZNF217 et/ou la voie BMP pourraient représenter des cibles thérapeutiques dans le traitement des cancers du sein ZNF217-positif / ZNF217 is an oncogene encoding for a Krüppel-like transcription factor. Our aims were to explore the roles of the ZNF217 oncogene in the development of breast cancer metastases to the bone and to decipher the prognostic value and the functions of a new ZNF217 isoform. Our work identified that high ZNF217 mRNA expression levels within the primitive breast tumor could represent an indicator for future recurrence to the bone. Further in vitro experiment demonstrated that ZNF217 is a new activator of the BMP pathway and that the inhibition of this pathway could inhibit the metastatic properties of ZNF217-positive breast cancer cells in vitro (migration, invasion, chemotaxis to bone cells). In vivo in mice, ZNF217-positive breast cancer cells developed osteolytic metastases very faster. In our second axis, we have proven the existence of the ZNF217-?E4 isoform and we found that this isoform possesses a prognostic significance associated with a poor prognosis in ER-a+ breast cancer. Furthermore, cells overexpressing ZNF217-?E4 developed a more aggressive phenotype than cells overexpressing ZNF217-WT (proliferation, paclitaxel resistance). Interestingly, ZNF217-?E4 seems to play a regulatory role regarding ZNF217-WT expression. In conclusion, ZNF217 and/or the BMP pathway could represent potential therapeutical targets in the management of ZNF217 positive breast cancer

Das Multiple Myelom (MM) ist eine hämatologische Erkrankung, welche sich durch eine Akkumulation von malignen Plasmazellen im Knochenmark auszeichnet und eine gestörte Hämatopoiese und Osteolyse zur Folge hat. Komplexe molekulare Interaktionen zwischen MM-Zellen und der Mikroumgebung/Nische im Knochenmark (bone marrow microenvironment, BMM) führen zu einer Aktivierung von verschiedenen Wachstums-, Überlebens- und anti-apoptotischen Signalwegen, die zur Entstehung bzw. Wirkstoffresistenz von MM-Zellen beitragen. IL-6R/STAT3, Ras/MAPK und PI3K/Akt sind die drei wichtigsten Signalwege, die mit dem Wachstum und der Entwicklung des MM assoziiert sind. Auf der anderen Seite sind Myelomzellen insensitiv gegenüber einer Blockade des IL6R/STAT3-Signalweges bzw. des Ras/MAPK-Signalwegs in der Gegenwart von Knochenmarksstromazellen (bone marrow stroma cells, BMSCs), was die Entbehrlichkeit dieser beiden Signalwege unter Ko-Kultur-Bedingungen nahelegt. Interessanterweise aber induziert die gleichzeitige Unterbrechung der IL6R/STAT3 und Ras/MAPK Signalwege Apoptose in MM-Zellen. Ziel der Arbeit war die Identifizierung und Analyse von Zielgenen, die von beiden Signalwegen und nicht durch einen Signalweg alleine reguliert werden. Genexpressionsanalysen zeigten eine deutliche Herunterregulierung der Proteine Hsp90alpha und Hsp90beta nach einer gleichzeitigen Inhibition der IL6R/STAT3 und Ras/MAPK Signalwege. In Hinblick auf die zentrale Rolle von Hsp90 in der Tumorbiologie fokussiert sich die vorliegende Arbeit auf die Erforschung der Rolle von Hsp90 im Multiplen Myelom. Die siRNA-vermittelte Herunterregulation der Proteinexpression von Hsp90-Proteinen zeigte, daß das Ausschalten von HsP90alpha alleine nur zu einer moderaten Apoptoseinduktion in INA-6- und MM.1s-Zellen führte. Die gleichzeitige Herunterregulation von HsP90beta hingegen führte zu einer Verstärkung dieses Effektes und deutet darauf hin, daß beide Proteine miteinander kooperieren. Die pharmakologische Inhibition der Hsp90-Funktion mittels eines neuen Hsp90-Inhibitors (17-DMAG) führte zu einer Verringerung von phospho-ERK1/2, zur Degradation von STAT3 und zu einem verminderten Überleben von MM-Zellen. Die pro-apoptotischen Effekte der gestörten Hsp90-Funktion konnten weder durch BMSCs und Osteoklasten noch durch ECs (??) abgeschwächt werden, obwohl für ECs beschrieben wurde, daß sie zum Wachstum und Überleben von MM-Zellen beitragen können. Diese Beobachtungen deuten auf einen positiven Rückkopplungskreislauf zwischen HsP90alpha/beta und den wichtigsten Signalwegen hin, welcher das Überleben von MM-Zellen gewährleistet. Desweiteren zeigten immunhistologische Analysen, daß Hsp90-Proteine im Vergleich zu MGUS (??) bzw. normalen Plasmazellen in MM-Plasmazellen hochreguliert sind. Zusammengefasst zeigen die Ergebnisse der vorliegenden Arbeit die essentielle Rolle von Hsp90-Proteinen für die Überlebensfähigkeit von MM-Zellen. Ein neuer Mechanismus der Hsp90-Regulation durch das Zusammenwirken der Signalwege IL6R/STAT3 und Ras/MAPK in MM-Zellen konnte gezeigt werden. Darüber hinaus deuten die Ergebnisse darauf hin, daß ein positiver Rückkopplungskreislauf zwischen Hsp90-Proteinen und den wichtigsten Signalwegen existiert, welcher zum Wachstum und zur Entwicklung von MM-Zellen beiträgt. Die Inhibition der Hsp90-Funktion durch den pharmakologischen Inhibitor 17-DMAG führte zum Absterben von MM-Zellen und der pro-apoptotische Effekt der Hsp90-Depletion konnte nicht durch unterstützende BMM-Zellen aufgehoben werden. Diese Beobachtungen untermauern die multifunktionelle Rolle von Hsp90 in der MM-Biologie und zeigen die Wichtigkeit der Entwicklung neuer therapeutischer Wirkstoffe zur Inhibition der Hsp90-Funktion bei der Behandlung des MM. / Multiple myeloma (MM) is a haematological malignancy characterised by the accumulation of malignant plasma cells in the bone marrow leading to impaired haematopoiesis and osteolytic bone destruction. Intricate molecular interactions between MM cells and the BMM activate a diverse set of growth, survival and anti-apoptotic signaling cascades that mediate tumor progression and drug resistance. IL-6R/STAT3, Ras/MAPK and PI3K/Akt are the three major signal transduction pathways that are associated with MM growth and progression. However, myeloma cells have shown independence from IL-6R/STAT3 blockade or insensitivity towards Ras/MAPK pathway inhibition in the presence of BMSCs, indicating the dispensability of both in co-culture conditions. Interestingly, concomitant disruption of both IL-6R/STAT3 and Ras/MAPK pathways was successful to drive MM cells into significant apoptosis. This study aimed to identify and analyse the downstream target genes that are regulated by both pathways and not by either pathway alone. Gene expression profiling revealed prominent downregulation of Hsp90alpha and Hsp90beta proteins after combined inhibition of the IL-6R/STAT3 and Ras/MAPK pathways. Owing to the important role played by Hsp90 in cancer biology, this study was narrowed down to investigate the role of Hsp90 in MM. Specific siRNA-mediated knockdown of Hsp90 proteins showed that although knockdown of Hsp90beta was sufficient to induce moderate apoptosis in INA-6 and MM.1s cells, the effect was more pronounced when both Hsp90 proteins were targeted, indicating co-operation between them. Pharmacological inhibition of Hsp90 function by using a novel Hsp90 inhibitor (17-DMAG) down-regulated the levels of pERK1/2 and led to degradation of STAT3 and decreased viability of MM cells. The pro-apoptotic effects of compromised Hsp90 function could not be alleviated by either BMSCs, OCs or ECs, which are well-known to support myeloma growth and survival. These observations point to the existence of a positive feedback loop consisting of Hsp90alpha/beta and major signaling pathways supporting MM cell survival. Furthermore, immunohistochemical analysis unveiled the up-regulated status of Hsp90 proteins in MM PCs as compared to MGUS or normal PCs. Taken together, the results of this study explain the critical contribution of Hsp90 proteins to MM cell survival. A novel mechanism of Hsp90 regulation by co-operation between the IL-6R/STAT3 and Ras/MAPK pathways was discovered in myeloma cells. There is also strong evidence of the existence of a positive feedback loop between Hsp90alpha/beta proteins and major signaling pathways supporting MM growth and progression. Inhibition of Hsp90 function by using the Hsp90 inhibitory drug 17-DMAG proved to be lethal for myeloma cells and the pro-apoptotic effects of Hsp90 blockade could not be reversed by the presence of cells from the supportive BMM. These observations highlight a multi-functional role of Hsp90 in MM biology and strongly strengthen the notion that therapeutic strategies targeting Hsp90 may open new perspectives for anti-myeloma drug development.