RESUMEN
Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.
Asunto(s)
Caspasa 9 , Movimiento Celular , Osteoblastos , Proteómica , Animales , Osteoblastos/metabolismo , Osteoblastos/citología , Ratones , Proteómica/métodos , Caspasa 9/metabolismo , Caspasa 9/genética , Línea Celular , Técnicas de Inactivación de GenesRESUMEN
Visualizing the differential distribution of carbon-carbon double bond (CâC db) positional isomers of unsaturated phospholipids (PL) in tissue sections by use of refined matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) technologies offers a high promise to deeper understand PL metabolism and isomer-specific functions in health and disease. Here we introduce an on-tissue ozonization protocol that enables a particular straightforward derivatization of unsaturated lipids in tissue sections. Collision-induced dissociation (CID) of MALDI-generated ozonide ions (with yields in the several ten percent range) produced the Criegee fragment ion pairs, which are indicative of CâC db position(s). We used our technique for visualizing the differential distribution of Δ9 and Δ11 isomers of phosphatidylcholines in mouse brain and in human colon samples with the desorption laser spot size 15 µm, emphasizing the potential of the technique to expose local isomer-specific metabolism of PLs.
Asunto(s)
Ozono/química , Fosfolípidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Carbono/química , Colon/diagnóstico por imagen , Colon/metabolismo , Humanos , Iones/química , Isomerismo , Ratones , Fosfolípidos/metabolismoRESUMEN
Neurodegenerative disorders present a broad group of neurological diseases and remain one of the greatest challenges and burdens to mankind. Maladies like amyotrophic lateral sclerosis, Alzheimer's disease, stroke or spinal cord injury commonly features astroglia involvement (astrogliosis) with signs of inflammation. Regenerative, paracrine and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) could target the above components, thus opening new therapeutic possibilities for regenerative medicine. A special interest should be given to hMSCs derived from the umbilical cord (UC) tissue, due to their origin, properties and lack of ethical paradigms. The aim of this study was to establish standard operating and scale-up good manufacturing practice (GMP) protocols of UC-hMSCs isolation, characterization, expansion and comparison of cells' properties when harvested on T-flasks versus using a large-scale bioreactor system. Human UC-hMSCs, isolated by tissue explant culture technique from Wharton's jelly, were harvested after reaching 75% confluence and cultured using tissue culture flasks. Obtained UC-hMSCs prior/after the cryopreservation and after harvesting in a bioreactor, were fully characterized for "mesenchymness" immunomodulatory, tumorigenicity and genetic stability, senescence and cell-doubling properties, as well as gene expression features. Our study demonstrates an efficient and simple technique for large scale UC-hMSCs expansion. Harvesting of UC-hMSCs' using classic and large scale methods did not alter UC-hMSCs' senescence, genetic stability or in vitro tumorigenicity features. We observed comparable growth and immunomodulatory capacities of fresh, frozen and expanded UC-hMSCs. We found no difference in the ability to differentiate toward adipogenic, osteogenic and chondrogenic lineages between classic and large scale UC-hMSCs expansion methods. Both, methods enabled derivation of genetically stabile cells with typical mesenchymal features. Interestingly, we found significantly increased mRNA expression levels of neural growth factor (NGF) and downregulated insulin growth factor (IGF) in UC-hMSCs cultured in bioreactor, while IL4, IL6, IL8, TGFb and VEGF expression levels remained at the similar levels. A culturing of UC-hMSCs using a large-scale automated closed bioreactor expansion system under the GMP conditions does not alter basic "mesenchymal" features and quality of the cells. Our study has been designed to pave a road toward translation of basic research data known about human UC-MSCs for the future clinical testing in patients with neurological and immunocompromised disorders. An industrial manufacturing of UC-hMSCs next will undergo regulatory approval following advanced therapy medicinal products (ATMP) criteria prior to clinical application and approval to be used in patients.
Asunto(s)
Reactores Biológicos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Enfermedades del Sistema Nervioso/terapia , Cordón Umbilical/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Trasplante de Células Madre Mesenquimatosas/tendencias , Enfermedades del Sistema Nervioso/patología , Cordón Umbilical/citología , Cordón Umbilical/trasplante , Gelatina de Wharton/citología , Gelatina de Wharton/fisiología , Gelatina de Wharton/trasplanteRESUMEN
Biological treatment of many cancers currently targets membrane bound receptors located on a cell surface. To identify novel membrane proteins associated with migration and metastasis of breast cancer cells, a more migrating subpopulation of MDA-MB-231 breast cancer cell line is selected and characterized. A high-resolution quantitative mass spectrometry with SILAC labeling is applied to analyze their surfaceome and it is compared with that of parental MDA-MB-231 cells. Among 824 identified proteins (FDR < 0.01), 128 differentially abundant cell surface proteins with at least one transmembrane domain are found. Of these, i) desmocollin-1 (DSC1) is validated as a protein connected with lymph node status of luminal A breast cancer, tumor grade, and Her-2 status by immunohistochemistry in the set of 96 primary breast tumors, and ii) catechol-O-methyltransferase is successfully verified as a protein associated with lymph node metastasis of triple negative breast cancer as well as with tumor grade by targeted data extraction from the SWATH-MS data of the same set of tissues. The findings indicate importance of both proteins for breast cancer development and metastasis and highlight the potential of biomarker validation strategy via targeted data extraction from SWATH-MS datasets.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Catecol O-Metiltransferasa/metabolismo , Movimiento Celular , Desmocolinas/metabolismo , Metástasis Linfática/patología , Proteómica , Neoplasias de la Mama/genética , Catecol O-Metiltransferasa/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular/genética , Desmocolinas/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Invasividad Neoplásica , Fenotipo , Receptor ErbB-2 , Análisis de Supervivencia , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Regulación hacia Arriba/genéticaRESUMEN
The cell surface glycoprotein Trop-2 is commonly overexpressed in carcinomas and represents an exceptional antigen for targeted therapy. Here, we provide evidence that surface Trop-2 expression is functionally connected with an epithelial phenotype in breast and prostate cell lines and in patient tumor samples. We further show that Trop-2 expression is suppressed epigenetically or through the action of epithelial-to-mesenchymal transition transcription factors and that deregulation of Trop-2 expression is linked with cancer progression and poor patient prognosis. Moreover, our data suggest that the cancer plasticity-driven intratumoral heterogeneity in Trop-2 expression may significantly contribute to response and resistance to therapies targeting Trop-2-expressing cells.
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Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/patología , Carcinoma/patología , Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/metabolismo , Neoplasias de la Próstata/patología , Animales , Antígenos CD/biosíntesis , Antígenos de Neoplasias/genética , Neoplasias de la Mama/mortalidad , Cadherinas/biosíntesis , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Metilación de ADN/genética , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Neoplasias de la Próstata/mortalidad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cathepsin D (CD), a ubiquitously expressed lysosomal aspartic protease, is upregulated in human breast carcinoma and many other tumor types. CD has been repeatedly reported to act as key mediator of apoptosis induced by various chemotherapeutics. However, there is still controversy over the role of enzymatic/proteolytic versus protein-protein interaction activities of CD in apoptotic signaling. The elucidation of molecular mechanism responsible for the effect of CD in the chemotherapy-induced cell death is crucial for development of an appropriate strategy to target this protease in cancer treatment. Therefore, the objective of this study was to investigate the molecular mechanism behind the CD-mediated regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. For this purpose, MDA-MB-231 breast carcinoma cells with an increased level of wt CD (CD) or mutant enzymatically inactive CD (ΔCD) were subjected to TRAIL and the frequency of apoptosis was determined. Our results show that CD facilitates the TRAIL-induced apoptosis of MDA-MB-231 breast cancer cells in enzymatic activity-dependent manner. Moreover, the importance of endosomal/lysosomal acidification in this process was documented. Analysis of the potential substrates specifically cleaved by CD during the TRAIL-induced apoptosis confirmed caspase-8 and Bid proteins as the CD targets. Moreover, in search for protein regulators of apoptosis that can be cleaved by CD at physiologically relevant pH, we identified the Bcl-2 protein as a suitable candidate. The modulatory role of CD in cell response to TRAIL was also confirmed in another breast cancer cell line SKBR3. These experiments identified the CD enzymatic activity as a new factor affecting sensitivity of breast cancer cells to TRAIL.
Asunto(s)
Adenocarcinoma/patología , Neoplasias de la Mama/patología , Catepsina D/fisiología , Proteínas de Neoplasias/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Adenocarcinoma/enzimología , Apoptosis/efectos de los fármacos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Neoplasias de la Mama/enzimología , Caspasa 8/metabolismo , Catepsina D/antagonistas & inhibidores , Catepsina D/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos , Endosomas/metabolismo , Activación Enzimática , Femenino , Humanos , Concentración de Iones de Hidrógeno , Lisosomas/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
The MYB gene codes for the c-Myb transcription factor maintaining proliferation of colon epithelial progenitors, thus controlling colon development and homeostasis. This gene is overexpressed in early phases of colorectal cancer (CRC) tumorigenesis. The aim of this study was to examine the expression of c-Myb in CRC tissue samples both at the messenger RNA (mRNA) and protein levels and to evaluate their associations with clinicopathological characteristics in a group of 108 CRC patients. Statistically significant negative association was found between the frequency of the c-Myb-positive tumor cells assessed by immunohistochemistry and the presence of distant metastases (p < 0.01) but not tumor differentiation, tumor stage, lymph node involvement, vascular invasion, tumor localization, age, and gender of the patients. Although the c-Myb protein level in the tumor tissue correlated with its mRNA level, no significant association between MYB mRNA and any clinicopathological characteristics was observed. We conclude that albeit overexpression of c-Myb is considered as an important factor contributing to early phases of CRC tumorigenesis, it may later have negative effect on tumor cell dissemination as observed recently in breast cancer as well. Further studies are required to explain the role of c-Myb during formation of CRC distant metastases.
Asunto(s)
Adenocarcinoma/secundario , Neoplasias Colorrectales/genética , Genes myb , Proteínas de Neoplasias/biosíntesis , Proteínas Proto-Oncogénicas c-myb/biosíntesis , Adenocarcinoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Neoplasias Colorrectales/patología , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-myb/genética , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesisRESUMEN
Doxorubicin is an effective chemotherapeutic drug, however, its toxicity is a significant limitation in therapy. Encapsulation of doxorubicin inside liposomes or ferritin cages decreases cardiotoxicity while maintaining anticancer potency. We synthesized novel apoferritin- and liposome-encapsulated forms of doxorubicin ("Apodox" and "lip-8-dox") and compared its toxicity with doxorubicin and Myocet on prostate cell lines. Three different prostatic cell lines PNT1A, 22Rv1, and LNCaP were chosen. The toxicity of the modified doxorubicin forms was compared to conventional doxorubicin using the MTT assay, real-time cell impedance-based cell growth method (RTCA), and flow cytometry. The efficiency of doxorubicin entrapment was 56% in apoferritin cages and 42% in the liposome carrier. The accuracy of the RTCA system was verified by flow-cytometric analysis of cell viability. The doxorubicin half maximal inhibition concentrations (IC50) were determined as 170.5, 234.0, and 169.0 nM for PNT1A, 22Rv1, and LNCaP, respectively by RTCA. Lip8-dox is less toxic on the non-tumor cell line PNT1A compared to doxorubicin, while still maintaining the toxicity to tumorous cell lines similar to doxorubicin or epirubicin (IC50 = 2076.7 nM for PNT1A vs. 935.3 and 729.0 nM for 22Rv1 and LNCaP). Apodox IC50 was determined as follows: 603.1, 1344.2, and 931.2 nM for PNT1A, 22Rv1, and LNCaP.
Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/toxicidad , Apoferritinas , Doxorrubicina/administración & dosificación , Doxorrubicina/toxicidad , Liposomas , Animales , Antibióticos Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/química , Portadores de Fármacos , Composición de Medicamentos , Caballos , Humanos , Concentración 50 InhibidoraRESUMEN
Fas ligand (FasL, CD178) belongs to classical apoptotic molecules, however, recent evidence expands the spectrum of FasL functions into non-apoptotic processes which also applies for the bone. Tgfb subfamily members (Tgfb1, Tgfb2, Tgfb3) represent major components in osteogenic pathways and extracellular matrix. Their possible association with FasL has not yet been investigated but can be postulated. To test such a hypothesis, FasL deficient (gld) calvaria-derived cells were examined with a focus on the expression of Tgfb receptor ligands. The qPCR analysis revealed significantly increased expression of Tgfb1, Tgfb2 and Tgfb3 in gld cells. To check the vice versa effect, the gld cells were stimulated by soluble FasL. As a consequence, a dramatic decrease in expression levels of all three ligands was observed. This phenomenon was also confirmed in IDG-SW3 (osteoblastic cells of endochondral origin). TFLink gateway identified Fosl2 as an exclusive candidate of FasL capable to impact expression of all three Tgfb ligands. However, Fosl2 siRNA did not cause any significant changes in expression of Tgfb ligands. Therefore, the upregulation of the three ligands is likely to occur separately. In this respect, we tested the only exclusive candidate transcription factor for Tgfb3, Prrx1. Additionally, an overlapping candidate for Tgfb1 and Tgfb2, Mef2c capable to modulate expression of sclerostin, was examined. Prrx1 as well as Mef2c were found upregulated in gld samples and their expression decreased after addition of FasL. The same effect of FasL treatment was observed in the IDG-SW3 model. Taken together, FasL deficiency causes an increase in the expression of Tgfb ligands and stimulation by FasL reduces Tgfb expression in osteoblastic cells. The candidates mediating the effect comprise Prrx1 for Tgfb3 and Mef2c for Tgfb1/2. These results indicate FasL as a novel cytokine interfering with Tgfb signaling and thus the complex osteogenic network. The emerging non-apoptotic functions of FasL in bone development and maintenance should also be considered in treatment strategies such as the anti-osteoporotic factor.
Asunto(s)
Proteína Ligando Fas , Osteoblastos , Transducción de Señal , Factor de Crecimiento Transformador beta3 , Proteína Ligando Fas/metabolismo , Osteoblastos/metabolismo , Animales , Ratones , Factor de Crecimiento Transformador beta3/metabolismo , Factor de Crecimiento Transformador beta3/genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta2/farmacología , Factores de Transcripción MEF2/metabolismo , Factores de Transcripción MEF2/genética , Factor de Crecimiento Transformador beta/metabolismo , Línea CelularRESUMEN
The transcription factor c-Myb is overexpressed in many different types of solid tumors, including colorectal cancer. However, its exact role in tumorigenesis is unclear. In this study, we show that tumor-intrinsic c-Myb expression in mouse models of colon cancer and melanoma suppresses tumor growth. Although no differences in proliferation, apoptosis, and angiogenesis of tumors were evident in tumors with distinct levels of c-Myb expression, we observed changes in intratumoral immune cell infiltrates. MC38 tumors with upregulated c-Myb expression showed increased numbers of CD103+ dendritic cells and eosinophils, but decreased tumor-associated macrophages (TAM). Concomitantly, an increase in the number of activated cytotoxic CD8+ T cells upon c-Myb upregulation was observed, which correlated with a pro-inflammatory tumor microenvironment and increased numbers of M1 polarized TAMs. Mechanistically, c-Myb upregulation in immunogenic MC38 colon cancer cells resulted in enhanced expression of immunomodulatory genes, including those encoding ß2-microglobulin and IFNß, and decreased expression of the gene encoding the chemokine receptor CCR2. The increased numbers of activated cytotoxic CD8+ T cells contributed to tumor growth attenuation. In poorly immunogenic CT26, LLC, and B16-BL6 tumor cells, c-Myb upregulation did not affect the immunomodulatory gene expression. Despite this, c-Myb upregulation led to reduced B16-BL6 tumor growth but it did not affect tumor growth of CT26 and LLC tumors. Altogether, we postulate that c-Myb functions as a tumor suppressor in a tumor cell-type specific manner and modulates antitumor immunity.
RESUMEN
BACKGROUND: The c-Myb transcription factor is essential for the maintenance of stem-progenitor cells in bone marrow, colon epithelia, and neurogenic niches. c-Myb malfunction contributes to several types of malignancies including breast cancer. However, the function of c-Myb in the metastatic spread of breast tumors remains unexplored. In this study, we report a novel role of c-Myb in the control of specific proteases that regulate the matrix-dependent invasion of breast cancer cells. RESULTS: Ectopically expressed c-Myb enhanced migration and ability of human MDA-MB-231 and mouse 4T1 mammary cancer cells to invade Matrigel but not the collagen I matrix in vitro. c-Myb strongly increased the expression/activity of cathepsin D and matrix metalloproteinase (MMP) 9 and significantly downregulated MMP1. The gene coding for cathepsin D was suggested as the c-Myb-responsive gene and downstream effector of the migration-promoting function of c-Myb. Finally, we demonstrated that c-Myb delayed the growth of mammary tumors in BALB/c mice and affected the metastatic potential of breast cancer cells in an organ-specific manner. CONCLUSIONS: This study identified c-Myb as a matrix-dependent regulator of invasive behavior of breast cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Catepsina D/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Animales , Neoplasias de la Mama/genética , Catepsina D/genética , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Immunoblotting , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/fisiopatología , Proteínas Proto-Oncogénicas c-myb/genética , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The MYB family of transcription activators has been associated with a high proliferation rate and an undifferentiated state of cells in a number of tissues. Recently emerging data suggest that these molecules may also play a role in differentiation. In this study, the pattern of expression of c-MYB was followed during postnatal stages of mouse molar odontogenesis using immunohistochemistry on serial sections. Along with an abundance of the c-MYB protein in proliferating zones, we confirmed the presence of this protein in differentiated ameloblasts, odontoblasts, and osteoblasts. In addition, c-MYB was also found in cementoblasts and alveolar fibroblasts. These findings suggest integration of c-MYB into regulatory networks during hard-tissue differentiation and mineralization.
Asunto(s)
Proceso Alveolar/citología , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genes myc/genética , Diente Molar/citología , Odontogénesis/genética , Proteínas Proto-Oncogénicas c-myb , Proceso Alveolar/crecimiento & desarrollo , Proceso Alveolar/metabolismo , Ameloblastos/metabolismo , Animales , Desarrollo Óseo/genética , Desarrollo Óseo/fisiología , Células del Tejido Conectivo/metabolismo , Cemento Dental/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Etiquetado Corte-Fin in Situ , Ratones , Diente Molar/crecimiento & desarrollo , Diente Molar/metabolismo , Proteínas Proto-Oncogénicas c-myb/análisis , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismoRESUMEN
TACSTD2 encodes a transmembrane glycoprotein Trop2 commonly overexpressed in carcinomas. While the Trop2 protein was discovered already in 1981 and first antibody-drug conjugate targeting Trop2 were recently approved for cancer therapy, the physiological role of Trop2 is still not fully understood. In this article, we show that TACSTD2/Trop2 expression is evolutionarily conserved in lungs of various vertebrates. By analysis of publicly available transcriptomic data we demonstrate that TACSTD2 level consistently increases in lungs infected with miscellaneous, but mainly viral pathogens. Single cell and subpopulation based transcriptomic data revealed that the major source of TACSTD2 transcript are lung epithelial cells and their progenitors and that TACSTD2 is induced directly in lung epithelial cells following infection. Increase in TACSTD2 expression may represent a mechanism to maintain/restore epithelial barrier function and contribute to regeneration process in infected/damaged lungs.
Asunto(s)
Antígenos de Neoplasias , Moléculas de Adhesión Celular , Animales , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/metabolismo , Pulmón/metabolismo , Regulación hacia ArribaRESUMEN
The transcription factor c-Myb is an oncoprotein promoting cell proliferation and survival when aberrantly activated/expressed, thus contributing to malignant transformation. Overexpression of c-Myb has been found in leukemias, breast, colon and adenoid cystic carcinoma. Recent studies revealed its expression also in osteosarcoma cell lines and suggested its functional importance during bone development. However, the relevance of c-Myb in control of osteosarcoma progression remains unknown. A retrospective clinical study was carried out to assess a relationship between c-Myb expression in archival osteosarcoma tissues and prognosis in a cohort of high-grade osteosarcoma patients. In addition, MYB was depleted in metastatic osteosarcoma cell lines SAOS-2 LM5 and 143B and their growth, chemosensitivity, migration and metastatic activity were determined. Immunohistochemical analysis revealed that high c-Myb expression was significantly associated with poor overall survival in the cohort and metastatic progression in young patients. Increased level of c-Myb was detected in metastatic osteosarcoma cell lines and its depletion suppressed their growth, colony-forming capacity, migration and chemoresistance in vitro in a cell line-dependent manner. MYB knock-out resulted in reduced metastatic activity of both SAOS-2 LM5 and 143B cell lines in immunodeficient mice. Transcriptomic analysis revealed the c-Myb-driven functional programs enriched for genes involved in the regulation of cell growth, stress response, cell adhesion and cell differentiation/morphogenesis. Wnt signaling pathway was identified as c-Myb target in osteosarcoma cells. Taken together, we identified c-Myb as a negative prognostic factor in osteosarcoma and showed its involvement in the regulation of osteosarcoma cell growth, chemosensitivity, migration and metastatic activity.
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Neoplasias Óseas , Osteosarcoma , Animales , Neoplasias Óseas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Osteosarcoma/patología , Pronóstico , Estudios Retrospectivos , Vía de Señalización WntRESUMEN
The transcription factor c-Myb can be involved in the activation of many genes with protumorigenic function; however, its role in breast cancer (BC) development is still under discussion. c-Myb is considered as a tumor-promoting factor in the early phases of BC, on the other hand, its expression in BC patients relates to a good prognosis. Previously, we have shown that c-Myb controls the capacity of BC cells to form spontaneous lung metastasis. Reduced seeding of BC cells to the lungs is linked to high expression of c-Myb and a decline in expression of a specific set of inflammatory genes. Here, we unraveled a c-Myb-IL1α-NF-κB signaling axis that takes place in tumor cells. We report that an overexpression of c-Myb interfered with the activity of NF-κB in several BC cell lines. We identified IL1α to be essential for this interference since it was abrogated in the IL1α-deficient cells. Overexpression of IL1α, as well as addition of recombinant IL1α protein, activated NF-κB signaling and restored expression of the inflammatory signature genes suppressed by c-Myb. The endogenous levels of c-Myb negatively correlated with IL1α on both transcriptional and protein levels across BC cell lines. We concluded that inhibition of IL1α expression by c-Myb reduces NF-κB activity and disconnects the inflammatory circuit, a potentially targetable mechanism to mimic the antimetastatic effect of c-Myb with therapeutic perspective.
Asunto(s)
Neoplasias de la Mama/metabolismo , Interleucina-1alfa/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/etiología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Estrés del Retículo Endoplásmico , Transición Epitelial-Mesenquimal , Femenino , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismoRESUMEN
Colorectal cancer (CRC) represents a serious challenge for oncologists due to high incidence and large heterogeneity. Prognostic factors are needed to stratify patients according to risk of disease progression. In this study, we report that high expression of c-Myb protein, determined by immunohistochemistry (IHC), associates with better overall and disease-free survival (OS, DFS) in a cohort of 103 patients. Although MYB has been previously considered to act as oncogene in CRC, our further analysis of datasets deposited in PrognoScan and SurvExpress databases confirmed that high MYB expression largely associates with good prognosis in CRC. As therapies targeting c-Myb have been developed and tested in preclinical studies, we believe that further studies are needed for detailed understanding of c-Myb function in CRC, before the c-Myb-targeted therapy enters clinical trials.
RESUMEN
Tumor-associated macrophages (TAMs) are prominent components of tumor stroma that promotes tumorigenesis. Many soluble factors participate in the deleterious cross-talk between TAMs and transformed cells; however mechanisms how tumors orchestrate their production remain relatively unexplored. c-Myb is a transcription factor recently described as a negative regulator of a specific immune signature involved in breast cancer (BC) metastasis. Here we studied whether c-Myb expression is associated with an increased presence of TAMs in human breast tumors. Tumors with high frequency of c-Myb-positive cells have lower density of CD68-positive macrophages. The negative association is reflected by inverse correlation between MYB and CD68/CD163 markers at the mRNA levels in evaluated cohorts of BC patients from public databases, which was found also within the molecular subtypes. In addition, we identified potential MYB-regulated TAMs recruiting factors that in combination with MYB and CD163 provided a valuable clinical multigene predictor for BC relapse. We propose that identified transcription program running in tumor cells with high MYB expression and preventing macrophage accumulation may open new venues towards TAMs targeting and BC therapy.
Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Macrófagos/inmunología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Microambiente Tumoral/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Mama/patología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Proliferación Celular , Estudios de Cohortes , Conjuntos de Datos como Asunto , Femenino , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Macrófagos/metabolismo , Persona de Mediana Edad , Pronóstico , Receptores de Superficie Celular/metabolismo , Microambiente Tumoral/genéticaRESUMEN
Increased levels of the chemokine CCL2 in cancer patients are associated with poor prognosis. Experimental evidence suggests that CCL2 correlates with inflammatory monocyte recruitment and induction of vascular activation, but the functionality remains open. Here, we show that endothelial Ccr2 facilitates pulmonary metastasis using an endothelial-specific Ccr2-deficient mouse model (Ccr2ecKO). Similar levels of circulating monocytes and equal leukocyte recruitment to metastatic lesions of Ccr2ecKO and Ccr2fl/fl littermates were observed. The absence of endothelial Ccr2 strongly reduced pulmonary metastasis, while the primary tumor growth was unaffected. Despite a comparable cytokine milieu in Ccr2ecKO and Ccr2fl/fl littermates the absence of vascular permeability induction was observed only in Ccr2ecKO mice. CCL2 stimulation of pulmonary endothelial cells resulted in increased phosphorylation of MLC2, endothelial cell retraction, and vascular leakiness that was blocked by an addition of a CCR2 inhibitor. These data demonstrate that endothelial CCR2 expression is required for tumor cell extravasation and pulmonary metastasis. IMPLICATIONS: The findings provide mechanistic insight into how CCL2-CCR2 signaling in endothelial cells promotes their activation through myosin light chain phosphorylation, resulting in endothelial retraction and enhanced tumor cell migration and metastasis.
Asunto(s)
Carcinoma Pulmonar de Lewis/metabolismo , Quimiocina CCL2/metabolismo , Células Endoteliales/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores CCR2/metabolismo , Animales , Permeabilidad Capilar , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/patología , Carcinoma Pulmonar de Lewis/secundario , Movimiento Celular/fisiología , Células Endoteliales/patología , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cadenas Ligeras de Miosina/metabolismo , Metástasis de la NeoplasiaRESUMEN
AIM: Pentose phosphate pathway (PPP) with key enzyme transketolase (TKT), represents a potentially 'protective' mechanism in hyperglycaemia. Diabetic kidney disease (DKD), a common complication of both type 1 and type 2 diabetes associated with significant morbidity and mortality, represents the most common cause of chronic kidney disease (CKD). We hypothesized that protective PPP action in diabetes and eventually even more severely in concomitant DKD might be compromised by limited intracellular availability of an active TKT cofactor thiamine diphosphate (TDP). METHODS: Effect of hyperglycaemia on gene expression and protein levels of key PPP loci was studied in vitro using human cell lines relevant to diabetes (HUVEC and HRGEC) and (together with measurement of TKT activity, plasma thiamine and erythrocyte TDP concentration) in vivo in diabetic vs. non-diabetic subjects with comparable renal function (n=83 in total). RESULTS: Hyperglycaemia significantly decreased protein levels of RFC-1, THTR1, THTR2 and TKT (P<0.05) in vitro. Analysis of blood samples from CKD patients with and without diabetes and from controls did not reveal any difference in gene expression and protein levels of thiamine transporters while TKT activity and TDP in erythrocytes gradually increased with decreasing kidney function being highest in patients with CKD3-4 of both diabetic and non-diabetic aetiology. Hyperglycaemia and uremic serum mimicking CKD in diabetes did not affect TKT activity in vitro (P<0.05). CONCLUSION: Both in vitro and human experiments showed decrease or unchanged expression, respectively, of thiamine transporters induced by hyperglycaemia while TKT activity in parallel with intracellular TDP was increased in CKD patients with or without diabetes. Therefore, lack of adaptive increase of thiamine transmembrane transport allowing further increase of TKT activity might contribute to compromised PPP function in diabetes and CKD and to the development of glycotoxic injury.