RESUMEN
We previously reported that transcription of the human IL1B gene, encoding the proinflammatory cytokine interleukin 1ß, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer-binding protein (C/EBPß) at a far-upstream enhancer. We have also reported that the C-terminal tail sequence beyond the C/EBPß leucine zipper is critical for its association with Spi1 via an exposed residue (Arg-232) located within a pocket at one end of the Spi1 DNA-recognition helix. Here, combining in vitro interaction studies with computational docking and molecular dynamics of existing X-ray structures for the Spi1 and C/EBPß DBDs, along with the C/EBPß C-terminal tail sequence, we found that the tail sequence is intimately associated with Arg-232 of Spi1. The Arg-232 pocket was computationally screened for small-molecule binding aimed at IL1B transcription inhibition, yielding l-arginine, a known anti-inflammatory amino acid, revealing a potential for disrupting the C/EBPß-Spi1 interaction. As evaluated by ChIP, cultured lipopolysaccharide (LPS)-activated THP-1 cells incubated with l-arginine had significantly decreased IL1B transcription and reduced C/EBPß's association with Spi1 on the IL1B promoter. No significant change was observed in direct binding of either Spi1 or C/EBPß to cognate DNA and in transcription of the C/EBPß-dependent IL6 gene in the same cells. These results support the notion that disordered sequences extending from a leucine zipper can mediate protein-protein interactions and can serve as druggable targets for regulating gene promoter activity.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Interleucina-1beta/genética , Mapas de Interacción de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Animales , Sitios de Unión , Proteína beta Potenciadora de Unión a CCAAT/química , Línea Celular , Cristalografía por Rayos X , Humanos , Ratones , Simulación del Acoplamiento Molecular , Regiones Promotoras Genéticas , Conformación Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/química , Transactivadores/químicaRESUMEN
Interleukin 1ß is a pro-inflammatory cytokine important for both normal immune responses and chronic inflammatory diseases. The regulation of the 31â¯kDa proIL-1ß precursor coded by the IL1B gene has been extensively studied in myeloid cells, but not in lymphoid-derived CD4 T cells. Surprisingly, we found that some CD4 T cell subsets express higher levels of proIL-1ß than unstimulated monocytes, despite relatively low IL1B mRNA levels. We observed a significant increase in IL1B transcription and translation in CD4 T cells upon ex vivo CD3/CD28 activation, and a similar elevation in the CCR5+ effector memory population compared to CCR5- T cells in vivo. The rapid and vigorous increase in IL1B gene transcription for stimulated monocytes has previously been associated with the presence of Spi-1/PU.1 (Spi1), a myeloid-lineage transcription factor, pre-bound to the promoter. In the case of CD4 T cells, this increase occurred despite the lack of detectable Spi1 at the IL1B promoter. Additionally, we found altered epigenetic regulation of the IL1B locus in CD3/CD28-activated CD4 T cells. Unlike monocytes, activated CD4 T cells possess bivalent H3K4me3+/H3K27me3+ nucleosome marks at the IL1B promoter, reflecting low transcriptional activity. These results support a model in which the IL1B gene in CD4 T cells is transcribed from a low-activity bivalent promoter independent of Spi1. Accumulated cytoplasmic proIL-1ß may ultimately be cleaved to mature 17â¯kDa bioactive IL-1ß, regulating T cell polarization and pathogenic chronic inflammation.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Interleucina-1beta/genética , Monocitos/fisiología , Transcripción Genética/genética , Biomarcadores/metabolismo , Antígenos CD28/genética , Complejo CD3/genética , Epigénesis Genética/genética , Regulación de la Expresión Génica/genética , Humanos , Nucleosomas/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Receptores CCR5/genética , Activación Transcripcional/genéticaRESUMEN
Multiple myeloma (MM) is an incurable B-cell malignancy in which the marrow microenvironment plays a critical role in our inability to cure MM. Marrow stromal cells in the microenvironment support homing, lodging, and growth of MM cells through activation of multiple signaling pathways in both MM and stromal cells. Recently, we identified annexin II (AXII) as a previously unknown factor produced by stromal cells and osteoclasts (OCL) that is involved in OCL formation, HSC and prostate cancer (PCa) homing to the BM as well as mobilization of HSC and PCa cells. AXII expressed on stromal cells supports PCa cell lodgment via the AXII receptor (AXIIR) on PCa cells, but the role of AXII and AXIIR in MM is unknown. In this study, we show that MM cells express AXIIR, that stromal/osteoblast-derived AXII facilitates adhesion of MM cells to stromal cells via AXIIR, and OCL-derived AXII enhances MM cell growth. Finally, we demonstrate that AXII activates the ERK1/2 and AKT pathways in MM cells to enhance MM cell growth. These results demonstrate that AXII and AXIIR play important roles in MM and that targeting the AXII/AXIIR axis may be a novel therapeutic approach for MM.
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Anexina A2/metabolismo , Médula Ósea/metabolismo , Proliferación Celular , Mieloma Múltiple/metabolismo , Receptores de Péptidos/metabolismo , Animales , Anexina A2/genética , Anexina A2/farmacología , Western Blotting , Células de la Médula Ósea/metabolismo , Adhesión Celular , Línea Celular Tumoral , Células Cultivadas , Microambiente Celular , Técnicas de Cocultivo , Humanos , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Osteoclastos/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Receptores de Péptidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Bone is a frequent site for breast cancer metastasis. Conditioning of the local tumor microenvironment (TME) through crosstalk between tumor cells and bone resident cells in the metastatic niche is a major driving force for bone colonization of breast cancer cells. The vast majority of breast cancer-associated metastasis is osteolytic in nature, and RANKL-induced differentiation of bone marrow-derived macrophages to osteoclasts (OCLs) is a key requirement for osteolytic metastatic growth of cancer cells. In this study, we demonstrate that breast cancer cell-secreted factors stimulate RANKL-induced OCL differentiation of BMDMs requiring the function of Myocardin-related transcription factor (MRTF) in tumor cells. This is partly attributed to the critical role of MRTF in maintaining the basal cellular expression of connective tissue growth factor (CTGF), a pro-osteoclastogenic matricellular factor known to promote bone metastasis in human breast cancer. Supporting these in vitro findings, bioinformatics analyses of multiple human breast cancer transcriptome datasets reveal a strong positive correlation between CTGF expression and MRTF gene signature further establishing the relevance of our findings in a human disease context. By Luminex analyses, we show that MRTF depletion in breast cancer cells has a broad impact on OCL-regulatory cell-secreted factors that extends beyond CTGF. These findings, taken together with demonstration of MRTF-dependence for bone colonization breast cancer cells in vivo, suggest that MRTF inhibition could be an effective strategy to diminish OCL formation and skeletal involvement in breast cancer. In summary, this study highlights a novel tumor-extrinsic function of MRTF relevant to breast cancer metastasis.
RESUMEN
Immunomodulatory derivatives of thalidomide (IMiD compounds), such as pomalidomide and lenalidomide, are highly active in multiple myeloma (MM) treatment. However, the precise mechanisms of action and resistance in MM are unresolved. Here we show that IMiD compounds down-regulate CCAAT/enhancer-binding protein-ß (C/EBPß) resulting in abrogation of cell proliferation. Overexpression of C/EBPß rescued MM cells from IMiD-induced inhibition of proliferation, indicating that C/EBPß is critical in mediating antiproliferative effects. IMiD-induced decrease of C/EBPß protein led to impaired transcription of interferon regulatory factor 4 (IRF4). Down-regulation of IRF4 by lenalidomide was confirmed by longitudinal studies of bone marrow samples from 23 patients obtained before and during lenalidomide treatment using CD138âº/IRF4⺠double labeling. In contrast to down-regulation of C/EBPß protein, IMiD compounds did not alter C/EBPß mRNA levels or protein stability, suggesting translational regulation of C/EBPß. We could demonstrate that C/EBPß protein expression is under eIF4E-translational control in MM. Furthermore, inhibition of the eIF4E-C/EBPß axis by IMiD compounds was not observed in IMiD-resistant MM cells. However, targeting translation at a different level by inhibiting eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation overcame resistance, suggesting that this pathway is critical and might be a target to overcome drug resistance.
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Proteína beta Potenciadora de Unión a CCAAT/biosíntesis , Factor 4E Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Factores Inmunológicos/farmacología , Mieloma Múltiple/metabolismo , Apoptosis/inmunología , Western Blotting , Separación Celular , Regulación hacia Abajo , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Factores Reguladores del Interferón/biosíntesis , Lenalidomida , Biosíntesis de Proteínas/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Talidomida/análogos & derivados , Talidomida/farmacología , TransfecciónRESUMEN
Protracted inhibition of osteoblast (OB) differentiation characterizes multiple myeloma (MM) bone disease and persists even when patients are in long-term remission. However, the underlying pathophysiology for this prolonged OB suppression is unknown. Therefore, we developed a mouse MM model in which the bone marrow stromal cells (BMSCs) remained unresponsive to OB differentiation signals after removal of MM cells. We found that BMSCs from both MM-bearing mice and MM patients had increased levels of the transcriptional repressor Gfi1 compared with controls and that Gfi1 was a novel transcriptional repressor of the critical OB transcription factor Runx2. Trichostatin-A blocked the effects of Gfi1, suggesting that it induces epigenetic changes in the Runx2 promoter. MM-BMSC cell-cell contact was not required for MM cells to increase Gfi1 and repress Runx2 levels in MC-4 before OBs or naive primary BMSCs, and Gfi1 induction was blocked by anti-TNF-α and anti-IL-7 antibodies. Importantly, BMSCs isolated from Gfi1(-/-) mice were significantly resistant to MM-induced OB suppression. Strikingly, siRNA knockdown of Gfi1 in BMSCs from MM patients significantly restored expression of Runx2 and OB differentiation markers. Thus, Gfi1 may have an important role in prolonged MM-induced OB suppression and provide a new therapeutic target for MM bone disease.
Asunto(s)
Neoplasias Óseas/metabolismo , Proteínas de Unión al ADN/metabolismo , Mieloma Múltiple/metabolismo , Osteoblastos/metabolismo , Células del Estroma/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Western Blotting , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Línea Celular Tumoral , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Expresión Génica , Humanos , Interleucina-7/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Osteoblastos/patología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/patología , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Multiple myeloma (MM) is an incapacitating hematological malignancy characterized by accumulation of cancerous plasma cells in the bone marrow (BM) and production of an abnormal monoclonal protein (M-protein). The BM microenvironment has a key role in myeloma development by facilitating the growth of the aberrant plasma cells, which eventually interfere with the homeostasis of the bone cells, exacerbating osteolysis and inhibiting osteoblast differentiation. Recent recognition that metabolic reprograming has a major role in tumor growth and adaptation to specific changes in the microenvironmental niche have led to consideration of the role of sphingolipids and the enzymes that control their biosynthesis and degradation as critical mediators of cancer since these bioactive lipids have been directly linked to the control of cell growth, proliferation, and apoptosis, among other cellular functions. In this review, we present the recent progress of the research investigating the biological implications of sphingolipid metabolism alterations in the regulation of myeloma development and its progression from the pre-malignant stage and discuss the roles of sphingolipids in in MM migration and adhesion, survival and proliferation, as well as angiogenesis and invasion. We introduce the current knowledge regarding the role of sphingolipids as mediators of the immune response and drug-resistance in MM and tackle the new developments suggesting the manipulation of the sphingolipid network as a novel therapeutic direction for MM.
RESUMEN
Multiple myeloma (MM) remains incurable for most patients due to the emergence of drug resistant clones. Here we report a p53-independent mechanism responsible for Growth Factor Independence-1 (GFI1) support of MM cell survival by its modulation of sphingolipid metabolism to increase the sphingosine-1-phosphate (S1P) level regardless of the p53 status. We found that expression of enzymes that control S1P biosynthesis, SphK1, dephosphorylation, and SGPP1 were differentially correlated with GFI1 levels in MM cells. We detected GFI1 occupancy on the SGGP1 gene in MM cells in a predicted enhancer region at the 5' end of intron 1, which correlated with decreased SGGP1 expression and increased S1P levels in GFI1 overexpressing cells, regardless of their p53 status. The high S1P:Ceramide intracellular ratio in MM cells protected c-Myc protein stability in a PP2A-dependent manner. The decreased MM viability by SphK1 inhibition was dependent on the induction of autophagy in both p53WT and p53mut MM. An autophagic blockade prevented GFI1 support for viability only in p53mut MM, demonstrating that GFI1 increases MM cell survival via both p53WT inhibition and upregulation of S1P independently. Therefore, GFI1 may be a key therapeutic target for all types of MM that may significantly benefit patients that are highly resistant to current therapies.
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The University of Pittsburgh Medical Center Hillman Cancer Center Academy (Hillman Academy) has the primary goal of reaching high school students from underrepresented and disadvantaged backgrounds and guiding them through a cutting-edge research and professional development experience that positions them for success in STEM. With this focus, the Hillman Academy has provided nearly 300 authentic mentored research internship opportunities to 239 students from diverse backgrounds over the past 13 years most of whom matriculated into STEM majors in higher education. These efforts have helped shape a more diverse generation of future scientists and clinicians, who will enrich these fields with their unique perspectives and lived experiences. In this paper, we describe our program and the strategies that led to its growth into a National Institutes of Health Youth Enjoy Science-funded program including our unique multi-site structure, tiered mentoring platform, multifaceted recruitment approach, professional and academic development activities, and a special highlight of a set of projects with Deaf and Hard of Hearing students. We also share student survey data from the past six years that indicate satisfaction with the program, self-perceived gains in key areas of scientific development, awareness of careers in STEM, and an increased desire to pursue advanced degrees in STEM.
RESUMEN
CCAAT/enhancer-binding protein beta (C/EBPbeta), also known as nuclear factor-interleukin-6 (NF-IL6), is a transcription factor that plays an important role in the regulation of growth and differentiation of myeloid and lymphoid cells. Mice deficient in C/EBPbeta show impaired generation of B lymphocytes. We show that C/EBPbeta regulates transcription factors critical for proliferation and survival in multiple myeloma. Multiple myeloma cell lines and primary multiple myeloma cells strongly expressed C/EBPbeta, whereas normal B cells and plasma cells had little or no detectable levels of C/EBPbeta. Silencing of C/EBPbeta led to down-regulation of transcription factors such as IRF4, XBP1, and BLIMP1 accompanied by a strong inhibition of proliferation. Further, silencing of C/EBPbeta led to a complete down-regulation of antiapoptotic B-cell lymphoma 2 (BCL2) expression. In chromatin immunoprecipitation assays, C/EBPbeta directly bound to the promoter region of IRF4, BLIMP1, and BCL2. Our data indicate that C/EBPbeta is involved in the regulatory network of transcription factors that are critical for plasma cell differentiation and survival. Targeting C/EBPbeta may provide a novel therapeutic strategy in the treatment of multiple myeloma.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proliferación Celular , Proteínas de Unión al ADN/biosíntesis , Regulación Neoplásica de la Expresión Génica , Factores Reguladores del Interferón/biosíntesis , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Represoras/biosíntesis , Factores de Transcripción/biosíntesis , Animales , Linfocitos B/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Femenino , Silenciador del Gen , Humanos , Factores Reguladores del Interferón/genética , Masculino , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/terapia , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Factores de Transcripción del Factor Regulador X , Proteínas Represoras/genética , Factores de Transcripción/genética , Proteína 1 de Unión a la X-BoxRESUMEN
The steroid receptor coactivator-1 (SRC-1) is a nuclear receptor co-activator, known to play key roles in both estrogen response in bone and in breast cancer metastases. We previously demonstrated that the P1272S single nucleotide polymorphism (SNP; P1272S; rs1804645) in SRC-1 decreases the activity of estrogen receptor in the presence of selective estrogen receptor modulators (SERMs) and that it is associated with a decrease in bone mineral density (BMD) after tamoxifen therapy, suggesting it may disrupt the agonist action of tamoxifen. Given such dual roles of SRC-1 in the bone microenvironment and in tumor cell-intrinsic phenotypes, we hypothesized that SRC-1 and a naturally occurring genetic variant, P1272S, may promote breast cancer bone metastases. We developed a syngeneic, knock-in mouse model to study if the SRC-1 SNP is critical for normal bone homeostasis and bone metastasis. Our data surprisingly reveal that the homozygous SRC-1 SNP knock-in increases tamoxifen-induced bone protection after ovariectomy. The presence of the SRC-1 SNP in mammary glands resulted in decreased expression levels of SRC-1 and reduced tumor burden after orthotopic injection of breast cancer cells not bearing the SRC-1 SNP, but increased metastases to the lungs in our syngeneic mouse model. Interestingly, the P1272S SNP identified in a small, exploratory cohort of bone metastases from breast cancer patients was significantly associated with earlier development of bone metastasis. This study demonstrates the importance of the P1272S SNP in both the effect of SERMs on BMD and the development of tumor in the bone.
Asunto(s)
Adenocarcinoma/secundario , Densidad Ósea/genética , Neoplasias Óseas/secundario , Neoplasias Mamarias Experimentales/patología , Coactivador 1 de Receptor Nuclear/fisiología , Adenocarcinoma/genética , Animales , Neoplasias Óseas/genética , Huesos/efectos de los fármacos , Huesos/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Técnicas de Sustitución del Gen , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/genética , Ratones Transgénicos , Polimorfismo de Nucleótido Simple , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacologíaRESUMEN
The tumor necrosis factor (TNF) receptor associated factor (TRAF) class of intracellular signal transducers is responsible for mediating many of the activation events initiated by TNF receptor (TNFR) and Toll-like/Interleukin-1, -17, and -18 receptor (TIR) families. Investigation of the mechanism by which TRAF6 is activated has demonstrated that two critical domains of the molecule required for activation and downstream signaling are involved in an interaction which renders the molecule inactive and structurally closed, as well as incapable of auto-ubiquitination. Contrary to its assumed role as a direct mediator of protein-protein interaction, TRAF auto-ubiquitination is a means of sustaining an open conformation active in downstream signaling. Furthermore, the inferred cis-function of TRAF auto-ubiquitination is now demonstrated to act in trans and requires both the RING-Zinc (RZ) fingers region and coiled-coil domain. We also observed that both the RZ fingers region and the MATH domain are targets for ubiquitination. Although TRAF6 ubiquitination has emerged as a hallmark of activation, trans-ubiquitination induced by two TRAF6 muteins is insufficient for NF-kappaB activation.
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Factor 6 Asociado a Receptor de TNF/química , Factor 6 Asociado a Receptor de TNF/metabolismo , Ubiquitinación/fisiología , Western Blotting , Línea Celular , Humanos , Inmunoprecipitación , FN-kappa B/metabolismo , Conformación Proteica , Transducción de Señal/fisiología , TransfecciónRESUMEN
Key osteoclast (OCL) regulatory gene promoters in bone marrow-derived monocytes harbor bivalent histone modifications that combine activating Histone 3 lysine 4 tri-methyl (H3K4me3) and repressive H3K27me3 marks, which upon RANKL stimulation resolve into repressive or activating architecture. Enhancer of zeste homologue 2 (EZH2) is the histone methyltransferase component of the polycomb repressive complex 2, which catalyzes H3K27me3 modifications. Immunofluorescence microscopy reveals that EZH2 localization during murine osteoclastogenesis is dynamically regulated. Using EZH2 knockdown and small molecule EZH2 inhibitor GSK126, we show that EZH2 plays a critical epigenetic role in OCL precursors (OCLp) during the first 24 hours of RANKL activation. RANKL triggers EZH2 translocation into the nucleus where it represses OCL-negative regulators MafB, Irf8, and Arg1. Consistent with its cytoplasmic localization in OCLp, EZH2 methyltransferase activity is required during early RANKL signaling for phosphorylation of AKT, resulting in downstream activation of the mTOR complex, which is essential for induction of OCL differentiation. Inhibition of RANKL-induced pmTOR-pS6RP signaling by GSK126 altered the translation ratio of the C/EBPß-LAP and C/EBPß-LIP isoforms and reduced nuclear translocation of the inhibitory C/EBPß-LIP, which is necessary for transcriptional repression of the OCL negative-regulatory transcription factor MafB. EZH2 in multinucleated OCL is primarily cytoplasmic and mature OCL cultured on bone segments in the presence of GSK126 exhibit defective cytoskeletal architecture and reduced resorptive activity. Here we present new evidence that EZH2 plays epigenetic and cytoplasmic roles during OCL differentiation by suppressing MafB transcription and regulating early phases of PI3K-AKT-mTOR-mediated RANKL signaling, respectively. Consistent with its cytoplasmic localization, EZH2 is required for cytoskeletal dynamics during resorption by mature OCL. Thus, EZH2 exhibits complex roles in supporting osteoclast differentiation and function. © 2019 American Society for Bone and Mineral Research.
Asunto(s)
Resorción Ósea , Osteoclastos , Animales , Resorción Ósea/genética , Diferenciación Celular , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Ratones , Fosfatidilinositol 3-QuinasasRESUMEN
Bone is the most preferred site for colonization of metastatic breast cancer cells for each subtype of the disease. The standard of therapeutic care for breast cancer patients with bone metastasis includes bisphosphonates (e.g., zoledronic acid), which have poor oral bioavailability, and a humanized antibody (denosumab). However, these therapies are palliative, and a subset of patients still develop new bone lesions and/or experience serious adverse effects. Therefore, a safe and orally bioavailable intervention for therapy of osteolytic bone resorption is still a clinically unmet need. This study demonstrates suppression of breast cancer-induced bone resorption by a small molecule (sulforaphane, SFN) that is safe clinically and orally bioavailable. In vitro osteoclast differentiation was inhibited in a dose-dependent manner upon addition of conditioned media from SFN-treated breast cancer cells representative of different subtypes. Targeted microarrays coupled with interrogation of The Cancer Genome Atlas data set revealed a novel SFN-regulated gene signature involving cross-regulation of runt-related transcription factor 2 (RUNX2) and nuclear factor-κB and their downstream effectors. Both RUNX2 and p65/p50 expression were higher in human breast cancer tissues compared with normal mammary tissues. RUNX2 was recruited at the promotor of NFKB1 Inhibition of osteoclast differentiation by SFN was augmented by doxycycline-inducible stable knockdown of RUNX2. Oral SFN administration significantly increased the percentage of bone volume/total volume of affected bones in the intracardiac MDA-MB-231-Luc model indicating in vivo suppression of osteolytic bone resorption by SFN. These results indicate that SFN is a novel inhibitor of breast cancer-induced osteolytic bone resorption in vitro and in vivo.
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Neoplasias Óseas/secundario , Resorción Ósea/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Redes Reguladoras de Genes/genética , Isotiocianatos/uso terapéutico , Animales , Femenino , Humanos , Isotiocianatos/farmacología , Ratones , SulfóxidosRESUMEN
A variety of tumor cells produce chemokines that promote tumor cell proliferation and chemotaxis. We previously reported that CXCL16 production is increased in aggressive prostate cancer cells compared with the less aggressive tumor cells and benign cells as identified in a cytokine antibody array. The functional contribution of CXCL16 in prostate cancer development has not yet been evaluated. Accordingly, mRNA expression of CXCL16 and its receptor, CXCR6, were determined by real-time reverse transcription-PCR in various cancer cell lines, including prostate cancer and tissues obtained from localized and metastatic prostate cancer. Consistent with our finding on CXCL16 protein production by prostate cancer cells, aggressive prostate cancer C4-2B and PC3 cells, as well as bone and liver metastatic tissues, expressed higher levels of both CXCL16 and CXCR6 mRNA compared with the less aggressive prostate cancer LNCaP cells, nonneoplastic PrEC and RWPE-1 cells, and benign prostate tissues, respectively. Furthermore, CXCR6 and CXCL16 protein expressions were examined in tissue specimens by immunohistochemistry. Immunohistochemical examination of CXCR6 expression showed strong epithelial staining that correlated with Gleason score, whereas CXCL16 staining was not. Finally, we found that both interleukin-1beta and tumor necrosis factor alpha significantly induced CXCL16 production by prostate epithelial cells, thereby indicating that inflammatory cytokines may play a role in the CXCL16 induction. CXCL16 was found to promote prostate cancer cell migration and invasion in vitro. Therefore, we concluded that CXCL16 functions, through CXCR6, as a novel chemotactic factor for prostate cancer cells.
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Quimiocinas CXC/metabolismo , Factores Quimiotácticos/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Depuradores/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL16 , Quimiocinas CXC/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Interleucina-1beta/farmacología , Masculino , Invasividad Neoplásica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CXCR6 , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Receptores Depuradores/genética , Receptores Virales/genética , Receptores Virales/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Amylin is a member of the calcitonin family of hormones cosecreted with insulin by pancreatic beta cells. Cell culture assays suggest that amylin could affect bone formation and bone resorption, this latter function after its binding to the calcitonin receptor (CALCR). Here we show that Amylin inactivation leads to a low bone mass due to an increase in bone resorption, whereas bone formation is unaffected. In vitro, amylin inhibits fusion of mononucleated osteoclast precursors into multinucleated osteoclasts in an ERK1/2-dependent manner. Although Amylin +/- mice like Amylin-deficient mice display a low bone mass phenotype and increased bone resorption, Calcr +/- mice display a high bone mass due to an increase in bone formation. Moreover, compound heterozygote mice for Calcr and Amylin inactivation displayed bone abnormalities observed in both Calcr +/- and Amylin +/- mice, thereby ruling out that amylin uses CALCR to inhibit osteoclastogenesis in vivo. Thus, amylin is a physiological regulator of bone resorption that acts through an unidentified receptor.
Asunto(s)
Amiloide/metabolismo , Resorción Ósea , Osteogénesis/fisiología , Receptores de Calcitonina/metabolismo , Amiloide/genética , Animales , Densidad Ósea , Huesos/anomalías , Huesos/citología , Huesos/metabolismo , Diferenciación Celular/fisiología , Polipéptido Amiloide de los Islotes Pancreáticos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteoclastos/citología , Osteoclastos/fisiología , FenotipoRESUMEN
Multiple myeloma (MM) bone disease is characterized by the development of osteolytic lesions, which cause severe complications affecting the morbidity, mortality, and treatment of myeloma patients. Myeloma tumors seeded within the bone microenvironment promote hyperactivation of osteoclasts and suppression of osteoblast differentiation. Because of this prolonged suppression of bone marrow stromal cells' (BMSCs) differentiation into functioning osteoblasts, bone lesions in patients persist even in the absence of active disease. Current antiresorptive therapy provides insufficient bone anabolic effects to reliably repair MM lesions. It has become widely accepted that myeloma-exposed BMSCs have an altered phenotype with pro-inflammatory, immune-modulatory, anti-osteogenic, and pro-adipogenic properties. In this review, we focus on the role of epigenetic-based modalities in the establishment and maintenance of myeloma-induced suppression of osteogenic commitment of BMSCs. We will focus on recent studies demonstrating the involvement of chromatin-modifying enzymes in transcriptional repression of osteogenic genes in MM-BMSCs. We will further address the epigenetic plasticity in the differentiation commitment of osteoprogenitor cells and assess the involvement of chromatin modifiers in MSC-lineage switching from osteogenic to adipogenic in the context of the inflammatory myeloma microenvironment. Lastly, we will discuss the potential of employing small molecule epigenetic inhibitors currently used in the MM research as therapeutics and bone anabolic agents in the prevention or repair of osteolytic lesions in MM. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
RESUMEN
Activating transcription factor 4 (ATF4) is essential for bone formation. However, the mechanism of its actions in bone is poorly understood. The present study examined the role for ATF4 in the regulation of proliferation and survival of primary mouse bone marrow stromal cells (BMSCs) and osteoblasts. Results showed that Atf4(-/-) cells display a severe proliferative defect as measured by multiple cell proliferation assays. Cell cycle progression of Atf4(-/-) BMSCs was largely delayed with significant G1 arrest. Expression of cyclin D1 was decreased both at the mRNA and protein level. A similar proliferation defect was observed in Atf4(-/-) calvarial periosteal osteoblasts when compared with wt control. Knocking down Atf4 mRNA by small interfering RNA in MC3T3-E1 subclone 4 preosteoblasts markedly reduced expression of cyclin D1 and cell proliferation. In contrast, overexpression of ATF4 increased cyclin D1 expression as well as cell proliferation in Atf4(-/-) BMSCs. In addition, apoptosis was significantly increased in Atf4(-/-) BMSCs and calvarial periosteal osteoblasts relative to wt controls. Taken together, these results for the first time demonstrate that ATF4 is a critical regulator of proliferation and survival in BMSCs and osteoblasts in vitro and in vivo.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Células de la Médula Ósea/citología , Proliferación Celular , Osteoblastos/citología , Factor de Transcripción Activador 4/genética , Animales , Apoptosis , Células de la Médula Ósea/metabolismo , Ciclo Celular , Supervivencia Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Ratones , Ratones Transgénicos , Osteoblastos/metabolismo , Cráneo/citología , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
Rapid induction of transcription is known to be mediated by factors which bind DNA following post-translational modification. We report here that non-tyrosine phosphorylated (NTP)-Stat1 is involved in a cooperative interaction with Spi-1/PU.1 and IRF8 to form a pre-associated, poised complex for IL1B gene induction. A double point mutation at a putative STAT binding site, which overlaps this composite Spi-1 x IRF8 site located in the LPS and IL-1 response element (LILRE), inhibited human IL1B LPS-dependent reporter activity to about 10 percent of the control wild type vector. Chromatin immunoprecipitation revealed stimulation-independent constitutive binding of IRF8, Spi-1 and NTP-Stat1 at the LILRE, while binding of C/EBP beta was activated at an adjacent C/EBP beta site after LPS stimulation. In contrast to Stat1, IRF8 was tyrosine phosphorylated following LPS treatment. Supporting the involvement of NTP-Stat1, LPS-induced IL1B reporter activity in monocytes was enhanced by ectopic expression of NTP-Stat1 Y701F. In contrast, co-expression of a Y211F IRF8 mutein functioned as a dominant-negative inhibitor of LPS-induced IL1B reporter activity. In vitro DNA binding using extracts from LPS-treated monocytes confirmed that the LILRE enhancer constitutively binds a trimolecular complex containing IRF8, Spi-1 and NTP-Stat1. Binding studies using in vitro-expressed proteins revealed that NTP-Stat1 enhanced the binding of Spi-1 and IRF8 to the LILRE. Co-expression of TRAF6, an LPS surrogate, with Spi-1 and IRF8 enhanced IL1B reporter activity in HEK293R cells, which was dramatically reduced when Y211F IRF8 was co-expressed. These results suggest that the rapid transcriptional induction of an important inflammatory gene is dependent upon constitutive cooperative binding of a Spi-1 x IRF8 x NTP-Stat1 complex to the LILRE, which primes the gene for immediate induction following IRF8 phosphorylation. Phosphorylation of chromatin pre-associated factors like IRF8 may be an important strategy for the rapid transcriptional activation of genes involved in innate immunity.
Asunto(s)
Factores Reguladores del Interferón/metabolismo , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Lipopolisacáridos/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción STAT1/fisiología , Transactivadores/metabolismo , Activación Transcripcional/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Humanos , Factores Reguladores del Interferón/fisiología , Ratones , Fosforilación , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas/fisiología , Elementos de Respuesta , Factor de Transcripción STAT1/metabolismo , Transactivadores/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Activación Transcripcional/genética , Tirosina/metabolismoRESUMEN
Multiple myeloma (MM) is the most frequent cancer to involve the skeleton with patients developing osteolytic bone lesions due to hyperactivation of osteoclasts and suppression of BMSCs differentiation into functional osteoblasts. Although new therapies for MM have greatly improved survival, MM remains incurable for most patients. Despite the major advances in current anti-MM and anti-resorptive treatments that can significantly improve osteolytic bone lysis, many bone lesions can persist even after therapeutic remission of active disease. Bone marrow mesenchymal stem cells (BMSCs) from MM patients are phenotypically distinct from their healthy counterparts and the mechanisms associated with the long-term osteogenic suppression are largely unknown. In this review we will highlight recent results of transcriptomic profiling studies that provide new insights into the establishment and maintenance of the persistent pathological alterations in MM-BMSCs that occur in MM. We will we discuss the role of genomic instabilities and senescence in propagating the chronically suppressed state and pro-inflammatory phenotype associated with MM-BMSCs. Lastly we describe the role of epigenetic-based mechanisms in regulating osteogenic gene expression to establish and maintain the pro-longed suppression of MM-BMSC differentiation into functional OBs.