RESUMEN
Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) in vitro and in vivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Transformación Celular Neoplásica/patología , Complejo I de Transporte de Electrón/metabolismo , Regulación Neoplásica de la Expresión Génica , Mitocondrias/patología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Complejo I de Transporte de Electrón/genética , Genoma Mitocondrial , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Invasividad Neoplásica , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-mdm2/genética , Transducción de Señal , Transcripción Genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Most high-grade ovarian carcinomas (HGOCs) are sensitive to carboplatin (CBP)-based chemotherapy but frequently recur within 24 months. Recurrent tumors remain CBP-sensitive and acquire resistance only after several treatment rounds. Recurrences arise from a small number of residual tumor cells not amenable to investigation in patients. We developed patient-derived xenografts (PDXs) that allow the study of these different stages of CBP-sensitive recurrence and acquisition of resistance. We generated PDX models from CBP-sensitive and intrinsically resistant HGOC. PDXs were CBP- or mock-treated and tumors were sampled, after treatment and at recurrence. We also isolated models with acquired-resistance from CBP-sensitive PDXs. Tumors were characterized at the histological and transcriptome levels. PDX models reproduced treatment response seen in the patients. CBP-sensitive residual tumors contained nonproliferating tumor cell clusters embedded in a fibrotic mesh. In nontreated PDX tumors and treated CBP-resistant tumors, fibrotic tissue was not prevalent. Residual tumors had marked differences in gene expression when compared to naïve and recurrent tumors, indicating downregulation of the cell cycle and proliferation and upregulation of interferon response and the epithelial-mesenchymal transition. This gene expression pattern resembled that described in embryonal diapause and 'drug-tolerant persister' states. Residual and acquired-resistance tumors share the overexpression of three genes: CEACAM6, CRYAB, and SOX2. Immunostaining analysis showed strong CEACAM6, CRYAB, and SOX2 protein expression in CBP-sensitive residual and acquired-resistance PDX, thus confirming the RNA profiling results. In HGOC PDX, CBP-sensitive recurrences arise from a small population of quiescent, drug-tolerant, residual cells embedded in a fibrotic mesh. These cells overexpress CEACAM6, CRYAB, and SOX2, whose overexpression is also associated with acquired resistance and poor patient prognosis. CEACAM6, CRYAB, and SOX2 may thus serve as a biomarker to predict recurrence and emergence of resistant disease in CBP-treated HGOC patients. © 2022 The Pathological Society of Great Britain and Ireland.
Asunto(s)
Antígenos CD , Carcinoma Epitelial de Ovario , Moléculas de Adhesión Celular , Proteínas Ligadas a GPI , Neoplasias Ováricas , Factores de Transcripción SOXB1 , Cadena B de alfa-Cristalina , Antígenos CD/biosíntesis , Antígenos CD/genética , Carboplatino/farmacología , Carboplatino/uso terapéutico , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Resistencia a Antineoplásicos , Femenino , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Humanos , Recurrencia Local de Neoplasia , Neoplasia Residual , Recurrencia , Factores de Transcripción SOXB1/biosíntesis , Factores de Transcripción SOXB1/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Cadena B de alfa-Cristalina/biosíntesis , Cadena B de alfa-Cristalina/genéticaRESUMEN
Molecular subtypes of breast cancer are defined on the basis of gene expression and genomic/epigenetic pattern differences. Different subtypes are thought to originate from distinct cell lineages, but the early activation of an oncogene could also play a role. It is difficult to discriminate the respective inputs of oncogene activation or cell type of origin. In this work, we wished to determine whether activation of distinct oncogenic pathways in human mammary epithelial cells (HMEC) could lead to different patterns of genetic and epigenetic changes. To this aim, we transduced shp53 immortalized HMECs in parallel with the CCNE1, WNT1 and RASv12 oncogenes which activate distinct oncogenic pathways and characterized them at sequential stages of transformation for changes in their genetic and epigenetic profiles. We show that initial activation of CCNE1, WNT1 and RASv12, in shp53 HMECs results in different and reproducible changes in mRNA and micro-RNA expression, copy number alterations (CNA) and DNA methylation profiles. Noticeably, HMECs transformed by RAS bore very specific profiles of CNAs and DNA methylation, clearly distinct from those shown by CCNE1 and WNT1 transformed HMECs. Genes impacted by CNAs and CpG methylation in the RAS and the CCNE1/WNT1 clusters showed clear differences, illustrating the activation of distinct pathways. Our data show that early activation of distinct oncogenic pathways leads to active adaptive events resulting in specific sets of CNAs and DNA methylation changes. We, thus, propose that activation of different oncogenes could have a role in reshaping the genetic landscape of breast cancer subtypes.
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Neoplasias de la Mama/genética , Glándulas Mamarias Humanas/fisiología , Oncogenes , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Ciclina E/biosíntesis , Ciclina E/genética , Metilación de ADN , Epigénesis Genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células Epiteliales/fisiología , Femenino , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Xenoinjertos , Humanos , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Ratones , Ratones Desnudos , Ratones SCID , Proteínas Oncogénicas/biosíntesis , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína Wnt1/biosíntesis , Proteína Wnt1/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer characterized by poor response to chemotherapy and radiotherapy due to the lack of efficient therapeutic tools and early diagnostic markers. We previously generated the nonligand competing anti-HER3 antibody 9F7-F11 that binds to pancreatic tumor cells and induces tumor regression in vivo in experimental models. Here, we asked whether coupling 9F7-F11 with a radiosensitizer, such as monomethylauristatin E (MMAE), by using the antibody-drug conjugate (ADC) technology could improve radiation therapy efficacy in PDAC. We found that the MMAE-based HER3 antibody-drug conjugate (HER3-ADC) was efficiently internalized in tumor cells, increased the fraction of cells arrested in G2/M, which is the most radiosensitive phase of the cell cycle, and promoted programmed cell death of irradiated HER3-positive pancreatic cancer cells (BxPC3 and HPAC cell lines). HER3-ADC decreased the clonogenic survival of irradiated cells by increasing DNA double-strand break formation (based on γH2AX level), and by modulating DNA damage repair. Tumor radiosensitization with HER3-ADC favored the inhibition of the AKT-induced survival pathway, together with more efficient caspase 3/PARP-mediated apoptosis. Incubation with HER3-ADC before irradiation synergistically reduced the phosphorylation of STAT3, which is involved in chemoradiation resistance. In vivo, the combination of HER3-ADC with radiation therapy increased the overall survival of mice harboring BxPC3, HPAC cell xenografts or patient-derived xenografts, and reduced proliferation (KI67-positive cells). Combining auristatin radiosensitizer delivery via an HER3-ADC with radiotherapy is a new promising therapeutic strategy in PDAC.
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Carcinoma Ductal Pancreático/terapia , Inmunoconjugados/administración & dosificación , Factores Inmunológicos/administración & dosificación , Neoplasias Pancreáticas/terapia , Animales , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Anticuerpos Monoclonales de Origen Murino/farmacología , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Quimioradioterapia , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacología , Factores Inmunológicos/farmacología , Ratones , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacología , Neoplasias Pancreáticas/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Factor de Transcripción STAT3/metabolismo , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: It remains presently unclear whether disease progression in colorectal carcinoma (CRC), from early, to invasive and metastatic forms, is associated to a gradual increase in genetic instability and to a scheme of sequentially occurring Copy Number Alterations (CNAs). METHODS: In this work we set to determine the existence of such links between CRC progression and genetic instability and searched for associations with patient outcome. To this aim we analyzed a set of 162 Chromosomal Instable (CIN) CRCs comprising 131 primary carcinomas evenly distributed through stage 1 to 4, 31 metastases and 14 adenomas by array-CGH. CNA profiles were established according to disease stage and compared. We, also, asked whether the level of genomic instability was correlated to disease outcome in stage 2 and 3 CRCs. Two metrics of chromosomal instability were used; (i) Global Genomic Index (GGI), corresponding to the fraction of the genome involved in CNA, (ii) number of breakpoints (nbBP). RESULTS: Stage 1, 2, 3 and 4 tumors did not differ significantly at the level of their CNA profiles precluding the conventional definition of a progression scheme based on increasing levels of genetic instability. Combining GGI and nbBP,we classified genomic profiles into 5 groups presenting distinct patterns of chromosomal instability and defined two risk classes of tumors, showing strong differences in outcome and hazard risk (RFS: p = 0.012, HR = 3; OS: p < 0.001, HR = 9.7). While tumors of the high risk group were characterized by frequent fractional CNAs, low risk tumors presented predominantly whole chromosomal arm CNAs. Searching for CNAs correlating with negative outcome we found that losses at 16p13.3 and 19q13.3 observed in 10% (7/72) of stage 2-3 tumors showed strong association with early relapse (p < 0.001) and death (p < 0.007, p < 0.016). Both events showed frequent co-occurrence (p < 1x10-8) and could, therefore, mark for stage 2-3 CRC susceptible to negative outcome. CONCLUSIONS: Our data show that CRC disease progression from stage 1 to stage 4 is not paralleled by increased levels of genetic instability. However, they suggest that stage 2-3 CRC with elevated genetic instability and particularly profiles with fractional CNA represent a subset of aggressive tumors.
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Inestabilidad Cromosómica/genética , Neoplasias Colorrectales/genética , Recurrencia Local de Neoplasia/genética , Pronóstico , Adulto , Anciano , Carcinoma in Situ/genética , Puntos de Rotura del Cromosoma , Neoplasias Colorrectales/patología , Hibridación Genómica Comparativa , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/patología , Resultado del TratamientoRESUMEN
OBJECTIVE: Mesenchymal stem cells (MSCs) represent a promising tool for cell therapy for several disorders, among them the osteoarticular diseases. For such clinical applications, intraarticular (IA) injection of MSCs may be favored for higher levels of safety and targeting of specific joints. Although the safety of intravenous (IV) administration of MSCs has been reported in a number of clinical trials, the safety and biodistribution of MSCs after IA injection have not been tested. Our objective was to assess the toxicity of clinical-grade human adipose-derived MSCs (AD-MSCs), as well as their biodistribution, after IA injection into SCID mice. METHODS: SCID mice received IA or IV administration of 10(6) human AD-MSCs. Several tissues were recovered at different time points and processed for histologic assessment or real-time polymerase chain reaction (PCR) analysis. A highly sensitive assay was used to monitor the distribution of AD-MSCs, based on amplification of human-specific Alu sequences. RESULTS: Absence of toxicity was observed after AD-MSC infusion. Alu PCR assay revealed a high sensitivity (1 human AD-MSC/10(5) murine cells), with a large linear range (1-5 × 10(4) /10(5) murine cells). Importantly, 15% of the IA-injected AD-MSCs were detectable in the joint for the first month and 1.5% of the AD-MSCs engrafted over the long term, at least 6 months. AD-MSCs were observed in the injected joints and in areas of tissue referred to as stem cell niches, such as the bone marrow, adipose tissue, and muscle. CONCLUSION: These data support the feasibility and safety of using IA delivery of human AD-MSCs in the treatment of rheumatic diseases that affect the joints.
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Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Tejido Adiposo/citología , Animales , Células Cultivadas , Femenino , Humanos , Infusiones Intravenosas , Inyecciones Intraarticulares , Masculino , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Ratones , Ratones SCIDRESUMEN
The non-receptor tyrosine kinase SRC is frequently deregulated in human colorectal cancer (CRC), and SRC increased activity has been associated with poor clinical outcomes. In nude mice engrafted with human CRC cells, SRC over-expression favors tumor growth and is accompanied by a robust increase in tyrosine phosphorylation in tumor cells. How SRC contributes to this tumorigenic process is largely unknown. We analyzed SRC oncogenic signaling in these tumors by means of a novel quantitative proteomic analysis. This method is based on stable isotope labeling with amino acids of xenograft tumors by the addition of [(13)C(6)]-lysine into mouse food. An incorporation level greater than 88% was obtained in xenograft tumors after 30 days of the heavy lysine diet. Quantitative phosphoproteomic analysis of these tumors allowed the identification of 61 proteins that exhibited a significant increase in tyrosine phosphorylation and/or association with tyrosine phosphorylated proteins upon SRC expression. These mainly included molecules implicated in vesicular trafficking and signaling and RNA binding proteins. Most of these proteins were specific targets of SRC signaling in vivo, as they were not identified by analysis via stable isotope labeling by amino acids in cell culture (SILAC) of the same CRC cells in culture. This suggests that oncogenic signaling induced by SRC in tumors significantly differs from that induced by SRC in cell culture. We next confirmed this notion experimentally with the example of the vesicular trafficking protein and SRC substrate TOM1L1. We found that whereas TOM1L1 depletion only slightly affected SRC-induced proliferation of CRC cells in vitro, it drastically decreased tumor growth in xenografted nude mice. We thus concluded that this vesicular trafficking protein plays an important role in SRC-induced tumor growth. Overall, these data show that SILAC analysis in mouse xenografts is a valuable approach for deciphering tyrosine kinase oncogenic signaling in vivo.
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Neoplasias Colorrectales/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aminoácidos/metabolismo , Animales , Isótopos de Carbono , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Humanos , Marcaje Isotópico , Espectrometría de Masas , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fosfoproteínas/análisis , Fosforilación , Proteoma/análisis , Transducción de Señal , Trasplante HeterólogoRESUMEN
One in three Triple Negative Breast Cancer (TNBC) is Homologous Recombination Deficient (HRD) and susceptible to respond to PARP inhibitor (PARPi), however, resistance resulting from functional HR restoration is frequent. Thus, pharmacologic approaches that induce HRD are of interest. We investigated the effectiveness of CDK-inhibition to induce HRD and increase PARPi sensitivity of TNBC cell lines and PDX models. Two CDK-inhibitors (CDKi), the broad range dinaciclib and the CDK12-specific SR-4835, strongly reduced the expression of key HR genes and impaired HR functionality, as illustrated by BRCA1 and RAD51 nuclear foci obliteration. Consequently, both CDKis showed synergism with olaparib, as well as with cisplatin and gemcitabine, in a range of TNBC cell lines and particularly in olaparib-resistant models. In vivo assays on PDX validated the efficacy of dinaciclib which increased the sensitivity to olaparib of 5/6 models, including two olaparib-resistant and one BRCA1-WT model. However, no olaparib response improvement was observed in vivo with SR-4835. These data support that the implementation of CDK-inhibitors could be effective to sensitize TNBC to olaparib as well as possibly to cisplatin or gemcitabine.
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Antineoplásicos , Piperazinas , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Resistencia a Antineoplásicos , Cisplatino/farmacología , Cisplatino/uso terapéutico , Gemcitabina , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Línea Celular TumoralRESUMEN
Background: About 15% of Triple-Negative-Breast-Cancer (TNBC) present silencing of the BRCA1 promoter methylation and are assumed to be Homologous Recombination Deficient (HRD). BRCA1-methylated (BRCA1-Me) TNBC could, thus, be eligible to treatment based on PARP-inhibitors or Platinum salts. However, their actual HRD status is discussed, as these tumors are suspected to develop resistance after chemotherapy exposure. Methods: We interrogated the sensitivity to olaparib vs. carboplatin of 8 TNBC Patient-Derived Xenografts (PDX) models. Four PDX corresponded to BRCA1-Me, of which 3 were previously exposed to NeoAdjuvant-Chemotherapy (NACT). The remaining PDX models corresponded to two BRCA1-mutated (BRCA1-Mut) and two BRCA1-wild type PDX that were respectively included as positive and negative controls. The HRD status of our PDX models was assessed using both genomic signatures and the functional BRCA1 and RAD51 nuclear foci formation assay. To assess HR restoration associated with olaparib resistance, we studied pairs of BRCA1 deficient cell lines and their resistant subclones. Results: The 3 BRCA1-Me PDX that had been exposed to NACT responded poorly to olaparib, likewise BRCA1-WT PDX. Contrastingly, 3 treatment-naïve BRCA1-deficient PDX (1 BRCA1-Me and 2 BRCA1-mutated) responded to olaparib. Noticeably, the three olaparib-responsive PDX scored negative for BRCA1- and RAD51-foci, whereas all non-responsive PDX models, including the 3 NACT-exposed BRCA1-Me PDX, scored positive for RAD51-foci. This suggested HRD in olaparib responsive PDX, while non-responsive models were HR proficient. These results were consistent with observations in cell lines showing a significant increase of RAD51-foci in olaparib-resistant subclones compared with sensitive parental cells, suggesting HR restoration in these models. Conclusion: Our results thus support the notion that the actual HRD status of BRCA1-Me TNBC, especially if previously exposed to chemotherapy, may be questioned and should be verified using the BRCA1- and RAD51-foci assay.
RESUMEN
In this study, we have analyzed the expression of TRIM24/TIF-1α, a negative regulator of various transcription factors (including nuclear receptors and p53) at the genomic, mRNA, and protein levels in human breast tumors. In breast cancer biopsy specimens, TRIM24/TIF-1α mRNA levels (assessed by Real-Time Quantitative PCR or microarray expression profiling) were increased as compared to normal breast tissues. At the genomic level, array comparative genomic hybridization analysis showed that the TRIM24/TIF-1α locus (7q34) exhibited both gains and losses that correlated with mRNA levels. By re-analyzing a series of 238 tumors, high levels of TRIM24/TIF-1α mRNA significantly correlated with various markers of poor prognosis (such as the molecular subtype) and were associated with worse overall survival. By using a rabbit polyclonal antibody for immunochemistry, the TRIM24/TIF-1α protein was detected in nuclei of normal luminal epithelial breast cells, but not in myoepithelial cells. Tissue microarray analysis confirmed that its expression was increased in epithelial cells from normal to breast infiltrating duct carcinoma and correlated with worse overall survival. Altogether, this work is the first study that shows that overexpression of the TRIM24/TIF-1α gene in breast cancer is associated with poor prognosis and worse survival, and it suggests that this transcription coregulator may play a role in mammary carcinogenesis and represent a novel prognostic marker.
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Neoplasias de la Mama/metabolismo , Proteínas Portadoras/biosíntesis , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/biosíntesis , Factores de Transcripción/biosíntesis , Mama/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Mapeo Cromosómico/métodos , Hibridación Genómica Comparativa , Epitelio/metabolismo , Femenino , Humanos , Inmunoquímica/métodos , Inmunohistoquímica/métodos , Pronóstico , ARN Mensajero/metabolismo , Resultado del TratamientoRESUMEN
BACKGROUND: The aim of this study was to develop an original method to extract sets of relevant molecular biomarkers (gene sequences) that can be used for class prediction and can be included as prognostic and predictive tools. MATERIALS AND METHODS: The method is based on sequential patterns used as features for class prediction. We applied it to classify breast cancer tumors according to their histological grade. RESULTS: We obtained very good recall and precision for grades 1 and 3 tumors, but, like other authors, our results were less satisfactory for grade 2 tumors. CONCLUSIONS: We demonstrated the interest of sequential patterns for class prediction of microarrays and we now have the material to use them for prognostic and predictive applications.
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Neoplasias de la Mama/patología , Minería de Datos/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Neoplasias de la Mama/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Estadificación de NeoplasiasRESUMEN
Glioblastomas (GBM) are high-grade brain tumors, containing cells with distinct phenotypes and tumorigenic potentials, notably aggressive and treatment-resistant multipotent glioblastoma stem cells (GSC). The molecular mechanisms controlling GSC plasticity and growth have only partly been elucidated. Contact with endothelial cells and the Notch1 pathway control GSC proliferation and fate. We used three GSC cultures and glioma resections to examine the expression, regulation, and role of two transcription factors, SLUG (SNAI2) and TAL1 (SCL), involved in epithelial to mesenchymal transition (EMT), hematopoiesis, vascular identity, and treatment resistance in various cancers. In vitro, SLUG and a truncated isoform of TAL1 (TAL1-PP22) were strongly upregulated upon Notch1 activation in GSC, together with LMO2, a known cofactor of TAL1, which formed a complex with truncated TAL1. SLUG was also upregulated by TGF-ß1 treatment and by co-culture with endothelial cells. In patient samples, the full-length isoform TAL1-PP42 was expressed in all glioma grades. In contrast, SLUG and truncated TAL1 were preferentially overexpressed in GBMs. SLUG and TAL1 are expressed in the tumor microenvironment by perivascular and endothelial cells, respectively, and to a minor extent, by a fraction of epidermal growth factor receptor (EGFR) -amplified GBM cells. Mechanistically, both SLUG and truncated TAL1 reduced GSC growth after their respective overexpression. Collectively, this study provides new evidence for the role of SLUG and TAL1 in regulating GSC plasticity and growth.
RESUMEN
Alterations to cell polarization or to intercellular junctions are often associated with epithelial cancer progression, including breast cancers (BCa). We show here that the loss of the junctional scaffold protein MAGI1 is associated with bad prognosis in luminal BCa, and promotes tumorigenesis. E-cadherin and the actin binding scaffold AMOTL2 accumulate in MAGI1 deficient cells which are subjected to increased stiffness. These alterations are associated with low YAP activity, the terminal Hippo-pathway effector, but with an elevated ROCK and p38 Stress Activated Protein Kinase activities. Blocking ROCK prevented p38 activation, suggesting that MAGI1 limits p38 activity in part through releasing actin strength. Importantly, the increased tumorigenicity of MAGI1 deficient cells is rescued in the absence of AMOTL2 or after inhibition of p38, demonstrating that MAGI1 acts as a tumor-suppressor in luminal BCa by inhibiting an AMOTL2/p38 stress pathway.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Angiomotinas/metabolismo , Neoplasias de la Mama/prevención & control , Carcinogénesis/patología , Moléculas de Adhesión Celular/metabolismo , Guanilato-Quinasas/metabolismo , Transducción de Señal , Estrés Fisiológico , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/metabolismo , Carcinogénesis/metabolismo , Moléculas de Adhesión Celular/deficiencia , Línea Celular Tumoral , Proliferación Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Guanilato-Quinasas/deficiencia , Humanos , Fenotipo , Unión Proteica , Proteínas Señalizadoras YAP/metabolismo , beta Catenina/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
AIMS AND BACKGROUND: The literature data show that the most frequently affected chromosomes in ovarian carcinogenesis are 1, 8 and 17. In the present study we aimed to define more precisely at a high resolution the genomic imbalances of these chromosomes in ovarian cancer and to determine genomic markers separating tumors of different histological types and stages. METHODS: Array comparative genomic hybridization (CGH) with a resolution of approximately 0.8 Mb was applied in 28 primary ovarian tumors. We identified regions of highly frequent gains or losses (affecting more than 40% of ovarian cancers) and determined sites showing alterations of elevated amplitude (amplifications or homozygous deletions). Doing this we also identified at least two adjacent changed clones. RESULTS: We determined anomalies strongly associated with the disease such as deletions at 8p21-23, 17p12-13, 1p35-36 or amplifications at 1q23, 17q12, 17q23.2, 8q13.2, 8q24. We defined more precisely the gains in 17q12-q24, finding as strong candidates for ovarian tumorigenesis the genes LASP1 (17q12), TGF11 (17q21.32), MUL (17q23.2), TBX2 (17q23.2), AXIN2 (17q24.3) and GRB2 (17q25.1). Of particular note was gain of 8q13.2, which occurred at a high frequency in ovarian cancer, especially in serous and late-stage tumors. We found that gains of 1q32-1q43, 8p11-p12, 8q11.23, 8q13.2, and 8q24.21-8q24.22 and losses of 1p36.21, 8p23.1-8p21.1 and 8q21.2 were associated with serous histology, whereas losses of 1q23 and 1q32-43 and gains of 17q11.2-12 and 17q25 were associated with mucinous histology. Gains of 1q23, 8q24, 17q23.2, 17q24.2 and losses of 1p35-36, 8p, 17p, and 17q were specific for late-stage ovarian cancers. CONCLUSIONS: Our study has identified potential genomic markers of interest on chromosomes 1, 8 and 17 in ovarian cancer. Tumors showed a wide variety in the patterns of alteration, suggesting that alternative mechanisms of genomic instability may play a role in this tumor type.
Asunto(s)
Cromosomas Humanos Par 17 , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 8 , Hibridación Genómica Comparativa , Marcadores Genéticos , Inestabilidad Genómica , Neoplasias Ováricas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adenocarcinoma Mucinoso/genética , Adulto , Anciano , Proteína Axina , Cistadenocarcinoma Seroso/genética , Proteínas del Citoesqueleto/genética , Femenino , Proteína Adaptadora GRB2/genética , Humanos , Proteínas con Dominio LIM , Persona de Mediana Edad , Proteínas Nucleares/genética , Neoplasias Ováricas/patología , Eliminación de Secuencia , Proteínas de Dominio T Box/genética , Factor de Crecimiento Transformador alfa/genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
PURPOSE: In 75% of ovarian cancer patients the tumor mass is completely eradicated by established surgical and cytotoxic treatment; however, the majority of the tumors recur within 24 months. Here we investigated the role of circulating tumor cells (CTCs) indicating occult tumor load, which remains inaccessible by established diagnostics. EXPERIMENTAL DESIGN: Blood was taken at diagnosis (baseline samples, n = 102) and six months after completion of adjuvant first-line chemotherapy (follow-up samples; n = 78). CTCs were enriched by density gradient centrifugation. A multi-marker immunostaining was established and further complemented by FISH on CTCs and tumor/metastasis tissues using probes for stem-cell like fusion genes MECOM and HHLA1. RESULTS: CTCs were observed in 26.5% baseline and 7.7% follow-up blood samples at a mean number of 12.4 and 2.8 CTCs per ml blood, respectively. Baseline CTCs indicated a higher risk of death in R0 patients with complete gross resection (univariate: HR 2.158, 95% CI 1.111-4.191, p = 0.023; multivariate: HR 2.720, 95% CI 1.340-5.522, p = 0.006). At follow-up, the presence of CTCs was associated with response to primary treatment as assessed using RECIST criteria. Chromosomal gains at MECOM and HHLA1 loci suggest that the observed cells were cancer cells and reflect pathophysiological decisive chromosomal aberrations of the primary and metastatic tumors. CONCLUSIONS: Our data suggest that CTCs detected by the multi-marker protein panel and/or MECOM/HHLA1 FISH represent minimal residual disease in optimally debulked ovarian cancer patients. The role of CTCs cells especially for clinical therapy stratification of the patients has to be validated in consecutive larger studies applying standardized treatment schemes.
RESUMEN
In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements that combine amplification at 8p11-12, break in the 8p12-21 region, and loss of 8p21-ter. Several studies have identified putative oncogenes in the 8p11-12 amplicon. However, discrepancies and the lack of knowledge on the structure of this amplification lead us to think that the actual identity of the oncogenes is not definitively established. We present here a comprehensive study combining genomic, expression, and chromosome break analyses of the 8p11-12 region in breast cell lines and primary breast tumors. We show the existence of four amplicons at 8p11-12 using array comparative genomic hybridization. Gene expression analysis of 123 samples using DNA microarrays identified 14 genes significantly overexpressed in relation to amplification. Using fluorescence in situ hybridization analysis on tissue microarrays, we show the existence of a cluster of breakpoints spanning a region just telomeric to and associated with the amplification. Finally, we show that 8p11-12 amplification has a pejorative effect on survival in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Aberraciones Cromosómicas , Cromosomas Humanos Par 8/genética , Amplificación de Genes , Oncogenes/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromosomas Humanos Par 8/metabolismo , Daño del ADN , Humanos , Hibridación Fluorescente in Situ , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Telómero/genética , Análisis de Matrices TisularesRESUMEN
Chromosome 17 is severely rearranged in breast cancer. Whereas the short arm undergoes frequent losses, the long arm harbors complex combinations of gains and losses. In this work we present a comprehensive study of quantitative anomalies at chromosome 17 by genomic array-comparative genomic hybridization and of associated RNA expression changes by cDNA arrays. We built a genomic array covering the entire chromosome at an average density of 1 clone per 0.5 Mb, and patterns of gains and losses were characterized in 30 breast cancer cell lines and 22 primary tumors. Genomic profiles indicated severe rearrangements. Compiling data from all samples, we subdivided chromosome 17 into 13 consensus segments: 4 regions showing mainly losses, 6 regions showing mainly gains, and 3 regions showing either gains or losses. Within these segments, smallest regions of overlap were defined (17 for gains and 16 for losses). Expression profiles were analyzed by means of cDNA arrays comprising 358 known genes at 17q. Comparison of expression changes with quantitative anomalies revealed that about half of the genes were consistently affected by copy number changes. We identified 85 genes overexpressed when gained (39 of which mapped within the smallest regions of overlap), 67 genes underexpressed when lost (32 of which mapped to minimal intervals of losses), and, interestingly, 32 genes showing reduced expression when gained. Candidate genes identified in this study belong to very diverse functional groups, and a number of them are novel candidates.
Asunto(s)
Neoplasias de la Mama/genética , Aberraciones Cromosómicas , Cromosomas Humanos Par 17/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Rotura Cromosómica , Dosificación de Gen , Perfilación de la Expresión Génica , Humanos , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Histone lysine acetylation is an epigenetic mark regulated by histone acetyltransferases and histone deacetylases (HDAC) which plays an important role in tumorigenesis. In this study, we observed a strong overexpression of class IIa HDAC9, at the mRNA and protein levels, in the most aggressive human breast cancer cell lines (i.e. in basal breast cancer cells vs luminal ones or in malignant vs begnin MCF10A breast epithelial cell lines). HDAC9 overexpression was associated with higher rates of gene transcription and increased epigenetic marks on the HDAC9 promoter. Ectopic expression of HDAC9 in MCF7 luminal breast cancer cells led to an increase in cell proliferation and to a decrease in apoptosis. These effects were associated with a deregulated expression of several genes controlled by HDAC inhibitors such as CDKN1A, BAX and TNFRSF10A. Inversely, knock-down of HDAC9 expression in MDA-MB436 basal breast cancer cells reduced cell proliferation. Moreover, high HDAC9 expression decreased the efficacy of HDAC inhibitors to reduce cell proliferation and to regulate CDKN1A gene expression. Interestingly, the gene encoding the transcription factor SOX9 was identified by a global transcriptomic approach as an HDAC9 target gene. In stably transfected MCF7 cells, SOX9 silencing significantly decreased HDAC9 mitogenic activity. Finally, in a large panel of breast cancer biopsies, HDAC9 expression was significantly increased in tumors of the basal subtype, correlated with SOX9 expression and associated with poor prognosis. Altogether, these results indicate that HDAC9 is a key factor involved in mammary carcinogenesis and in the response to HDAC inhibitors.
Asunto(s)
Neoplasias de la Mama/enzimología , Proliferación Celular/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Proteínas Represoras/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Células MCF-7 , Microscopía Fluorescente , Interferencia de ARN , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismoRESUMEN
ERBB2 overexpression in human breast cancer leads to invasive carcinoma but the mechanism is not clearly understood. Here we report that TOM1L1 is co-amplified with ERBB2 and defines a subgroup of HER2(+)/ER(+) tumours with early metastatic relapse. TOM1L1 encodes a GAT domain-containing trafficking protein and is a SRC substrate that negatively regulates tyrosine kinase signalling. We demonstrate that TOM1L1 upregulation enhances the invasiveness of ERBB2-transformed cells. This pro-tumoural function does not involve SRC, but implicates membrane-bound membrane-type 1 MMP (MT1-MMP)-dependent activation of invadopodia, membrane protrusions specialized in extracellular matrix degradation. Mechanistically, ERBB2 elicits the indirect phosphorylation of TOM1L1 on Ser321. The phosphorylation event promotes GAT-dependent association of TOM1L1 with the sorting protein TOLLIP and trafficking of the metalloprotease MT1-MMP from endocytic compartments to invadopodia for tumour cell invasion. Collectively, these results show that TOM1L1 is an important element of an ERBB2-driven proteolytic invasive programme and that TOM1L1 amplification potentially enhances the metastatic progression of ERBB2-positive breast cancers.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Receptor ErbB-2/metabolismo , Células 3T3 , Animales , Línea Celular Tumoral , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Invasividad NeoplásicaRESUMEN
DNA amplification at band q13 of chromosome 11 is common in breast cancer, and CCND1 and EMS1 remain the strongest candidate genes. However, amplification patterns are consistent with the existence of four cores of amplification, suggesting the involvement of additional genes. Here we present evidence strongly suggesting the involvement of the recently characterized EMSY gene in the formation of the telomeric amplicon. EMSY maps at 11q13.5, 100 kb centromeric to the GARP gene, which has been mapped within the core of the distal amplicon. The EMSY protein was shown to interact with BRCA2 and has a role in chromatin remodeling. This makes EMSY a strong candidate oncogene for the 11q13.5 amplicon. DNA amplification was studied in a total of 940 primary breast tumors and 39 breast cancer cell lines. Amplification profiles were consistent with the EMSY-GARP locus being amplified independently of CCND1 and/or EMS1. EMSY RNA expression levels were studied along with those of five other genes located at 11q13.5 by real-time quantitative PCR in the 39 cell lines and a subset of 65 tumors. EMSY overexpression correlated strongly with DNA amplification in both primary tumors and cell lines. In a subset of 296 patients, EMSY amplification was found by both uni- and multivariate analyses to correlate with shortened disease-free survival. These data indicate that EMSY is a strong candidate oncogene for the 11q13.5 amplicon.