BACKGROUND:Tumor cell lines that can be tracked in vivo during tumorigenesis and metastasis provide vital tools for studying the specific cellular mechanisms that mediate these processes as well as investigating therapeutic targets to inhibit them. The goal of this study was to engineer imageable mouse mammary tumor cell lines with discrete propensities to metastasize to bone in vivo. Two novel luciferase expressing cell lines were developed and characterized for use in the study of breast cancer metastasis to bone in a syngeneic mouse model.RESULTS:The 4 T1.2 luc3 and 66c14 luc2 cell lines were shown to have high levels of bioluminescence intensity in vitro and in vivo after orthotopic injection into mouse mammary fat pads. The 4 T1.2 luc3 cell line was found to closely model the sites of metastases seen in human patients including lung, liver, and bone. Specifically, 4 T1.2 luc3 cells demonstrated a high incidence of metastasis to spine, with an ex-vivo BLI intensity three orders of magnitude above the commercially available 4 T1 luc2 cells. 66c14 luc2 cells also demonstrated metastasis to spine, which was lower than that of 4 T1.2 luc3 cells but higher than 4 T1 luc2 cells, in addition to previously unreported metastases in the liver. High osteolytic activity of the 4 T1.2 luc3 cells in vivo in the bone microenvironment was also detected.CONCLUSIONS:The engineered 4 T1.2 luc3 and 66c14 luc2 cell lines described in this study are valuable tools for studying the cellular events moderating the metastasis of breast tumor cells to bone.

O 153Sm-EDTMP é um radiofármaco utilizado em medicina nuclear com resultados promissores no alívio da dor metastática. No entanto, pouco se sabe sobre os efeitos do 153Sm-EDTMP em nível celular. O presente trabalho foi conduzido com o intuito de avaliar os efeitos citogenéticos do 153Sm-EDTMP em linfócitos periféricos de pacientes com metástases ósseas (com e sem radio e/ou quimioterapias anteriores) pela da técnica de detecção de aberrações cromossômicas, tanto in vivo como in vitro. Para tanto, as amostras sangüíneas foram coletadas antes e 1 hora após a administração endovenosa do 153Sm- EDTMP (atividade média de 42,53 + 5,31 MBq/kg de peso corpóreo), levando-se em consideração o rápido clearance sangüíneo. Os principais tipos de aberrações cromossômicas estruturais encontrados foram os gaps e quebras, fragmentos acêntricos, anéis cêntricos, double minutes e dicêntricos. A análise estatística mostrou que o único grupo de pacientes que apresentou uma diferença significativa na freqüência de aberrações cromossômicas 1 hora após o tratamento foi o que recebeu prévio tratamento radio e quimioterápico antes da terapia com 153Sm-EDTMP. Quanto a averiguação do número modal de cromossomos e da cinética do ciclo celular, a análise estatística mostrou que não houve diferença significativa entre os grupos analisados, sugerindo que o tratamento com 153Sm-EDTMP não influenciou nesses parâmetros. A molécula carreadora, EDTMP, não teve qualquer influência na indução de aberrações cromossômicas. Em relação aos ensaios in vitro, os dados obtidos de linfócitos periféricos submetidos às diferentes concentrações radioativas de 153Sm-EDTMP (0,046 – 1,110 MBq/mL) de doadores sadios e de pacientes sem prévio tratamento se ajustaram melhor ao modelo de regressão linear (Y=A+BX). O dano cromossômico induzido pelo 153Sm-EDTMP observado in vitro foi cerca de 2 vezes maior do que o encontrado in vivo para o grupo de pacientes sem prévio tratamento. Os dados obtidos mostraram que a terapia com 153Sm-EDTMP induziu uma pequena quantidade de danos citogenéticos em linfócitos periféricos de pacientes 1 hora após sua administração, embora, teoricamente, um efeito estocástico a longo prazo não possa ser descartado. / The 153Sm-EDTMP is a radiopharmaceutical used in nuclear medicine with promising results for the relief of metastatic pain. Therefore, there are few knowledge about the effects of 153Sm-EDTMP at cellular level. The present study was conducted with the aim of evaluating the cytogenetic effects of 153Sm-EDTMP in peripheral lymphocytes from patients with bone metastasis (with and without previous radio and/or chemotherapy) by the chromosome aberration technique, either in vivo or in vitro. For that, the blood samples were collected before and one hour after the endovenous administrations of 153Sm-EDTMP (mean activity of 42.53 + 5.31 MBq/kg body weight), taking into account the rapid blood clearance. The principal types of structural chromosome aberrations found gaps and breaks, acentric fragments centric rings, double minutes and dicentrics. The statistical analysis showed that the group submitted to previous radio and chemotherapy before153Sm-EDTMP administration showed significant difference in chromosome aberrations frequency one hour after the treatment. The analysis of the chromosome modal number and the kinetics of cellular cycle showed no statistical difference among the groups, suggesting that the treatment with 153Sm-EDTMP, did not influence these parameters. The carrier molecule, EDTMP, did not influence the induction of chromosome aberration. In relation to the in vitro assays, the obtained data of peripheral lymphocytes of healthy donors and patients with no previous treatment exposed to different radioactive concentration of 153Sm-EDTMP (0.046 – 1.110 MBq/mL) were better adjusted by linear regression model (Y=A+BX). The chromosome damage induced by 153Sm-EDTMP observed in vitro was about 2 fold higher than that found in vivo for the group of patients with no previous treatment. The obtained data showed that the therapy with 153Sm- EDTMP induced a few quantity of cytogenetic damages in peripheral lymphocytes one hour after its administration in patients, although, theoretically, a long term stochastic effect cannot be disregarded.

O 153Sm-EDTMP é um radiofármaco utilizado em medicina nuclear com resultados promissores no alívio da dor metastática. No entanto, pouco se sabe sobre os efeitos do 153Sm-EDTMP em nível celular. O presente trabalho foi conduzido com o intuito de avaliar os efeitos citogenéticos do 153Sm-EDTMP em linfócitos periféricos de pacientes com metástases ósseas (com e sem radio e/ou quimioterapias anteriores) pela da técnica de detecção de aberrações cromossômicas, tanto in vivo como in vitro. Para tanto, as amostras sangüíneas foram coletadas antes e 1 hora após a administração endovenosa do 153Sm- EDTMP (atividade média de 42,53 + 5,31 MBq/kg de peso corpóreo), levando-se em consideração o rápido clearance sangüíneo. Os principais tipos de aberrações cromossômicas estruturais encontrados foram os gaps e quebras, fragmentos acêntricos, anéis cêntricos, double minutes e dicêntricos. A análise estatística mostrou que o único grupo de pacientes que apresentou uma diferença significativa na freqüência de aberrações cromossômicas 1 hora após o tratamento foi o que recebeu prévio tratamento radio e quimioterápico antes da terapia com 153Sm-EDTMP. Quanto a averiguação do número modal de cromossomos e da cinética do ciclo celular, a análise estatística mostrou que não houve diferença significativa entre os grupos analisados, sugerindo que o tratamento com 153Sm-EDTMP não influenciou nesses parâmetros. A molécula carreadora, EDTMP, não teve qualquer influência na indução de aberrações cromossômicas. Em relação aos ensaios in vitro, os dados obtidos de linfócitos periféricos submetidos às diferentes concentrações radioativas de 153Sm-EDTMP (0,046 – 1,110 MBq/mL) de doadores sadios e de pacientes sem prévio tratamento se ajustaram melhor ao modelo de regressão linear (Y=A+BX). O dano cromossômico induzido pelo 153Sm-EDTMP observado in vitro foi cerca de 2 vezes maior do que o encontrado in vivo para o grupo de pacientes sem prévio tratamento. Os dados obtidos mostraram que a terapia com 153Sm-EDTMP induziu uma pequena quantidade de danos citogenéticos em linfócitos periféricos de pacientes 1 hora após sua administração, embora, teoricamente, um efeito estocástico a longo prazo não possa ser descartado. / The 153Sm-EDTMP is a radiopharmaceutical used in nuclear medicine with promising results for the relief of metastatic pain. Therefore, there are few knowledge about the effects of 153Sm-EDTMP at cellular level. The present study was conducted with the aim of evaluating the cytogenetic effects of 153Sm-EDTMP in peripheral lymphocytes from patients with bone metastasis (with and without previous radio and/or chemotherapy) by the chromosome aberration technique, either in vivo or in vitro. For that, the blood samples were collected before and one hour after the endovenous administrations of 153Sm-EDTMP (mean activity of 42.53 + 5.31 MBq/kg body weight), taking into account the rapid blood clearance. The principal types of structural chromosome aberrations found gaps and breaks, acentric fragments centric rings, double minutes and dicentrics. The statistical analysis showed that the group submitted to previous radio and chemotherapy before153Sm-EDTMP administration showed significant difference in chromosome aberrations frequency one hour after the treatment. The analysis of the chromosome modal number and the kinetics of cellular cycle showed no statistical difference among the groups, suggesting that the treatment with 153Sm-EDTMP, did not influence these parameters. The carrier molecule, EDTMP, did not influence the induction of chromosome aberration. In relation to the in vitro assays, the obtained data of peripheral lymphocytes of healthy donors and patients with no previous treatment exposed to different radioactive concentration of 153Sm-EDTMP (0.046 – 1.110 MBq/mL) were better adjusted by linear regression model (Y=A+BX). The chromosome damage induced by 153Sm-EDTMP observed in vitro was about 2 fold higher than that found in vivo for the group of patients with no previous treatment. The obtained data showed that the therapy with 153Sm- EDTMP induced a few quantity of cytogenetic damages in peripheral lymphocytes one hour after its administration in patients, although, theoretically, a long term stochastic effect cannot be disregarded.

Pain is the most feared symptom of cancer and can impact patients' lives more than the cancer itself. Despite improvements in cancer prevention and detection, pain is often the first sign of cancer, with an estimated 70-75% of advanced stage cancer patients presenting with skeletal metastases. Cancer metastasis to the bone is associated with persistent pain that increases in intensity over time. Current treatments follow the World Health Organization (WHO) analgesic ladder for cancer pain management suggesting non-steroidal anti-inflammatory drugs (NSAIDs) for mild to moderate pain and opioids for moderate to severe pain. However, estimates indicate as many as 50-80% of cancer patients worldwide receive inadequate pain management. Moreover, opioid doses required for these patients are associated with adverse side effects further diminishing quality of life. Development of improved non-opioid therapies is dependent on increased understanding of mechanisms driving cancer pain and its relief. The objective of this dissertation was to characterize a rat model of cancer-induced bone pain, to develop approaches to measure both ongoing and breakthrough pain and to investigate the contribution of underlying inflammatory mechanisms to pain, bone destruction and bone remodeling. Using female Fischer F344/NhSD rats, histocompatible MAT B III mammary adenocarcinoma cells were sealed into the intramedullary space of the right rear tibia for a time course of 13 days. Ongoing pain was characterized based on the WHO 3-step ladder for pain management utilizing novel behavioral and neurochemical assays. Morphine and peripheral nerve block were sufficient to control ongoing pain, whereas NSAID treatment failed to provide pain relief. Cancer-bearing rats selectively displayed movement-induced breakthrough pain to a background of morphine-controlled ongoing pain. Furthermore, we determined that breakthrough pain is initiated, but not maintained, by peripheral afferent input from the tumor-bearing tibia using lidocaine administration prior to or following movement. For the final part of this study, we investigated the role of transient receptor potential vanilloid 1 (TRPV1) and interleukin-6 (IL-6) blockade, as these have been shown to be important mediators in animal models CIBP. Acute blockade of TRPV1 channels by AMG9810 selectively reversed inflammatory-induced pain, but failed to control evoked or ongoing CIBP. Acute blockade of interleukin-6 signaling by TB-2-081, an IL-6 receptor antagonist, successfully reversed evoke pain responses, but like AMG9810, failed to control ongoing pain. Sustained administration of TB-2-081 reversed cancer-induced tactile hypersensitivity and tumor-induced bone remodeling of the tibia. Further in vitro analysis revealed TB-2-081 functions by inhibiting the Jak/STAT cascade on both tumor cells and osteoblasts, suggesting that blockade of IL-6 signaling can effectively modulate the bone microenvironment to reduce tumor burden and pain. Combined, our data introduce a rat model of breast cancer bone metastasis, in which the underlying mechanisms of ongoing and breakthrough CIBP can be effectively studied. From this, novel therapeutic agents can be developed and investigated to help improve quality of life in patients suffering from this disease.

Thesis--Copenhagen. / Summary in Danish. Includes bibliographical references (p. 202-221) and index.

Les métastases osseuses sont des complications fréquentes du cancer du sein, responsables sur le plan clinique d'hypercalcémie, fractures osseuses et douleurs, pour lesquelles, il n'existe que des traitements palliatifs. Les cellules tumorales de carcinomes mammaires qui métastasent au site osseux expriment des gènes qui favorisent le tropisme osseux de ces cellules ainsi que leur ancrage et développement dans la moelle osseuse. Les mécanismes moléculaires sous-jacents à ces processus sont contrôlés par l'expression génique des cellules tumorales qui interagissent avec le microenvironnement et les cellules osseuses. Dans ce contexte, les microARNs en tant que régulateur endogène de l'expression génique, interfèrent avec les différentes étapes de la formation des métastases osseuses, incluant l'échappement des cellules tumorales de la tumeur primaire, la dissémination et l'invasion du site osseux, ainsi que l'apparition de lésions ostéolytiques. Les profils transcriptomiques des microARNs de cellules tumorales mammaires à caractère ostéotropique montrent que l'expression de la famille de microARNs-30s (miRs-30) est inhibée dans ces cellules. En clinique, la faible expression des miRs-30 est associée à un mauvais diagnostique de rechute et au statut hormono-résistant. Dans un modèle animal de métastases osseuses, l'expression forcée des miRs-30 dans des cellules tumorales qui métastasent fortement et spécifiquement à l'os, inhibe la formation des métastases osseuses. Nous montrons que les miRs-30 inhibent l'invasion et stimulent l'ostéoblastogenèse, in vitro et réduisent la charge tumorale et l'ostéoclastogenèse, in vivo. En accord avec ces résultats, l'expression de gènes qui stimulent les métastases osseuses est inhibée par les miRs-30. Parmi ces gènes, l'expression du CTGF (connective tissue growth factor) est augmentée dans les métastases osseuses humaines. Les étapes précoces des métastases osseuses sont étudiées par inoculation de cellules tumorales métastatiques murines dans la glande mammaire de souris. Dans ce modèle, les miRs-30 n'altèrent pas la croissance tumorale et la dissémination métastatique à l'os. Cependant, les miRs-30 inhibent l'invasion et le caractère de cellules souches tumorales de ces cellules métastatiques. Ces résultats suggèrent que les miRs-30, en régulant négativement les métastases osseuses, représentent une thérapie potentielle pour réprimer des gènes cibles qui stimulent les métastases osseuses / Bone metastasis is a common complication of advanced breast cancers and is clinically responsible of bone fractures, hypercalcemia and pain for which only palliative therapies are proposed. Breast tumor cells that preferentially invade bone express a set of deregulated genes that enhance bone tropism and facilitate bone marrow engraftment which may lead to the formation of overt osteolytic lesions. Molecular pathways underlining these steps are regulated through the tight control of genes expressed by cancer cells interacting with cells from the bone microenvironment. In this context, microRNAs act as regulators of gene expression and control multiple aspects of bone metastasis, including tumor cell escape from the primary site, dissemination, invasion of the bone marrow and secondary outgrowth. MicroRNA transcriptomic profiling of osteotropic breast cancer cell lines identified drastic down-regulation of the miR-30 family (miRs-30). In the clinic, low expression of miRs-30 in breast primary tumors is associated with poor distant metastasis-free survival and hormoneinsensitive status. In a model of human bone metastasis in vivo, the forced expression of miRs-30 in a breast cancer cell line that is highly and specifically metastatic to bone inhibited bone metastasis. We demonstrated that miRs-30 inhibit tumor cell invasiveness and stimulate osteoblastogenesis, in vitro, and reduces tumor burden and osteoclast activity, in vivo. Consistent with that, the expression of several genes that promote bone metastasis were inhibited by miRs- 30. Among these, expression of connective tissue growth factor (CTGF) was up-regulated in human bone metastasis. The early steps of bone metastasis were studied in a mouse model using spontaneously metastatic mouse breast cancer cell lines inoculated in the mammary gland. In this model, miRs-30 did not alter tumor growth or metastatic dissemination to bone. However, miRs-30 inhibited cell invasiveness and cancer stem cell-like phenotype of these metastatic cells. We conclude that miRs-30, by interfering negatively with bone metastasis, represent a potential therapy to repress gene targets that promote bone metastasis

Les taux de guérison du cancer du sein sont en progression constante, toutefois l’apparition de métastases osseuses contribue à la morbidité et à la mortalité des femmes à un stade avancé et les traitements actuels ne sont que palliatifs. Afin de prévenir les métastases osseuses, il est essentiel de mieux comprendre les mécanismes moléculaires qui contrôlent les événements cellulaires précédant l'apparition de lésions squelettiques. Dans ce but, une analyse transcriptomique comparative a été réalisée entre une lignée de cellules de cancer du sein humaines MDA-MB-231 qui dissémine dans plusieurs organes sans site préférentiel, et une sous-population ostéotropique, les cellules B02, qui métastasent uniquement dans les os. Par comparaison à la lignée MDA-MB-231, les cellules B02 surexpriment Roundabout 4 (ROBO4), codant un récepteur cellulaire membranaire, appartenant à la famille des récepteurs de guidage axonal Roundabout. Physiologiquement, ROBO4 est impliqué dans l'angiogenèse, l'intégrité des vaisseaux sanguins et la migration des cellules souches hématopoïétiques dans la niche ostéoblastique. Afin de déterminer si ROBO4 peut, également, jouer un rôle de médiateur dans la localisation des cellules tumorales au sein de la moelle osseuse, nous avons invalidé ROBO4 dans les cellules B02 en utilisant une stratégie CRISPR / Cas9. In vivo, l'injection orthotopique de cellules B02 KO ROBO4 a entraîné la formation de tumeurs plus petites par rapport à celles formées par les cellules B02. De même, dans les os, on observe une forte diminution de la croissance tumorale et de l’étendue de l’ostéolyse. Des protocoles à court-terme de formation de micro-métastases ont également montré un rôle de ROBO4 dans les étapes précoces de la colonisation osseuse, probablement en agissant sur l'ancrage et/ou la survie des cellules tumorales. De plus, la colonisation d’une niche ostéoblastique humanisée implantée en sous-cutanée chez des souris NOD / SCID, par les cellules B02 KO ROBO4 a également été nettement réduite en comparaison aux cellules B02. Par conséquent, ROBO4 intervient dans la dissémination osseuse précoce de cellules cancéreuses du sein et dans la formation tumorale. In vitro, l’interaction des cellules B02 KO ROBO4 avec les cellules résidentes de la moelle osseuse est réduite par rapport à celle observée avec les cellules parentales B02. Les co-cultures de cellules B02 ou B02 KO ROBO4 en suspension avec les cellules de la moelle osseuse, conduisent à la formation de mammosphères hétérotypiques. Cependant, leurs tailles sont considérablement réduites suite à l'inhibition de ROBO4. Des résultats similaires sont observés lors de la culture homotypique de cellules B02 ou B02 KO ROBO4. On observe également une importante réduction de l’agrégation cellulaire lorsque ROBO4 est inhibée, suggérant un rôle de ROBO4 dans l’interaction cellulaire. De plus, les cellules B02 KO ROBO4 sont plus petites et plus isolées que les cellules B02 parentales, comme le montre l'analyse du cytosquelette d'actine. Ces résultats nous permettent d’émettre l’hypothèse que ROBO4 est une molécule d’adhérence jouant un rôle important dans la cohésion des cellules tumorales et dans l’interaction des cellules cancéreuses avec les cellules du microenvironnement osseux. De façon intéressante, l’inhibition de ROBO4, par utilisation d’un anticorps anti-ROBO4, réduit également la formation de sphéroïdes et la colonisation osseuse des cellules B02. L’ensemble de ces résultats suggèrent que ROBO4 pourrait être une nouvelle cible thérapeutique dans le traitement de tumeur primitive du sein et des métastases osseuses / The cure rates of breast cancer are steadily increasing; however, the occurrence of bone metastases contributes to the morbidity and mortality of women at an advanced stage and current treatments are only palliative. In order to prevent bone metastasis, it is vital to increase our understanding of the molecular mechanisms that control cellular events preceding the development of overt skeletal lesions. A comparative transcriptional analysis was performed between human MDA-MB-231 breast cancer cells that spread to several organs in animals, and a sub-population of the MDA-MB-231 cell line (B02) that metastasis only to bones. Compared to MDA-MB-231, B02 cells overexpress Roundabout 4 (ROBO4), encoding a cell surface receptor belonging to the Roundabout family of axonal guidance receptors. ROBO4 is involved in angiogenesis, blood vessel integrity and homing of hematopoietic stem cells in the osteoblastic niche. To determine whether ROBO4 could also mediate the homing of tumour cells in the bone marrow, we invalidate ROBO4 in B02 cells using a CRISPR/Cas9 strategy (KO ROBO4 B02). In vivo, orthotopic injection of KO ROBO4 B02 cells leads to smaller primary tumours compared to the ones formed by B02 control cell line. Moreover, in bone, ROBO4 invalidation induces a strong decrease of tumour growth, osteolysis occurrence and micrometastasis formation. Short-term protocols show the involvement of ROBO4 in the early-step of bone colonisation, probably by acting in tumour cell anchorage and/or survival. Furthermore, compared to B02 cells, the homing of KO ROBO4 B02 cells in a humanized osteoblastic niche implanted subcutaneously in NOD/SCID mice is also markedly reduced. Therefore, ROBO4 is involved in the early bone marrow dissemination of breast cancer cells and in tumor formation. According to this result, in vitro, the interaction of KO ROBO4 B02 cells with bone marrow resident cells is decreased, compared to what is observe with parental B02 cells. After the co-culture of B02 or KO ROBO4 cells in suspension with bone marrow resident cells, they both form heterotypic mammospheres. However, their size is substantially reduced in the absence of ROBO4. Similar findings were observed in homotypic culture of B02 or KO ROBO4 cells. An important decrease of tumor cell aggregation is also observed when ROBO4 is inhibited, suggesting a role of ROBO4 in cell interaction. Moreover, KO ROBO4 B02 cells are smaller and more isolated than parental B02 cells, as shown in actin cytoskeletal analysis. Altogether, these results provide strong evidence that ROBO4 is an adherent molecule which plays a crucial role in tumour cell cohesion and in the interaction of cancer cells with bone cells component. Interestingly, ROBO4 inhibition, using an anti-ROBO4 antibody, also reduces spheroid formation and bone colonization of B02 cells. Taken together, these results suggest ROBO4 as a new potential therapeutic target to treat primary breast tumours and bone metastasis

No description available.

La périostine est une protéine matricellulaire préférentiellement exprimée aux sites de contraintes mécaniques, notamment le périoste, et dans le stroma associé à de nombreux types de cancers. En premier lieu, nous nous sommes attachés à évaluer la pertinence de cette protéine en tant que biomarqueur du métabolisme osseux et de la réaction stromale dans les métastases osseuses. Nous avons développé le premier dosage ELISA de la périostine circulante chez la souris présentant des caractéristiques analytiques (spécificité, précision) conformes aux exigences réglementaires. Ce dosage nous a permis de préciser l’implication de la périostine dans le métabolisme osseux et les métastases osseuses de cancer du sein. Nos données in vitro et in vivo suggèrent que la périostine n’est pas un indice direct du remodelage osseux, contrairement aux marqueurs biologiques conventionnels, mais une composante de l’ossification primaire. Nous avons aussi montré dans les métastases osseuses d’origine mammaire que la périostine est surexprimée par les cellules stromales de la métastase, comme cela a pu être observé au niveau des tumeurs primaires. Enfin, nous avons confirmé par une approche bioinformatique la relation étroite entre périostine et réaction stromale dans la plupart des tumeurs chez l’Homme. La périostine et d’autres protéines conjointement exprimées pourraient donc constituer un panel de marqueurs biologiques de la progression tumorale, certains pouvant se révéler comme nouvelles cibles thérapeutiques en oncologie. / Periostin is a matricellular protein preferentially expressed at sites subjected to mechanical constraints, including the periosteum, and in the stroma associated to several tumor types. We first aimed to evaluate the relevance of periostin as a biomarker of bone metabolism or stromal reaction in bone metastases. We developed the first ELISA for serum periostin in mouse with analytical characteristics (specificity, precision) that are in accordance with regulatory standards. This ELISA allowed us to specify further the involvement of periostin in bone metabolism and breast cancer bone metastases. Our in vitro and in vivo data suggested that periostin is a component of primary ossification rather than a direct index of bone remodeling, unlike conventional bone markers. In breast cancer bone metastases, we also showed that periostin is overexpressed by stromal cells associated with bone metastasis, in agreement with its localization in the stroma of primary tumors. Finally, using bioinformatics analyses of large datasets from various tumors in human, we confirmed the close relationship between periostin and the stromal reaction. Periostin and other co-expressed proteins could therefore constitute a set of biological markers of cancer progression, and/or appear as potential therapeutic targets.

This study focused on the role of RANKL in prostate cancer EMT progression and metastasis. Activation of RANK, a receptor activator of NF-kB, by its ligand RANKL, in a paracrine manner is responsible for osteoclast differentiation and bone remodeling. RANK activation in cancer cells, however, is thought to be promoted by both autocrine and paracrine mechanisms because RANKL has been shown to be derived from either tumor or its microenvironment, such as osteoblasts, infiltrating inflammatory cells and stromal fibroblasts. In the present study, we demonstrated that autocrine and paracrine RANKL-RANK signaling could be responsible for driving prostate cancer bone metastasis by promoting epithelial to mesenchymal transition (EMT). We further characterized a novel converging RANKL-c-Met signaling network in which the activation of RANKL was found to promote the expression of both RANKL and c-Met in an autocrine manner in prostate cancer cells. The induced RANKL and c-Met in prostate cancer cells is biologically functional and contributes to increased osteoclastogenesis, epithelial to mesenchymal transition (EMT), cell motility, migration and invasion and conferred bone and soft tissue metastases. Remarkably, RANKL expression by 1,000 prostate cancer cells can provoke bone and soft tissue metastases of a “dormant” population of prostate cancer cells which by themselves failed to form tumors and colonize mouse skeleton, suggesting RANKL can serve as a factor in “reawakening” cancer dormancy to initiate the re-growth and metastasis of cancer cells. We also showed that RANKL-induced RANKL feed-forward autocrine regulation is mediated through cMyc transactivation, allowing the establishment of a “vicious cycle” further promoting prostate cancer growth and metastasis. The converging RANKL-c-Met signaling network is therefore a novel target that could be further manipulated for delaying the lethal progression of castration-resistant human prostate cancer bone metastasis.