RESUMEN
Tumors represent ecosystems where subclones compete during tumor growth. While extensively investigated, a comprehensive picture of the interplay of clonal lineages during dissemination is still lacking. Using patient-derived pancreatic cancer cells, we created orthotopically implanted clonal replica tumors to trace clonal dynamics of unperturbed tumor expansion and dissemination. This model revealed the multifaceted nature of tumor growth, with rapid changes in clonal fitness leading to continuous reshuffling of tumor architecture and alternating clonal dominance as a distinct feature of cancer growth. Regarding dissemination, a large fraction of tumor lineages could be found at secondary sites each having distinctive organ growth patterns as well as numerous undescribed behaviors such as abortive colonization. Paired analysis of primary and secondary sites revealed fitness as major contributor to dissemination. From the analysis of pro- and nonmetastatic isogenic subclones, we identified a transcriptomic signature able to identify metastatic cells in human tumors and predict patients' survival.
Asunto(s)
Ecosistema , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Perfilación de la Expresión Génica , TranscriptomaRESUMEN
Inflammation is a major risk factor for pancreatic ductal adenocarcinoma (PDAC). When occurring in the context of pancreatitis, KRAS mutations accelerate tumor development in mouse models. We report that long after its complete resolution, a transient inflammatory event primes pancreatic epithelial cells to subsequent transformation by oncogenic KRAS. Upon recovery from acute inflammation, pancreatic epithelial cells display an enduring adaptive response associated with sustained transcriptional and epigenetic reprogramming. Such adaptation enables the reactivation of acinar-to-ductal metaplasia (ADM) upon subsequent inflammatory events, thereby limiting tissue damage through a rapid decrease of zymogen production. We propose that because activating mutations of KRAS maintain an irreversible ADM, they may be beneficial and under strong positive selection in the context of recurrent pancreatitis.
Asunto(s)
Células Acinares/patología , Carcinogénesis , Carcinoma Ductal Pancreático/patología , Genes ras , Páncreas/patología , Pancreatitis/fisiopatología , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/fisiopatología , Transformación Celular Neoplásica , Células Cultivadas , Reprogramación Celular , Cromatina/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Precursores Enzimáticos/metabolismo , Epigénesis Genética , Células Epiteliales/patología , Células Epiteliales/fisiología , Femenino , Sistema de Señalización de MAP Quinasas , Masculino , Metaplasia , Ratones , Mutación , Páncreas/metabolismo , Pancreatitis/genética , Pancreatitis/inmunología , Esferoides Celulares , TranscriptomaRESUMEN
Capitalizing on the inherent multiplexing capability of AsCpf1, we developed a multiplexed, high-throughput screening strategy that minimizes library size without sacrificing gene targeting efficiency. We demonstrated that AsCpf1 can be used for functional genomics screenings and that an AsCpf1-based multiplexed library performs similarly as compared to currently available monocistronic CRISPR/Cas9 libraries, with only one vector required for each gene. We construct the smallest whole-genome CRISPR knock-out library, Mini-human, for the human genome (n = 17,032 constructs targeting 16,977 protein-coding genes), which performs favorably compared to conventional Cas9 libraries.
Asunto(s)
Sistemas CRISPR-Cas/genética , Biblioteca de Genes , Proteína 9 Asociada a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Edición Génica , Humanos , ARN Guía de Kinetoplastida/químicaRESUMEN
Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro cultures and in vivo tumors are maintained by a common set of tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of adaptive responses to therapeutics, we uncovered a multitude of functionally heterogeneous subpopulations of cells with differential degrees of drug sensitivity. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying functionally diverse subpopulations. Molecular annotation of gemcitabine-naive clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy, representing a potential biomarker to stratify patients with pancreatic cancer.
Asunto(s)
Resistencia a Antineoplásicos , Heterogeneidad Genética , Neoplasias Pancreáticas/genética , Transcriptoma , Anciano , Animales , Antimetabolitos Antineoplásicos/farmacología , Células Cultivadas , Evolución Clonal , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/patología , GemcitabinaRESUMEN
Myxoid/round-cell liposarcoma (MLS/RCLS) is characterized by either the fusion gene FUS-DDIT3 or the less commonly occurring EWSR1-DDIT3 and most cases carry few or no additional cytogenetic changes. There are conflicting reports concerning the status and role of TP53 in MLS/RCLS. Here we analysed four MLS/RCLS derived cell lines for TP53 mutations, expression and function. Three SV40 transformed cell lines expressed normal TP53 proteins. Irradiation caused normal posttranslational modifications of TP53 and induced P21 expression in two of these cell lines. Transfection experiments showed that the FUS-DDIT3 fusion protein had no effects on irradiation induced TP53 responses. Ion Torrent AmpliSeq screening, using the Cancer Hotspot panel, showed no dysfunctional or disease associated alleles/mutations. In conclusion, our results suggest that most MLS/RCLS cases carry functional TP53 genes and this is consistent with the low numbers of secondary mutations observed in this tumor entity.
Asunto(s)
Liposarcoma Mixoide/genética , Mutación , Proteína p53 Supresora de Tumor/genética , Línea Celular Transformada , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inestabilidad Genómica , Humanos , Liposarcoma Mixoide/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
OBJECTIVE: To determine and compare the frequency of cancer-associated genetic abnormalities in esophageal metaplasia biopsies with and without goblet cells. BACKGROUND: Barrett's esophagus is associated with increased risk of esophageal adenocarcinoma (EAC), but the appropriate histologic definition of Barrett's esophagus is debated. Intestinal metaplasia (IM) is defined by the presence of goblet cells whereas nongoblet cell metaplasia (NGM) lacks goblet cells. Both have been implicated in EAC risk but this is controversial. Although IM is known to harbor genetic changes associated with EAC, little is known about NGM. We hypothesized that if NGM and IM infer similar EAC risk, then they would harbor similar genetic aberrations in genes associated with EAC. METHODS: Ninety frozen NGM, IM, and normal tissues from 45 subjects were studied. DNA copy number abnormalities were identified using microarrays and fluorescence in situ hybridization. Targeted sequencing of all exons from 20 EAC-associated genes was performed on metaplasia biopsies using Ion AmpliSeq DNA sequencing. RESULTS: Frequent copy number abnormalities targeting cancer-associated genes were found in IM whereas no such changes were observed in NGM. In 1 subject, fluorescence in situ hybridization confirmed loss of CDKN2A and amplification of chromosome 8 in IM but not in a nearby NGM biopsy. Targeted sequencing revealed 11 nonsynonymous mutations in 16 IM samples and 2 mutations in 19 NGM samples. CONCLUSIONS: This study reports the largest and most comprehensive comparison of DNA aberrations in IM and NGM genomes. Our results show that IM has a much higher frequency of cancer-associated mutations than NGM.
Asunto(s)
Adenocarcinoma/genética , Esófago de Barrett/genética , ADN de Neoplasias/genética , Neoplasias Esofágicas/genética , Genes p16/fisiología , Células Caliciformes/patología , Mutación , Lesiones Precancerosas , Adenocarcinoma/patología , Anciano , Esófago de Barrett/patología , Biopsia , Análisis Mutacional de ADN , Neoplasias Esofágicas/patología , Esófago/patología , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Metaplasia , Reacción en Cadena de la Polimerasa , Estudios RetrospectivosRESUMEN
High-throughput RNA sequencing enables quantification of transcripts (both known and novel), exon/exon junctions and fusions of exons from different genes. Discovery of gene fusions-particularly those expressed with low abundance- is a challenge with short- and medium-length sequencing reads. To address this challenge, we implemented an RNA-Seq mapping pipeline within the LifeScope software. We introduced new features including filter and junction mapping, annotation-aided pairing rescue and accurate mapping quality values. We combined this pipeline with a Suffix Array Spliced Read (SASR) aligner to detect chimeric transcripts. Performing paired-end RNA-Seq of the breast cancer cell line MCF-7 using the SOLiD system, we called 40 gene fusions among over 120,000 splicing junctions. We validated 36 of these 40 fusions with TaqMan assays, of which 25 were expressed in MCF-7 but not the Human Brain Reference. An intra-chromosomal gene fusion involving the estrogen receptor alpha gene ESR1, and another involving the RPS6KB1 (Ribosomal protein S6 kinase beta-1) were recurrently expressed in a number of breast tumor cell lines and a clinical tumor sample.