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1.
Proc Natl Acad Sci U S A ; 116(47): 23625-23635, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31690663

RESUMO

Myocardin-related transcription factor B (MRTFB) is a candidate tumor-suppressor gene identified in transposon mutagenesis screens of the intestine, liver, and pancreas. Using a combination of cell-based assays, in vivo tumor xenograft assays, and Mrtfb knockout mice, we demonstrate here that MRTFB is a human and mouse colorectal cancer (CRC) tumor suppressor that functions in part by inhibiting cell invasion and migration. To identify possible MRTFB transcriptional targets, we performed whole transcriptome RNA sequencing in MRTFB siRNA knockdown primary human colon cells and identified 15 differentially expressed genes. Among the top candidate tumor-suppressor targets were melanoma cell adhesion molecule (MCAM), a known tumor suppressor, and spindle apparatus coiled-coil protein 1 (SPDL1), which has no confirmed role in cancer. To determine whether these genes play a role in CRC, we knocked down the expression of MCAM and SPDL1 in human CRC cells and showed significantly increased invasion and migration of tumor cells. We also showed that Spdl1 expression is significantly down-regulated in Mrtfb knockout mouse intestine, while lower SPDL1 expression levels are significantly associated with reduced survival in CRC patients. Finally, we show that depletion of MCAM and SPDL1 in human CRC cells significantly increases tumor development in xenograft assays, further confirming their tumor-suppressive roles in CRC. Collectively, our findings demonstrate the tumor-suppressive role of MRTFB in CRC and identify several genes, including 2 tumor suppressors, that act downstream of MRTFB to regulate tumor growth and survival in CRC patients.


Assuntos
Adenocarcinoma/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/fisiologia , Fatores de Transcrição/fisiologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Antígeno CD146/metabolismo , Movimento Celular , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Células HCT116 , Células HT29 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Interferência de RNA , RNA Interferente Pequeno/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
2.
Proc Natl Acad Sci U S A ; 116(31): 15635-15644, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31300537

RESUMO

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide Sleeping Beauty transposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations (Apc and Kras mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that Acvr1b, Acvr2a, and Arid2 could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53 in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-ß (TGF-ß) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets.


Assuntos
Sistemas CRISPR-Cas , Neoplasias Colorretais , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Proteínas de Neoplasias , Organoides , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Organoides/metabolismo , Organoides/patologia
4.
Cancer Cell ; 35(1): 10-15, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30645969

RESUMO

We propose that initiating truncal mutations plays a special role in tumor formation by both enhancing the survival of the initiating cancer cell and by selecting for secondary mutations that contribute to tumor progression, and that these mutations often act in a tissue-preferred fashion. Here, we explain why inherited mutations often have different tissue specificities compared with spontaneous mutations in the same gene. Initiating truncal mutations make excellent neo-antigens for immunotherapy, and understanding why one mutation selects for a second mutation in a particular tissue type could one day aid in the design of gene-targeted combination therapies.


Assuntos
Mutação , Neoplasias/genética , Animais , Progressão da Doença , Desenho de Fármacos , Predisposição Genética para Doença , Humanos , Imunoterapia , Especificidade de Órgãos
5.
Proc Natl Acad Sci U S A ; 115(44): E10417-E10426, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30327349

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is the fastest rising cause of hepatocellular carcinoma (HCC) in Western countries; however, the molecular mechanisms that cause NAFLD-HCC remain elusive. To identify molecular drivers of NAFLD-HCC, we performed Sleeping Beauty (SB) transposon mutagenesis screens in liver-specific Pten knockout and in high-fat diet-fed mice, which are murine models of NAFLD-HCC. SB mutagenesis accelerated liver tumor formation in both models and identified 588 and 376 candidate cancer genes (CCGs), respectively; 257 CCGs were common to both screens and were enriched in signaling pathways known to be important for human HCC. Comparison of these CCGs with those identified in a previous SB screen of hepatitis B virus-induced HCC identified a core set of 141 CCGs that were mutated in all screens. Forty-one CCGs appeared specific for NAFLD-HCC, including Sav1, a component of the Hippo signaling pathway and the most frequently mutated gene identified in both NAFLD-HCC screens. Liver-specific deletion of Sav1 was found to promote hepatic lipid accumulation, apoptosis, and fibrogenesis, leading to the acceleration of hepatocarcinogenesis in liver-specific Pten mutant mice. Sav1/Pten double-mutant livers also showed a striking up-regulation of markers of liver progenitor cells (LPCs), along with synergistic activation of Yap, which is a major downstream effector of Hippo signaling. Lastly, Yap activation, in combination with Pten inactivation, was found to accelerate cell growth and sphere formation of LPCs in vitro and induce their malignant transformation in allografts. Our forward genetic screens in mice have thus identified pathways and genes driving the development of NAFLD-HCC.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Elementos de DNA Transponíveis/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Animais , Apoptose/genética , Carcinogênese/genética , Carcinogênese/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Dieta Hiperlipídica/efeitos adversos , Fígado/patologia , Camundongos , Mutagênese/genética , Oncogenes/genética , Transdução de Sinais/genética , Regulação para Cima/genética
6.
Cytoskeleton (Hoboken) ; 75(9): 395-409, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29979496

RESUMO

The actin-based motor myosin Va transports numerous cargos, including the smooth endoplasmic reticulum (SER) in cerebellar Purkinje neurons (PNs) and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Toward that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the immunoglobulin G-binding domain of protein A, a tobacco etch virus cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of myosin Va (TAP-MyoVa) functions normally in terms of SER transport in PNs and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it together with associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va-binding partners like dynein light chain were identified. Importantly, numerous novel interacting proteins were also tentatively identified, including guanine nucleotide-binding protein G(o) subunit alpha (Gnao1), a biomarker for schizophrenia. Consistently, an antibody to Gnao1 immunoprecipitates myosin Va, and Gnao1's localization to PN dendritic spines depends on myosin Va. The mouse model created here should facilitate the identification of novel myosin Va-binding partners, which in turn should advance our understanding of the roles played by this important myosin in vivo.


Assuntos
Cerebelo/fisiologia , Camundongos Transgênicos/metabolismo , Miosina Tipo V/metabolismo , Animais , Camundongos
7.
Nucleic Acids Res ; 46(16): e94, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-29846651

RESUMO

Cancer driver prioritization for functional analysis of potential actionable therapeutic targets is a significant challenge. Meta-analyses of mutated genes across different human cancer types for driver prioritization has reaffirmed the role of major players in cancer, including KRAS, TP53 and EGFR, but has had limited success in prioritizing genes with non-recurrent mutations in specific cancer types. Sleeping Beauty (SB) insertional mutagenesis is a powerful experimental gene discovery framework to define driver genes in mouse models of human cancers. Meta-analyses of SB datasets across multiple tumor types is a potentially informative approach to prioritize drivers, and complements efforts in human cancers. Here, we report the development of SB Driver Analysis, an in-silico method for defining cancer driver genes that positively contribute to tumor initiation and progression from population-level SB insertion data sets. We demonstrate that SB Driver Analysis computationally prioritizes drivers and defines distinct driver classes from end-stage tumors that predict their putative functions during tumorigenesis. SB Driver Analysis greatly enhances our ability to analyze, interpret and prioritize drivers from SB cancer datasets and will continue to substantially increase our understanding of the genetic basis of cancer.


Assuntos
Transformação Celular Neoplásica/genética , Elementos de DNA Transponíveis/genética , Mutagênese Insercional , Neoplasias/genética , Oncogenes/genética , Proteínas Supressoras de Tumor/genética , Algoritmos , Animais , Predisposição Genética para Doença/genética , Humanos , Camundongos , Modelos Genéticos , Neoplasias/patologia
8.
Front Mol Neurosci ; 11: 103, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29674952

RESUMO

Structural and functional plasticity of synapses are critical neuronal mechanisms underlying learning and memory. While activity-dependent regulation of synaptic strength has been extensively studied, much less is known about the transcriptional control of synapse maintenance and plasticity. Hippocampal mossy fiber (MF) synapses connect dentate granule cells to CA3 pyramidal neurons and are important for spatial memory formation and consolidation. The transcription factor Bcl11b/Ctip2 is expressed in dentate granule cells and required for postnatal hippocampal development. Ablation of Bcl11b/Ctip2 in the adult hippocampus results in impaired adult neurogenesis and spatial memory. The molecular mechanisms underlying the behavioral impairment remained unclear. Here we show that selective deletion of Bcl11b/Ctip2 in the adult mouse hippocampus leads to a rapid loss of excitatory synapses in CA3 as well as reduced ultrastructural complexity of remaining mossy fiber boutons (MFBs). Moreover, a dramatic decline of long-term potentiation (LTP) of the dentate gyrus-CA3 (DG-CA3) projection is caused by adult loss of Bcl11b/Ctip2. Differential transcriptomics revealed the deregulation of genes associated with synaptic transmission in mutants. Together, our data suggest Bcl11b/Ctip2 to regulate maintenance and function of MF synapses in the adult hippocampus.

9.
Nucleic Acids Res ; 46(D1): D1011-D1017, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29059366

RESUMO

Large-scale oncogenomic studies have identified few frequently mutated cancer drivers and hundreds of infrequently mutated drivers. Defining the biological context for rare driving events is fundamentally important to increasing our understanding of the druggable pathways in cancer. Sleeping Beauty (SB) insertional mutagenesis is a powerful gene discovery tool used to model human cancers in mice. Our lab and others have published a number of studies that identify cancer drivers from these models using various statistical and computational approaches. Here, we have integrated SB data from primary tumor models into an analysis and reporting framework, the Sleeping Beauty Cancer Driver DataBase (SBCDDB, http://sbcddb.moffitt.org), which identifies drivers in individual tumors or tumor populations. Unique to this effort, the SBCDDB utilizes a single, scalable, statistical analysis method that enables data to be grouped by different biological properties. This allows for SB drivers to be evaluated (and re-evaluated) under different contexts. The SBCDDB provides visual representations highlighting the spatial attributes of transposon mutagenesis and couples this functionality with analysis of gene sets, enabling users to interrogate relationships between drivers. The SBCDDB is a powerful resource for comparative oncogenomic analyses with human cancer genomics datasets for driver prioritization.


Assuntos
Bases de Dados Genéticas , Genes Neoplásicos , Neoplasias Experimentais/genética , Animais , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Camundongos , Mutagênese Insercional
10.
J Clin Invest ; 127(12): 4462-4476, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29130932

RESUMO

p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an "obligatory" haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Apc loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3-dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors α-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.


Assuntos
Proteína da Polipose Adenomatosa do Colo , Cateninas , Haploinsuficiência , Neoplasias Intestinais , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Cateninas/genética , Cateninas/metabolismo , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Camundongos , Camundongos Knockout , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
11.
Proc Natl Acad Sci U S A ; 114(35): E7301-E7310, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28811376

RESUMO

Epithelial ovarian cancer (EOC) is a deadly cancer, and its prognosis has not been changed significantly during several decades. To seek new therapeutic targets for EOC, we performed an in vivo dropout screen in human tumor xenografts using a pooled shRNA library targeting thousands of druggable genes. Then, in follow-up studies, we performed a second screen using a genome-wide CRISPR/Cas9 library. These screens identified 10 high-confidence drug targets that included well-known oncogenes such as ERBB2 and RAF1, and novel oncogenes, notably KPNB1, which we investigated further. Genetic and pharmacological inhibition showed that KPNB1 exerts its antitumor effects through multiphase cell cycle arrest and apoptosis induction. Mechanistically, proteomic studies revealed that KPNB1 acts as a master regulator of cell cycle-related proteins, including p21, p27, and APC/C. Clinically, EOC patients with higher expression levels of KPNB1 showed earlier recurrence and worse prognosis than those with lower expression levels of KPNB1. Interestingly, ivermectin, a Food and Drug Administration-approved antiparasitic drug, showed KPNB1-dependent antitumor effects on EOC, serving as an alternative therapeutic toward EOC patients through drug repositioning. Last, we found that the combination of ivermectin and paclitaxel produces a stronger antitumor effect on EOC both in vitro and in vivo than either drug alone. Our studies have thus identified a combinatorial therapy for EOC, in addition to a plethora of potential drug targets.


Assuntos
Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , beta Carioferinas/genética , beta Carioferinas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Epitelial do Ovário , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Ivermectina/farmacologia , Mutação com Perda de Função/genética , Neoplasias Epiteliais e Glandulares/metabolismo , Oncogenes , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , RNA Interferente Pequeno/genética
12.
Cancer Res ; 2017 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-28724549

RESUMO

Although genomic sequencing has provided a better understating of the genetic landmarks in triple-negative breast cancer (TNBC), functional validation of candidate cancer genes (CCG) remains unsolved. In this study, we used a transposon mutagenesis strategy based on a two-step Sleeping Beauty (SB) forward genetic screen to identify and validate new tumor suppressors (TS) in this disease. We generated 120 siRNAs targeting 40 SB-identified candidate breast cancer TS genes and used them to downregulate expression of these genes in four human TNBC cell lines. Among CCG whose SB-mediated genetic mutation resulted in increased cellular proliferation in all cell lines tested, the genes ADNP, AP2B1, TOMM70A and ZNF326 showed tumor suppressor (TS) activity in tumor xenograft studies. Subsequent studies showed that ZNF326 regulated expression of multiple EMT and cancer stem cell (CSC) pathway genes. It also modulated expression of TS genes involved in the regulation of migration and cellular invasion and was a direct transcriptional activator of genes that regulate CSC self-renewal. ZNF326 expression associated with TNBC patient survival, with ZNF326 protein levels showing a marked reduction in TNBC. Our validation of several new tumor suppressor genes in TNBC demonstrate the utility of two-step forward genetic screens in mice, and offer an invaluable tool to identify novel candidate therapeutic pathways and targets.

13.
Proc Natl Acad Sci U S A ; 114(11): E2215-E2224, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28251929

RESUMO

Robust prognostic gene signatures and therapeutic targets are difficult to derive from expression profiling because of the significant heterogeneity within breast cancer (BC) subtypes. Here, we performed forward genetic screening in mice using Sleeping Beauty transposon mutagenesis to identify candidate BC driver genes in an unbiased manner, using a stabilized N-terminal truncated ß-catenin gene as a sensitizer. We identified 134 mouse susceptibility genes from 129 common insertion sites within 34 mammary tumors. Of these, 126 genes were orthologous to protein-coding genes in the human genome (hereafter, human BC susceptibility genes, hBCSGs), 70% of which are previously reported cancer-associated genes, and ∼16% are known BC suppressor genes. Network analysis revealed a gene hub consisting of E1A binding protein P300 (EP300), CD44 molecule (CD44), neurofibromin (NF1) and phosphatase and tensin homolog (PTEN), which are linked to a significant number of mutated hBCSGs. From our survival prediction analysis of the expression of human BC genes in 2,333 BC cases, we isolated a six-gene-pair classifier that stratifies BC patients with high confidence into prognostically distinct low-, moderate-, and high-risk subgroups. Furthermore, we proposed prognostic classifiers identifying three basal and three claudin-low tumor subgroups. Intriguingly, our hBCSGs are mostly unrelated to cell cycle/mitosis genes and are distinct from the prognostic signatures currently used for stratifying BC patients. Our findings illustrate the strength and validity of integrating functional mutagenesis screens in mice with human cancer transcriptomic data to identify highly prognostic BC subtyping biomarkers.


Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Elementos de DNA Transponíveis , Estudos de Associação Genética , Predisposição Genética para Doença , Mutagênese Insercional , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Biologia Computacional/métodos , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Knockout , Mutação , Prognóstico , Reprodutibilidade dos Testes , Risco , Transdução de Sinais , Análise de Sobrevida , Transcriptoma
14.
Proc Natl Acad Sci U S A ; 113(48): E7749-E7758, 2016 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-27849608

RESUMO

Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1 Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC.


Assuntos
Adenocarcinoma/genética , Elementos de DNA Transponíveis , Neoplasias Mamárias Experimentais/genética , PTEN Fosfo-Hidrolase/genética , Adenocarcinoma/secundário , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Genes Supressores de Tumor , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares , Neoplasias Mamárias Experimentais/patologia , Camundongos Transgênicos , Mutagênese , Mutação de Sentido Incorreto , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologia
15.
Pharmacol Ther ; 168: 29-42, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27595930

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease with a high mortality rate. Genetic and biochemical studies have shown that RAS signaling mediated by KRAS plays a pivotal role in disease initiation, progression and drug resistance. RAS signaling affects several cellular processes in PDAC, including cellular proliferation, migration, cellular metabolism and autophagy. 90% of pancreatic cancer patients harbor somatic oncogenic point mutations in KRAS, which lead to constitutive activation of the molecule. Pancreatic cancers lacking KRAS mutations show activation of RAS via upstream signaling through receptor mediated tyrosine kinases, like EGFR, and in a small fraction of patients, oncogenic activation of the downstream B-RAF molecule is detected. RAS-stimulated signaling of RAF/MEK/ERK, PI3K/AKT/mTOR and RalA/B is active in human pancreatic cancers, cancer cell lines and mouse models of PDAC, although activation levels of each signaling arm appear to be variable across different tumors and perhaps within different subclones of single tumors. Recently, several targeted therapies directed towards MEK, ERK, PI3K and mTOR have been assayed in pancreatic cancer cell lines and in mouse models of the disease with promising results for their ability to impede cellular growth or delay tumor formation, and several inhibitors are currently in clinical trials. However, therapy-induced cross activation of RAS effector molecules has elucidated the complexities of targeting RAS signaling. Combinatorial therapies are now being explored as an approach to overcome RAS-induced therapeutic resistance in pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Mutação Puntual , Transdução de Sinais
16.
Nat Biotechnol ; 34(9): 962-72, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27479497

RESUMO

A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor's major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.


Assuntos
DNA de Neoplasias/genética , Genes Neoplásicos/genética , Hibridização In Situ/métodos , Leucemia Mieloide/genética , Mutagênese Insercional/genética , Análise de Sequência de DNA/métodos , Algoritmos , Animais , Biomarcadores Tumorais/genética , Elementos de DNA Transponíveis , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mieloide/patologia , Masculino , Camundongos , Proteínas de Neoplasias/genética , Software , Transposases/genética
17.
Nat Biotechnol ; 34(8): 845-51, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27398792

RESUMO

Refined cancer models are needed to bridge the gaps between cell line, animal and clinical research. Here we describe the engineering of an organotypic colon cancer model by recellularization of a native human matrix that contains cell-populated mucosa and an intact muscularis mucosa layer. This ex vivo system recapitulates the pathophysiological progression from APC-mutant neoplasia to submucosal invasive tumor. We used it to perform a Sleeping Beauty transposon mutagenesis screen to identify genes that cooperate with mutant APC in driving invasive neoplasia. We identified 38 candidate invasion-driver genes, 17 of which, including TCF7L2, TWIST2, MSH2, DCC, EPHB1 and EPHB2 have been previously implicated in colorectal cancer progression. Six invasion-driver genes that have not, to our knowledge, been previously described were validated in vitro using cell proliferation, migration and invasion assays and ex vivo using recellularized human colon. These results demonstrate the utility of our organoid model for studying cancer biology.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Colo/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Perfilação da Expressão Gênica/métodos , Proteínas de Neoplasias/metabolismo , Carcinogênese/genética , Sistema Livre de Células/metabolismo , Células Cultivadas , Colo/patologia , Genes Neoplásicos/genética , Humanos , Organogênese , Engenharia Tecidual/métodos
18.
Proc Natl Acad Sci U S A ; 113(24): E3384-93, 2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27247392

RESUMO

Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets.


Assuntos
Carcinoma Hepatocelular , Elementos de DNA Transponíveis , Transição Epitelial-Mesenquimal/genética , Neoplasias Hepáticas , Mutagênese , Proteínas de Neoplasias , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimento Celular/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Nus , Camundongos Transgênicos , Proteínas de Neoplasias/genética
19.
Gastroenterology ; 151(2): 324-337.e12, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27178121

RESUMO

BACKGROUND & AIMS: High-throughput sequencing technologies have identified thousands of infrequently mutated genes in hepatocellular carcinomas (HCCs). However, high intratumor and intertumor heterogeneity, combined with large numbers of passenger mutations, have made it difficult to identify driver mutations that contribute to the development of HCC. We combined transposon mutagenesis with a high-throughput screen of a small-hairpin RNA (shRNA) library to identify genes and pathways that contribute to HCC development. METHODS: Sleeping beauty transposons were mobilized in livers of transgenic mice predisposed to develop hepatocellular adenoma and HCC owing to expression of the hepatitis B virus surface antigen. This whole-genome mutagenesis technique was used to generate an unbiased catalogue of candidate cancer genes (CCGs). Pooled shRNA libraries targeting 250 selected CCGs then were introduced into immortalized mouse liver cells and the cells were monitored for their tumor-forming ability after injection into nude mice. RESULTS: Transposon-mediated mutagenesis identified 1917 high-confident CCGs and highlighted the importance of Ras signaling in the development of HCC. Subsequent pooled shRNA library screening of 250 selected CCGs validated 27 HCC tumor-suppressor genes. Individual shRNA knockdown of 4 of these genes (Acaa2, Hbs1l, Ralgapa2, and Ubr2) increased the proliferation of multiple human HCC cell lines in culture and accelerated the formation of xenograft tumors in nude mice. The ability of Ralgapa2 to promote HCC cell proliferation and tumor formation required its inhibition of Rala and Ralb. Dual inhibition of Ras signaling via Ral and Raf, using a combination of small-molecule inhibitor RBC8 and sorafenib, reduced the proliferation of HCC cells in culture and completely inhibited their growth as xenograft tumors in nude mice. CONCLUSIONS: In a 2-step forward genetic screen in mice, we identified members of the Ral guanosine triphosphatase-activating protein pathway and other proteins as suppressors of HCC cell proliferation and tumor growth. These proteins might serve as therapeutic targets for liver cancer.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas Ativadoras de GTPase/fisiologia , Genes Supressores de Tumor , Neoplasias Hepáticas Experimentais/genética , Proteínas ral de Ligação ao GTP/fisiologia , Animais , Proliferação de Células/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Transdução de Sinais/genética
20.
Proc Natl Acad Sci U S A ; 113(14): E2057-65, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-27006499

RESUMO

Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-ß, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC.


Assuntos
Elementos de DNA Transponíveis/genética , Mutagênese , Proteína Smad4/genética , Neoplasias Gástricas/genética , Transposases/genética , Junções Aderentes/genética , Animais , Cromatina/metabolismo , Humanos , Camundongos , Camundongos Knockout , Oncogenes , Neoplasias Gástricas/patologia
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