RESUMO
Bone metastasis is a key event responsible for morbidity in prostate cancer patients. Interactions between prostate cancer cells and the bone microenvironment facilitate survival of tumor cells and alter bone turnover, a process that is thought to enhance the growth of metastases in this site. This study aimed to test the hypothesis that the presence of tumors cells increases transforming growth factor beta (TGF-ß) signaling in bone and that this regulates the proliferation and differentiation of osteoblastic lineage cells in metastatic sites. Initial studies showed that factors produced by prostate cancer cells increased the proliferation of osteoblastic cells and suppressed the early phase of their differentiation. We subsequently showed that interactions between prostate cancer and osteoblastic cells affected the expression of TGF-ß superfamily genes in the latter. Noggin was expressed and secreted by prostate cancer cells but expressed at very low levels by osteoblastic cells when these cells were grown alone. This pattern changed when osteoblasic cells were treated with conditioned medium derived from prostate cancer cells or were cocultured with the latter, with strong induction of Noggin being demonstrated. Immunohistochemical examination of prostate cancer xenografts showed strong Noggin protein staining on endosteal bone surfaces and in bone lining cells in close proximity to tumor foci. These studies support previous work that suggest Noggin is an important suppressor of the differentiation of osteoblast lineage cells in bone metastases. Importantly, we have now also shown that this protein can be induced in bone cells themselves by factors derived from prostate cancer cells.
Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas de Transporte/biossíntese , Osteoblastos/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Diferenciação Celular , Proliferação de Células , Humanos , Masculino , Fator de Crescimento Transformador beta1/metabolismo , Células Tumorais CultivadasRESUMO
Breast cancer cells colonize the skeleton by homing to specific niches, but the involvement of osteoblasts in tumour cell seeding, colonization, and progression is unknown. We used an in vivo model to determine how increasing the number of cells of the osteoblast lineage with parathyroid hormone (PTH) modified subsequent skeletal colonization by breast cancer cells. BALB/c nude mice were injected for five consecutive days with PBS (control) or PTH and then injected with DiD-labelled breast cancer cells via the intra-cardiac route. Effects of PTH on the bone microenvironment and tumour cell colonization and growth was analyzed using bioluminescence imaging, two-photon microscopy, and histological analysis. PTH treatment caused a significant, transient increase in osteoblast numbers compared to control, whereas bone volume/structure in the tibia was unaffected. There were no differences in the number of tumour cells seeding to the tibias, or in the number of tumours in the hind legs, between the control and PTH group. However, animals pre-treated with PTH had a significantly higher number of tumour colonies distributed throughout skeletal sites outside the hind limbs. This is the first demonstration that PTH-induced stimulation of osteoblastic cells may result in alternative skeletal sites becoming available for breast cancer cell colonization.
Assuntos
Osso e Ossos/efeitos dos fármacos , Neoplasias da Mama/patologia , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Animais , Apoptose/efeitos dos fármacos , Osso e Ossos/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência por Excitação Multifotônica , Tíbia/efeitos dos fármacos , Tíbia/patologia , Transplante HeterólogoRESUMO
This study aimed to identify subpopulations of prostate cancer cells that are responsible for the initiation of bone metastases. Using rapidly dividing human prostate cancer cell lines, we identified mitotically quiescent subpopulations (<1%), which we compared with the rapidly dividing populations for patterns of gene expression and for their ability to migrate to the skeletons of athymic mice. The study used 2-photon microscopy to map the presence/distribution of fluorescently labeled, quiescent cells and luciferase expression to determine the presence of growing bone metastases. We showed that the mitotically quiescent cells were very significantly more tumorigenic in forming bone metastases than fast-growing cells (55 vs. 15%) and had a unique gene expression profile. The quiescent cells were not uniquely stem cell like, with no expression of CD133 but had the same level expression of other putative prostate stem cell markers (CD44 and integrins α2/ß1), when compared to the rapidly proliferating population. In addition, mitotic quiescence was associated with very high levels of C-X-C chemokine receptor type 4 (CXCR4) production. Inhibition of CXCR4 activity altered the homing of quiescent tumor cells to bone. Our studies suggest that mitotic dormancy is a unique phenotype that facilitates tumor cell colonization of the skeleton in prostate cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/patologia , Mitose/fisiologia , Neoplasias da Próstata/patologia , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Neoplasias Ósseas/metabolismo , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Cadeias alfa de Integrinas/metabolismo , Cadeias beta de Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Peptídeos/metabolismo , Fenótipo , Neoplasias da Próstata/metabolismo , Receptores CXCR4/metabolismo , Células Tumorais CultivadasRESUMO
Dormant disseminated tumour cells can be detected in the bone marrow of breast cancer patients several years after resection of the primary tumour. The majority of these patients will remain asymptomatic, however, â¼ 15% will go on to develop overt bone metastases and this condition is currently incurable. The reason why these dormant cells are stimulated to proliferate and form bone tumours in some patients and not others remains to be elucidated. We have recently shown that in an in vivo model, increasing bone turnover by ovariectomy stimulated proliferation of disseminated tumour cells, resulting in formation of bone metastasis. We now show for the first time that osteoclast mediated mechanisms induce growth of tumours from dormant MDA-MB-231 cells disseminated in the bone. We also show that disruption of RANK-RANKL interactions following administration of OPG-Fc inhibits growth of these dormant tumour cells in vivo. Our data support early intervention with anti-resorptive therapy in a low-oestrogen environment to prevent development of bone metastases.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Neoplasias da Mama/patologia , Osteoprotegerina/administração & dosagem , Ovariectomia/efeitos adversos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/cirurgia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos , Osteoclastos , Osteoprotegerina/imunologia , Ligante RANK/metabolismoRESUMO
Micrometastasis is a barrier to the development of effective cancer therapies for prostate cancer metastasis to bone. The mechanisms remain incompletely characterised, primarily due to an inability to adequately monitor the initial metastatic events in vivo. This study aimed to establish a new model, allowing the tracking of prostate cancer cells homing to bone, and furthermore, to evaluate the response of this approach to therapeutic modulation, using the integrin antagonist GLPG0187. A single murine metatarsal was engrafted into a dorsal skinfold chamber implanted on a SCID mouse. Fluorescently-labeled human prostate (PC3-GFP) or oral (SCC4-GFP) cancer cells were administered via intracardiac (i.c) injection, with simultaneous daily GLPG0187 or vehicle-control treatment (i.p. 100 mg/kg/day) for the experimental duration. Metatarsal recordings were taken every 48 h for up to 4 weeks. Tissue was harvested and processed for microCT, multiphoton analysis, histology and immunohistochemistry. Cell viability, proliferation and migration in vitro were also quantified following treatment with GLPG0187. Metatarsals rapidly revascularised by inosculation with the host vasculature (day 5-7). PC3-GFP cells adhered to the microvascular endothelium and/or metatarsal matrix 3 days after administration, with adhesion maintained for the experimental duration. GLPG0187 treatment significantly (p < 0.05) reduced PC3 cell number within the metatarsal in vivo and reduced migration (p < 0.05) and proliferation (p < 0.05) but not cell viability in vitro. This new model allows evaluation of the early events of tumour-cell homing and localisation to the bone microenvironment, in addition to determining responses to therapeutic interventions.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/secundário , Integrinas/antagonistas & inibidores , Neoplasias da Próstata/patologia , Animais , Antineoplásicos/administração & dosagem , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The number of cells positive for the α-6 and α-2 integrin subunits and the c-Met receptor in primary tumors and bone biopsies from prostate cancer patients has been correlated with metastasis and disease progression. The objective of this study was to quantify disseminated tumour cells present in bone marrow in prostate cancer patients using specific markers and determine their correlation with metastasis and survival. METHODS: Patients were included at different stage of prostate cancer disease, from localised to metastatic castration-resistant prostate cancer. Healthy men were used as a control group. Bone marrow samples were collected and nucleated cells separated. These were stained for CD45, α-2, α-6 integrin subunits and c-Met and samples were processed for analysis and quantification of CD45-/α2+/α6+/c-met + cells using flow cytometry. Clinical and pathological parameters were assessed and survival measured. Statistical analyses were made of associations between disease specific parameters, bone marrow flow cytometry data, prostate-specific antigen (PSA) progression free survival and bone metastases progression free survival. RESULTS: For all markers, the presence of more than 0.1% positive cells in bone marrow aspirates was significantly associated with the risk of biochemical progression, the risk of developing metastasis and death from prostate cancer. CONCLUSIONS: Quantification of cells carrying putative stem cell markers in bone marrow is a potential indicator of disease progression. Functional studies on isolated cells are needed to show more specifically their property for metastatic spread in prostate cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Medula Óssea/metabolismo , Neoplasias Ósseas/secundário , Progressão da Doença , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Adulto , Biópsia , Medula Óssea/patologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/mortalidade , Estudos de Casos e Controles , Humanos , Integrina alfa2/metabolismo , Integrina alfa6/metabolismo , Estimativa de Kaplan-Meier , Antígenos Comuns de Leucócito/metabolismo , Masculino , Metástase Neoplásica/patologia , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/mortalidade , Proteínas Proto-Oncogênicas c-met/metabolismo , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
BACKGROUND: A number of putative stem cell markers have been associated with aggressiveness of prostate cancer, including alpha 2 and alpha 6 integrin and c-met. The study aimed to test the hypothesis that the development of bone metastasis correlates with the proportion of prostate cancer stem cell-like cells present in the primary tumor. METHODS: Prostate tissue samples were obtained from patients with high-risk prostatic adenocarcinoma. Prostate cancer tumor tissue samples underwent immunohistochemical staining for alpha 2 and alpha 6 integrin and c-met; positive and negative controls were included. Samples were scored as positive if >5% of cells within the sample stained positively. Survival and bone metastasis-free survival curves on the patient cohort were estimated by the actuarial method of Kaplan-Meier. RESULTS: A total of 62 patients were included in the study. Bone metastases progression rate was 46% at 105 months with a median time of 46 months (95% CI: 1-62.5 months); prostate cancer-specific survival was 33% at 122 months with a median survival time of 69.4 months (95% CI: 63.5-109.4 months). Survival curves show that c-met-, alpha 2, and alpha 6 integrin-positive tumors were positively associated with the occurrence of bone metastasis-free survival. There was a higher level of significance when at least c-met and either alpha 2 or alpha 6 integrin was positive. CONCLUSION: It can be concluded that percentage of stem cell-like prostate cancer cells has a prognostic impact especially on the risk of metastatic bone progression.
Assuntos
Adenocarcinoma/secundário , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/secundário , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/cirurgia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Neoplasias Ósseas/metabolismo , Estudos de Coortes , Progressão da Doença , Intervalo Livre de Doença , Humanos , Integrina alfa2/análise , Integrina alfa6/análise , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/cirurgia , Proteínas Proto-Oncogênicas c-met/análiseRESUMO
BACKGROUND: Tumour cells with a stem cell-like phenotype have recently been identified in prostate tumors and it has been suggested that this population may be responsible for the diversity of cell types within tumors and also for the initiation of metastases. These cells carry a number of defined markers: they are cd133 and cd44+ve and express high levels of alpha2beta1 integrin. In this study we have, for the first time, assessed matched primary and bone marrow biopsies from prostate cancer patients for the distribution of cells carrying these and a number of other putative stem cell markers. METHODS: Eleven matched (primary and bone metastasis) specimens from prostate cancer patients were assessed for the presence of cd133, cd44, alpha2beta1 integrin, CXCR4, c-met, alpha6 integrin, and nestin using immunohistochemistry and stain intensity and distribution scored. RESULTS: In the bone metastases, tumor cells staining positively for cd133 were detected at low frequency in approximately 50% of samples. Staining for nestin was confined to endothelium. Positive staining of tumor cells for the other antigens was present at variable frequency in >70% of metastases with the exception of CXCR4 which was absent from all but 2 specimens. Where positive staining of tumor cells was present in the metastasis, cells staining for each antigen were present in the matched primary with the exception of cd44 which was absent in all but 2/11 matched primary tissues. CONCLUSIONS: In established metastases no single or combination of marker expression profiles identify the established metastatic phenotype, although cd44 expression was shown to be more frequent in metastases that in primary cancers.
Assuntos
Adenocarcinoma/secundário , Neoplasias Ósseas/secundário , Células-Tronco Neoplásicas/citologia , Neoplasias da Próstata/patologia , Adenocarcinoma/metabolismo , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/metabolismo , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Contagem de Células , Humanos , Imuno-Histoquímica , Integrinas/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Células-Tronco Neoplásicas/patologia , Proteínas do Tecido Nervoso/metabolismo , Nestina , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismoRESUMO
BACKGROUND: Due to the heterogeneity in the biological behavior of prostate cancer, biomarkers that can reliably distinguish indolent from aggressive disease are urgently needed to inform treatment choices. METHODS: We employed 8-plex isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to profile the proteomes of two distinct panels of isogenic prostate cancer cells with varying growth and metastatic potentials, in order to identify novel biomarkers associated with progression. The LNCaP, LNCaP-Pro5, and LNCaP-LN3 panel of cells represent a model of androgen-responsive prostate cancer, while the PC-3, PC-3M, and PC-3M-LN4 panel represent a model of androgen-insensitive disease. RESULTS: Of the 245 unique proteins identified and quantified (>or=95% confidence; >or=2 peptides/protein), 17 showed significant differential expression (>or=+/-1.5), in at least one of the variant LNCaP cells relative to parental cells. Similarly, comparisons within the PC-3 panel identified 45 proteins to show significant differential expression in at least one of the variant PC-3 cells compared with parental cells. Differential expression of selected candidates was verified by Western blotting or immunocytochemistry, and corresponding mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Immunostaining of prostate tissue microarrays for ERp5, one of the candidates identified, showed a significant higher immunoexpression in pre-malignant lesions compared with non-malignant epithelium (P < 0.0001, Mann-Whitney U-test), and in high Gleason grade (4-5) versus low grade (2-3) cancers (P < 0.05). CONCLUSIONS: Our study provides proof of principle for the application of an 8-plex iTRAQ approach to uncover clinically relevant candidate biomarkers for prostate cancer progression.
Assuntos
Antígeno Prostático Específico/genética , Neoplasias da Próstata/patologia , Animais , Western Blotting , Progressão da Doença , Variação Genética , Proteínas de Choque Térmico/genética , Histonas/genética , Humanos , Imuno-Histoquímica , Incidência , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias da Próstata/epidemiologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/mortalidade , Receptores de Estrogênio/análise , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , Transcetolase/genética , Células Tumorais CultivadasRESUMO
Factors that aid survival of prostate cancer cells in the presence of the various categories of cytotoxic cytokines present in tumors in vivo are largely unknown. Osteoprotegerin (OPG) is a decoy receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that inhibits TRAIL-induced apoptosis. In relation to this activity, we hypothesized that the ability to produce OPG by prostate cancer cells would confer a survival advantage on these cells. In this study we have demonstrated that high levels of OPG are produced by the hormone-insensitive prostate cancer cell lines PC3 and Du145, whereas the hormone-sensitive cell line LNCaP produced 10-20-fold less OPG under the same conditions. A strong negative correlation was observed between levels of endogenously produced OPG in the medium and the capacity of TRAIL to induce apoptosis in cells that produced high levels of OPG. The antiapoptotic effect of OPG was reversed by coadministration of 100-fold molar excess of receptor-activator of nuclear factor-kappaB ligand, another protein that selectively binds OPG. These observations suggest that prostate cancer-derived OPG may be an important survival factor in hormone-resistant prostate cancer cells.
Assuntos
Glicoproteínas/biossíntese , Neoplasias da Próstata/metabolismo , Receptores Citoplasmáticos e Nucleares/biossíntese , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Sobrevivência Celular/fisiologia , Interações Medicamentosas , Glicoproteínas/imunologia , Glicoproteínas/farmacologia , Glicoproteínas/fisiologia , Humanos , Masculino , Glicoproteínas de Membrana/farmacologia , Osteoprotegerina , Neoplasias da Próstata/patologia , Receptores Citoplasmáticos e Nucleares/imunologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: While both preclinical and clinical studies suggest that the frequency of growing skeletal metastases is elevated in individuals with higher bone turnover, it is unclear whether this is a result of increased numbers of tumour cells arriving in active sites or of higher numbers of tumour cells being induced to divide by the bone micro-environment. Here we have investigated how the differences in bone turnover affect seeding of tumour cells and/or development of overt osteolytic bone metastasis using in vivo models of hormone-independent breast and prostate cancer. METHODS: Cohorts of 6 (young) and 16 (mature)-week old BALB/c nude mice were culled 1, 7 and 21 days after received intracardiac injection of luciferase expressing human prostate (PC3) or breast cancer (MDA-MB-231) cell lines labelled with a fluorescent cell membrane dye (Vybrant DiD). The presence of growing bone metastases was determined by bioluminescence using an in vivo imaging system (IVIS) and followed by anatomical confirmation of tumour metastatic sites post mortem, while the presence of individual fluorescently labelled tumour cells was evaluated using two-photon microscopy ex vivo. The bone remodelling activities were compared between young and mature naïve mice (both male and female) using micro-CT analysis, ELISA and bone histomorphometry. RESULTS: Both prostate and breast cancer cells generated higher numbers of overt skeletal lesions in young mice (~80%) than in mature mice (~20%). Although mature mice presented with fewer overt bone metastases, the number of tumour cells arriving/colonizing in the tibias was comparable between young and mature animals. Young naïve mice had lower bone volume but higher bone formation and resorption activities compared to mature animals. CONCLUSIONS: Our studies suggest that higher frequencies of growing osteolytic skeletal metastases in these models are linked to increased bone turnover and not to the initial number of tumour cells entering the bone microenvironment.
Assuntos
Neoplasias Ósseas/patologia , Neoplasias da Mama/patologia , Células Neoplásicas Circulantes/patologia , Osteólise/patologia , Neoplasias da Próstata/patologia , Animais , Neoplasias Ósseas/secundário , Contagem de Células , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Transplante HeterólogoRESUMO
Multiple myeloma is largely incurable, despite development of therapies that target myeloma cell-intrinsic pathways. Disease relapse is thought to originate from dormant myeloma cells, localized in specialized niches, which resist therapy and repopulate the tumour. However, little is known about the niche, and how it exerts cell-extrinsic control over myeloma cell dormancy and reactivation. In this study, we track individual myeloma cells by intravital imaging as they colonize the endosteal niche, enter a dormant state and subsequently become activated to form colonies. We demonstrate that dormancy is a reversible state that is switched 'on' by engagement with bone-lining cells or osteoblasts, and switched 'off' by osteoclasts remodelling the endosteal niche. Dormant myeloma cells are resistant to chemotherapy that targets dividing cells. The demonstration that the endosteal niche is pivotal in controlling myeloma cell dormancy highlights the potential for targeting cell-extrinsic mechanisms to overcome cell-intrinsic drug resistance and prevent disease relapse.
Assuntos
Remodelação Óssea/fisiologia , Mieloma Múltiplo/metabolismo , Osteoclastos/fisiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Pessoa de Meia-Idade , Osteoblastos/fisiologiaRESUMO
UNLABELLED: Tumor-derived OPG has recently been shown to protect prostate cancer cells from apoptosis. This study has confirmed that bone marrow stromal cell-derived OPG also suppresses cytokine-induced apoptosis in this tumor type, suggesting that it may be the presence of bone-derived OPG that is responsible for the observed preference of these cells in colonizing the skeleton. INTRODUCTION: Metastasis to the skeleton occurs in around 70% of patients with advanced prostate cancer (CaP), suggesting that the bone microenvironment may provide factors that favor the growth and survival of prostate cancer cells. Osteoprotegerin (OPG) is a molecule involved in bone remodeling, where it acts as an inhibitor of osteoclastogenesis, but it is also a decoy receptor for TRAIL/Apo 2L, a member of the TNF family of pro-apoptotic cytokines. The aim of this study was to determine whether OPG produced by human bone marrow stromal cells could protect prostate cancer cells from TRAIL-induced apoptosis. MATERIALS AND METHODS: Human bone marrow stromal cell cultures were generated from bone biopsies taken from newly diagnosed untreated CaP patients with (M1) or without (M0) bony metastasis. The stromal origin of these cells was confirmed by Western blot analysis using antibodies raised to stromal and epithelial markers. Media were conditioned over the cultures of these cells for 4 days, and levels of OPG were determined using an ELISA. The human prostate cancer cell line PC3 was challenged with TRAIL (50 ng/ml) in fresh media or in media supplemented with 50% conditioned media, and apoptosis was assessed using DAPI stain. The effects of specific removal of OPG activity by immunoprecipitation or by co-treatment of cultures with an alternative ligand for OPG (RANKL) were also tested. RESULTS AND CONCLUSIONS: The presence of stromal cell conditioned media in PC3 culture significantly reduced TRAIL-induced apoptosis. All stromal cell lines isolated were shown to express OPG and to release this protein into the conditioned media. Immunoprecipitation of OPG and co-treatment of cultures with sRANKL reversed the protective effects of the conditioned media. These data suggest that at least part of the survival advantage gained by CaP cells in colonizing bone may be caused by the production of OPG by tumor-associated stromal cells.
Assuntos
Apoptose , Células da Medula Óssea/fisiologia , Glicoproteínas/metabolismo , Neoplasias da Próstata/patologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Células Estromais/fisiologia , Proteínas Reguladoras de Apoptose , Neoplasias Ósseas/prevenção & controle , Neoplasias Ósseas/secundário , Proteínas de Transporte/farmacologia , Células Cultivadas , Humanos , Imuno-Histoquímica , Masculino , Glicoproteínas de Membrana/farmacologia , Osteoprotegerina , Ligante RANK , RNA Mensageiro/metabolismo , Receptor Ativador de Fator Nuclear kappa-B , Receptores do Fator de Necrose Tumoral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Metastasis to bone is a common feature in advanced prostate cancer patients. Current treatments, while effective in suppressing tumour growth and relieving tumour associated bone pain, do not provide long term remission or 'cure' for the disease. A greater understanding of prostate cancer metastasis is required if new treatment strategies are to be developed. Growth of tumour foci in skeletal sites is a major cause of morbidity in advanced prostate cancer and has required the development of specialised approaches to treatment, including the use of bisphosphonates. These drugs inhibit tumour induced osteoclastic bone resorption, thereby preventing skeletal related events and treatment induced bone loss. Zoledronic acid is currently the only bisphosphonate with proven benefit in prostate cancer. Bisphosphonates may also modify the bone microenvironment so that it becomes less favourable for the growth and survival of metastases. The most recent developments in our understanding of the advantages for growth and survival gained by metastatic prostate cancer cells in the skeleton are reviewed, along with the clinical evidence supporting the use of bisphosphonates in advanced prostate cancer.
Assuntos
Neoplasias Ósseas/fisiopatologia , Neoplasias Ósseas/secundário , Difosfonatos/uso terapêutico , Neoplasias da Próstata/patologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Glicoproteínas/metabolismo , Humanos , Interleucina-6/metabolismo , Masculino , Osteoprotegerina , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/fisiopatologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Up to 90% of patients with castrate-resistant prostate cancer develop bone metastases, and the majority of these men have received androgen deprivation therapy known to cause bone loss. Whether this treatment-induced change to the bone microenvironment affects disseminated tumour cells, potentially stimulating development of bone metastasis, remains to be determined. The objective of this study was to use an in vivo model mimicking androgen ablation to establish the effects of this intervention on disseminated prostate cancer cells in bone. We mimicked the effects of androgen deprivation on bone metastasis by castrating 12-week-old BALB/c nude mice that had disseminated, hormone-insensitive PC3 prostate cancer cells present in the long bones. Castration caused increased bone resorption and loss of bone volume, compared with sham operation. In addition, castration triggered growth of disseminated PC3 cells to form bone metastasis in 70% of animals. In contrast, only 10% of sham-operated animals had detectable long bone tumours. Weekly administration of 100âµg/kg zoledronic acid (ZOL) prevented castration-induced tumour growth in bone and increased bone volume, but did not eliminate the disseminated tumour cells. ZOL had no effect on tumour growth in the sham-operated animals, despite causing a significant increase in bone volume. This is the first demonstration that, in a model of prostate cancer bone metastasis, mimicking androgen ablation results in growth of disseminated tumour cells in bone through osteoclast-mediated mechanisms. We provide the first biological evidence supporting the administration of ZOL to prostate cancer patients at the time of androgen ablation to prevent subsequent relapse in bone.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Neoplasias Ósseas/secundário , Reabsorção Óssea/prevenção & controle , Castração , Difosfonatos/farmacologia , Imidazóis/farmacologia , Neoplasias da Próstata/patologia , Animais , Reabsorção Óssea/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos Endogâmicos BALB C , Carga Tumoral , Ácido ZoledrônicoRESUMO
PURPOSE: Clinical trials in early breast cancer have suggested that benefits of adjuvant bone-targeted treatments are restricted to women with established menopause. We developed models that mimic pre- and postmenopausal status to investigate effects of altered bone turnover on growth of disseminated breast tumor cells. Here, we report a differential antitumor effect of zoledronic acid (ZOL) in these two settings. EXPERIMENTAL DESIGN: Twleve-week-old female Balb/c-nude mice with disseminated MDA-MB-231 breast tumor cells in bone underwent sham operation or ovariectomy (OVX), mimicking the pre- and postmenopausal bone microenvironment, respectively. To determine the effects of bone-targeted therapy, sham/OVX animals received saline or 100 µg/kg ZOL weekly. Tumor growth was assessed by in vivo imaging and effects on bone by real-time PCR, micro-CT, histomorphometry, and measurements of bone markers. Disseminated tumor cells were detected by two-photon microscopy. RESULTS: OVX increased bone resorption and induced growth of disseminated tumor cells in bone. Tumors were detected in 83% of animals following OVX (postmenopausal model) compared with 17% following sham operation (premenopausal model). OVX had no effect on tumors outside of bone. OVX-induced tumor growth was completely prevented by ZOL, despite the presence of disseminated tumor cells. ZOL did not affect tumor growth in bone in the sham-operated animals. ZOL increased bone volume in both groups. CONCLUSIONS: This is the first demonstration that tumor growth is driven by osteoclast-mediated mechanisms in models that mimic post- but not premenopausal bone, providing a biologic rationale for the differential antitumor effects of ZOL reported in these settings. Clin Cancer Res; 20(11); 2922-32. ©2014 AACR.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Neoplasias Ósseas/secundário , Osso e Ossos/efeitos dos fármacos , Neoplasias da Mama/patologia , Microambiente Celular , Difosfonatos/farmacologia , Imidazóis/farmacologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Confocal , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Ovariectomia , Pós-Menopausa/efeitos dos fármacos , Pós-Menopausa/metabolismo , Pré-Menopausa/efeitos dos fármacos , Pré-Menopausa/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Microtomografia por Raio-X , Ensaios Antitumorais Modelo de Xenoenxerto , Ácido ZoledrônicoRESUMO
It has been suggested that metastasis-initiating cells gain a foothold in bone by homing to a metastastatic microenvironment (or "niche"). Whereas the precise nature of this niche remains to be established, it is likely to contain bone cell populations including osteoblasts and osteoclasts. In the mouse tibia, the distribution of osteoblasts on endocortical bone surfaces is non-uniform, and we hypothesize that studying co-localization of individual tumor cells with resident cell populations will reveal the identity of critical cellular components of the niche. In this study, we have mapped the distribution of three human prostate cancer cell lines (PC3-NW1, LN-CaP, and C4 2B4) colonizing the tibiae of athymic mice following intracardiac injection and evaluated their interaction with potential metastatic niches. Prostate cancer cells labeled with the fluorescent cell membrane dye (Vybrant DiD) were found by two-photon microscopy to be engrafted in the tibiae in close proximity (â¼40 µm) to bone surfaces and 70% more cancer cells were detected in the lateral compared to the medial endocortical bone regions. This was associated with a 5-fold higher number of osteoblasts and 7-fold higher bone formation rate on the lateral endocortical bone surface compared to the medial side. By disrupting cellular interactions mediated by the chemokine (C-X-C motif) receptor 4 (CXCR4)/chemokine ligand 12 (CXCL12) axis with the CXCR4 inhibitor AMD3100, the preferential homing pattern of prostate cancer cells to osteoblast-rich bone surfaces was disrupted. In this study, we map the location of prostate cancer cells that home to endocortical regions in bone and our data demonstrate that homing of prostate cancer cells is associated with the presence and activity of osteoblast lineage cells, and suggest that therapies targeting osteoblast niches should be considered to prevent development of incurable prostate cancer bone metastases.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias Experimentais/metabolismo , Osteoblastos/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Quimiocina CXCL12/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Neoplasias Experimentais/patologia , Osteoblastos/patologia , Neoplasias da Próstata/patologia , Receptores CXCR4/metabolismoRESUMO
A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (nâ=â5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer 'BPH', (ii) localised cancer with no evidence of progression, 'non-progressing' (iii) localised cancer with evidence of biochemical progression, 'progressing', and (iv) bone metastasis at presentation 'metastatic'. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and 'panels' of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (pâ=â0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/metabolismo , Osteossarcoma/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Proteômica , Espectrometria de Massas em Tandem , Idoso , Biomarcadores Tumorais/genética , Western Blotting , Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Progressão da Doença , Humanos , Técnicas Imunoenzimáticas , Masculino , Gradação de Tumores , Osteossarcoma/genética , Osteossarcoma/secundário , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Estudos Prospectivos , Antígeno Prostático Específico/sangue , Hiperplasia Prostática/genética , Hiperplasia Prostática/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais CultivadasRESUMO
Metastatic progression of advanced prostate cancer is a major clinical problem. Identifying the cell(s) of origin in prostate cancer and its distant metastases may permit the development of more effective treatment and preventive therapies. In this study, aldehyde dehydrogenase (ALDH) activity was used as a basis to isolate and compare subpopulations of primary human prostate cancer cells and cell lines. ALDH-high prostate cancer cells displayed strongly elevated clonogenicity and migratory behavior in vitro. More strikingly, ALDH-high cells readily formed distant metastases with strongly enhanced tumor progression at both orthotopic and metastatic sites in preclinical models. Several ALDH isoforms were expressed in human prostate cancer cells and clinical specimens of primary prostate tumors with matched bone metastases. Our findings suggest that ALDH-based viable cell sorting can be used to identify and characterize tumor-initiating and, more importantly perhaps, metastasis-initiating cells in human prostate cancer.
Assuntos
Aldeído Desidrogenase/metabolismo , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/secundário , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Animais , Biomarcadores Tumorais/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Progressão da Doença , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Próstata/enzimologia , Próstata/patologiaRESUMO
Prostate cancer (PCa) is the most common cancer diagnosis and the second most common cause of cancer-related deaths in men. Currently, serum prostate-specific antigen (PSA) is the only biomarker widely used in the diagnosis and management of patients with PCa. However, PSA lacks diagnostic sensitivity and specificity, leading to false-negative and false-positive test results. PSA cannot distinguish indolent from aggressive disease, leading to many patients being over-treated with associated side-effects. Furthermore, PSA is unable to identify which tumors are likely to become unresponsive to treatment at an early stage. Thus, there is an urgent need for clinically validated biomarkers which will improve the diagnosis and management of PCa. Given the heterogeneity of PCa it is likely that a panel of biomarkers will be required. In the quest for PCa biomarkers, a wide range of samples including urine, serum, tissues, and cell lines have been studied using proteomic approaches such as 2-DE, SELDI-TOF, SILAC, ICAT, iTRAQ, and MALDI-IMS. The value of these technologies, and other emerging platforms such as selected reaction monitoring (SRM) and multiple reaction monitoring (MRM), are discussed in the context of biomarker discovery, validation and addressing the "bottle-necks" that exist prior to clinical translation.