RESUMO
Quiescent prostate cancer (PCa) cells are common in tumors but are often resistant to chemotherapy. Quiescent PCa cells are also enriched for a stem-like tumor initiating population, and can lead to recurrence after dormancy. Unfortunately, quiescent PCa cells are difficult to identify and / or target with treatment in part because the relevant markers are intracellular and regulated by protein stability. We addressed this problem by utilizing PCa cells expressing fluorescent markers for CDKN1B (p27) and CDT1, which can separate viable PCa cells into G0, G1, or combined S/G2/M populations. We used FACS to collect G1 and G0 PC3 PCa cells, isolated membrane proteins, and analyzed protein abundance in G0 vs G1 cells by gas chromatography mass spectrometry. Enrichment analysis identified nucleocytoplasmic transport as the most significantly different pathway. To identify cell surface proteins potentially identifying quiescent PCa cells for future patient samples or for antibody based therapeutic research, we focused on differentially abundant plasma membrane proteins, and identified ERBB2 (HER2) as a cell surface protein enriched on G0 PCa cells. High HER2 on the cell membrane is associated with quiescence in PCa cells and likely induced by the bone microenvironment. Using a drug conjugated anti-HER2 antibody (trastuzumab emtansine) in a mouse PCa xenograft model delayed metastatic tumor growth, suggesting approaches that target HER2-high cells may be beneficial in treating PCa. We propose that HER2 is deserving of further study in PCa as a target on quiescent cells to prevent recurrence, decrease chemotherapy resistance, or eradicate minimal residual disease.
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The proliferation-quiescence decision is a dynamic process that remains incompletely understood. Live-cell imaging with fluorescent cell cycle sensors now allows us to visualize the dynamics of cell cycle transitions and has revealed that proliferation-quiescence decisions can be highly heterogeneous, even among clonal cell lines in culture. Under normal culture conditions, cells often spontaneously enter non-cycling G0 states of varying duration and depth. This also occurs in cancer cells and G0 entry in tumors may underlie tumor dormancy and issues with cancer recurrence. Here we show that a cell cycle indicator previously shown to indicate G0 upon serum starvation, mVenus-p27K-, can also be used to monitor spontaneous quiescence in untransformed and cancer cell lines. We find that the duration of spontaneous quiescence in untransformed and cancer cells is heterogeneous and that a portion of this heterogeneity results from asynchronous proliferation-quiescence decisions in pairs of daughters after mitosis, where one daughter cell enters or remains in temporary quiescence while the other does not. We find that cancer dormancy signals influence both entry into quiescence and asynchronous proliferation-quiescence decisions after mitosis. Finally, we show that spontaneously quiescent prostate cancer cells exhibit altered expression of components of the Hippo pathway and are enriched for the stem cell markers CD133 and CD44. This suggests a hypothesis that dormancy signals could promote cancer recurrence by increasing the proportion of quiescent tumor cells poised for cell cycle re-entry with stem cell characteristics in cancer.
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Prostate cancer (PCa) commonly metastasizes to the bone where the cells frequently undergo dormancy. The escape of disseminated tumor cells from cellular dormancy is a major cause of recurrence in marrow. Abscisic acid (ABA), a phytohormone, is known to regulate dormancy of plant seeds and to regulate other stress responses in plants. Recently, ABA was found to be synthesized by mammals cells and has been linked to human disease. Yet the role of ABA in regulating tumor dormancy or reactivation is unknown. We found that ABA is produced by human marrow cells, and exogenous ABA inhibits PCa cell proliferation while increasing the expression of p27, p21, and p16 and decreasing the expression of the proliferation marker, Ki67. Further, ABA significantly increased the percentage of PCa cells in the G0 phase of the cell cycle as well as the duration the cells were arrested in G0. We found that ABA regulates an increase of PPARγ receptor expression and suppressed phosphorylation of mTOR/p70S6K signaling and resulting in the induction of the cellular dormancy. We then confirmed that ABA regulates G0 cell cycle arrest through PPARγ receptor signaling in vitro and under co-culture conditions with osteoblasts. Finally, we demonstrate that ABA regulates PCa dormancy in vivo following intratibial injection in an animal model. Together these data suggest that the ABA and PPARγ signaling pathways contribute to the establishment of PCa cellular dormancy in the bone marrow microenvironment. These findings may suggest critical pathways for targeting metastatic disease.
Assuntos
Ácido Abscísico/farmacologia , Medula Óssea/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Neoplasias da Próstata/patologia , Animais , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Metástase Neoplásica , PPAR gama/metabolismo , Fase de Repouso do Ciclo Celular , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
BACKGROUND: Disseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs. METHODS: We used fluorescence-activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA-Seq) and whole-exome sequencing after whole genome amplification. We also enriched for BM DTCs with α-EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT-PCR) for PCa markers. RESULTS: At a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients' plasma PSA values. RNA-Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole-exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA-Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B-cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT-PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%). CONCLUSION: These data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.
Assuntos
Células da Medula Óssea/patologia , Medula Óssea/patologia , Metástase Neoplásica/patologia , Neoplasias da Próstata/patologia , Idoso , Biomarcadores Tumorais/análise , Separação Celular/métodos , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Polimorfismo de Nucleotídeo Único/genética , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/genética , Análise de Sequência de RNA , Sequenciamento do ExomaRESUMO
Congenital cardiac malformations are among the most common birth defects in humans. Here we show that Trim33, a member of the Tif1 subfamily of tripartite domain containing transcriptional cofactors, is required for appropriate differentiation of the pre-cardiogenic mesoderm during a narrow time window in late gastrulation. While mesoderm-specific Trim33 mutants did not display noticeable phenotypes, epiblast-specific Trim33 mutant embryos developed ventricular septal defects, showed sparse trabeculation and abnormally thin compact myocardium, and died as a result of cardiac failure during late gestation. Differentiating embryoid bodies deficient in Trim33 showed an enrichment of gene sets associated with cardiac differentiation and contractility, while the total number of cardiac precursor cells was reduced. Concordantly, cardiac progenitor cell proliferation was reduced in Trim33-deficient embryos. ChIP-Seq performed using antibodies against Trim33 in differentiating embryoid bodies revealed more than 4000 peaks, which were significantly enriched close to genes implicated in stem cell maintenance and mesoderm development. Nearly half of the Trim33 peaks overlapped with binding sites of the Ctcf insulator protein. Our results suggest that Trim33 is required for appropriate differentiation of precardiogenic mesoderm during late gastrulation and that it will likely mediate some of its functions via multi-protein complexes, many of which include the chromatin architectural and insulator protein Ctcf.
Assuntos
Embrião de Mamíferos/embriologia , Gastrulação , Mesoderma/embriologia , Miocárdio/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Embrião de Mamíferos/citologia , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Mesoderma/citologia , Camundongos , Camundongos Transgênicos , Células-Tronco/citologia , Fatores de Transcrição/genéticaRESUMO
The discovery of anticancer therapeutics effective in eliminating dormant cells is a significant challenge in cancer biology. Here, we describe new synthetic polymer-based anticancer agents that mimic the mode of action of anticancer peptides. These anticancer polymers developed here are designed to capture the cationic, amphiphilic traits of anticancer peptides. The anticancer polymers are designed to target anionic lipids exposed on the cancer cell surfaces and act by disrupting the cancer cell membranes. Because the polymer mechanism is not dependent on cell proliferation, we hypothesized that the polymers were active against dormant cancer cells. The polymers exhibited cytotoxicity to proliferating prostate cancer. Importantly, the polymer killed dormant prostate cancer cells that were resistant to docetaxel. This study demonstrates a new approach to discover novel anticancer therapeutics.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Polímeros/química , Polímeros/farmacologia , Biomimética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Estrutura Molecular , Neoplasias da Próstata/metabolismoRESUMO
There is evidence that cancer stem-like cells (CSC) and neuroendocrine behavior play critical roles in the pathogenesis and clinical course of metastatic castration-resistant prostate cancer (m-CRPC). However, there is limited mechanistic understanding of how CSC and neuroendocrine phenotypes impact the development of m-CRPC. In this study, we explored the role of the intracellular chemokine CXCL12γ in CSC induction and neuroendocrine differentiation and its impact on m-CRPC. CXCL12γ expression was detected in small-cell carcinoma of metastatic tissues and circulating tumor cells from m-CRPC patients and in prostate cancer cells displaying an neuroendocrine phenotype. Mechanistic investigations demonstrated that overexpression of CXCL12γ induced CSC and neuroendocrine phenotypes in prostate cancer cells through CXCR4-mediated PKCα/NFκB signaling, which promoted prostate tumor outgrowth, metastasis, and chemoresistance in vivo Together, our results establish a significant function for CXCL12γ in m-CRPC development and suggest it as a candidate therapeutic target to control aggressive disease.Significance: Expression of CXCL12γ induces the expression of a cancer stem cell and neuroendocrine phenotypes, resulting in the development of aggressive m-CRPC. Cancer Res; 78(8); 2026-39. ©2018 AACR.
Assuntos
Neoplasias Ósseas/secundário , Reprogramação Celular , Quimiocina CXCL12/fisiologia , Células-Tronco Neoplásicas/patologia , Células Neuroendócrinas/patologia , Neoplasias de Próstata Resistentes à Castração/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos SCID , NF-kappa B/metabolismo , Fenótipo , Proteína Quinase C-alfa/metabolismo , Receptores CXCR4/metabolismo , Transdução de SinaisRESUMO
Clinical observations have identified an association between psychologic stress and cancer relapse, suggesting that the sympathetic nervous system/norepinephrine (NE) plays a role in reactivation of dormant disseminated tumor cells (DTC) in the bone marrow niche. Here, the mechanism by which NE regulates prostate cancer DTCs in the marrow is explored. NE directly stimulated prostate cancer cell proliferation through ß2-adrenergic receptors (ADRB2). NE also altered prostate cancer proliferation in the marrow niche by indirectly downregulating the secretion of the dormancy inducing molecule growth arrest specific-6 (GAS6) expressed by osteoblasts. These observations were confirmed in cocultures of prostate cancer cells expressing the fluorescent ubiquitination-based cell-cycle reporters (FUCCI) and osteoblasts isolated from GAS6-deficient (GAS6-/-) animals. A novel ex vivo model system, using femurs harvested from GAS6+/+ or GAS6-/- mice, was used to confirm these results. As in coculture, when prostate cancer cells were injected into the marrow cavities of GAS6+/+ femurs, NE altered the prostate cancer cell cycle. However, NE had less of an impact on prostate cancer cells in femur explants isolated from GAS6-/- mice. Together, this study demonstrates that NE reactivates prostate cancer cell cycling through both a direct action on prostate cancer cells and indirectly on adjacent niche cells.Implications: Identification of mechanisms that target DTCs may provide novel therapeutic approaches to prevent or treat cancer metastases more effectively. Mol Cancer Res; 15(12); 1644-55. ©2017 AACR.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Norepinefrina/genética , Neoplasias da Próstata/genética , Receptores Adrenérgicos beta 3/genética , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Norepinefrina/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Recidiva , Estresse Psicológico/genética , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/patologiaRESUMO
Many prostate cancer (PCa) recurrences are thought to be due to reactivation of disseminated tumor cells (DTCs). We previously found a role of the TAM family of receptor tyrosine kinases TYRO3, AXL, and MERTK in PCa dormancy regulation. However, the mechanism and contributions of the individual TAM receptors is largely unknown. Knockdown of MERTK, but not AXL or TYRO3 by shRNA in PCa cells induced a decreased ratio of P-Erk1/2 to P-p38, increased expression of p27, NR2F1, SOX2, and NANOG, induced higher levels of histone H3K9me3 and H3K27me3, and induced a G1/G0 arrest, all of which are associated with dormancy. Similar effects were also observed with siRNA. Most importantly, knockdown of MERTK in PCa cells increased metastasis free survival in an intra-cardiac injection mouse xenograft model. MERTK knockdown also failed to inhibit PCa growth in vitro and subcutaneous growth in vivo, which suggests that MERTK has specificity for dormancy regulation or requires a signal from the PCa microenvironment. The effects of MERTK on the cell cycle and histone methylation were reversed by p38 inhibitor SB203580, which indicates the importance of MAP kinases for MERTK dormancy regulation. Overall, this study shows that MERTK stimulates PCa dormancy escape through a MAP kinase dependent mechanism, also involving p27, pluripotency transcription factors, and histone methylation. J. Cell. Biochem. 118: 891-902, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Silenciamento de Genes , Xenoenxertos , Histonas/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos SCID , Recidiva Local de Neoplasia/enzimologia , Recidiva Local de Neoplasia/patologia , Neoplasias da Próstata/secundário , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Evasão Tumoral , Microambiente Tumoral , c-Mer Tirosina QuinaseRESUMO
Disseminated prostate cancer (PCa) cells in the marrow survive for years without evidence of proliferation, while maintaining the capacity to develop into metastatic lesions. These dormant disseminated tumor cells (DTCs) may reside in close proximity to osteoblasts, while expressing high levels of Axl, one of the tyrosine kinase receptors for growth arrest specific 6 (Gas6). Yet how Axl regulates DTC proliferation in marrow remains undefined. Here, we explored the impact of the loss of Axl in PCa cells (PC3 and DU145) on the induction of their dormancy when they are co-cultured with a pre-osteoblastic cell line, MC3T3-E1. MC3T3-E1 cells dramatically decrease the proliferation of PCa cells, however this suppressive effect of osteoblasts is significantly reduced by the reduction of Axl expression in PCa cells. Interestingly, expression of both TGF-ß and its receptors were regulated by Axl expression in PCa cells, while specific blockade of TGF-ß signaling limited the ability of the osteoblasts to induce dormancy of PCa cells. Finally, we found that both Gas6 and Axl are required for TGF-ß2-mediated cell growth suppression. Taken together, these data suggest that a loop between the Gas6/Axl axis and TGF-ß2 signaling plays a significant role in the induction of PCa cell dormancy.
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Medula Óssea/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Masculino , Camundongos , Osteoblastos/metabolismo , Próstata/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Cancer metastasis is a multistep process associated with the induction of an epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs). Although significant progress has been made in understanding the molecular mechanisms regulating EMT and the CSC phenotype, little is known of how these processes are regulated by epigenetics. Here we demonstrate that reduced expression of DNA methyltransferase 1 (DNMT1) plays an important role in the induction of EMT and the CSC phenotype by prostate cancer (PCa) cells, with enhanced tumorigenesis and metastasis. First, we observed that reduction of DNMT1 by 5-azacitidine (5-Aza) promotes EMT induction as well as CSCs and sphere formation in vitro. Reduced expression of DNMT1 significantly increased PCa migratory potential. We showed that the increase of EMT and CSC activities by reduction of DNMT1 is associated with the increase of protein kinase C. Furthermore, we confirmed that silencing DNMT1 is correlated with enhancement of the induction of EMT and the CSC phenotype in PCa cells. Additionally, chromatin immunoprecipitation assay reveals that reduction of DNMT1 promotes the suppression of H3K9me3 and H3K27me3 on the Zeb2 and KLF4 promoter region in PCa cells. Critically, we found in an animal model that significant tumor growth and more disseminated tumor cells in most osseous tissues were observed following injection of 5-Aza pretreated-PCa cells compared with vehicle-pretreated PCa cells. Our results suggest that epigenetic alteration of histone demethylation regulated by reduction of DNMT1 may control induction of EMT and the CSC phenotype, which facilitates tumorigenesis in PCa cells and has important therapeutic implications in targeting epigenetic regulation.
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DNA (Citosina-5-)-Metiltransferases/genética , Transição Epitelial-Mesenquimal/genética , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Biomarcadores , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , Modelos Animais de Doenças , Inativação Gênica , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Masculino , Metilação , Camundongos , Metástase Neoplásica , Regiões Promotoras Genéticas , Neoplasias da Próstata/tratamento farmacológico , Proteína Quinase C-alfa/metabolismo , Interferência de RNA , Proteínas Repressoras/genética , Esferoides Celulares , Carga Tumoral , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Homeobox 2 de Ligação a E-box com Dedos de ZincoRESUMO
Dissemination of cancer stem cells (CSCs) serves as the basis of metastasis. Recently, we demonstrated that circulating prostate cancer targets the hematopoietic stem cell (HSCs) 'niche' in marrow during dissemination. Once in the niche, disseminated tumor cells (DTCs) may remain dormant for extended periods. As the major function of the HSC niche is to maintain stem cell functions, we hypothesized that the niche regulates CSC activities of DTCs. Here we show that DTCs recovered from marrow were significantly enriched for a CSC phenotype. Critically, the conversion of DTCs to CSCs is regulated by niche-derived GAS6 through the Mer/mTOR; molecules previously shown to regulate dormancy. The data demonstrate that the niche plays a significant role in maintaining tumor-initiating prostate cancer in marrow and suggests a functional relationship between CSCs and dormancy. Understanding how the marrow niche regulates the conversion of DTCs to CSCs is critical for the development of therapeutics specifically targeting skeletal bone metastasis and dormancy.
Assuntos
Adenocarcinoma/patologia , Medula Óssea/patologia , Neoplasias Ósseas/secundário , Células-Tronco Hematopoéticas/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/patologia , Nicho de Células-Tronco/fisiologia , Animais , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células-Tronco Hematopoéticas/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Metástase Neoplásica , Células-Tronco Neoplásicas/fisiologia , FenótipoRESUMO
Prostate cancer (PCa) is known to develop resistance to chemotherapy. Growth arrest-specific 6 (GAS6), plays a role in tumor progression by regulating growth in many cancers. Here, we explored how GAS6 regulates the cell cycle and apoptosis of PCa cells in response to chemotherapy. We found that GAS6 is sufficient to significantly increase the fraction of cells in G1 and the duration of phase in PCa cells. Importantly, the effect of GAS6 on G1 is potentiated during docetaxel chemotherapy. GAS6 altered the levels of several key cell cycle regulators, including the downregulation of Cyclin B1 (G2 /M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry), while the upregulation of cell cycle inhibitors p27 and p21, Cyclin D1, and CDK4. Importantly, these changes became further accentuated during docetaxel treatment in the presence of GAS6. Moreover, GAS6 alters the apoptotic response of PCa cells during docetaxel chemotherapy. Docetaxel induced PCa cell apoptosis is efficiently suppressed in PCa cell culture in the presence of GAS6 or GAS6 secreted from co-cultured osteoblasts. Similarly, the GAS6-expressing bone environment protects PCa cells from apoptosis within primary tumors in vivo studies. Docetaxel induced significant levels of Caspase-3 and PARP cleavage in PCa cells, while GAS6 protected PCa cells from docetaxel-induced apoptotic signaling. Together, these data suggest that GAS6, expressed by osteoblasts in the bone marrow, plays a significant role in the regulation of PCa cell survival during chemotherapy, which will have important implications for targeting metastatic disease. J. Cell. Biochem. 117: 2815-2824, 2016. © 2016 Wiley Periodicals, Inc.
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Apoptose/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neoplasias da Próstata/patologia , Fase S/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Docetaxel , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxoides/farmacologiaRESUMO
UNLABELLED: GAS6 and its receptors (Tryo 3, Axl, Mer or "TAM") are known to play a role in regulating tumor progression in a number of settings. Previously we have demonstrated that GAS6 signaling regulates invasion, proliferation, chemotherapy-induced apoptosis of prostate cancer (PCa) cells. We have also demonstrated that GAS6 secreted from osteoblasts in the bone marrow environment plays a critical role in establishing prostate tumor cell dormancy. Here we investigated the role that endogenous GAS6 and Mer receptor signaling plays in establishing prostate cancer stem cells in the bone marrow microenvironment.We first observed that high levels of endogenous GAS6 are expressed by disseminated tumor cells (DTCs) in the bone marrow, whereas relatively low levels of endogenous GAS6 are expressed in PCa tumors grown in a s.c. SETTING: Interestingly, elevated levels of endogenous GAS6 were identified in putative cancer stem cells (CSCs, CD133+/CD44+) compared to non-CSCs (CD133-/CD44-) isolated from PCa/osteoblast cocultures in vitro and in DTCs isolated from the bone marrow 24 hours after intracardiac injection. Moreover, we found that endogenous GAS6 expression is associated with Mer receptor expression in growth arrested (G1) PCa cells, which correlates with the increase of the CSC populations. Importantly, we found that overexpression of GAS6 activates phosphorylation of Mer receptor signaling and subsequent induction of the CSC phenotype in vitro and in vivo.Together these data suggest that endogenous GAS6 and Mer receptor signaling contribute to the establishment of PCa CSCs in the bone marrow microenvironment, which may have important implications for targeting metastatic disease.
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Neoplasias Ósseas/secundário , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/patologia , c-Mer Tirosina Quinase/metabolismo , Animais , Medula Óssea/patologia , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos SCID , Metástase Neoplásica , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Transforming growth factor-beta3 (TGF-ß3) plays a critical role in palatal epithelial cells by inducing palatal epithelial fusion, failure of which results in cleft palate, one of the most common birth defects in humans. Recent studies have shown that Smad-dependent and Smad-independent pathways work redundantly to transduce TGF-ß3 signaling in palatal epithelial cells. However, detailed mechanisms by which this signaling is mediated still remain to be elucidated. Here we show that TGF-ß activated kinase-1 (Tak1) and Smad4 interact genetically in palatal epithelial fusion. While simultaneous abrogation of both Tak1 and Smad4 in palatal epithelial cells resulted in characteristic defects in the anterior and posterior secondary palate, these phenotypes were less severe than those seen in the corresponding Tgfb3 mutants. Moreover, our results demonstrate that Trim33, a novel chromatin reader and regulator of TGF-ß signaling, cooperates with Smad4 during palatogenesis. Unlike the epithelium-specific Smad4 mutants, epithelium-specific Tak1:Smad4- and Trim33:Smad4-double mutants display reduced expression of Mmp13 in palatal medial edge epithelial cells, suggesting that both of these redundant mechanisms are required for appropriate TGF-ß signal transduction. Moreover, we show that inactivation of Tak1 in Trim33:Smad4 double conditional knockouts leads to the palatal phenotypes which are identical to those seen in epithelium-specific Tgfb3 mutants. To conclude, our data reveal added complexity in TGF-ß signaling during palatogenesis and demonstrate that functionally redundant pathways involving Smad4, Tak1 and Trim33 regulate palatal epithelial fusion.
Assuntos
MAP Quinase Quinase Quinases/metabolismo , Palato/embriologia , Palato/metabolismo , Transdução de Sinais , Proteína Smad4/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Animais , Apoptose/genética , Fusão Celular , Proliferação de Células , Cruzamentos Genéticos , Embrião de Mamíferos/metabolismo , Ativação Enzimática , Células Epiteliais/metabolismo , Epitélio/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Metaloproteinase 13 da Matriz/metabolismo , Camundongos Knockout , Modelos Biológicos , Mutação/genética , Especificidade de Órgãos , Palato/anormalidades , Palato/enzimologiaRESUMO
UNLABELLED: Annexin 2 (ANXA2) plays a critical role in hematopoietic stem cell (HSC) localization to the marrow niche. In part, ANXA2 supports HSCs by serving as an anchor for stromal-derived factor-1 (CXCL12/SDF-1). Recently, it was demonstrated that prostate cancer cells, like HSCs, use ANXA2 to establish metastases in marrow. The present study determined the capacity of ANXA2 expression by bone marrow stromal cells (BMSC) to facilitate tumor recruitment and growth through ANXA2-CXCL12 interactions. Significantly more CXCL12 was expressed by BMSC(Anxa2) (+/+) than by BMSC(Anxa2) (-/-) resulting in more prostate cancer cells migrating and binding to BMSC(Anxa2) (+/+) than BMSC(Anxa2) (-/-), and these activities were reduced when CXCL12 interactions were blocked. To further confirm that BMSC signaling through ANXA2-CXCL12 plays a critical role in tumor growth, immunocompromised SCID mice were subcutaneously implanted with human prostate cancer cells mixed with BMSC(Anxa2) (+/+) or BMSC(Anxa2) (-/-). Significantly larger tumors grew in the mice when the tumors were established with BMSC(Anxa2) (+/+) compared with the tumors established with BMSC(Anxa2) (-/-). In addition, fewer prostate cancer cells underwent apoptosis when cocultured with BMSC(Anxa2) (+/+) compared with BMSC(Anxa2) (-/-), and similar results were obtained in tumors grown in vivo. Finally, significantly more vascular structures were observed in the tumors established with the BMSC(Anxa2) (+/+) compared with the tumors established with BMSC(Anxa2) (-/-). Thus, ANXA2-CXCL12 interactions play a crucial role in the recruitment, growth, and survival of prostate cancer cells in the marrow. IMPLICATIONS: The tumor microenvironment interaction between ANXA2-CXCL12 is critical for metastatic phenotypes and may impact chemotherapeutic potential.
Assuntos
Anexina A2/genética , Quimiocina CXCL12/genética , Células-Tronco Mesenquimais/metabolismo , Neoplasias da Próstata/genética , Animais , Anexina A2/biossíntese , Quimiocina CXCL12/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Camundongos , Metástase Neoplásica , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Transdução de SinaisRESUMO
Tgfb3 is strongly and specifically expressed in the epithelial tips of pre-fusion palatal shelves where it plays a critical non-redundant role in palatal fusion in both medial edge epithelial (MEE) cells and in a thin layer of flattened peridermal cells that covers the MEE. It is not known how Tgfb3 expression is regulated in these specific cell types. Using comparative genomics and transgenic reporter assays, we have identified cis-regulatory elements that could control Tgfb3 expression during palatogenesis. Our results show that a 61-kb genomic fragment encompassing the Tgfb3 gene drives remarkably specific reporter expression in the MEE and adjacent periderm. Within this fragment, we identified two small, non-coding, evolutionarily conserved regions in intron 2 of the neighboring Ift43 gene, and a larger region in the intervening sequence between the Ift43 and Tgfb3 genes, each of which could target reporter activity to the tips of pre-fusion/fusing palatal shelves. Identification of the cis-regulatory sequences controlling spatio-temporal Tgfb3 expression in palatal shelves is a key step toward understanding upstream regulation of Tgfb3 expression during palatogenesis and should enable the development of improved tools to investigate palatal epithelial fusion.