RESUMEN
Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.
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Carcinoma de Pulmón de Células no Pequeñas/inmunología , Neoplasias Colorrectales/inmunología , Neoplasias Pulmonares/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos T Reguladores/inmunología , Anciano , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Separación Celular , Neoplasias Colorrectales/mortalidad , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pronóstico , TranscriptomaRESUMEN
The adipocyte-like morphology of clear cell renal cell carcinoma (ccRCC) cells results from a grade-dependent neutral lipid accumulation; however, the molecular mechanism and role in renal cancer progression have yet to be clarified. ccRCC shows a gene expression signature consistent with adipogenesis, and the phospholipid-binding protein annexin A3 (AnxA3), a negative regulator of adipocyte differentiation, is down-regulated in RCC and shows a differential expression pattern for two isoforms of 36 and 33 kDa. Using primary cell cultures and cell lines, we investigated the involvement of AnxA3 isoforms in lipid storage modulation of ccRCC cells. We found that the increased accumulation of lipids into ccRCC cells correlated with a decrease of the 36/33 isoform ratio. Treatment with adipogenic medium induced a significant increment of lipid storage in ccRCC cells that had a low 36-kDa AnxA3 expression and 36/33 ratio. The 36-kDa AnxA3 silencing in ccRCC cells increased lipid storage induced by adipogenic medium. These data suggest that 36-kDa AnxA3 negatively modulates the response to adipogenic treatment and may act as negative regulator of lipid storage in ccRCC cells. The subcellular distribution of AnxA3 in the cellular endocytic compartment suggests its involvement in modulation of vesicular trafficking, and it might serve as a putative mechanism of lipid storage regulation in ccRCC cells, opening novel translational outcomes.
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Anexina A3/metabolismo , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/metabolismo , Metabolismo de los Lípidos , Proteínas de Neoplasias/metabolismo , Anciano , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Femenino , Humanos , Isoenzimas/metabolismo , Neoplasias Renales/patología , MasculinoRESUMEN
In end-stage chronic kidney disease, the option of organ transplantation is limited because of the scarce availability of kidneys. The combination of stem cell research, regenerative medicine, and tissue engineering seems a promising approach to produce new transplantable kidneys. Currently, the possibility to repopulate naturally obtained scaffolds with cells of different sources is advancing. Our aim was to test, for the first time, whether the nephrosphere (NS) cells, composed by renal stem/progenitor-like cells, were able to repopulate different nephron portions of renal extracellular matrix scaffolds obtained after decellularization of human renal tissue slices. Our decellularization protocol enabled us to obtain a completely acellular renal scaffold while maintaining the extracellular matrix structure and composition in terms of collagen IV, laminin, and fibronectin. NS cells, cultured on decellularized renal scaffolds with basal medium, differentiated into proximal and distal tubules as well as endothelium, as highlighted by histology and by the specific expression of epithelial cytokeratin 8.18, proximal tubular CD10, distal tubular cytokeratin 7, and endothelial von Willebrand factor markers. Endothelial medium promoted the differentiation toward the endothelium, whereas epithelial medium promoted the differentiation toward the epithelium. NS cells seem to be a good tool for scaffold repopulation, paving the way for experimental investigations focused on whole-kidney reconstruction.
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Diferenciación Celular/fisiología , Riñón/citología , Andamios del Tejido , Anciano , Anciano de 80 o más Años , Células Cultivadas , Colágeno Tipo IV/metabolismo , Matriz Extracelular/metabolismo , Femenino , Fibronectinas/metabolismo , Humanos , Laminina/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Renal tubular cells are involved in the tubular interstitial fibrosis observed in diabetic nephropathy. It is debated whether epithelial-mesenchymal transition (EMT) affects tubular cells, which under high-glucose conditions overproduce transforming growth factor-ß (TGF-ß), a fibrogenic cytokine involved in interstitial fibrosis development. Our study investigated the involvement of non-receptor tyrosine kinase Arg (also called Abl2) in TGF-ß production. Human primary tubular cell cultures exposed to high-glucose conditions were used. These cells showed an elongated morphology, stress fibers and vimentin increment but maintained most of the epithelial marker expression and distribution. In these cells exposed to high glucose, which overexpressed and secreted active TGF-ß1, Arg protein and activity was downregulated. A further TGF-ß1 increase was induced by Arg silencing with siRNA, as with the Arg tyrosine kinase inhibitor Imatinib. In the cells exposed to high glucose, reactive oxygen species (ROS)-dependent Arg kinase downregulation induced both RhoA activation, through p190RhoGAPA (also known as ARHGAP35) modulation, and proteasome activity inhibition. These data evidence a new specific involvement of Arg kinase into the regulation of TGF-ß1 expression in tubular cells under high-glucose conditions and provide cues for new translational approaches in diabetic nephropathy.
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Glucosa/farmacología , Túbulos Renales/citología , Proteínas Tirosina Quinasas/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Adulto , Animales , Biomarcadores/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Silenciador del Gen/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Mesilato de Imatinib/farmacología , Ratones , Células 3T3 NIH , Fenotipo , Fosfotirosina/metabolismo , Inhibidores de Proteasoma/farmacología , Proteolisis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Fibras de Estrés/efectos de los fármacos , Fibras de Estrés/metabolismo , Ubiquitina/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Human clear cell renal cell carcinoma (ccRCC) is therapy resistant; therefore, it is worthwhile studying in depth the molecular aspects of its progression. In ccRCC the biallelic inactivation of the VHL gene leads to stabilization of hypoxia-inducible factors (HIFs). Among the targets of HIF-1α transcriptional activity is the LOX gene, which codes for the inactive proenzyme (Pro-Lox) from which, after extracellular secretion and proteolysis, derives the active enzyme (Lox) and the propeptide (Lox-PP). By increasing stiffness of extracellular matrix by collagen crosslinking, Lox promotes tumor progression and metastasis. Lox and Lox-PP can reenter the cells where Lox promotes cell proliferation and invasion, whereas Lox-PP acts as tumor suppressor because of its Ras recision and apoptotic activity. Few data are available concerning LOX in ccRCC. Using an in vitro model of ccRCC primary cell cultures, we performed, for the first time in ccRCC, a detailed study of endogenous LOX and also investigated their transcriptomic profile. We found that endogenous LOX is overexpressed in ccRCC, is involved in a positive-regulative loop with HIF-1α, and has a major action on ccRCC progression through cellular adhesion, migration, and collagen matrix stiffness increment; however, the oncosuppressive action of Lox-PP was not found to prevail. These findings may suggest translational approaches for new therapeutic strategies in ccRCC.
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Carcinoma de Células Renales/patología , Colágeno/metabolismo , Neoplasias Renales/patología , Proteína-Lisina 6-Oxidasa/metabolismo , Anciano , Anciano de 80 o más Años , Western Blotting , Carcinoma de Células Renales/enzimología , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Progresión de la Enfermedad , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Neoplasias Renales/enzimología , Masculino , Microscopía de Fuerza Atómica , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Cultivo Primario de Células , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Células Tumorales CultivadasRESUMEN
The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness.
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Movimiento Celular/genética , Núcleo Celular/enzimología , Citoesqueleto/enzimología , Citosol/enzimología , Proteínas Tirosina Quinasas/fisiología , Animales , Células COS , Forma de la Célula/genética , Chlorocebus aethiops , Citoesqueleto/genética , Adhesiones Focales/genética , Adhesiones Focales/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/fisiología , Transporte de Proteínas/genética , Proteínas Tirosina Quinasas/genética , Transfección , Células Tumorales CultivadasRESUMEN
Frailty is an age-related syndrome that drives multiple physiological system impairments in some older adults, and its pathophysiological mechanisms remain unclear. We evaluated whether frailty-related biological processes could impair stem cell compartments, specifically the renal stem compartment, given that kidney dysfunctions are frequent in frailty. A well-characterized in vitro nephrosphere model of human adult renal stem/progenitor cells has been instrumental to and was appropriate for verifying this hypothesis in our current research. Evaluating the effects of plasma from older individuals with frailty (frail plasma) on allogeneic renal stem/progenitor cells, we showed significant functional impairment and nuclear DNA damage in the treated cells of the renal stem compartment. The analysis of the frail plasma revealed mitochondrial functional impairment associated with the activation of oxidative stress and a unique inflammatory mediator profile in frail individuals. In addition, the plasma of frail subjects also contained the highest percentage of DNA-damaged autologous circulating hematopoietic progenitor/stem cells. The integration of both molecular and functional data obtained allowed us to discern patterns associated with frailty status, irrespective of the comorbidities present in the frail individuals. The data obtained converged toward biological conditions that in frailty caused renal and hematopoietic impairment of stem cells, highlighting the possibility of concomitant exhaustion of several stem compartments.
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Clear cell Renal Cell Carcinoma (ccRCC) is the most common and metastatic urological cancer. Molecular players of ccRCC progression and metastasis are not completely known. Here, using primary cell cultures from patients' specimens, we found that TGFß1/Smad signalling is more activated in high versus low grade ccRCC and inversely correlates with Abl2 tyrosine kinase protein expression. TGFß1 treatment increased ubiquitination and degradation of Abl2 protein in ccRCC cell lines by TGFß1/Smad pathway activation and reactive oxygen species production. 3D invasion and matrix degradation assays showed that Abl2 promoted TGFß1-induced ccRCC cell invasion and maturation of invadopodia, a hallmark of tumour invasion and metastasis. Our findings define Abl2 as a new downstream molecule of TGFß1 signalling and putative target to counteract advanced ccRCC.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/patología , Neoplasias Renales/patología , Transducción de Señal , Proteínas Tirosina Quinasas/metabolismo , Línea Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Proliferación CelularRESUMEN
Tubulointerstitial fibrosis is observed in diabetic nephropathy. It is still debated whether tubular cells, undergoing epithelial-mesenchymal transition (EMT) in high glucose (HG) conditions, may contribute to interstitial fibrosis development. In this study, we investigated the phenotypic and molecular EMT-like changes and the alteration of inflammatory and fibrogenic secretome induced by HG in human primary tubular cell cultures. Taking advantage of this in vitro cell model composed of proximal and distal tubular cells, we showed that HG-treated tubular cells acquired a fibroblast-like morphology with increased cytoplasmic stress fibers, maintaining the expression of the epithelial markers specific of proximal and distal tubular cells. HG increased Snail1, miRNA210 and Vimentin mesenchymal markers, decreased N-cadherin expression and migration ability of primary tubular cells, while E-cadherin expression and focal adhesion distribution were not affected. Furthermore, HG treatment of tubular cells altered the inflammatory cytokine secretion creating a secretome able to enhance the proliferation and migration of fibroblasts. Our findings show that HG promotes an activated state of partial EMT in human tubular primary cells and induces a pro-inflammatory and pro-fibrogenic microenvironment, supporting the active role of tubular cells in diabetic nephropathy onset.
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Nefropatías Diabéticas , Humanos , Nefropatías Diabéticas/metabolismo , Transición Epitelial-Mesenquimal , Células Epiteliales/metabolismo , Glucosa/metabolismo , Fibrosis , Técnicas de Cultivo de CélulaRESUMEN
Frailty is an age-related syndrome that exposes individuals to increased vulnerability. Although it is potentially reversible, in most cases it leads to negative outcomes, including mortality. The different methods proposed identify frailty after the onset of clinical manifestations. An early diagnosis might make it possible to manage the frailty progression better. The frailty pathophysiology is still unclear although mechanisms, in particular, those linked to inflammation and immunosenescence, have been investigated. A common feature of several clinical aspects involved in senescent organisms is the increase of oxidative stress, described as one of the major causes of deoxyribonucleic acid (DNA) damage accumulation in aged cells including the adult stem cell compartment. Likely, this accumulation is implicated in frailty status. The oxidative status of our frail, pre-frail, and non-frail population was characterized. In addition, the DNA damage in hematopoietic cells was evidenced by analyzing the peripheral blood mononuclear cell and their T lymphocyte, monocyte, circulating hematopoietic progenitor stem cell (cHPSC) subpopulations. The phosphorylation of C-terminal of histone H2AX at amino acid Ser 139 (γ-H2AX), which occurs at the DNA double-strand break focus, was evaluated. In our frail population, increased oxidative stress and a high level of DNA damage in cHPSC were found. This study may have potential implications because the increment of DNA damage in cHPSC could be suggestive of an organism impairment preceding the evident frailty. In addition, it may open the possibility for attenuation of frailty progression throughout specific drugs acting on preventing DNA damage or removing damaged cells.
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Fragilidad , Anciano , Biomarcadores , ADN , Daño del ADN , Anciano Frágil , Fragilidad/epidemiología , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucocitos Mononucleares/metabolismoRESUMEN
Primary cell cultures from renal cell carcinoma (RCC) and normal renal cortex tissue of 60 patients have been established, with high efficiency (more than 70%) and reproducibility, and extensively characterized. These cultures composed of more than 90% of normal or tumor tubular cells have been instrumental for molecular characterization of Annexin A3 (AnxA3), never extensively studied before in RCC cells although AnxA3 has a prognostic relevance in some cancer and it has been suggested to be involved in the hypoxia-inducible factor-1 pathway. Western blot analysis of 20 matched cortex/RCC culture lysates showed two AnxA3 protein bands of 36 and 33 kDa, and two-dimensional Western blot evidenced several specific protein spots. In RCC cultures the 36-kDa isoform was significantly down-regulated and the 33-kDa isoform up-regulated. Furthermore, the inversion of the quantitative expression pattern of two AnxA3 isoforms in tumor cultures correlate with hypoxia-inducible factor-1alpha expression. The total AnxA3 protein is down-regulated in RCC cultures as confirmed also in tissues by tissue microarray. Two AnxA3 transcripts that differ for alternative splicing of exon III have been also detected. Real-time PCR quantification in 19 matched cortex/RCC cultures confirms the down-regulation of longer isoform in RCC cells. The characteristic expression pattern of AnxA3 in normal and tumor renal cells, documented in our primary cultures, may open new insight in RCC management.
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Anexina A3/biosíntesis , Carcinoma de Células Renales/metabolismo , Regulación Neoplásica de la Expresión Génica , Corteza Renal/patología , Neoplasias Renales/metabolismo , Isoformas de Proteínas , Adulto , Anciano , Anciano de 80 o más Años , Regulación hacia Abajo , Femenino , Humanos , Hipoxia , Corteza Renal/metabolismo , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. METHODS: We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). RESULTS: A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. CONCLUSIONS: ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches aimed to study genes or pathways involved in ccRCC etiopathogenesis and to identify novel clinical markers or therapeutic targets. Moreover, SNP array technology proved to be a powerful tool to better define the cell composition and homogeneity of RCC primary cultures.
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Carcinoma de Células Renales/patología , Línea Celular Tumoral/citología , Neoplasias Renales/patología , Carcinoma de Células Renales/química , Carcinoma de Células Renales/genética , Línea Celular Tumoral/química , Línea Celular Tumoral/ultraestructura , Forma de la Célula , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Dosificación de Gen , Perfilación de la Expresión Génica , Inestabilidad Genómica , Genotipo , Humanos , Inmunofenotipificación , Neoplasias Renales/química , Neoplasias Renales/genética , Pérdida de Heterocigocidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Polimorfismo de Nucleótido Simple , Eliminación de SecuenciaRESUMEN
The mechanism upon which human kidneys undergo regeneration is debated, though different lineage-tracing mouse models have tried to explain the cellular types and the mechanisms involved. Different sources of human renal progenitors have been proposed, but it is difficult to argue whether these populations have the same capacities that have been described in mice. Using the nephrosphere (NS) model, we isolated the quiescent population of adult human renal stem-like PKHhigh/CD133+/CD24- cells (RSC). The aim of this study was to deepen the RSC in vitro multipotency capacity. RSC, not expressing endothelial markers, generated secondary nephrospheres containing CD31+/vWf+ cells and cytokeratin positive cells, indicating the coexistence of endothelial and epithelial commitment. RSC cultured on decellularized human renal scaffolds generated endothelial structures together with the proximal and distal tubular structures. CD31+ endothelial committed progenitors sorted from nephrospheres generated spheroids with endothelial-like sprouts in Matrigel. We also demonstrated the double commitment toward endothelial and epithelial lineages of single RSC. The ability of the plastic RSC population to recapitulate the development of tubular epithelial and endothelial renal lineages makes these cells a good tool for the creation of organoids with translational relevance for studying the parenchymal and endothelial cell interactions and developing new therapeutic strategies.
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Antígeno AC133/metabolismo , Antígeno CD24/metabolismo , Colorantes Fluorescentes/metabolismo , Riñón/citología , Células Madre Multipotentes/metabolismo , Compuestos Orgánicos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Materiales Biocompatibles/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Colágeno/metabolismo , Combinación de Medicamentos , Femenino , Humanos , Laminina/metabolismo , Masculino , Persona de Mediana Edad , Proteoglicanos/metabolismoRESUMEN
Clear cell renal cell carcinoma (ccRCC) is fundamentally a metabolic disease. Given the importance of lipids in many cellular processes, in this study we delineated a lipidomic profile of human ccRCC and integrated it with transcriptomic data to connect the variations in cancer lipid metabolism with gene expression changes. Untargeted lipidomic analysis was performed on 20 ccRCC and 20 paired normal tissues, using LC-MS and GC-MS. Different lipid classes were altered in cancer compared to normal tissue. Among the long chain fatty acids (LCFAs), significant accumulations of polyunsaturated fatty acids (PUFAs) were found. Integrated lipidomic and transcriptomic analysis showed that fatty acid desaturation and elongation pathways were enriched in neoplastic tissue. Consistent with these findings, we observed increased expression of stearoyl-CoA desaturase(SCD1) and FA elongase 2 and 5 in ccRCC. Primary renal cancer cells treated with a small molecule SCD1 inhibitor (A939572) proliferated at a slower rate than untreated cancer cells. In addition, after cisplatin treatment, the death rate of tumor cells treated with A939572 was significantly greater than that of untreated cancer cells. In conclusion, our findings delineate a ccRCC lipidomic signature and showed that SCD1 inhibition significantly reduced cancer cell proliferation and increased cisplatin sensitivity, suggesting that this pathway can be involved in ccRCC chemotherapy resistance.
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Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1-negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in SETBP1, ASXL1 and ETNK1 genes, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. To provide a comprehensive genomic characterization of aCML we applied a high-throughput sequencing strategy to 43 aCML samples, including both whole-exome and RNA-sequencing data. Our dataset identifies ASXL1, SETBP1, and ETNK1 as the most frequently mutated genes with a total of 43.2%, 29.7 and 16.2%, respectively. We characterized the clonal architecture of 7 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur early in the clonal evolution history of aCML, while SETBP1 mutations often represent a late event. The presence of actionable mutations conferred both ex vivo and in vivo sensitivity to specific inhibitors with evidence of strong in vitro synergism in case of multiple targeting. In one patient, a clinical response was obtained. Stratification based on RNA-sequencing identified two different populations in terms of overall survival, and differential gene expression analysis identified 38 significantly overexpressed genes in the worse outcome group. Three genes correctly classified patients for overall survival.
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The fibrotic tissue and the stroma adjacent to cancer cells are characterised by the presence of activated fibroblasts (myofibroblasts) which play a role in creating a supportive tissue characterised by abundant extracellular matrix (ECM) secretion. The myofibroblasts remodel this tissue through secreted molecules and modulation of their cytoskeleton and specialized contractile structures. The non-receptor protein tyrosine kinase Arg (also called Abl2) has the unique ability to bind directly to the actin cytoskeleton, transducing diverse extracellular signals into cytoskeletal rearrangements. In this study we analysed the 1ALCTL and 1BLCTL Arg isoforms in Arg-/- murine embryonal fibroblasts (MEF) cell line, focusing on their capacity to activate fibroblasts and to remodel ECM. The results obtained showed that Arg isoform 1BLCTL has a major role in proliferation, migration/invasion of MEF and in inducing a milieu able to modulate tumour cell morphology, while 1ALCTL isoform has a role in MEF adhesion maintaining active focal adhesions. On the whole, the presence of Arg in MEF supports the proliferation, activation, adhesion, ECM contraction and stiffness, while the absence of Arg affected these myofibroblast features.This article has an associated First Person interview with the first author of the paper.
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The human Arg (Abl2) nonreceptor tyrosine kinase has a role in cytoskeletal rearrangements by its C-terminal F-actin- and microtubule-binding sequences. We have previously identified Arg transcripts with different 5'- and 3'-ends, named respectively long and short 1A and 1B (1AL, 1AS, 1BL, 1BS) and long and short C-termini (CTL and CTS), that have different expression patterns in various cell types. The combination of the different ends permits to predict eight putative full-length Arg transcripts and corresponding proteins. By Reverse Transcription-Long PCR we show here that all eight full-length transcripts are endogenously expressed in Caki-1 cells and the two bands, approximately 10 kDa different, shown by 1-D Western blots of Hek293T and Caki-1 lysates correspond to the full-length Arg protein isoforms with different C-termini. 2-D Western blot analysis evidenced different high molecular weight and slight acidic specific spots in Hek293T and Caki-1 lysates. The cellular localization of two Arg isoforms (1BLCTL and 1BLCTS) transfected in Caki-1 and Hek293T cells was cytoplasmic, and some differences in cytoskeleton interactions have been evidenced. Moreover, in Hek293T cells only the transfected 1BLCTS isoform gives rise to a large intracytoplasmic cylindrical structure containing phalloidin-positive amorphous actin aggregates. The presence of eight full-length Arg isoforms with different cellular expression may imply a diverse functional role in normal and neoplastic cells.
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Proteínas Tirosina Quinasas/genética , Línea Celular Tumoral , Células Cultivadas , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopía Fluorescente , Proteínas Tirosina Quinasas/metabolismo , TransfecciónRESUMEN
We verified the feasibility of plasma bound method for detecting renal cell carcinoma (RCC) combining the study of plasma DNA concentration and microsatellite alterations (LOH). Plasma DNA concentration was evaluated with real-time PCR in 54 patients with renal neoplasm before surgery and in 20 of these patients during a 26-64 month follow-up. Microsatellite study was performed on tumour tissue DNA of 33 RCC clear cell (RCCcc) and on plasma DNA of 14 RCCcc patients during preoperative and/or follow-up period. Patients had a significantly high (26.4+/-48.3 ng/ml versus controls 3.2+/-1.5 ng/ml; p=0.003) preoperative plasma DNA concentration that decreased after nephrectomy. During follow-up, plasma DNA increased in 12 patients without evidence of neoplasia; 3 patients successively relapsed. Tumour tissue DNA of 25 RCCcc patients (75.8%) displayed microsatellite LOH. Preoperative plasma DNA of 9 patients harboured LOH in 5 cases (55.6%). Augmented plasma DNA of 7 patients displayed LOH in 3 cases (42.9%) at follow-up, and in 1 case preceded the recurrence of disease. Plasma DNA concentration combined with microsatellite LOH in plasma DNA may predict disease recurrence in RCC patients.