RESUMEN
Animal experimentation is a vast ecosystem that tries to make different issues such as legislative, ethical and scientific coexist. Research in animal experimentation has made many strides thanks to the 3Rs principle and the attached legislative decrees, but for this very reason, it needs to be evenly implemented both among the countries that have adhered to the decrees and among the team members who design and execute the experimental practice. In this article, we emphasize the importance of the 3Rs principle's application, with a particular focus on the concept of Reduction and related key aspects that can best be handled with the contribution of experts from different fields.
RESUMEN
An immunosuppressive microenvironment in lung concurs to pre-malignant lesions progression to cancer. Here, we explore if perturbing lung microbiota, which contribute to immunosuppression, by antibiotics or probiotic aerosol interferes with lung cancer development in a mouse carcinogen-induced tumor model. Urethane-injected mice were vancomycin/neomycin (V/N)-aerosolized or live or dead L. rhamnosus GG (L.RGG)-aerosolized, and tumor development was evaluated. Transcriptional profiling of lungs and IHC were performed. Tumor nodules number, diameter and area were reduced by live or heat-killed L.RGG, while only a decrease in nodule diameter was observed in V/N-treated lungs. Both L.RGG and V/N reduced Tregs in the lung. In L.RGG-treated groups, the gene encoding the joining chain (J chain) of immunoglobulins was increased, and higher J chain protein and IgA levels were observed. An increased infiltration of B, NK and myeloid-derived cells was predicted by TIMER 2.0. The Kaplan-Meier plotter revealed an association between high levels of J chain mRNA and good prognosis in lung adenocarcinoma patients that correlated with increased B and CD4 T cells and reduced Tregs and M2 macrophages. This study highlights L.RGG aerosol efficacy in impairing lung cancer growth by promoting local immunity and points to this non-invasive strategy to treat individuals at risk of lung cancer.
Asunto(s)
Adenoma , Lacticaseibacillus rhamnosus , Neoplasias Pulmonares , Probióticos , Ratones , Animales , Carcinógenos , Calor , Neoplasias Pulmonares/patología , Probióticos/uso terapéutico , Probióticos/farmacología , Modelos Animales de Enfermedad , Microambiente TumoralRESUMEN
Two non-inbred mouse lines, phenotypically selected for maximal (AIRmin) and minimal (AIRmax) acute inflammatory response, show differential susceptibility/resistance to the development of several chemically-induced tumor types. An intercross pedigree of these mice was generated and treated with the chemical carcinogen dimethylhydrazine, which induces lung and intestinal tumors. Genome wide high-density genotyping with the Restriction Site-Associated DNA genotyping (2B-RAD) technique was used to map genetic loci modulating individual genetic susceptibility to both lung and intestinal cancer. Our results evidence new common quantitative trait loci (QTL) for those phenotypes and provide an improved understanding of the relationship between genomic variation and individual genetic predisposition to tumorigenesis in different organs.
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Neoplasias del Colon , Sitios de Carácter Cuantitativo , Animales , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/genética , Predisposición Genética a la Enfermedad , Pulmón , Ratones , Ratones EndogámicosRESUMEN
Inflammation plays a critical role in thyroid cancer onset and progression. We previously characterized the in vitro interplay between macrophages and senescent human thyrocytes and thyroid tumor-derived cell lines, modeling the early and the late thyroid tumor phases, respectively. We reported that both models are able to induce pro-tumoral M2-like macrophage polarization, through the activation of the COX2-PGE2 axis. Here, we investigated the presence of macrophage infiltrating cells in mouse xenografts derived from the above described cells models. We showed that subcutaneous injection in immunodeficient mice of both senescent human thyrocytes and thyroid tumor-derived cell lines elicits macrophage recruitment. Furthermore, considering the type of macrophage infiltrate, we observed a stronger infiltration of Arginase I positive cells (M2-like). Overall, these results demonstrate the in vivo capability of senescent and tumor thyroid cells to recruit and polarize macrophages, suggesting that the promotion of a pro-tumoral microenvironment through tumor associated macrophages may occurs in late as well as in early thyroid tumor stages, favoring tumor onset and progression.
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Nano- and microsized extracellular vesicles (EVs) are naturally occurring cargo-bearing packages of regulatory macromolecules, and recent studies are increasingly showing that EVs are responsible for physiological intercellular communication. Nanoparticles encapsulating anti-tumor theranostics represent an attractive "exosome-interfering" strategy for cancer therapy. Methods: Herein, by labeling plasma-derived EVs with indocyanine green (ICG) and following their biodistribution by in vivo and ex vivo imaging, we demonstrate the existence of nanoparticles with a highly selective cancer tropism in the blood of colorectal cancer (CRC) patients but not in that of healthy volunteers. Results: In CRC patient-derived xenograft (PDX) mouse models, we show that transplanted EVs recognize tumors from the cognate nanoparticle-generating individual, suggesting the theranostic potential of autologous EVs encapsulating tumor-interfering molecules. In large canine breeds bearing spontaneous malignant skin and breast tumors, the same autologous EV transplantation protocol shows comparable safety and efficacy profiles. Conclusions: Our data show the existence of an untapped resource of intercellular communication present in the blood of cancer patients, which represents an efficient and highly biocompatible way to deliver molecules directly to the tumor with great precision. The novel EV-interfering approach proposed by our study may become a new research direction in the complex interplay of modern personalized cancer therapy.
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Neoplasias de la Mama/terapia , Neoplasias Colorrectales/terapia , Vesículas Extracelulares/trasplante , Neoplasias Hepáticas/terapia , Animales , Apoptosis , Neoplasias de la Mama/patología , Estudios de Casos y Controles , Proliferación Celular , Neoplasias Colorrectales/patología , Perros , Femenino , Humanos , Neoplasias Hepáticas/secundario , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Distribución Tisular , Trasplante Autólogo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIRmax and AIRmin mouse strains phenotypically selected for high and low acute inflammatory responsiveness (AIR) are, respectively, susceptible or resistant to developing hepatocellular carcinoma (HCC) induced by the chemical carcinogens urethane and diethylnitrosamine (DEN). Early production of TNF-α, IL-1ß, and IL-6 in the liver after DEN treatment correlated with tumor development in AIRmax mice. Transcriptome analysis of livers from untreated AIRmax and AIRmin mice showed specific gene expression profiles in each line, which might play a role in their differential susceptibility to HCC. Linkage analysis with SNP markers in F2 (AIRmax×AIRmin) intercross mice revealed two quantitative trait loci (QTL) in chromosomes 2 and 9, which are significantly associated with the number and progression of urethane-induced liver tumors. An independent linkage analysis with an intercross population from A/J and C57BL/6J inbred mice mapped regions in chromosomes 1 and 7 associated with the progression of urethane-induced liver tumors, evidencing the heterogeneity of HCC genetic control.
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Animales no Consanguíneos , Carcinoma Hepatocelular/genética , Predisposición Genética a la Enfermedad , Inflamación/inmunología , Neoplasias Hepáticas/genética , Alelos , Animales , Carcinogénesis/genética , Carcinoma Hepatocelular/inmunología , Modelos Animales de Enfermedad , Ligamiento Genético , Endogamia , Inflamación/genética , Interleucina-1beta/genética , Interleucina-6/genética , Neoplasias Hepáticas/inmunología , Ratones , Ratones Endogámicos C57BL , Fenotipo , Sitios de Carácter Cuantitativo , Transcriptoma , Factores de Necrosis Tumoral/genéticaRESUMEN
Inhibitor of apoptosis (IAP) proteins constitute a family of conserved molecules that regulate both apoptosis and receptor signaling. They are often deregulated in cancer cells and represent potential targets for therapy. In our work, we investigated the effect of IAP inhibition in vivo to identify novel downstream genes expressed in an IAP-dependent manner that could contribute to cancer aggressiveness. To this end, immunocompromised mice engrafted subcutaneously with the triple-negative breast cancer MDA-MB231 cell line were treated with SM83, a Smac mimetic that acts as a pan-IAP inhibitor, and tumor nodules were profiled for gene expression. SM83 reduced the expression of Snai2, an epithelial-to-mesenchymal transition factor often associated with increased stem-like properties and metastatic potential especially in breast cancer cells. By testing several breast cancer cell lines, we demonstrated that Snai2 downregulation prevents cell motility and that its expression is promoted by cIAP1. In fact, the chemical or genetic inhibition of cIAP1 blocked epidermal growth factor receptor (EGFR)-dependent activation of the mitogen-activated protein kinase (MAPK) pathway and caused the reduction of Snai2 transcription levels. In a number of breast cancer cell lines, cIAP1 depletion also resulted in a reduction of EGFR protein levels which derived from the decrease of its gene transcription, though, paradoxically, the silencing of cIAP1 promoted EGFR protein stability rather than its degradation. Finally, we provided evidence that IAP inhibition displays an anti-tumor and anti-metastasis effect in vivo. In conclusion, our work indicates that IAP-targeted therapy could contribute to EGFR inhibition and to the reduction of its downstream mediators. This approach could be particularly effective in tumors characterized by high levels of EGFR and Snai2, such as triple-negative breast cancer.
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Proteínas Inhibidoras de la Apoptosis/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Administración Intravenosa , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/deficiencia , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos NOD , Ratones SCID , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacología , Factores de Transcripción de la Familia Snail/antagonistas & inhibidores , Factores de Transcripción de la Familia Snail/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Células Tumorales CultivadasRESUMEN
Somatic KRAS mutations are common in human lung adenocarcinomas and are associated with worse prognosis. In mice, Kras is frequently mutated in both spontaneous and experimentally induced lung tumors, although the pattern of mutation varies among strains, suggesting that such mutations are not random events. We tested if the occurrence of Kras mutations is under genetic control in two mouse intercrosses. Codon 61 mutations were prevalent, but the patterns of nucleotide changes differed between the intercrosses. Whole genome analysis with SNPs in (A/J x C57BL/6)F4 mice revealed a significant linkage between a locus on chromosome 19 and 2 particular codon 61 variants (CTA and CGA). In (AIRmax × AIRmin) F2 mice, there was a significant linkage between SNPs located on distal chromosome 6 (around 135 Mbp) and the frequency of codon 61 mutation. These results reveal the presence of two loci, on chromosomes 6 and 19, that modulate Kras mutation frequency in different mouse intercrosses. These findings indicate that somatic mutation frequency and type are not simple random events, but are under genetic control.
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Adenocarcinoma/genética , Predisposición Genética a la Enfermedad/genética , Neoplasias Pulmonares/genética , Mutación , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Codón/genética , Cruzamientos Genéticos , Femenino , Frecuencia de los Genes , Masculino , Ratones Endogámicos C57BL , Ratones EndogámicosRESUMEN
Thyroid carcinoma is generally associated with good prognosis, but no effective treatments are currently available for aggressive forms not cured by standard therapy. To find novel therapeutic targets for this tumor type, we had previously performed a siRNA-based functional screening to identify genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same extent for the viability of normal cells (non-oncogene addiction paradigm). Among those, we found the coatomer protein complex ζ1 (COPZ1) gene, which is involved in intracellular traffic, autophagy and lipid homeostasis. In this paper, we investigated the mechanisms through which COPZ1 depletion leads to thyroid tumor cell death. We showed that siRNA-mediated COPZ1 depletion causes abortive autophagy, endoplasmic reticulum stress, unfolded protein response and apoptosis. Interestingly, we observed that mouse tumor xenografts, locally treated with siRNA targeting COPZ1, showed a significant reduction of tumor growth. On the whole, we demonstrated for the first time the crucial role of COPZ1 in the viability of thyroid tumor cells, suggesting that it may be considered an attractive target for novel therapeutic approaches for thyroid cancer.
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Proteína Coatómero/metabolismo , Tratamiento con ARN de Interferencia , Neoplasias de la Tiroides/terapia , Animales , Apoptosis , Autofagia , Línea Celular Tumoral , Supervivencia Celular , Proteína Coatómero/genética , Estrés del Retículo Endoplásmico , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Desnudos , Interferencia de ARN , Transducción de Señal , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Factores de Tiempo , Transfección , Carga Tumoral , Respuesta de Proteína Desplegada , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The SM/J mouse strain is resistant to chemically-induced lung tumorigenesis despite having a haplotype, in the pulmonary adenoma susceptibility locus (Pas1) locus, that confers tumor susceptibility in other strains. To clarify this inconsistent genotype-phenotype correlation, we crossed SM/J mice with another resistant strain and conducted genome-wide linkage analysis in the (C57BL/6J × SM/J)F2 progeny exposed to urethane to induce lung tumors. Overall, >80% of F2 mice of both sexes developed from 1 to 20 lung tumors. Genotyping of 372 F2 mice for 744 informative non-redundant SNPs dispersed over all autosomal chromosomes revealed four quantitative trait loci (QTLs) affecting lung tumor multiplicity, on chromosomes 3 (near rs13477379), 15 (rs6285067), 17 (rs33373629) and 18 (rs3706601), all with logarithm of the odds (LOD) scores >5. Four QTLs modulated total lung tumor volume, on chromosome 3 (rs13477379), 10 (rs13480702), 15 (rs6285067) and 17 (rs3682923), all with LOD scores >4. No QTL modulating lung tumor multiplicity or total volume was detected in Pas1 on chromosome 6. The present study demonstrates that the SM/J strain carries, at the Pas1 locus, the resistance allele: a finding that will facilitate identification of the Pas1 causal element. More generally, it demonstrates that lung tumorigenesis is under complex polygenic control even in a pedigree with low susceptibility to this neoplasia, suggesting that the genetics of lung tumorigenesis is much more complex than evidenced by the pulmonary adenoma susceptibility and resistance loci that have, so far, been mapped in a small number of crosses between a few inbred strains.
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Carcinogénesis/genética , Estudios de Asociación Genética , Sitios Genéticos/genética , Predisposición Genética a la Enfermedad , Neoplasias Pulmonares/genética , Alelos , Animales , Modelos Animales de Enfermedad , Femenino , Genotipo , Haplotipos/genética , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo/genéticaRESUMEN
BACKGROUND: Strategies aimed at obtaining a complete cytoreduction are needed to improve long-term survival for patients with colorectal cancer peritoneal carcinomatosis (CRC-pc). METHODS: We established organoid models from peritoneal metastases of two naïve CRC patients. A standard paraffin inclusion was conducted to compare their 3D structure and immunohistochemical profile with that of the corresponding surgical samples. RNA expression levels of the CRC stem cell marker LGR5 was measured by in situ hybridization. The secretome of organoids was profiled by mass spectrometry. Energy homeostasis of organoids was interfered with 4-IPP and metformin. Biochemical and metabolic changes after drug treatments were investigated by western blot and mass spectrometry. Mitochondria impairment was evaluated by electron microscopy and mitotraker staining. RESULTS: The two organoids recapitulated their corresponding clinical samples in terms of 3D structure and immmunoistochemical profile and were positive for the cancer stem cells marker LGR5. Proteomic analyses of organoids highlighted their strong dependence on energy producing pathways, which suggest that their targeting could be an effective therapeutic approach. To test this hypothesis, we treated organoids with two drugs that target metabolism acting on AMP-activated protein kinase (AMPK), the main regulator of cellular energy homeostasis, which may act as metabolic tumour suppressor in CRC. Organoids were treated with 4-IPP, an inhibitor of MIF/CD74 signalling axis which activates AMPK function, or metformin that inhibits mitochondrial respiratory chain complex I. As a new finding we observed that treatment with 4-IPP downregulated AMPK signalling activity, reduced AKT phosphorylation and activated a JNK-mediated stress-signalling response, thus generating mitochondrial impairment and cell death. Metformin treatment enhanced AMPK activation, decreasing the activity of the anabolic factors ribosomal protein S6 and p4EBP-1 and inducing mitochondrial depolarization. CONCLUSION: We provide evidence that the modulation of AMPK activity may be a strategy for targeting metabolism of CRC-pc organoids.
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Antígenos de Diferenciación de Linfocitos B/metabolismo , Neoplasias del Colon/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Metformina/farmacología , Neoplasias Peritoneales/secundario , Pirimidinas/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Metabolismo Energético/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Peritoneales/genética , Proteómica , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Retina-derived POU domain Factor 1 (RPF-1), a member of POU transcription factor family, is encoded by POU6F2 gene, addressed by interstitial deletions at chromosome 7p14 in Wilms tumor (WT). Its expression has been detected in developing kidney and nervous system, suggesting an early role for this gene in regulating development of these organs. To investigate into its functions and determine its role in transcriptional regulation, we generated an inducible stable transfectant from HEK293 cells. RPF-1 showed nuclear localization, elevated stability, and transactivation of promoters featuring POU consensus sites, and led to reduced cell proliferation and in vivo tumor growth. By addressing the whole transcriptome regulated by its induction, we could detect a gross alteration of gene expression that is consistent with promoter occupancy predicted by genome-wide Chip-chip analysis. Comparison of bound regulatory regions with differentially expressed genes allowed identification of 217 candidate targets. Enrichment of divergent octamers in predicted regulatory regions revealed promiscuous binding to bipartite POUS and POUH consensus half-sites with intervening spacers. Gel-shift competition assay confirmed the specificity of RPF-1 binding to consensus motifs, and demonstrated that the Ser-rich region upstream of the POU domain is indispensable to achieve DNA-binding. Promoter-reporter activity addressing a few target genes indicated a dependence by RPF-1 on transcriptional response. In agreement with its expression in developing kidney and nervous system, the induced transcriptome appears to indicate a function for this protein in early renal differentiation and neuronal cell fate, providing a resource for understanding its role in the processes thereby regulated.
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Regulación del Desarrollo de la Expresión Génica , Riñón/crecimiento & desarrollo , Neuronas/citología , Factores del Dominio POU/metabolismo , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Núcleo Celular/metabolismo , Proliferación Celular , Secuencia de Consenso , Células HEK293 , Humanos , Transcripción GenéticaRESUMEN
Melanoma is a highly heterogeneous tumor for which recent evidence supports a model of dynamic stemness. Melanoma cells might temporally acquire tumor-initiating properties or switch from a status of tumor-initiating cells (TICs) to a more differentiated one depending on the tumor context. However, factors driving these functional changes are still unknown. We focused on the role of cyto/chemokines in shaping TICs isolated directly from tumor specimens of two melanoma patients, namely Me14346S and Me15888S. We analyzed the secretion profile of TICs and of their corresponding melanoma differentiated cells and we tested the ability of cyto/chemokines to influence TIC self-renewal and differentiation. We found that TICs, grown in vitro as melanospheres, had a complex secretory profile as compared to their differentiated counterparts. Some factors, such as CCL-2 and IL-8, also produced by adherent melanoma cells and melanocytes did not influence TIC properties. Conversely, IL-6, released by differentiated cells, reduced TIC self-renewal and induced TIC differentiation while IL-10, produced by Me15888S, strongly promoted TIC self-renewal through paracrine/autocrine actions. Complete neutralization of IL-10 activity by gene silencing and antibody-mediated blocking of the IL-10Rα was required to sensitize Me15888S to IL-6-induced differentiation. For the first time these results show that functional heterogeneity of melanoma could be directly influenced by inflammatory and suppressive soluble factors, with IL-6 favoring TIC differentiation, and IL-10 supporting TIC self-renewal. Thus, understanding the tumor microenvironment (TME) role in modulating melanoma TIC phenotype is fundamental to identifying novel therapeutic targets to achieve long-lasting regression of metastatic melanoma. Stem Cells 2016;34:2449-2460.
Asunto(s)
Linaje de la Célula/efectos de los fármacos , Factores Inmunológicos/farmacología , Melanoma/patología , Células Madre Neoplásicas/patología , Comunicación Autocrina/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Autorrenovación de las Células/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Melanoma/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Pruebas de Neutralización , Comunicación Paracrina/efectos de los fármacos , Fenotipo , Receptores de Quimiocina/metabolismo , Esferoides Celulares/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gene expression modulates cellular functions in both physiologic and pathologic conditions. Herein, we carried out a genetic linkage study on the transcriptome of lung tumors induced by urethane in an (A/J x C57BL/6)F4 intercross population, whose individual lung tumor multiplicity (Nlung) is linked to the genotype at the Pulmonary adenoma susceptibility 1 (Pas1) locus. We found that expression levels of 1179 and 1579 genes are modulated by an expression quantitative trait locus (eQTL) in cis and in trans, respectively (LOD score > 5). Of note, the genomic area surrounding and including the Pas1 locus regulated 14 genes in cis and 857 genes in trans. In lung tumors of the same (A/J x C57BL/6)F4 mice, we found 1124 genes whose transcript levels associated with Nlung (FDR < 0.001). The expression levels of about a third of these genes (n = 401) were regulated by the genotype at the Pas1 locus. Pathway analysis of the sets of genes associated with Nlung and regulated by Pas1 revealed a set of 14 recurrently represented genes that are components or targets of the Ras-Erk and Pi3k-Akt signaling pathways. Altogether our results illustrate the architecture of germline control of gene expression in mouse lung cancer: they highlight the importance of Pas1 as a tumor-modifier locus, attribute to it a novel role as a major regulator of transcription in lung tumor nodules and strengthen the candidacy of the Kras gene as the effector of this locus.
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Ligamiento Genético , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Alelos , Animales , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Genotipo , Células Germinativas , Humanos , Neoplasias Pulmonares/patología , Ratones , Proteínas de Neoplasias/biosíntesis , Fenotipo , Fosfatidilinositol 3-Quinasas/genética , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , TranscriptomaRESUMEN
Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is over-expressed in several human neoplastic cells. When MIF binds its receptor (CD74) and co-receptor (CD44), it initiates signaling cascades that orchestrate cell proliferation and survival, and it can directly modulate the activity of AMPK. These activities indicate that MIF potentially regulates cell survival and metabolism. We found that MIF was primarily co-expressed with CD74 in 16 out of 23 papillary thyroid carcinoma (PTC) and in all the 27 available anaplastic thyroid carcinoma (ATC) biopsy samples. MIF and CD74 were co-expressed in TPC-1 and HTC-C3 cell lines. The selective MIF inhibitor, 4-iodo-6-phenylpyrimidine (4-IPP), blocked MIF/CD74 internalization, activated JNK, and dose-dependently inhibited proliferation inducing apoptosis and mitotic cell death. In two CD74-negative cell lines, NIM-1 and K1, 4-IPP treatment partially reduced proliferation. Coordinated MIF and CD74 expression appeared to confer in tumor cells the plasticity necessary to escape cell cycle regulation, metabolic changes, and stress conditions. MIF/CD74 signaling removal made cells susceptible to apoptosis and mitotic cell death. This finding suggests a possible avenue for targeting DNA endoreduplication, thus preventing the proliferation of therapy-resistant cell subpopulations. This study highlights MIF/CD74 axis as an important player in the biology of aggressive thyroid neoplasms.
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Carcinoma/patología , Indoles/farmacología , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Metaboloma , Mitosis/efectos de los fármacos , Proteoma/análisis , Neoplasias de la Tiroides/patología , Apoptosis , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinoma/tratamiento farmacológico , Carcinoma/metabolismo , Carcinoma Papilar , Ciclo Celular , Proliferación Celular , Cromatografía Liquida/métodos , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Receptores Inmunológicos/metabolismo , Espectrometría de Masas en Tándem/métodos , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/metabolismo , Células Tumorales CultivadasRESUMEN
Target-specific agents used in melanoma are not curative, and chemokines are being implicated in drug-resistance to target-specific agents. Thus, the use of conventional agents in rationale combinations may result in optimization of therapy. Because histone deacetylases participate in tumor development and progression, the combination of the pan-inhibitor SAHA and temozolomide might provide a therapeutic advantage. Here, we show synergism between the two drugs in mutant BRAF cell lines, in association with decreased phosphorylation of cell survival proteins (e.g., C-Jun-N-terminal-kinase, JNK). In the spontaneous ret transgenic mouse melanoma model, combination therapy produced a significant disease onset delay and down-regulation of Chemokine (C-C motif) ligand 2 (CCL2), JNK, and of Myeloid-derived suppressor cell recruitment. Co-incubation with a CCL2-blocking-antibody enhanced in vitro cell sensitivity to temozolomide. Conversely, recombinant CCL2 activated JNK in human tumor melanoma cells. In keeping with these results, the combination of a JNK-inhibitor with temozolomide was synergistic. By showing that down-regulation of CCL2-driven signals by SAHA and temozolomide via JNK contributes to reduce melanoma growth, we provide a rationale for the therapeutic advantage of the drug combination. This combination strategy may be effective because of interference both with tumor cell and tumor microenvironment.
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Quimiocinas/metabolismo , Dacarbazina/análogos & derivados , Inhibidores de Histona Desacetilasas/metabolismo , Melanoma/genética , Animales , Apoptosis , Supervivencia Celular , Dacarbazina/metabolismo , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Humanos , Ratones , Ratones Transgénicos , Temozolomida , Microambiente TumoralRESUMEN
Pulmonary adenoma susceptibility 1 (Pas1) is the major locus responsible for lung tumor susceptibility in mice; among the six genes mapping in this locus, Kras is considered the best candidate for Pas1 function although how it determines tumor susceptibility remains unknown. In an (A/J × C57BL/6)F4 intercross population treated with urethane to induce lung tumors, Pas1 not only modulated tumor susceptibility (LOD score = 48, 69% of phenotypic variance explained) but also acted, in lung tumor tissue, as an expression quantitative trait locus (QTL) for Kras-4A, one of two alternatively spliced Kras transcripts, but not Kras-4B. Additionally, Kras-4A showed differential allelic expression in lung tumor tissue of (A/J × C57BL/6)F4 heterozygous mice, with significantly higher expression from the A/J-derived allele; these results suggest that cis-acting elements control Kras-4A expression. In normal lung tissue from untreated mice of the same cross, Kras-4A levels were also highly linked to the Pas1 locus (LOD score = 23.2, 62% of phenotypic variance explained) and preferentially generated from the A/J-derived allele, indicating that Pas1 is an expression QTL in normal lung tissue as well. Overall, the present findings shed new light on the genetic mechanism by which Pas1 modulates the susceptibility to lung tumorigenesis, through the fine control of Kras isoform levels.
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Adenoma/genética , Predisposición Genética a la Enfermedad/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Sitios de Carácter Cuantitativo/genética , Alelos , Animales , Mapeo Cromosómico/métodos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
BACKGROUND: Chordomas are rare and indolent bone tumors that arise in the skull base and mobile spine. Distant metastases occur in >20% of cases, but morbidity and mortality are mainly related to local relapses that affect the majority of patients. Standard chemotherapy has modest activity, whereas new targeted therapies (alone or in combination) have some activity in controlling disease progression. However, the scarcity of preclinical models capable of testing in vivo responses to these therapies hampers the development of new medical strategies. METHODS: In this study, 8 chordoma samples taken from 8 patients were implanted in nude mice. Four engrafted successfully and gave rise to tumor masses that were analyzed histologically, by means of fluorescence in situ hybridization and biochemical techniques. The data relating to each of the mouse tumors were compared with those obtained from the corresponding human tumor. RESULTS: All 4 engraftments retained the histological, genetic and biochemical features of the human tumors they came from. In one epidermal growth factor receptor(EGFR)-positive xenograft, responsiveness to lapatinib was evaluated by comparing the pre- and post treatment findings. The treatment induced a low-level, heterogeneous switching off of EGFR and its downstream signaling effectors. CONCLUSIONS: Overall, this model is very close to human chordoma and represents a new means of undertaking preclinical investigations and developing tailored therapies.
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Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/análisis , Neoplasias Óseas/tratamiento farmacológico , Cordoma/tratamiento farmacológico , Quinazolinas/uso terapéutico , Sacro/patología , Animales , Western Blotting , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Cordoma/metabolismo , Cordoma/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Humanos , Técnicas para Inmunoenzimas , Hibridación Fluorescente in Situ , Lapatinib , Imagen por Resonancia Magnética , Ratones , Ratones Desnudos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Sacro/efectos de los fármacos , Sacro/metabolismo , Transducción de Señal/efectos de los fármacos , Trasplante Heterólogo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lung adenocarcinoma patients of similar clinical stage and undergoing the same treatments often have marked interindividual variations in prognosis. These clinical discrepancies may be due to the genetic background modulating an individual's predisposition to fighting cancer. Herein, we hypothesized that the lung microenvironment, as reflected by its expression profile, may affect lung adenocarcinoma patients' survival. The transcriptome of non-involved lung tissue, excised from a discovery series of 204 lung adenocarcinoma patients, was evaluated using whole-genome expression microarrays (with probes corresponding to 28 688 well-annotated coding sequences). Genes associated with survival status at 60 months were identified by Cox regression analysis (adjusted for gender, age and clinical stage) and retested in a validation series of 78 additional cases. RNA-Seq analysis from non-involved lung tissue of 12 patients was performed to characterize the different isoforms of candidate genes. Ten genes for which the loge-transformed hazard ratios expressed the same direction of effect in the discovery (P < 1.0 × 10(-3)) and validation series comprised the gene expression signature associated with survival: CNTNAP1, PKNOX1, FAM156A, FRMD8, GALNTL1, TXNDC12, SNTB1, PPP3R1, SNX10 and SERPINH1. RNA sequencing highlighted the complex expression pattern of these genes in non-involved lung tissue from different patients and permitted the detection of a read-through gene fusion between PPP3R1 and the flanking gene (CNRIP1) as well as a novel isoform of CNTNAP1. Our findings support the hypothesis that individual genetic characteristics, evidenced by the expression pattern of non-involved tissue, influence the outcome of lung adenocarcinoma patients.
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Adenocarcinoma/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Pulmonares/genética , Transcriptoma/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Pulmón/patología , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Pronóstico , ARN Mensajero/genéticaRESUMEN
CONTEXT: Papillary thyroid carcinoma (PTC) is the most frequent thyroid tumor and is responsible for the overall increase in thyroid cancer incidence. S100A11 (calgizzarin), a member of the S100 Ca(2+)-binding protein family, is involved in several different biological processes. S100A11 has been found up-regulated in PTC, both at the mRNA and protein levels. OBJECTIVE: Through a combination of expression analysis and functional in vitro and in vivo studies, we have attempted to gain insight into the relevance of S100A11 overexpression in PTC biology. DESIGN: The expression of the S100A11 gene in PTC was investigated in several gene expression data sets. The effect of S100A11 silencing on the hallmarks of the malignant phenotype of several PTC-derived cell lines was investigated. In NIH3T3 cells, the cooperation of S100A11 with the different PTC-specific oncogenes was assessed. RESULTS: We found that the S100A11 gene expression is frequently up-regulated in PTC, anaplastic thyroid carcinoma, but not in follicular thyroid carcinoma. S100A11 overexpression was also detected in PTC-derived cell lines, which were then used for functional studies. S100A11 silencing in PTC-derived cell lines did not affect cell proliferation, whereas it reduced the loss of contact inhibition, anchorage-independent growth, and resistance to anoikis. Cotransfection experiments in NIH3T3 cells showed that overexpression of the S100A11 gene was able to enhance the transforming capabilities of the different PTC-associated oncogenes by affecting the loss of contact inhibition, anchorage-independent growth, and in vivo tumor formation. CONCLUSION: Our data indicate that S100A11 overexpression exerts a protumoral functional role in PTC pathogenesis.