RESUMEN
The blood-nerve-barrier (BNB) that regulates peripheral nerve homeostasis is formed by endoneurial capillaries and perineurial cells surrounding the Schwann cell (SC)-rich endoneurium. Barrier dysfunction is common in human tumorigenesis, including in some nerve tumors. We identify barrier disruption in human NF1 deficient neurofibromas, which were characterized by reduced perineurial cell glucose transporter 1 (GLUT1) expression and increased endoneurial fibrin(ogen) deposition. Conditional Nf1 loss in murine SCs recapitulated these alterations and revealed decreased tight junctions and decreased caveolin-1 (Cav1) expression in mutant nerves and in tumors, implicating reduced Cav1-mediated transcytosis in barrier disruption and tumorigenesis. Additionally, elevated receptor tyrosine kinase activity and genetic deletion of Cav1 increased endoneurial fibrin(ogen), and promoted SC tumor formation. Finally, when SC lacked Nf1, genetic loss or pharmacological inhibition of P2RY14 rescued Cav1 expression and barrier function. Thus, loss of Nf1 in SC causes dysfunction of the BNB via P2RY14-mediated G-protein coupled receptor (GPCR) signaling.
RESUMEN
Voltage-sensitive potassium channels play an important role in controlling membrane potential and ionic homeostasis in the gut and have been implicated in gastrointestinal (GI) cancers. Through large-scale analysis of 897 patients with gastro-oesophageal adenocarcinomas (GOAs) coupled with in vitro models, we find KCNQ family genes are mutated in â¼30% of patients, and play therapeutically targetable roles in GOA cancer growth. KCNQ1 and KCNQ3 mediate the WNT pathway and MYC to increase proliferation through resultant effects on cadherin junctions. This also highlights novel roles of KCNQ3 in non-excitable tissues. We also discover that activity of KCNQ3 sensitises cancer cells to existing potassium channel inhibitors and that inhibition of KCNQ activity reduces proliferation of GOA cancer cells. These findings reveal a novel and exploitable role of potassium channels in the advancement of human cancer, and highlight that supplemental treatments for GOAs may exist through KCNQ inhibitors.
Asunto(s)
Adenocarcinoma , Canales de Potasio KCNQ , Humanos , Canales de Potasio KCNQ/genética , Canales de Potasio KCNQ/metabolismo , Canal de Potasio KCNQ3/genética , Canal de Potasio KCNQ3/metabolismo , Canal de Potasio KCNQ2/fisiología , Adenocarcinoma/genéticaRESUMEN
Cancer has been a leading cause of death for decades. This dismal statistic has increased efforts to prevent the disease or to detect it early, when treatment is less invasive, relatively inexpensive and more likely to cure. But precisely how tissues are transformed continues to provoke controversy and debate, hindering cancer prevention and early intervention strategies. Various theories of cancer origins have emerged, including the suggestion that it is 'bad luck': the inevitable consequence of random mutations in proliferating stem cells. In this Review, we discuss the principal theories of cancer origins and the relative importance of the factors that underpin them. The body of available evidence suggests that developing and ageing tissues 'walk a tightrope', retaining adequate levels of cell plasticity to generate and maintain tissues while avoiding overstepping into transformation. Rather than viewing cancer as 'bad luck', understanding the complex choreography of cell intrinsic and extrinsic factors that characterize transformation holds promise to discover effective new ways to prevent, detect and stop cancer before it becomes incurable.
Asunto(s)
Neoplasias , Humanos , Neoplasias/genética , Neoplasias/prevención & control , Oncogenes , Mutación , Células Madre , EnvejecimientoRESUMEN
We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium-a NALCN channel blocker-similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies.
Asunto(s)
Neoplasias , Humanos , Ratones , Animales , Canales Iónicos/genética , Proteínas de la Membrana/genéticaRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.1609.].
RESUMEN
We previously identified ZNF217 as an oncogenic driver of a subset of osteosarcomas using the Sleeping Beauty (SB) transposon system. Here, we followed up by investigating the genetic role of ZNF217 in osteosarcoma initiation and progression through the establishment of a novel genetically engineered mouse model, in vitro assays, orthotopic mouse studies, and paired these findings with preclinical studies using a small-molecule inhibitor. Throughout, we demonstrate that ZNF217 is coupled to numerous facets of osteosarcoma transformation, including proliferation, cell motility, and anchorage independent growth, and ultimately promoting osteosarcoma growth, progression, and metastasis in part through positive modulation of PI3K-AKT survival signaling. Pharmacologic blockade of AKT signaling with nucleoside analogue triciribine in ZNF217+ orthotopically injected osteosarcoma cell lines reduced tumor growth and metastasis. Our data demonstrate that triciribine treatment may be a relevant and efficacious therapeutic strategy for patients with osteosarcoma with ZNF217+ and p-AKT rich tumors. With the recent revitalization of triciribine for clinical studies in other solid cancers, our study provides a rationale for further evaluation preclinically with the purpose of clinical evaluation in patients with incurable, ZNF217+ osteosarcoma.
Asunto(s)
Biomarcadores de Tumor , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Transactivadores/genética , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Expresión Génica Ectópica , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Modelos Biológicos , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/etiología , Osteosarcoma/metabolismo , Osteosarcoma/patología , Transducción de Señal/efectos de los fármacos , Transactivadores/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis. We examined CSF1R and CSF1 expression in OSAs. We utilized gain-of-function and loss-of-function studies (GOF/LOF) to evaluate properties of cellular transformation, downstream signaling, and mechanisms of CSF1R-CSF1 action. Genetic perturbation of CSF1R in immortalized osteoblasts and human OSA cell lines significantly altered oncogenic properties, which were dependent on the CSF1R-CSF1 autocrine/paracrine signaling. These functional alterations were associated with changes in the known CSF1R downstream ERK effector pathway and mitotic cell cycle arrest. We evaluated the recently FDA-approved CSF1R inhibitor Pexidartinib (PLX3397) in OSA cell lines in vitro and in vivo in cell line and patient-derived xenografts. Pharmacological inhibition of CSF1R signaling recapitulated the in vitro genetic alterations. Moreover, in orthotopic OSA cell line and subcutaneous patient-derived xenograft (PDX)-injected mouse models, PLX3397 treatment significantly inhibited local OSA tumor growth and lessened metastatic burden. In summary, CSF1R is utilized by OSA cells to promote tumorigenesis and may represent a new molecular target for therapy.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos , Osteosarcoma , Aminopiridinas , Animales , Carcinogénesis , Ratones , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Pirroles , Receptores de Factor Estimulante de Colonias de Granulocitos y MacrófagosRESUMEN
Semaphorins, specifically type IV, are important regulators of axonal guidance and have been increasingly implicated in poor prognoses in a number of different solid cancers. In conjunction with their cognate PLXNB family receptors, type IV members have been increasingly shown to mediate oncogenic functions necessary for tumor development and malignant spread. In this study, we investigated the role of semaphorin 4C (SEMA4C) in osteosarcoma growth, progression, and metastasis. We investigated the expression and localization of SEMA4C in primary osteosarcoma patient tissues and its tumorigenic functions in these malignancies. We demonstrate that overexpression of SEMA4C promotes properties of cellular transformation, while RNAi knockdown of SEMA4C promotes adhesion and reduces cellular proliferation, colony formation, migration, wound healing, tumor growth, and lung metastasis. These phenotypic changes were accompanied by reductions in activated AKT signaling, G1 cell cycle delay, and decreases in expression of mesenchymal marker genes SNAI1, SNAI2, and TWIST1. Lastly, monoclonal antibody blockade of SEMA4C in vitro mirrored that of the genetic studies. Together, our results indicate a multi-dimensional oncogenic role for SEMA4C in metastatic osteosarcoma and more importantly that SEMA4C has actionable clinical potential.
Asunto(s)
Neoplasias Óseas/patología , Progresión de la Enfermedad , Osteosarcoma/patología , Semaforinas/metabolismo , Carcinogénesis , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia , Semaforinas/deficiencia , Semaforinas/genéticaRESUMEN
Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. SIGNIFICANCE: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Cerebelosas/genética , Meduloblastoma/genética , Tumores Neuroectodérmicos Primitivos/genética , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Transformación Celular Neoplásica/genética , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Elementos Transponibles de ADN/genética , Femenino , Factores de Transcripción Forkhead/genética , Proteínas Activadoras de GTPasa/biosíntesis , Proteínas Activadoras de GTPasa/genética , Humanos , Masculino , Meduloblastoma/metabolismo , Meduloblastoma/patología , Proteínas de la Membrana/genética , Ratones , Ratones Desnudos , Mutagénesis Insercional/métodos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Tumores Neuroectodérmicos Primitivos/metabolismo , Tumores Neuroectodérmicos Primitivos/patología , PronósticoRESUMEN
Follicular lymphoma and diffuse large B-cell lymphoma (DLBCL) are the most common non-Hodgkin lymphomas distinguishable by unique mutations, chromosomal rearrangements, and gene expression patterns. Here, it is demonstrated that early B-cell progenitors express 2',3'-cyclic-nucleotide 3' phosphodiesterase (CNP) and that when targeted with Sleeping Beauty (SB) mutagenesis, Trp53R270H mutation or Pten loss gave rise to highly penetrant lymphoid diseases, predominantly follicular lymphoma and DLBCL. In efforts to identify the genetic drivers and signaling pathways that are functionally important in lymphomagenesis, SB transposon insertions were analyzed from splenomegaly specimens of SB-mutagenized mice (n = 23) and SB-mutagenized mice on a Trp53R270H background (n = 7) and identified 48 and 12 sites with statistically recurrent transposon insertion events, respectively. Comparison with human data sets revealed novel and known driver genes for B-cell development, disease, and signaling pathways: PI3K-AKT-mTOR, MAPK, NFκB, and B-cell receptor (BCR). Finally, functional data indicate that modulating Ras-responsive element-binding protein 1 (RREB1) expression in human DLBCL cell lines in vitro alters KRAS expression, signaling, and proliferation; thus, suggesting that this proto-oncogene is a common mechanism of RAS/MAPK hyperactivation in human DLBCL. IMPLICATIONS: A forward genetic screen identified new genetic drivers of human B-cell lymphoma and uncovered a RAS/MAPK-activating mechanism not previously appreciated in human lymphoid disease. Overall, these data support targeting the RAS/MAPK pathway as a viable therapeutic target in a subset of human patients with DLBCL.
Asunto(s)
Proteínas de Unión al ADN/genética , Linfoma de Células B Grandes Difuso/genética , Factores de Transcripción/genética , Animales , Línea Celular Tumoral , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Transgénicos , Mutagénesis Insercional , Mutación , Proto-Oncogenes MasRESUMEN
Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma.
Asunto(s)
Neoplasias Óseas/genética , Osteosarcoma/genética , Animales , Neoplasias Óseas/patología , Carcinogénesis/genética , Línea Celular Tumoral , Elementos Transponibles de ADN , Perros , Predisposición Genética a la Enfermedad , Inestabilidad Genómica , Humanos , Ratones Transgénicos , Mutagénesis Insercional , Osteosarcoma/secundario , Fosfohidrolasa PTEN/genética , Semaforinas/genética , Semaforinas/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations in Dck in both Ara-C resistant cell lines. CRISPR and TALEN-based KO of Dck dramatically increased the IC50 of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance.
Asunto(s)
Citarabina/farmacología , Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas Serina-Treonina Quinasas/genética , Células 3T3 , Animales , Antimetabolitos Antineoplásicos/farmacología , Secuencia de Bases , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Interferencia de ARN , ARN Interferente Pequeño , Análisis de Secuencia de ARNRESUMEN
The advent of Transcription Activator-Like Effector Nucleases (TALENs), and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO). Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for genetic studies and therapeutics based on gene modified human cells. We have developed and validated two methods for simple enrichment and isolation of single or multiplex gene KO's in transformed, immortalized, and human progenitor cells. These methods rely on selection of a phenotypic change such as resistance to a particular drug or ability to grow in a selective environment. The first method, termed co-transposition, utilizes integration of a piggyBac transposon vector encoding a drug resistance gene. The second method, termed co-targeting, utilizes TALENs to KO any gene that when lost induces a selectable phenotype. Using these methods we also show removal of entire genes and demonstrate that TALENs function in human CD34+ progenitor cells. Further, co-transposition can be used to generate conditional KO cell lines utilizing an inducible cDNA rescue transposon vector. These methods allow for robust enrichment and isolation of KO cells in a rapid and efficient manner.
Asunto(s)
Separación Celular/métodos , Elementos Transponibles de ADN , Endonucleasas , Técnicas de Inactivación de Genes/métodos , Vectores Genéticos , Células Madre , Línea Celular , Endonucleasas/biosíntesis , Endonucleasas/genética , Humanos , Células Madre/citología , Células Madre/metabolismoRESUMEN
Malignant peripheral nerve sheath tumors (MPNSTs) are genetically diverse, aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 1 syndrome. Reduced TP53 gene expression and amplification/overexpression of the epidermal growth factor receptor (EGFR) gene occur in MPNST formation. We focused on determining the cooperativity between reduced TP53 expression and EGFR overexpression for Schwann cell transformation in vitro (immortalized human Schwann cells) and MPNST formation in vivo (transgenic mice). Human gene copy number alteration data, microarray expression data, and TMA analysis indicate that TP53 haploinsufficiency and increased EGFR expression co-occur in human MPNST samples. Concurrent modulation of EGFR and TP53 expression in HSC1λ cells significantly increased proliferation and anchorage-independent growth in vitro. Transgenic mice heterozygous for a Trp53-null allele and overexpressing EGFR in Schwann cells had a significant increase in neurofibroma and grade 3 PNST (MPNST) formation compared with single transgenic controls. Histological analysis of tumors identified a significant increase in pAkt expression in grade 3 PNSTs compared with neurofibromas. Array comparative genome hybridization analysis of grade 3 PNSTs identified recurrent focal regions of chromosomal gains with significant enrichment in genes involved in extracellular signal-regulated kinase 5 signaling. Collectively, altered p53 expression cooperates with overexpression of EGFR in Schwann cells to enhance in vitro oncogenic properties and tumorigenesis and progression in vivo.
Asunto(s)
Carcinogénesis/genética , Receptores ErbB/metabolismo , Haploinsuficiencia , Neoplasias de la Vaina del Nervio/genética , Células de Schwann/patología , Proteína p53 Supresora de Tumor/genética , Animales , Transformación Celular Neoplásica/genética , Células Cultivadas , Receptores ErbB/genética , Humanos , Ratones Transgénicos , Neoplasias de la Vaina del Nervio/patología , Sarcoma/genética , Sarcoma/patologíaRESUMEN
Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that occur spontaneously, or from benign plexiform neurofibromas, in the context of the genetic disorder Neurofibromatosis Type 1 (NF1). The current standard treatment includes surgical resection, high-dose chemotherapy, and/or radiation. To date, most targeted therapies have failed to demonstrate effectiveness against plexiform neurofibromas and MPNSTs. Recently, several studies suggested that the mTOR and MAPK pathways are involved in the formation and progression of MPNSTs. Everolimus (RAD001) inhibits the mTOR and is currently FDA approved for several types of solid tumors. PD-0325901 (PD-901) inhibits MEK, a component of the MAPK pathway, and is currently in clinical trials. Here, we show in vitro than MPNST cell lines are more sensitive to inhibition of cellular growth by Everolimus and PD-901 than immortalized human Schwann cells. In combination, these drugs synergistically inhibit cell growth and induce apoptosis. In two genetically engineered mouse models of MPNST formation, modeling both sporadic and NF1-associated MPNSTs, Everolimus, or PD-901 treatment alone each transiently reduced tumor burden and size, and extended lifespan. However, prolonged treatment of each single agent resulted in the development of resistance and reactivation of target pathways. Combination therapy using Everolimus and PD-901 had synergistic effects on reducing tumor burden and size, and increased lifespan. Combination therapy allowed persistent and prolonged reduction in signaling through both pathways. These data suggest that co-targeting mTOR and MEK may be effective in patients with sporadic or NF1-associated MPNSTs.
Asunto(s)
Benzamidas/farmacología , Difenilamina/análogos & derivados , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neurilemoma/prevención & control , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Sirolimus/análogos & derivados , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Difenilamina/farmacología , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Everolimus , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Inmunosupresores/farmacología , Ratones , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Clasificación del Tumor , Neurilemoma/genética , Neurilemoma/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Células de Schwann/patología , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Malignant peripheral nerve sheath tumors (MPNSTs) are sarcomas of Schwann cell lineage origin that occur sporadically or in association with the inherited syndrome neurofibromatosis type 1. To identify genetic drivers of MPNST development, we used the Sleeping Beauty (SB) transposon-based somatic mutagenesis system in mice with somatic loss of transformation-related protein p53 (Trp53) function and/or overexpression of human epidermal growth factor receptor (EGFR). Common insertion site (CIS) analysis of 269 neurofibromas and 106 MPNSTs identified 695 and 87 sites with a statistically significant number of recurrent transposon insertions, respectively. Comparison to human data sets identified new and known driver genes for MPNST formation at these sites. Pairwise co-occurrence analysis of CIS-associated genes identified many cooperating mutations that are enriched in Wnt/ß-catenin, PI3K-AKT-mTOR and growth factor receptor signaling pathways. Lastly, we identified several new proto-oncogenes, including Foxr2 (encoding forkhead box R2), which we functionally validated as a proto-oncogene involved in MPNST maintenance.
Asunto(s)
Transformación Celular Neoplásica/genética , Genes Relacionados con las Neoplasias , Pruebas Genéticas/métodos , Neoplasias de la Vaina del Nervio/genética , Animales , Línea Celular Tumoral , Análisis Mutacional de ADN , Elementos Transponibles de ADN/genética , Genes Relacionados con las Neoplasias/fisiología , Estudios de Asociación Genética , Humanos , Ratones , Ratones Transgénicos , Mutación/fisiología , Neurofibroma/genética , Proto-Oncogenes Mas , Transducción de Señal/genéticaRESUMEN
Genetic changes required for the formation and progression of human Schwann cell tumors remain elusive. Using a Sleeping Beauty forward genetic screen, we identified several genes involved in canonical Wnt signaling as potential drivers of benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). In human neurofibromas and MPNSTs, activation of Wnt signaling increased with tumor grade and was associated with downregulation of ß-catenin destruction complex members or overexpression of a ligand that potentiates Wnt signaling, R-spondin 2 (RSPO2). Induction of Wnt signaling was sufficient to induce transformed properties in immortalized human Schwann cells, and downregulation of this pathway was sufficient to reduce the tumorigenic phenotype of human MPNST cell lines. Small-molecule inhibition of Wnt signaling effectively reduced the viability of MPNST cell lines and synergistically induced apoptosis when combined with an mTOR inhibitor, RAD-001, suggesting that Wnt inhibition represents a novel target for therapeutic intervention in Schwann cell tumors.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Neoplasias de la Vaina del Nervio/metabolismo , Neoplasias de la Vaina del Nervio/patología , Células de Schwann/metabolismo , Células de Schwann/patología , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Progresión de la Enfermedad , Regulación hacia Abajo , Humanos , Ratones , Neoplasias de la Vaina del Nervio/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Studying complex biological processes such as cancer development, stem cell induction and transdifferentiation requires the modulation of multiple genes or pathways at one time in a single cell. Herein, we describe straightforward methods for rapid and efficient assembly of bacterial marker free multigene cassettes containing up to six complementary DNAs/short hairpin RNAs. We have termed this method RecWay assembly, as it makes use of both Cre recombinase and the commercially available Gateway cloning system. Further, because RecWay assembly uses truly modular components, it allows for the generation of randomly assembled multigene vector libraries. These multigene vectors are integratable, and later excisable, using the highly efficient piggyBac (PB) DNA transposon system. Moreover, we have dramatically improved the expression of stably integrated multigene vectors by incorporation of insulator elements to prevent promoter interference seen with multigene vectors. We demonstrate that insulated multigene PB transposons can stably integrate and faithfully express up to five fluorescent proteins and the puromycin-thymidine kinase resistance gene in vitro, with up to 70-fold higher gene expression compared with analogous uninsulated vectors. RecWay assembly of multigene transposon vectors allows for widely applicable modelling of highly complex biological processes and can be easily performed by other research laboratories.
Asunto(s)
Elementos Transponibles de ADN , Vectores Genéticos , Animales , Células Cultivadas , ADN Complementario/metabolismo , Expresión Génica , Humanos , Integrasas/metabolismo , Ratones , Neoplasias Experimentales/genética , ARN Interferente Pequeño/metabolismo , TransfecciónRESUMEN
L1 retrotransposons comprise 17% of the human genome and are its only autonomous mobile elements. Although L1-induced insertional mutagenesis causes Mendelian disease, their mutagenic load in cancer has been elusive. Using L1-targeted resequencing of 16 colorectal tumor and matched normal DNAs, we found that certain cancers were excessively mutagenized by human-specific L1s, while no verifiable insertions were present in normal tissues. We confirmed de novo L1 insertions in malignancy by both validating and sequencing 69/107 tumor-specific insertions and retrieving both 5' and 3' junctions for 35. In contrast to germline polymorphic L1s, all insertions were severely 5' truncated. Validated insertion numbers varied from up to 17 in some tumors to none in three others, and correlated with the age of the patients. Numerous genes with a role in tumorigenesis were targeted, including ODZ3, ROBO2, PTPRM, PCM1, and CDH11. Thus, somatic retrotransposition may play an etiologic role in colorectal cancer.