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1.
Genes Dev ; 35(15-16): 1079-1092, 2021 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-34266888

RESUMEN

Chromosome gains and losses are a frequent feature of human cancers. However, how these aberrations can outweigh the detrimental effects of aneuploidy remains unclear. An initial comparison of existing chromosomal instability (CIN) mouse models suggests that aneuploidy accumulates to low levels in these animals. We therefore developed a novel mouse model that enables unprecedented levels of chromosome missegregation in the adult animal. At the earliest stages of T-cell development, cells with random chromosome gains and/or losses are selected against, but CIN eventually results in the expansion of progenitors with clonal chromosomal imbalances. Clonal selection leads to the development of T-cell lymphomas with stereotypic karyotypes in which chromosome 15, containing the Myc oncogene, is gained with high prevalence. Expressing human MYC from chromosome 6 (MYCChr6) is sufficient to change the karyotype of these lymphomas to include universal chromosome 6 gains. Interestingly, while chromosome 15 is still gained in MYCChr6 tumors after genetic ablation of the endogenous Myc locus, this chromosome is not efficiently gained after deletion of one copy of Rad21, suggesting a synergistic effect of both MYC and RAD21 in driving chromosome 15 gains. Our results show that the initial detrimental effects of random missegregation are outbalanced by clonal selection, which is dictated by the chromosomal location and nature of certain genes and is sufficient to drive cancer with high prevalence.


Asunto(s)
Aneuploidia , Inestabilidad Cromosómica , Animales , Transformación Celular Neoplásica/genética , Inestabilidad Cromosómica/genética , Aberraciones Cromosómicas , Cariotipo , Ratones , Prevalencia , Células Madre
2.
Immunity ; 48(2): 227-242.e8, 2018 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-29466755

RESUMEN

How chromatin reorganization coordinates differentiation and lineage commitment from hematopoietic stem and progenitor cells (HSPCs) to mature immune cells has not been well understood. Here, we carried out an integrative analysis of chromatin accessibility, topologically associating domains, AB compartments, and gene expression from HSPCs to CD4+CD8+ T cells. We found that abrupt genome-wide changes at all three levels of chromatin organization occur during the transition from double-negative stage 2 (DN2) to DN3, accompanying the T lineage commitment. The transcription factor BCL11B, a critical regulator of T cell commitment, is associated with increased chromatin interaction, and Bcl11b deletion compromised chromatin interaction at its target genes. We propose that these large-scale and concerted changes in chromatin organization present an energy barrier to prevent the cell from reversing its fate to earlier stages or redirecting to alternatives and thus lock the cell fate into the T lineages.


Asunto(s)
Linaje de la Célula , Núcleo Celular/fisiología , Cromatina/fisiología , Linfocitos T/fisiología , Animales , Diferenciación Celular , Humanos , Proteínas Represoras/fisiología , Proteínas Supresoras de Tumor/fisiología
3.
Mol Cell ; 71(1): 129-141.e8, 2018 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-29979962

RESUMEN

The enhancer regions of the myogenic master regulator MyoD give rise to at least two enhancer RNAs. Core enhancer eRNA (CEeRNA) regulates transcription of the adjacent MyoD gene, whereas DRReRNA affects expression of Myogenin in trans. We found that DRReRNA is recruited at the Myogenin locus, where it colocalizes with Myogenin nascent transcripts. DRReRNA associates with the cohesin complex, and this association correlates with its transactivating properties. Despite being expressed in undifferentiated cells, cohesin is not loaded on Myogenin until the cells start expressing DRReRNA, which is then required for cohesin chromatin recruitment and maintenance. Functionally, depletion of either cohesin or DRReRNA reduces chromatin accessibility, prevents Myogenin activation, and hinders muscle cell differentiation. Thus, DRReRNA ensures spatially appropriate cohesin loading in trans to regulate gene expression.


Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Proteínas Cromosómicas no Histona/biosíntesis , Elementos de Facilitación Genéticos , Músculo Esquelético/metabolismo , Miogenina/biosíntesis , ARN no Traducido/metabolismo , Transcripción Genética , Animales , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Células HEK293 , Humanos , Ratones , Músculo Esquelético/citología , Proteína MioD/biosíntesis , Proteína MioD/genética , Miogenina/genética , ARN no Traducido/genética , Cohesinas
4.
Genes Chromosomes Cancer ; 60(6): 434-446, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33527590

RESUMEN

Renal cell carcinoma (RCC) is not a single disease but is made up of several different histologically defined subtypes that are associated with distinct genetic alterations which require subtype specific management and treatment. Papillary renal cell carcinoma (pRCC) is the second most common subtype after conventional/clear cell RCC (ccRCC), representing ~20% of cases, and is subcategorized into type 1 and type 2 pRCC. It is important for preclinical studies to have cell lines that accurately represent each specific RCC subtype. This study characterizes seven cell lines derived from both primary and metastatic sites of type 1 pRCC, including the first cell line derived from a hereditary papillary renal carcinoma (HPRC)-associated tumor. Complete or partial gain of chromosome 7 was observed in all cell lines and other common gains of chromosomes 16, 17, or 20 were seen in several cell lines. Activating mutations of MET were present in three cell lines that all demonstrated increased MET phosphorylation in response to HGF and abrogation of MET phosphorylation in response to MET inhibitors. CDKN2A loss due to mutation or gene deletion, associated with poor outcomes in type 1 pRCC patients, was observed in all cell line models. Six cell lines formed tumor xenografts in athymic nude mice and thus provide in vivo models of type 1 pRCC. These type 1 pRCC cell lines provide a comprehensive representation of the genetic alterations associated with pRCC that will give insight into the biology of this disease and be ideal preclinical models for therapeutic studies.


Asunto(s)
Carcinoma de Células Renales/genética , Autenticación de Línea Celular/métodos , Neoplasias Renales/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Inestabilidad Cromosómica , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Humanos , Neoplasias Renales/patología , Ratones , Mutación , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo
5.
Nat Methods ; 15(9): 741-747, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30150754

RESUMEN

Long-range chromatin interactions play critical roles in genome organization and regulation of transcription. We now report transposase-mediated analysis of chromatin looping (Trac-looping) for simultaneous detection of multiscale genome-wide chromatin interactions among regulatory elements and chromatin accessibility. With this technique, a bivalent oligonucleotide linker is inserted between two interacting regions such that the chromatin interactions are captured without prior chromatin fragmentation and proximity-based ligation. Application of Trac-looping to human CD4+ T cells revealed substantial reorganization of enhancer-promoter interactions associated with changes in gene expression after T cell receptor stimulation.


Asunto(s)
Cromatina/metabolismo , Genoma Humano , Cromatina/química , Biología Computacional , Elementos de Facilitación Genéticos , Humanos , Regiones Promotoras Genéticas , Transcripción Genética
6.
Genes Chromosomes Cancer ; 59(8): 472-483, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32259323

RESUMEN

Renal medullary carcinoma (RMC) is a rare, aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses with a median overall survival of 13 months. Patients are typically young (median age-22) and male (male:female ratio of 2:1) and tumors are characterized by complete loss of expression of the SMARCB1 tumor suppressor protein. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to better understand the pathogenesis of this disease and to identify effective forms of therapy. Two novel cell line models, UOK353 and UOK360, were derived from primary RMCs that both demonstrated the characteristic SMARCB1 loss. Both cell lines overexpressed EZH2 and other members of the polycomb repressive complex and EZH2 inhibition in RMC tumor spheroids resulted in decreased viability. High throughput drug screening of both cell lines revealed several additional candidate compounds, including bortezomib that had both in vitro and in vivo antitumor activity. The activity of bortezomib was shown to be partially dependent on increased oxidative stress as addition of the N-acetyl cysteine antioxidant reduced the effect on cell proliferation. Combining bortezomib and cisplatin further decreased cell viability both in vitro and in vivo that single agent bortezomib treatment. The UOK353 and UOK360 cell lines represent novel preclinical models for the development of effective forms of therapy for RMC patients.


Asunto(s)
Carcinoma Medular/patología , Neoplasias Renales/patología , Cultivo Primario de Células/métodos , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Bortezomib/farmacología , Bortezomib/uso terapéutico , Carcinoma Medular/tratamiento farmacológico , Carcinoma Medular/genética , Autenticación de Línea Celular/métodos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Cisplatino/uso terapéutico , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Ratones , Ratones Desnudos , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Células Tumorales Cultivadas
7.
Int J Cancer ; 144(7): 1561-1573, 2019 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-30229897

RESUMEN

Colorectal adenomas are common precancerous lesions with the potential for malignant transformation to colorectal adenocarcinoma. Endoscopic polypectomy provides an opportunity for cancer prevention; however, recurrence rates are high. We collected formalin-fixed paraffin-embedded tissue of 15 primary adenomas with recurrence, 15 adenomas without recurrence, and 14 matched pair samples (primary adenoma and the corresponding recurrent adenoma). The samples were analysed by array-comparative genomic hybridisation (aCGH) and single-cell multiplex interphase fluorescence in situ hybridisation (miFISH) to understand clonal evolution, to examine the dynamics of copy number alterations (CNAs) and to identify molecular markers for recurrence prediction. The miFISH probe panel consisted of 14 colorectal carcinogenesis-relevant genes (COX2, PIK3CA, APC, CLIC1, EGFR, MYC, CCND1, CDX2, CDH1, TP53, HER2, SMAD7, SMAD4 and ZNF217), and a centromere probe (CEP10). The aCGH analysis confirmed the genetic landscape typical for colorectal tumorigenesis, that is, CNAs of chromosomes 7, 13q, 18 and 20q. Focal aberrations (≤10 Mbp) were mapped to chromosome bands 6p22.1-p21.33 (33.3%), 7q22.1 (31.4%) and 16q21 (29.4%). MiFISH detected gains of EGFR (23.6%), CDX2 (21.8%) and ZNF217 (18.2%). Most adenomas exhibited a major clone population which was accompanied by multiple smaller clone populations. Gains of CDX2 were exclusively seen in primary adenomas with recurrence (25%) compared to primary adenomas without recurrence (0%). Generation of phylogenetic trees for matched pair samples revealed four distinct patterns of clonal dynamics. In conclusion, adenoma development and recurrence are complex genetic processes driven by multiple CNAs whose evaluations by miFISH, with emphasis on CDX2, might serve as a predictor of recurrence.


Asunto(s)
Adenoma/genética , Factor de Transcripción CDX2/genética , Neoplasias Colorrectales/genética , Recurrencia Local de Neoplasia/genética , Análisis de la Célula Individual/métodos , Anciano , Biomarcadores de Tumor/genética , Aberraciones Cromosómicas , Evolución Clonal , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad
8.
FASEB J ; 32(7): 3502-3517, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29452566

RESUMEN

A considerable proportion of tumors exhibit aneuploid karyotypes, likely resulting from the progressive loss of chromosomes after whole-genome duplication. Here, by using isogenic diploid and near-tetraploid (4N) single-cell-derived clones from the same parental cell lines, we aimed at exploring how polyploidization affects cellular functions and how tetraploidy generates chromosome instability. Gene expression profiling in 4N clones revealed a significant enrichment of transcripts involved in cell cycle and DNA replication. Increased levels of replication stress in 4N cells resulted in DNA damage, impaired proliferation caused by a cell cycle delay during S phase, and higher sensitivity to S phase checkpoint inhibitors. In fact, increased levels of replication stress were also observed in nontransformed, proliferative posttetraploid RPE1 cells. Additionally, replication stress promoted higher levels of intercellular genomic heterogeneity and ongoing genomic instability, which could be explained by high rates of mitotic defects, and was alleviated by the supplementation of exogenous nucleosides. Finally, our data found that 4N cancer cells displayed increased migratory and invasive capacity, both in vitro and in primary colorectal tumors, indicating that tetraploidy can promote aggressive cancer cell behavior.-Wangsa, D., Quintanilla, I., Torabi, K., Vila-Casadesús, M., Ercilla, A., Klus, G., Yuce, Z., Galofré, C., Cuatrecasas, M., Lozano, J. J., Agell, N., Cimini, D., Castells, A., Ried, T., Camps, J. Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness.


Asunto(s)
Movimiento Celular , Inestabilidad Cromosómica , Daño del ADN , Neoplasias/genética , Tetraploidía , Línea Celular Tumoral , Replicación del ADN , Humanos , Fase S
9.
Methods ; 142: 30-38, 2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29408376

RESUMEN

The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.


Asunto(s)
Mapeo Cromosómico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Hibridación Fluorescente in Situ/métodos , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Mapeo Cromosómico/instrumentación , Cromosomas Humanos Par 18/genética , Cromosomas Humanos Par 18/metabolismo , Cromosomas Humanos X/genética , Cromosomas Humanos X/metabolismo , Cromosomas Humanos Y/genética , Cromosomas Humanos Y/metabolismo , Femenino , Fibroblastos , Humanos , Procesamiento de Imagen Asistido por Computador/instrumentación , Hibridación Fluorescente in Situ/instrumentación , Masculino , Cultivo Primario de Células/métodos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Piel/citología , Coloración y Etiquetado/instrumentación , Coloración y Etiquetado/métodos
10.
Carcinogenesis ; 39(8): 993-1005, 2018 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-29800151

RESUMEN

Intratumor heterogeneity is a major challenge in cancer treatment. To decipher patterns of chromosomal heterogeneity, we analyzed six colorectal cancer cell lines by multiplex interphase FISH (miFISH). The mismatch-repair-deficient cell lines DLD-1 and HCT116 had the most stable copy numbers, whereas aneuploid cell lines (HT-29, SW480, SW620 and H508) displayed a higher degree of instability. We subsequently assessed the clonal evolution of single cells in two colorectal carcinoma cell lines, SW480 and HT-29, which both have aneuploid karyotypes but different degrees of chromosomal instability. The clonal compositions of the single cell-derived daughter lines, as assessed by miFISH, differed for HT-29 and SW480. Daughters of HT-29 were stable, clonal, with little heterogeneity. Daughters of SW480 were more heterogeneous, with the single cell-derived daughter lines separating into two distinct populations with different ploidy (hyper-diploid and near-triploid), morphology, gene expression and tumorigenicity. To better understand the evolutionary trajectory for the two SW480 populations, we constructed phylogenetic trees which showed ongoing instability in the daughter lines. When analyzing the evolutionary development over time, most single cell-derived daughter lines maintained their major clonal pattern, with the exception of one daughter line that showed a switch involving a loss of APC. Our meticulous analysis of the clonal evolution and composition of these colorectal cancer models shows that all chromosomes are subject to segregation errors, however, specific net genomic imbalances are maintained. Karyotype evolution is driven by the necessity to arrive at and maintain a specific plateau of chromosomal copy numbers as the drivers of carcinogenesis.


Asunto(s)
Carcinogénesis/genética , Neoplasias Colorrectales/genética , Evolución Molecular , Línea Celular Tumoral , Inestabilidad Cromosómica , Aberraciones Cromosómicas , Evolución Clonal , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Cariotipo , Filogenia
11.
Chromosoma ; 126(5): 655-667, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28343235

RESUMEN

Human chromosomes occupy distinct territories in the interphase nucleus. Such chromosome territories (CTs) are positioned according to gene density. Gene-rich CTs are generally located in the center of the nucleus, while gene-poor CTs are positioned more towards the nuclear periphery. However, the association between gene expression levels and the radial positioning of genes within the CT is still under debate. In the present study, we performed three-dimensional fluorescence in situ hybridization experiments in the colorectal cancer cell lines DLD-1 and LoVo using whole chromosome painting probes for chromosomes 8 and 11 and BAC clones targeting four genes with different expression levels assessed by gene expression arrays and RT-PCR. Our results confirmed that the two over-expressed genes, MYC on chromosome 8 and CCND1 on chromosome 11, are located significantly further away from the center of the CT compared to under-expressed genes on the same chromosomes, i.e., DLC1 and SCN3B. When CCND1 expression was reduced after silencing the major transcription factor of the WNT/ß-catenin signaling pathway, TCF7L2, the gene was repositioned and mostly detected in the interior of the CT. Thus, we suggest a non-random distribution in which over-expressed genes are located more towards the periphery of the respective CTs.


Asunto(s)
Núcleo Celular/metabolismo , Cromosomas Humanos/metabolismo , Interfase , Transducción de Señal , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Línea Celular Tumoral , Núcleo Celular/genética , Cromosomas Humanos/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ
12.
Int J Cancer ; 138(1): 98-109, 2016 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-26175310

RESUMEN

Oral tongue squamous cell carcinoma (OTSCC) is associated with poor prognosis. To improve prognostication, we analyzed four gene probes (TERC, CCND1, EGFR and TP53) and the centromere probe CEP4 as a marker of chromosomal instability, using fluorescence in situ hybridization (FISH) in single cells from the tumors of sixty-five OTSCC patients (Stage I, n = 15; Stage II, n = 30; Stage III, n = 7; Stage IV, n = 13). Unsupervised hierarchical clustering of the FISH data distinguished three clusters related to smoking status. Copy number increases of all five markers were found to be correlated to non-smoking habits, while smokers in this cohort had low-level copy number gains. Using the phylogenetic modeling software FISHtrees, we constructed models of tumor progression for each patient based on the four gene probes. Then, we derived test statistics on the models that are significant predictors of disease-free and overall survival, independent of tumor stage and smoking status in multivariate analysis. The patients whose tumors were modeled as progressing by a more diverse distribution of copy number changes across the four genes have poorer prognosis. This is consistent with the view that multiple genetic pathways need to become deregulated in order for cancer to progress.


Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidad , Variaciones en el Número de Copia de ADN , Filogenia , Neoplasias de la Lengua/genética , Neoplasias de la Lengua/mortalidad , Adulto , Anciano , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Femenino , Papillomavirus Humano 16 , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Infecciones por Papillomavirus , Pronóstico , Factores de Riesgo , Análisis de Supervivencia , Neoplasias de la Lengua/patología , Neoplasias de la Lengua/virología , Adulto Joven
13.
Bioinformatics ; 31(12): i258-67, 2015 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-26072490

RESUMEN

MOTIVATION: Phylogenetic algorithms have begun to see widespread use in cancer research to reconstruct processes of evolution in tumor progression. Developing reliable phylogenies for tumor data requires quantitative models of cancer evolution that include the unusual genetic mechanisms by which tumors evolve, such as chromosome abnormalities, and allow for heterogeneity between tumor types and individual patients. Previous work on inferring phylogenies of single tumors by copy number evolution assumed models of uniform rates of genomic gain and loss across different genomic sites and scales, a substantial oversimplification necessitated by a lack of algorithms and quantitative parameters for fitting to more realistic tumor evolution models. RESULTS: We propose a framework for inferring models of tumor progression from single-cell gene copy number data, including variable rates for different gain and loss events. We propose a new algorithm for identification of most parsimonious combinations of single gene and single chromosome events. We extend it via dynamic programming to include genome duplications. We implement an expectation maximization (EM)-like method to estimate mutation-specific and tumor-specific event rates concurrently with tree reconstruction. Application of our algorithms to real cervical cancer data identifies key genomic events in disease progression consistent with prior literature. Classification experiments on cervical and tongue cancer datasets lead to improved prediction accuracy for the metastasis of primary cervical cancers and for tongue cancer survival. AVAILABILITY AND IMPLEMENTATION: Our software (FISHtrees) and two datasets are available at ftp://ftp.ncbi.nlm.nih.gov/pub/FISHtrees.


Asunto(s)
Evolución Molecular , Dosificación de Gen , Modelos Genéticos , Neoplasias/genética , Algoritmos , Progresión de la Enfermedad , Femenino , Genómica , Humanos , Filogenia , Programas Informáticos , Neoplasias del Cuello Uterino/genética
14.
PLoS Genet ; 9(2): e1003251, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23408900

RESUMEN

Deletion of tumor suppressor genes in stromal fibroblasts induces epithelial cancer development, suggesting an important role of stroma in epithelial homoeostasis. However, the underlying mechanisms remain to be elucidated. Here we report that deletion of the gene encoding TGFß receptor 2 (Tgfbr2) in the stromal fibroblasts (Tgfbr2(fspKO)) induces inflammation and significant DNA damage in the neighboring epithelia of the forestomach. This results in loss or down-regulation of cyclin-dependent kinase inhibitors p15, p16, and p21, which contribute to the development of invasive squamous cell carcinoma (SCC). Anti-inflammation treatment restored p21 expression, delayed tumorigenesis, and increased survival of Tgfbr2(fspKO) mice. Our data demonstrate for the first time that inflammation is a critical player in the epigenetic silencing of p21 in tumor progression. Examination of human esophageal SCC showed a down-regulation of TGFß receptor 2 (TßRII) in the stromal fibroblasts, as well as increased inflammation, DNA damage, and loss or decreased p15/p16 expression. Our study suggests anti-inflammation may be a new therapeutic option in treating human SCCs with down-regulation of TßRII in the stroma.


Asunto(s)
Neoplasias de la Mama , Carcinoma de Células Escamosas , Transformación Celular Neoplásica/genética , Neoplasias Esofágicas , Proteínas Serina-Treonina Quinasas , Receptores de Factores de Crecimiento Transformadores beta , Factor de Crecimiento Transformador beta , Animales , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo , Epigénesis Genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago , Femenino , Fibroblastos , Humanos , Inflamación/genética , Ratones , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Células del Estroma/citología , Células del Estroma/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo
15.
FASEB J ; 28(8): 3423-34, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24732130

RESUMEN

Nuclear lamin B1 (LMNB1) constitutes one of the major structural proteins in the lamina mesh. We silenced the expression of LMNB1 by RNA interference in the colon cancer cell line DLD-1 and showed a dramatic redistribution of H3K27me3 from the periphery to a more homogeneous nuclear dispersion. In addition, we observed telomere attrition and an increased frequency of micronuclei and nuclear blebs. By 3D-FISH analyses, we demonstrated that the volume and surface of chromosome territories were significantly larger in LMNB1-depleted cells, suggesting that LMNB1 is required to maintain chromatin condensation in interphase nuclei. These changes led to a prolonged S phase due to activation of Chk1. Finally, silencing of LMNB1 resulted in extensive changes in alternative splicing of multiple genes and in a higher number of enlarged nuclear speckles. Taken together, our results suggest a mechanistic role of the nuclear lamina in the organization of chromosome territories, maintenance of genome integrity and proper gene splicing.


Asunto(s)
Lamina Tipo B/fisiología , Fase S/fisiología , Adenocarcinoma/patología , Línea Celular Tumoral , Núcleo Celular/ultraestructura , Cromosomas Humanos Par 18/ultraestructura , Cromosomas Humanos Par 19/ultraestructura , Neoplasias del Colon/patología , Heterocromatina/fisiología , Histonas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Interfase , Lamina Tipo B/deficiencia , Metilación , Procesamiento Proteico-Postraduccional , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Fase S/efectos de los fármacos , Acortamiento del Telómero/efectos de los fármacos
16.
Nucleic Acids Res ; 41(8): 4535-48, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23470994

RESUMEN

During the CD4(-)CD8(-) (DN) stage of T-cell development, RAG-dependent DNA breaks and V(D)J recombination occur at three T-cell receptor (TCR) loci: TCRß, TCRγ and TCRδ. During this stage, abnormal trans-rearrangements also take place between TCR loci, occurring at increased frequency in absence of the DNA damage response mediator ataxia telangiectasia mutated (ATM). Here, we use this model of physiologic trans-rearrangement to study factors that predispose to rearrangement and the role of ATM in preventing chromosomal translocations. The frequency of DN thymocytes with DNA damage foci at multiple TCR loci simultaneously is increased 2- to 3-fold in the absence of ATM. However, trans-rearrangement is increased 10 000- to 100 000-fold, indicating that ATM function extends beyond timely resolution of DNA breaks. RAG-mediated synaptic complex formation occurs between recombination signal sequences with unequal 12 and 23 base spacer sequences (12/23 rule). TCR trans-rearrangements violate this rule, as we observed similar frequencies of 12/23 and aberrant 12/12 or 23/23 recombination products. This suggests that trans-rearrangements are not the result of trans-synaptic complex formation, but they are instead because of unstable cis synaptic complexes that form simultaneously at distinct TCR loci. Thus, ATM suppresses trans-rearrangement primarily through stabilization of DNA breaks at TCR loci.


Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas de Unión al ADN/fisiología , Reordenamiento Génico de Linfocito T , Proteínas Serina-Treonina Quinasas/fisiología , Timocitos/inmunología , Proteínas Supresoras de Tumor/fisiología , Recombinación V(D)J , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , ADN/metabolismo , Roturas del ADN , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Linfocitos T/inmunología , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética
17.
bioRxiv ; 2024 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-38496539

RESUMEN

Chromosomal aberrations are prevalent in cancer genomes, yet it remains challenging to resolve the long-range structure of rearranged chromosomes. A key problem is to determine the chromosomal origin of rearranged genomic segments, which requires chromosome-length haplotype information. Here we describe refLinker, a new computational method for whole-chromosome haplotype inference using external reference panels and Hi-C. We show that refLinker ensures consistent long-range phasing accuracy in both diploid human genomes and aneuploid cancers, including regions with loss-of-heterozygosity and high-level focal amplification. We further demonstrate the feasibility of complex genome reconstruction using haplotype-specific Hi-C contacts, revealing new karyotype features in two widely studied cancer cell lines. Together, these findings provide a new framework for the complete resolution of long-range chromosome structure in complex genomes and highlight the unique advantages of Hi-C data for reconstructing cancer genomes with chromosome-scale continuity.

18.
bioRxiv ; 2024 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-39416100

RESUMEN

Metastasis is the leading cause of cancer-related deaths, yet its regulatory mechanisms are not fully understood. Small-cell lung cancer (SCLC) is the most metastatic form of lung cancer, with most patients presenting with widespread disease, making it an ideal model for studying metastasis. However, the lack of suitable preclinical models has limited such studies. We utilized well-annotated rapid autopsy-derived tumors to develop xenograft models that mimic key features of SCLC, including histopathology, rapid and widespread development of metastasis to the liver, brain, adrenal, bone marrow, and kidneys within weeks, and response to chemotherapy. By integrating in vivo lineage selection with comprehensive transcriptomic and epigenomic analyses, we identified critical cellular programs driving metastatic organotropism to the liver and brain, the most common sites of SCLC metastasis. Our findings reveal the key role of nuclear-cytoskeletal interactions in SCLC liver metastasis. Specifically, the loss of the nuclear envelope protein lamin A/C, encoded by the LMNA gene, increased nuclear deformability and significantly increased the incidence of liver metastasis. Human liver metastases exhibited reduced LMNA expression compared to other metastatic sites, correlating with poorer patient outcomes and increased mortality. This study introduces novel preclinical models for SCLC metastasis and highlights pathways critical for organ-specific metastasis, offering new avenues for the development of targeted therapies to prevent or treat metastatic disease.

19.
Cancer Res ; 83(11): 1768-1781, 2023 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-36971511

RESUMEN

SIGNIFICANCE: Multimers of the HPV genome are generated in cervical tumors replicating as extrachromosomal episomes, which is associated with deletion and rearrangement of the HPV genome and provides a mechanism for oncogenesis without integration.


Asunto(s)
Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Femenino , Humanos , Virus del Papiloma Humano , Infecciones por Papillomavirus/complicaciones , Cuello del Útero , Neoplasias del Cuello Uterino/genética , Plásmidos , Transformación Celular Neoplásica , Papillomaviridae/genética
20.
Cancer Discov ; 13(4): 910-927, 2023 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-36715691

RESUMEN

The human papillomavirus (HPV) genome is integrated into host DNA in most HPV-positive cancers, but the consequences for chromosomal integrity are unknown. Continuous long-read sequencing of oropharyngeal cancers and cancer cell lines identified a previously undescribed form of structural variation, "heterocateny," characterized by diverse, interrelated, and repetitive patterns of concatemerized virus and host DNA segments within a cancer. Unique breakpoints shared across structural variants facilitated stepwise reconstruction of their evolution from a common molecular ancestor. This analysis revealed that virus and virus-host concatemers are unstable and, upon insertion into and excision from chromosomes, facilitate capture, amplification, and recombination of host DNA and chromosomal rearrangements. Evidence of heterocateny was detected in extrachromosomal and intrachromosomal DNA. These findings indicate that heterocateny is driven by the dynamic, aberrant replication and recombination of an oncogenic DNA virus, thereby extending known consequences of HPV integration to include promotion of intratumoral heterogeneity and clonal evolution. SIGNIFICANCE: Long-read sequencing of HPV-positive cancers revealed "heterocateny," a previously unreported form of genomic structural variation characterized by heterogeneous, interrelated, and repetitive genomic rearrangements within a tumor. Heterocateny is driven by unstable concatemerized HPV genomes, which facilitate capture, rearrangement, and amplification of host DNA, and promotes intratumoral heterogeneity and clonal evolution. See related commentary by McBride and White, p. 814. This article is highlighted in the In This Issue feature, p. 799.


Asunto(s)
Neoplasias Orofaríngeas , Infecciones por Papillomavirus , Humanos , Virus del Papiloma Humano , Reordenamiento Génico , Evolución Clonal/genética , Integración Viral/genética , Papillomaviridae/genética
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