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1.
Mol Cancer ; 19(1): 126, 2020 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-32799870

RESUMEN

This article has been retracted. Please see the retraction notice for more detail: https://doi.org/10.1186/s12943-015-0389-y.

2.
Int J Mol Sci ; 20(10)2019 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-31121879

RESUMEN

Hepatocyte growth factor (HGF) and transforming growth factor ß1 (TGFß1) are biological stimuli of the micro-environment which affect bone metastasis phenotype through transcription factors, but their influence on the growth is scarcely known. In a xenograft model prepared with 1833 bone metastatic cells, derived from breast carcinoma cells, we evaluated mice survival and Twist and Snail expression and localization after competitive inhibition of HGF with NK4, or after blockade of TGFß1-type I receptor (RI) with SB431542: in the latter condition HGF was also measured. To explain the in vivo data, in 1833 cells treated with SB431542 plus TGFß1 we measured HGF formation and the transduction pathway involved. Altogether, HGF seemed relevant for bone-metastatic growth, being hampered by NK4 treatment, which decreased Twist more than Snail in the metastasis bulk. TGFß1-RI blockade enhanced HGF in metastasis and adjacent bone marrow, while reducing prevalently Snail expression at the front and bulk of bone metastasis. The HGF accumulation in 1833 cells depended on an auxiliary signaling pathway, triggered by TGFß1 under SB431542, which interfered in the transcription of HGF activator inhibitor type 1 (HAI-1) downstream of TGFß-activated kinase 1 (TAK1): HGF stimulated Twist transactivation. In conclusion, the impairment of initial outgrowth with NK4 seemed therapeutically promising more than SB431542 chemotherapy; a functional correlation between Twist and Snail in bone metastasis seemed to be influenced by the biological stimuli of the micro-environment, and the targeting of these phenotype biomarkers might inhibit metastasis plasticity and colonization, even if it would be necessary to consider the changes of HGF levels in bone metastases undergoing TGFß1-RI blockade.


Asunto(s)
Benzamidas/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Dioxoles/uso terapéutico , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Receptor Tipo I de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Neoplasias Óseas/patología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Femenino , Humanos , Ratones
3.
Int J Mol Sci ; 19(1)2018 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-29337876

RESUMEN

We examined the influence of microenvironment stimuli on molecular events relevant to the biological functions of 1833-bone metastatic clone and the parental MDA-MB231 cells. (i) In both the cell lines, hepatocyte growth factor (HGF) and the osteoblasts' biological products down regulated nuclear Ets-1-protein level in concomitance with endogenous miR-125b accumulation. In contrast, under hypoxia nuclear Ets-1 was unchanged, notwithstanding the miR-125b increase. (ii) Also, the 1833-cell invasiveness and the expression of Endothelin-1, the target gene of Ets-1/HIF-1, showed opposite patterns under HGF and hypoxia. We clarified the molecular mechanism(s) reproducing the high miR-125b levels with the mimic in 1833 cells. Under hypoxia, the miR-125b mimic maintained a basal level and functional Ets-1 protein, as testified by the elevated cell invasiveness. However, under HGF ectopic miR-125b downregulated Ets-1 protein and cell motility, likely involving an Ets-1-dominant negative form sensible to serum conditions; Ets-1-activity inhibition by HGF implicated HIF-1α accumulation, which drugged Ets-1 in the complex bound to the Endothelin-1 promoter. Altogether, 1833-cell exposure to HGF would decrease Endothelin-1 transactivation and protein expression, with the possible impairment of Endothelin-1-dependent induction of E-cadherin, and the reversion towards an invasive phenotype: this was favoured by Ets-1 overexpression, which inhibited HIF-1α expression and HIF-1 activity. (iii) In MDA-MB231 cells, HGF strongly and rapidly decreased Ets-1, hampering invasiveness and reducing Ets-1-binding to Endothelin-1 promoter; HIF-1α did not form a complex with Ets-1 and Endothelin-1-luciferase activity was unchanged. Overall, depending on the microenvironment conditions and endogenous miR-125b levels, bone-metastatic cells might switch from Ets-1-dependent motility towards colonization/growth, regulated by the balance between Ets-1 and HIF-1.


Asunto(s)
Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Factor de Crecimiento de Hepatocito/farmacología , MicroARNs/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Microambiente Tumoral , Neoplasias Óseas/metabolismo , Neoplasias de la Mama/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , ADN/metabolismo , Endotelina-1/metabolismo , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , MicroARNs/genética , Modelos Biológicos , Invasividad Neoplásica , Activación Transcripcional/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos
4.
Carcinogenesis ; 38(5): 492-503, 2017 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-28334277

RESUMEN

The highlight of the molecular basis and therapeutic targets of the bone-metastatic process requires the identification of biomarkers of metastasis colonization. Here, we studied miR-34a-5p expression, and Met-receptor expression and localization in bone metastases from ductal breast carcinomas, and in ductal carcinomas without history of metastasis (20 cases). miR-34a-5p was elevated in non-metastatic breast carcinoma, intermediate in the adjacent tissue and practically absent in bone metastases, opposite to pair-matched carcinoma. Met-receptor biomarker was highly expressed and inversely correlated with miR-34a-5p using the same set of bone-metastasis tissues. The miR-34a-5p silencing might depend on aberrant-epigenetic mechanisms of plastic-bone metastases, since in 1833 cells under methyltransferase blockade miR-34a-5p augmented. In fact, 1833 cells showed very low endogenous miR-34a-5p, in respect to parental MDA-MB231 breast carcinoma cells, and the restoration of miR-34a-5p with the mimic reduced Met and invasiveness. Notably, hepatocyte growth factor (HGF)-dependent Met stabilization was observed in bone-metastatic 1833 cells, consistent with Met co-distribution with the ligand HGF at plasma membrane and at nuclear levels in bone metastases. Met-protein level was higher in non-metastatic (low grade) than in metastatic (high grade) breast carcinomas, notwithstanding miR-34a-5p-elevated expression in both the specimens. Thus, mostly in non-metastatic carcinomas the elevated miR-34a-5p unaffected Met, important for invasive/mesenchymal phenotype, while possibly targeting some stemness biomarkers related to metastatic phenotype. In personalized therapies against bone metastasis, we suggest miR-34a-5p as a suitable target of epigenetic reprogramming leading to the accumulation of miR-34a-5p and the down-regulation of Met-tyrosine kinase, a key player of the bone-metastatic process.


Asunto(s)
Neoplasias Óseas/genética , Neoplasias Óseas/secundario , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Proteínas Proto-Oncogénicas c-met/genética , Biomarcadores de Tumor , Neoplasias Óseas/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Progresión de la Enfermedad , Femenino , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Inmunohistoquímica , Modelos Biológicos , Proteínas Proto-Oncogénicas c-met/metabolismo
5.
Biochim Biophys Acta ; 1863(1): 64-76, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26481505

RESUMEN

The pathogenesis of bone metastasis is unclear, and much focus in metastatic biology and therapy relays on epigenetic alterations. Since DNA-methyltransferase blockade with 5-aza-2'-deoxycytidine (dAza) counteracts tumour growth, here we utilized dAza to clarify whether molecular events undergoing epigenetic control were critical for bone metastatization. In particular, we investigated the patterns of secreted-protein acidic and rich in cysteine (SPARC) and of Endothelin 1, affected by DNA methyltransferases in tumours, with the hypothesis that in bone metastasis a coordinate function of SPARC and Endothelin 1, if any occurs, was orchestrated by DNA methylation. To this purpose, we prepared a xenograft model with the clone 1833, derived from human-MDA-MB231 cells, and dAza administration slowed-down metastasis outgrowth. This seemed consequent to the reductions of SPARC and Endothelin 1 at invasive front and in the bone marrow, mostly due to loss of Twist. In the metastasis bulk Snail, partly reduced by dAza, might sustain Endothelin 1-SPARC cooperativity. Both SPARC and Endothelin 1 underwent post-translational control by miRNAs, a molecular mechanism that might explain the in vivo data. Ectopic miR29a reduced SPARC expression also under long-term dAza exposure, while Endothelin 1 down-regulation occurred in the presence of endogenous-miR98 expression. Notably, dAza effects differed depending on in vivo and in vitro conditions. In 1833 cells exposed to 30-days dAza, SPARC-protein level was practically unaffected, while Endothelin 1 induction depended on the 3'-UTR functionality. The blockade of methyltransferases leading to SPARC reduction in vivo, might represent a promising strategy to hamper early steps of the metastatic process affecting the osteogenic niche.


Asunto(s)
Neoplasias Óseas/metabolismo , Neoplasias de la Mama/metabolismo , Metilación de ADN , ADN de Neoplasias/metabolismo , Microambiente Tumoral , Animales , Azacitidina/farmacología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , ADN de Neoplasias/genética , Endotelina-1/biosíntesis , Endotelina-1/genética , Femenino , Humanos , Ratones , Ratones Desnudos , MicroARNs/biosíntesis , MicroARNs/genética , Metástasis de la Neoplasia , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Osteonectina/biosíntesis , Osteonectina/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética
6.
Int J Mol Sci ; 18(1)2017 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-28045433

RESUMEN

Epigenetic mechanisms influence molecular patterns important for the bone-metastatic process, and here we highlight the role of WW-domain containing oxidoreductase (Wwox). The tumor-suppressor Wwox lacks in almost all cancer types; the variable expression in osteosarcomas is related to lung-metastasis formation, and exogenous Wwox destabilizes HIF-1α (subunit of Hypoxia inducible Factor-1, HIF-1) affecting aerobic glycolysis. Our recent studies show critical functions of Wwox present in 1833-osteotropic clone, in the corresponding xenograft model, and in human bone metastasis from breast carcinoma. In hypoxic-bone metastatic cells, Wwox enhances HIF-1α stabilization, phosphorylation, and nuclear translocation. Consistently, in bone-metastasis specimens Wwox localizes in cytosolic/perinuclear area, while TAZ (transcriptional co-activator with PDZ-binding motif) and HIF-1α co-localize in nuclei, playing specific regulatory mechanisms: TAZ is a co-factor of HIF-1, and Wwox regulates HIF-1 activity by controlling HIF-1α. In vitro, DNA methylation affects Wwox-protein synthesis; hypoxia decreases Wwox-protein level; hepatocyte growth factor (HGF) phosphorylates Wwox driving its nuclear shuttle, and counteracting a Twist program important for the epithelial phenotype and metastasis colonization. In agreement, in 1833-xenograft mice under DNA-methyltransferase blockade with decitabine, Wwox increases in nuclei/cytosol counteracting bone metastasis with prolongation of the survival. However, Wwox seems relevant for the autophagic process which sustains metastasis, enhancing more Beclin-1 than p62 protein levels, and p62 accumulates under decitabine consistent with adaptability of metastasis to therapy. In conclusion, Wwox methylation as a bone-metastasis therapeutic target would depend on autophagy conditions, and epigenetic mechanisms regulating Wwox may influence the phenotype of bone metastasis.


Asunto(s)
Neoplasias Óseas/secundario , Huesos/patología , Neoplasias de la Mama/patología , Mama/patología , Epigénesis Genética , Oxidorreductasas/genética , Proteínas Supresoras de Tumor/genética , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Huesos/metabolismo , Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Vía de Señalización Hippo , Humanos , Oxidorreductasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Oxidorreductasa que Contiene Dominios WW
7.
Exp Cell Res ; 330(2): 287-299, 2015 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-25447306

RESUMEN

The present study deals with the molecular mechanisms involved in the regulation of E-cadherin expression under hypoxia, because the adjustment of the amount of E-cadherin due to physical stimuli of the microenvironment might influence the colonization of metastasis to skeleton. We analyzed the effect of 1% oxygen tension, that is similar to that encountered in the bone marrow by metastatic cells spreading from breast carcinoma. The purpose was to evaluate the hypoxia-orchestrated control of E-cadherin transactivation via hypoxia inducible factor-1 (HIF-1) and peroxisome proliferator activated receptor-γ (PPARγ), and the involvement of Hippo pathway members, as regulators of transcription factors. To give a translational significance to the study, we took into consideration human pair-matched ductal breast carcinoma and bone metastasis: E-cadherin and Wwox were expressed in bone metastasis but not in breast carcinoma, while HIF-1α and TAZ seemed localized principally in nuclei of metastasis and were found in all cell compartments of breast carcinoma. A close examination of the regulatory mechanisms underlying E-cadherin expression in bone metastasis was done in 1833 clone derived from MDA-MB231 cells. Hypoxia induced E-cadherin only in 1833 clone, but not in parental cells, through HIF-1 and PPARγ activities, while Wwox decreased. Since Wwox was highly expressed in bone metastasis, the effect of ectopic Wwox was evaluated, and we showed E-cadherin transactivation and enhanced invasiveness in WWOX transfected 1833 cells. Also, hypoxia was additive with ectopic Wwox remarkably enhancing HIF-1α nuclear shuttle and accumulation due to the lengthening of the half-life of HIF-1α protein; under this experimental condition HIF-1α appeared as a slower migrated band compared with control, in agreement with the phosphorylation state. The in vitro data strongly supported the almost exclusive presence of HIF-1α in nuclei of human-bone metastasis. Thus, we identified Wwox as a novel molecule in the HIF-1α-HDM2 regulatory loop, necessary for the dynamic regulation of the HIF-1α amount, and we suggested that the reduction of endogenous Wwox free pool under hypoxia might also be due to the interaction with HDM2, sequestering the E3 ubiquitin ligase. We highlighted the importance of nuclear HIF-1α in the biology of metastasis for the mesenchymal-epithelial transition: this phenotype was regulated by Wwox plus hypoxia through E-cadherin target gene, playing a pivotal role in bone metastasis colonization.


Asunto(s)
Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Cadherinas/genética , Carcinoma Ductal de Mama/secundario , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Oxidorreductasas/genética , Proteínas Supresoras de Tumor/genética , Aciltransferasas , Cadherinas/biosíntesis , Cadherinas/metabolismo , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica , Vía de Señalización Hippo , Humanos , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/biosíntesis , PPAR gamma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/biosíntesis , Ubiquitina-Proteína Ligasas/metabolismo , Oxidorreductasa que Contiene Dominios WW
9.
Int J Mol Sci ; 17(5)2016 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-27187355

RESUMEN

Bone metastatic cells release bone microenvironment proteins, such as the matricellular protein SPARC (secreted protein acidic and rich in cysteine), and share a cell signaling typical of the bone metabolism controlled by Runx2. The megakaryocytes in the bone marrow engrafted by the metastases seem to be one of the principal microenvironment sources of the biological stimuli, implicated in the formation of an osteoblastic niche, and affecting metastasis phenotype and colonization. Educated platelets in the circulation might derive from megakaryocytes in bone metastasis. The evaluation of predictive markers in the circulating platelets might be useful for the stratification of patients for therapeutic purposes. The hypoxic environment in bone metastasis is one of the key regulators of the network of the biological soluble and structural components of the matrix. In bone metastatic cells under hypoxia, similar patterns of Runx2 and SPARC are observed, both showing downregulation. Conversely, hypoxia induces Endothelin 1, which upregulates SPARC, and these biological stimuli may be considered prognostic markers of bone metastasis in breast carcinoma patients.


Asunto(s)
Neoplasias Óseas/metabolismo , Oxígeno/metabolismo , Transducción de Señal , Microambiente Tumoral , Animales , Neoplasias Óseas/secundario , Hipoxia de la Célula , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo
10.
Int J Mol Sci ; 17(4): 443, 2016 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-27023526

RESUMEN

Autophagy favours metastatic growth through fuelling energy and nutrients and resistance to anoikis, typical of disseminated-tumour cells. The autophagic process, mediated by a unique organelle, the autophagosome, which fuses with lysosomes, is divided into three steps. Several stages, especially early omegasome formation and isolation-membrane initiation, remain controversial; molecular mechanisms involve the small-GTPase Rab5a, which regulates vesicle traffic for autophagosome formation. We examined Rab5a involvement in the function of key members of ubiquitin-conjugation systems, Atg7 and LC3-lipidated, interacting with the scaffold-protein p62. Immunohistochemistry of Rab5a was performed in human specimens of bone metastasis and pair-matched breast carcinoma; the autophagic-molecular mechanisms affected by Rab5a were evaluated in human 1833 bone metastatic cells, derived from breast-carcinoma MDA-MB231 cells. To clarify the role of Rab5a, 1833 cells were transfected transiently with Rab5a-dominant negative, and/or stably with the short-hairpin RNA Atg7, were exposed to two inhibitors of autolysosome function, and LC3II and p62 expression was measured. We showed basal autophagy in bone-metastatic cells and the pivotal role of Rab5a together with Beclin 1 between the early stages, elongation of isolation membrane/closed autophagosome mediated by Atg7, and the late-degradative stages. This regulatory network might occur in bone-metastasis and in high-grade dysplastic lesions, preceding invasive-breast carcinoma and conferring phenotypic characteristics for dissemination.


Asunto(s)
Neoplasias Óseas/patología , Neoplasias de la Mama/patología , Proteínas de Unión al GTP rab5/metabolismo , Autofagia , Proteína 7 Relacionada con la Autofagia/antagonistas & inhibidores , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Beclina-1/genética , Beclina-1/metabolismo , Western Blotting , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Estadificación de Neoplasias , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al GTP rab5/genética
11.
Biochim Biophys Acta ; 1843(4): 815-26, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24373848

RESUMEN

The present study was undertaken to clarify the function(s) of Endothelin-1 and its receptors ETAR and ETBR in osteolytic-bone metastasis from breast cancer, and their regulation by hepatocyte and transforming growth factors (HGF, TGF-ß) and hypoxia. The aim was to evaluate the adaptability of bone metastasis to microenvironmental stimuli through Endothelin-1-mediated epithelial-mesenchymal transition (EMT), or the reverse process MET, and through osteomimicry possible key features for bone colonization. We compared low (MCF-7) and high (MDA-MB231) invasive-breast carcinoma cells, and 1833-bone metastatic clone, with human pair-matched primary breast-carcinomas and bone metastases. Parental MDA-MB231 and the derived 1833-clone responded oppositely to the stimuli. In 1833 cells, TGF-ß and hypoxia increased Endothelin-1 release, altogether reducing invasiveness important for engraftment, while Endothelin-1 enhanced MDA-MB231 cell invasiveness. The Endothelin-1-autocrine loop contributed to the cooperation of intracellular-signaling pathways and extracellular stimuli triggering MET in 1833 cells, and EMT in MDA-MB231 cells. Only in 1833 cells, HGF negatively influenced transactivation and release of Endothelin-1, suggesting a temporal sequence of these stimuli with an initial role of HGF-triggered Wnt/ß-catenin pathway in metastatization. Then, Endothelin-1/ETAR conferred MET and osteomimetic phenotypes, with Runt-related transcription factor 2 activation and metalloproteinase 9 expression, contributing to colonization and osteolysis. Findings with human pair-matched primary ductal carcinomas and bone metastases gave a translational significance to the molecular study. Endothelin-1, ETAR and ETBR correlated with the acquisition of malignant potential, because of high expression already in the in situ carcinoma. These molecular markers might be used as predictive index of aggressive behavior and invasive/metastatic phenotype.


Asunto(s)
Neoplasias Óseas/genética , Neoplasias de la Mama/genética , Endotelina-1/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Endotelina-1/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Invasividad Neoplásica/genética , Receptor de Endotelina A , Receptor de Endotelina B , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral , Vía de Señalización Wnt/genética
12.
Mol Cancer ; 14: 112, 2015 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-26041563

RESUMEN

BACKGROUND: Much effort has been devoted to determining how metastatic cells and microenvironment reciprocally interact. However, the role of biological stimuli of microenvironment in controlling molecular events in bone metastasis from breast carcinoma for mesenchymal-epithelial transition (MET) is largely unknown. The purpose of the present paper was to clarify (1) the influence of hepatocyte-growth factor (HGF) and transforming growth factorß1 (TGFß1) on the phenotype of bone-metastatic 1833 and parental MDA-MB231 cells; (2) the hierarchic response of Twist and Snail controlled by Wwox co-factor, that might be critical for the control of 1833-adhesive properties via E-cadherin. METHODS: We studied under HGF and TGFß1 the gene profiles-responsible for epithelial-mesenchymal transition (EMT), versus the revertant MET phenotype-making the correspondence with 1833 morphology and the relation to HGF-dependent control of TGFß1 signalling. In particular, the activation of Twist program and the underlying molecular mechanisms were investigated, considering the role of endogenous and exogenous Wwox with siRNAWWOX and the expression vector transfection, to clarify whether Twist affected E-cadherin transactivation through a network of transcription factors and regulators. RESULTS: HGF and TGFß1 oppositely affected the expression of Wwox in 1833 cells. Under HGF, endogenous Wwox decreased concomitant with Twist access to nuclei and its phosphorylation via PI3K/Akt pathway. Twist activated by HGF did not influence the gene profile through an E-box mechanism, but participated in the interplay of PPARγ/Ets1/NF-kB-transcription factors, triggering E-cadherin transactivation. Altogether, HGF conferred MET phenotype to 1833 cells, even if this was transient since followed by TGFß1-signalling activation. TGFß1 induced Snail in both the cell lines, with E-cadherin down-regulation only in 1833 cells because in MDA-MB231 cells E-cadherin was practically absent. Exogenous Wwox activated metastatic HIF-1, with Twist as co-factor. CONCLUSIONS: HGF and TGFß1 of bone-metastasis microenvironment acted co-ordinately, influencing non redundant pathways regulated by Twist program or Snail-transcription factor, with reversible MET switch. This process implicated different roles for Wwox in the various steps of the metastatic process including colonization, with microenvironmental/exogenous Wwox that activated HIF-1, important for E-cadherin expression. Interfering with the Twist program by targeting the pre-metastatic niche stimuli could be an effective anti-bone metastasis therapy.


Asunto(s)
Neoplasias Óseas/secundario , Neoplasias de la Mama/metabolismo , Transición Epitelial-Mesenquimal , Factor de Crecimiento de Hepatocito/metabolismo , Proteínas Nucleares/metabolismo , Oxidorreductasas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Animales , Neoplasias Óseas/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Cadherinas/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , ADN de Neoplasias/metabolismo , Regulación hacia Abajo , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Ratones Desnudos , Modelos Biológicos , Fosforilación , Unión Proteica , Transducción de Señal , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Oxidorreductasa que Contiene Dominios WW
13.
Int J Mol Sci ; 16(12): 28108-22, 2015 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-26703564

RESUMEN

In order to become established in the skeleton, metastatic cells disseminating from the breast carcinoma need to acquire organ-specific traits. There are no effective predictors for who will develop bone metastasis to guide long-term predictive therapy. Our purpose was to individuate events critical for bone colonization to make a molecular classification of breast carcinoma useful for bone-metastasis outcome. In dysplasia adjacent to carcinoma and in pair-matched specimens of bone metastasis we examined SPARC expression and localization as well as Endothelin 1/ETAR signals by immunohistochemistry, and the evaluation of plasma levels of SPARC by ELISA was also performed. In patients with breast carcinoma metastasizing to bone, SPARC and Endothelin 1/ETAR axis were highly expressed from dysplasia until bone metastasis, but the SPARC plasma level was as low as that of normal women, in contrast to patients that never develop bone metastasis, suggesting that circulating SPARC was counter adhesive. Altogether, the early identification of SPARC/Endothelin 1/ETAR in dysplastic lesions would be important to devise therapies preventing metastasis engraftment, since often carcinoma cells spread to distant organs at the time or even before patients present with cancer.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/diagnóstico , Neoplasias de la Mama/sangre , Carcinoma/sangre , Osteonectina/metabolismo , Adulto , Biomarcadores de Tumor/sangre , Neoplasias Óseas/sangre , Neoplasias Óseas/secundario , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/patología , Estudios de Casos y Controles , Endotelina-1/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Osteonectina/sangre , Receptor de Endotelina A/metabolismo
14.
Exp Cell Res ; 319(12): 1865-1874, 2013 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-23660387

RESUMEN

Here, we report a complex regulation of endothelin-1 (ET-1) axis driven by epigenetic reactions in 1833-bone metastatic cells, emphasizing the importance in skeletal metastasis from breast carcinoma. Inhibitors of histone deacetylases, trichostatin A (TSA), and of DNA methylases, 5'-Azacytidine (Aza), caused, respectively, reduction and increase in 1833 cell invasiveness, without affecting the basal migration of parental MDA-MB231 cells. Of note, in the two cell lines exposed to Aza the blockade of the ET-1 receptor ETAR with BQ-123 oppositely changed invasive properties. Even if in MDA-MB231 cells the ET-1 axis was scarcely influenced by epigenetic reactions, ETAR remarkably decreased after Aza. In contrast, in 1833 cells Aza exposure enhanced ET-1 coupled to ETAR wild type, being also ETAR truncated form increased, and invasiveness was stimulated. Under demethylation, the increase in ET-1 steady state protein level in 1833 clone seemed regulated at transcriptional level principally via Ets1 transcription factor. In fact, actinomycin D almost completely prevented ET-1 mRNA induction due to Aza. Only in 1833 cells, TSA exposure inactivated ET-1 axis, with reduction of the expression of ET-1 and ETAR mutated form, in agreement with Matrigel invasion decrease. This treatment favoured the ET-1 repressional control, taking place at the level of mRNA stability due to the 3'-untranslated region in the ET-1 gene, and also decreased transcription via NF-kB. Environmental conditions that alter the balance between epigenetic reactions might, therefore, affect metastasis migratory mode influencing ET-1 axis.


Asunto(s)
Neoplasias Óseas/secundario , Neoplasias de la Mama/genética , Carcinoma/genética , Movimiento Celular , Endotelina-1/genética , Epigénesis Genética , Azacitidina/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/secundario , Línea Celular Tumoral , Colágeno/metabolismo , Metilación de ADN , Dactinomicina/farmacología , Combinación de Medicamentos , Antagonistas de los Receptores de la Endotelina A , Endotelina-1/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Laminina/metabolismo , Mutación , Invasividad Neoplásica/genética , Péptidos Cíclicos/farmacología , Proteoglicanos/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Transcripción Genética
15.
Biochim Biophys Acta ; 1813(10): 1767-76, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21741415

RESUMEN

Metastatic cells switch between different modes of migration through supramolecular plasticity mechanism(s) still largely unknown. The aim of the present paper was to clarify some molecular aspects of the epigenetic control of migration of 1833-bone metastatic cells compared to MDA-MB231-parental mammary carcinoma cells. Active c-Src overexpression enhanced 1833-cell spontaneous migration and CXCR4-mediated chemoinvasion toward CXCL12 ligand. Only in metastatic cells, in fact, c-Src seemed to stabilize nuclear CXCR4-protein receptor possibly due to tyrosine phosphorylation, by impairing protein-degradative smear and causing instead an electrophoretic-mobility shift; the cytosolic steady-state level of CXCR4 was enhanced, and the protein appeared also phosphorylated. These findings suggested the triggering of unique signaling pathways in metastasis for homing of breast-cancer cells to congenial environment of specific organs. Microenvironmental stimuli activating c-Src might influence Ets1 binding to CXCR4 promoter and consequent transactivation, as well as CXCR4 post-translational regulatory mechanisms such as phosphorylation. Enhancement of Ets1 activity and CXCR4 induction by c-Src overexpression were prevented by histone deacetylase (HDAC) blockade. In contrast, HDAC inhibition with trichostatin A increased cytosolic phosphorylated CXCR4 expression in MDA-MB231 cells, but Ets1 involvement was practically unneeded. c-Src might be suggested as a bio-marker predicting metastasis sensitivity patterns to HDAC inhibitors. Rationally designed and individualized therapy may become possible as more is learned about the target molecules of HDAC's inhibitory agents and their roles, as undertaken for CXCR4 that is likely to be crucial for homing, angiogenesis and survival in a c-Src-dependent manner in bone-metastatic mammary cells.


Asunto(s)
Acetiltransferasas/fisiología , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Carcinoma/patología , Proteínas Tirosina Quinasas/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Acetilación/efectos de los fármacos , Acetiltransferasas/metabolismo , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteína Tirosina Quinasa CSK , Carcinoma/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Humanos , Modelos Biológicos , Invasividad Neoplásica , Metástasis de la Neoplasia , Fenotipo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Familia-src Quinasas
16.
Biochem Biophys Res Commun ; 428(2): 271-7, 2012 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-23085045

RESUMEN

The human adipose-tissue derived stem/stromal cells (hASCs) are an interesting source for bone-tissue engineering applications. Our aim was to clarify in hASCs the role of acetylation in the control of Runt-related transcription factor 2 (Runx2) and Peroxisome proliferator activated receptor (PPAR) γ. These key osteogenic and adipogenic transcription factors are oppositely involved in osteo-differentiation. The hASCs, committed or not towards bone lineage with osteoinductive medium, were exposed to HDACs chemical blockade with Trichostatin A (TSA) or were genetically silenced for HDACs. Alkaline phosphatase (ALP) and collagen/calcium deposition, considered as early and late osteogenic markers, were evaluated concomitantly as index of osteo-differentiation. TSA pretreatment, useful experimental protocol to analyse pan-HDAC-chemical inhibition, and switch to osteogenic medium induced early-osteoblast maturation gene Runx2, while transiently decreased PPARγ and scarcely affected late-differentiation markers. Time-dependent effects were observed after knocking-down of HDAC1 and 3: Runx2 and ALP underwent early activation, followed by late-osteogenic markers increase and by PPARγ/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPARγ target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal a role for HDACs in orchestrating osteo-differentiation of hASCs at transcriptional level, and might provide new insights into the modulation of hASCs-based regenerative therapy.


Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/fisiología , Histona Desacetilasas/fisiología , Osteogénesis/fisiología , Células Madre/citología , Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Adipogénesis/fisiología , Tejido Adiposo/efectos de los fármacos , Adulto , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Histona Desacetilasa 1/genética , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Humanos , Ácidos Hidroxámicos/farmacología , Persona de Mediana Edad , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , PPAR gamma/metabolismo , Medicina Regenerativa , Células Madre/efectos de los fármacos , Supresión Genética
17.
Breast Cancer Res Treat ; 129(2): 433-50, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21069452

RESUMEN

The aim of this article is to identify nuclear co-localization of COX-2 and HIF-1α in human-bone metastasis of breast cancer, index of transcriptionally activated cells and functional for gene expression. In particular, we verified whether hypoxia exerted a direct role on metastasis-gene expression or through COX-2 signaling, due to the relevance for clinical implications to individuate molecular targets for diagnosis and therapy. The experiments were performed in vitro with two metastatic clones, 1833 and MDA-231BO, and the parental MDA-MB231 cells, in vivo (1833-xenograft model), and in human-bone metastasis specimens. In 1833 cells in vitro, COX-2 signaling pathway was critical for nuclear HIF-1α-protein expression/translocation, mechanisms determining HIF-1 activity and gene expression. The data were corroborated by immunohistochemistry in human-bone metastasis specimens. COX-2 and HIF-1α showed wide co-localization in the nucleus, indicative of COX-2-nuclear import in transcriptionally activated metastatic cells and consistent with COX-2-HIF-1α functional interaction. A network of microenvironmental signals controlled COX-2 induction and HIF-1 activation downstream. In fact, hypoxia through HGF and TGF-ß1 autoregulatory loops triggered a specific array of transcription factors responsible for COX-2 transactivation. The novelty was that HGF and TGF-ß1 biological signals were produced by hypoxic metastatic cells and, therefore, the microenvironment seemed to be modified by metastatic-cell engraftment in the bone. In agreement, HIF-1α expression in bone marrow supportive cells occurred in metastasis-bearing animals. Altogether, the data supported the pre-metastatic-niche theory. Our observations might be useful to design therapies against bone metastasis, by affecting the phenotype changes of metastatic cells occurring at the secondary growth site through COX-2-HIF-1 interaction.


Asunto(s)
Neoplasias Óseas/enzimología , Neoplasias de la Mama/enzimología , Carcinoma/enzimología , Núcleo Celular/enzimología , Ciclooxigenasa 2/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Animales , Western Blotting , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma/genética , Carcinoma/secundario , Hipoxia de la Célula , Línea Celular Tumoral , Núcleo Celular/patología , Ciclooxigenasa 2/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunohistoquímica , Ratones , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Transfección , Factor de Crecimiento Transformador beta1/genética , Microambiente Tumoral
18.
J Cell Physiol ; 225(3): 757-66, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20568106

RESUMEN

Loss of articular cartilage through injury or disease presents major clinical challenges also because cartilage has very poor regenerative capacity, giving rise to the development of biological approaches. As autologous blood product, platelet-rich plasma (PRP) provides a promising alternative to surgery by promoting safe and natural healing. Here we tested the possibility that PRP might be effective as an anti-inflammatory agent, providing an attractive basis for regeneration of articular cartilage, and two principal observations were done. First, activated PRP in chondrocytes reduced the transactivating activity of NF-κB, critical regulator of the inflammatory process, and decreased the expression of COX-2 and CXCR4 target genes. By analyzing a panel of cytokines with different biological significance, in activated PRP we observed increases in hepatocyte growth factor (HGF), interleukin-4 and tumor necrosis factor-α (TNF-α). HGF and TNF-α, by disrupting NF-κB-transactivating activity, were important for the anti-inflammatory function of activated PRP. The key molecular mechanisms involved in PRP-inhibitory effects on NF-κB activity were for HGF the enhanced cellular IkBα expression, that contributed to NF-κB-p65 subunit retention in the cytosol and nucleo-cytoplasmic shuttling, and for TNF-α the p50/50 DNA-binding causing inhibition of target-gene expression. Second, activated PRP in U937-monocytic cells reduced chemotaxis by inhibiting chemokine transactivation and CXCR4-receptor expression, thus possibly controlling local inflammation in cartilage. In conclusion, activated PRP is a promising biological therapeutic agent, as a scaffold in micro-invasive articular cartilage regeneration, not only for its content of proliferative/differentiative growth factors, but also for the presence of anti-inflammatory agents including HGF.


Asunto(s)
Plaquetas/metabolismo , Condrocitos/metabolismo , Factor de Crecimiento de Hepatocito/sangre , Inflamación/prevención & control , FN-kappa B/metabolismo , Transfusión de Plaquetas , Transporte Activo de Núcleo Celular , Plaquetas/inmunología , Quimiocina CXCL12/metabolismo , Quimiotaxis , Condrocitos/inmunología , Ciclooxigenasa 2/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Inflamación/sangre , Inflamación/inmunología , Mediadores de Inflamación/sangre , Interleucina-4/sangre , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Fosforilación , Receptores CXCR4/metabolismo , Factor de Transcripción ReIA/metabolismo , Activación Transcripcional , Transfección , Factor de Necrosis Tumoral alfa/sangre , Células U937
19.
Carcinogenesis ; 30(6): 937-45, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19357348

RESUMEN

Hepatocyte growth factor (HGF)/Met system is deregulated in tumors and is implicated in different aspects of invasive growth. Here, we report that in the highly aggressive MDA-MB231 breast carcinoma cells, Met cytosolic fragments [C-terminal fragment (CTF)] were present in the nuclei. They were constitutively active because tyrosine phosphorylated at regulatory and catalytic domains and endowed with transactivating activity independently of HGF exposure. In fact, various constructs containing juxtamembrane (Jxtm) Met fragments, fused with Gal4 DNA-binding domain, transactivated Gal4Luc activity. MDA-MB231 cells were devoid of WW domain-containing oxidoreductase (Wwox) tumor suppressor. Exogenous Wwox protein expression negatively regulated Jxtm3-transactivating activity and decreased spontaneous migration of MDA-MB231 cells. Also, we demonstrate that the lack of endogenous Wwox in MDA-MB231 cells represented a molecular mechanism for intranuclear Met-CTF accumulation and for the decrease of full-length Met stability. Yes-associated proteins maintained constitutively activated nuclear Met fragments that played a role as transcription factors regulating genes probably including those for motile phenotype. The difference with low invasive MCF-7 cells was evident because the latter did not show intranuclear Met and the transfected constructs-containing Jxtm fragments were inactive also in the presence of HGF. The constitutive activation of nuclear Met-signaling pathway in MDA-MB231 cells, possibly determined at genetic or epigenetic levels of WWOX gene, might participate in breast carcinoma progression influencing invasive/metastatic phenotype. Wwox/Met system can be suggested as a potential target to impair breast carcinoma progression.


Asunto(s)
Neoplasias de la Mama/metabolismo , Núcleo Celular/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Epigénesis Genética , Femenino , Humanos , Invasividad Neoplásica , Oxidorreductasas/metabolismo , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-met , Proteínas Proto-Oncogénicas c-yes/metabolismo , Receptores de Factores de Crecimiento/genética , Transducción de Señal , Factores de Transcripción , Activación Transcripcional , Proteínas Supresoras de Tumor/metabolismo , Oxidorreductasa que Contiene Dominios WW , Proteínas Señalizadoras YAP
20.
Mol Cancer Res ; 5(8): 833-45, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17699109

RESUMEN

Hepatocyte growth factor (HGF), a cytokine of tumor microenvironment, exerts opposite effects on CXCR4 expression in MCF-7 (low invasive) and MDA-MB231 (highly invasive) breast carcinoma cells, and here, we show that completely different molecular mechanisms downstream of c-Src activation were involved. As experimental models, we used cells transfected with two CXCR4 promoter constructs and treated with HGF or cotransfected with c-Src wild-type (Srcwt) expression vector; phospho-c-Src formation was enhanced in both cell lines. In MCF-7 cells, consistent with activations of CXCR4Luc constructs after HGF treatment and Srcwt expression, Ets1 and nuclear factor-kappaB (NF-kappaB) transcription factors were activated. In contrast, in MDA-MB231 cells, CXCR4Luc construct, Ets1 and NF-kappaB activities decreased. The divergence point seemed to be downstream of HGF/c-Src and consisted in the interaction between c-Src and the substrate histone deacetylase 3 (HDAC3). Only in MDA-MB231 cells, HDAC3 level was enhanced in membranes and nuclei 30 min after HGF and colocalized/coimmunoprecipitated with phospho-c-Src and phosphotyrosine. Thus, the CXCR4 induction by HGF in MCF-7 cells required NF-kappaB and Ets1 activations, downstream of phosphoinositide-3-kinase/Akt, whereas in HGF-treated MDA-MB231 cells, HDAC3 activation via c-Src probably caused a reduction of transcription factor activities, such as that of NF-kappaB. These results indicate possible roles of HGF in invasive growth of breast carcinomas. By enhancing CXCR4 in low invasive tumor cells, HGF probably favors their homing to secondary sites, whereas by suppressing CXCR4 in highly invasive cells, HGF might participate to retain them in the metastatic sites.


Asunto(s)
Neoplasias de la Mama/patología , Factor de Crecimiento de Hepatocito/farmacología , Histona Desacetilasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptores CXCR4/metabolismo , Western Blotting , Neoplasias de la Mama/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Humanos , Inmunoprecipitación , FN-kappa B/genética , FN-kappa B/metabolismo , Invasividad Neoplásica , Fosforilación , Regiones Promotoras Genéticas/genética , Receptores CXCR4/genética , Activación Transcripcional
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