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1.
Cell ; 181(2): 424-441.e21, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32234521

RESUMO

KRAS mutant pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic response that promotes hypovascularity, immunosuppression, and resistance to chemo- and immunotherapies. We show that a combination of MEK and CDK4/6 inhibitors that target KRAS-directed oncogenic signaling can suppress PDAC proliferation through induction of retinoblastoma (RB) protein-mediated senescence. In preclinical mouse models of PDAC, this senescence-inducing therapy produces a senescence-associated secretory phenotype (SASP) that includes pro-angiogenic factors that promote tumor vascularization, which in turn enhances drug delivery and efficacy of cytotoxic gemcitabine chemotherapy. In addition, SASP-mediated endothelial cell activation stimulates the accumulation of CD8+ T cells into otherwise immunologically "cold" tumors, sensitizing tumors to PD-1 checkpoint blockade. Therefore, in PDAC models, therapy-induced senescence can establish emergent susceptibilities to otherwise ineffective chemo- and immunotherapies through SASP-dependent effects on the tumor vasculature and immune system.


Assuntos
Envelhecimento/fisiologia , Carcinoma Ductal Pancreático/patologia , Remodelação Vascular/fisiologia , Animais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/microbiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Genes ras/genética , Humanos , Imunoterapia/métodos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Neoplasias Pancreáticas/patologia , Proteína do Retinoblastoma/imunologia , Transdução de Sinais/genética , Microambiente Tumoral , Remodelação Vascular/genética
3.
Cell ; 161(7): 1539-1552, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-26091037

RESUMO

The adenomatous polyposis coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. To determine whether Apc disruption is required for tumor maintenance, we developed a mouse model of CRC whereby Apc can be conditionally suppressed using a doxycycline-regulated shRNA. Apc suppression produces adenomas in both the small intestine and colon that, in the presence of Kras and p53 mutations, can progress to invasive carcinoma. In established tumors, Apc restoration drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. Tumor regression is accompanied by the re-establishment of normal crypt-villus homeostasis, such that once aberrantly proliferating cells reacquire self-renewal and multi-lineage differentiation capability. Our study reveals that CRC cells can revert to functioning normal cells given appropriate signals and provide compelling in vivo validation of the Wnt pathway as a therapeutic target for treatment of CRC.


Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Intestino Grosso/patologia , Intestino Delgado/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Proliferação de Células , Neoplasias Colorretais/patologia , Doxiciclina/administração & dosagem , Genes p53 , Pólipos Intestinais/metabolismo , Pólipos Intestinais/patologia , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , Via de Sinalização Wnt
4.
Cell ; 153(2): 449-60, 2013 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-23562644

RESUMO

The p53 tumor suppressor can restrict malignant transformation by triggering cell-autonomous programs of cell-cycle arrest or apoptosis. p53 also promotes cellular senescence, a tumor-suppressive program that involves stable cell-cycle arrest and secretion of factors that modify the tissue microenvironment. In the presence of chronic liver damage, we show that ablation of a p53-dependent senescence program in hepatic stellate cells increases liver fibrosis and cirrhosis associated with reduced survival and enhances the transformation of adjacent epithelial cells into hepatocellular carcinoma. p53-expressing senescent stellate cells release factors that skew macrophage polarization toward a tumor-inhibiting M1-state capable of attacking senescent cells in culture, whereas proliferating p53-deficient stellate cells secrete factors that stimulate polarization of macrophages into a tumor-promoting M2-state and enhance the proliferation of premalignant cells. Hence, p53 can act non-cell autonomously to suppress tumorigenesis by promoting an antitumor microenvironment, in part, through secreted factors that modulate macrophage function.


Assuntos
Transformação Celular Neoplásica , Senescência Celular , Células Estreladas do Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Microambiente Celular , Fibrose/patologia , Células Estreladas do Fígado/citologia , Humanos , Inflamação/metabolismo , Células de Kupffer/metabolismo , Células de Kupffer/patologia , Fígado/citologia , Fígado/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , NF-kappa B
5.
Cell ; 134(4): 657-67, 2008 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-18724938

RESUMO

Cellular senescence acts as a potent mechanism of tumor suppression; however, its functional contribution to noncancer pathologies has not been examined. Here we show that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lacking key senescence regulators, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell-cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix-degrading enzymes, and enhanced immune surveillance. Accordingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the resolution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage.


Assuntos
Senescência Celular , Cirrose Hepática/imunologia , Fígado/citologia , Animais , Tetracloreto de Carbono , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Fígado/fisiologia , Cirrose Hepática/metabolismo , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/imunologia , Cirrose Hepática Experimental/metabolismo , Camundongos
6.
Proc Natl Acad Sci U S A ; 109(21): 8212-7, 2012 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-22566646

RESUMO

The large chromosomal deletions frequently observed in cancer genomes are often thought to arise as a "two-hit" mechanism in the process of tumor-suppressor gene (TSG) inactivation. Using a murine model system of hepatocellular carcinoma (HCC) and in vivo RNAi, we test an alternative hypothesis, that such deletions can arise from selective pressure to attenuate the activity of multiple genes. By targeting the mouse orthologs of genes frequently deleted on human 8p22 and adjacent regions, which are lost in approximately half of several other major epithelial cancers, we provide evidence suggesting that multiple genes on chromosome 8p can cooperatively inhibit tumorigenesis in mice, and that their cosuppression can synergistically promote tumor growth. In addition, in human HCC patients, the combined down-regulation of functionally validated 8p TSGs is associated with poor survival, in contrast to the down-regulation of any individual gene. Our data imply that large cancer-associated deletions can produce phenotypes distinct from those arising through loss of a single TSG, and as such should be considered and studied as distinct mutational events.


Assuntos
Carcinoma Hepatocelular/genética , Deleção de Genes , Genes Supressores de Tumor/fisiologia , Genômica/métodos , Neoplasias Hepáticas Experimentais/genética , Monossomia , Animais , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Transformada , Linhagem Celular Tumoral , Cromossomos Humanos Par 8 , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Haploinsuficiência/genética , Humanos , Fígado/citologia , Neoplasias Hepáticas Experimentais/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Interferência de RNA , Células-Tronco/citologia
7.
Proc Natl Acad Sci U S A ; 108(17): 7113-8, 2011 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-21482754

RESUMO

RNAi has revolutionized loss-of-function genetics by enabling sequence-specific suppression of virtually any gene. Furthermore, tetracycline response elements (TRE) can drive expression of short hairpin RNAs (shRNAs) for inducible and reversible target gene suppression. Here, we demonstrate the feasibility of transgenic inducible RNAi for suppression of essential genes. We set out to directly target cell proliferation by screening an RNAi library against DNA replication factors and identified multiple shRNAs against Replication Protein A, subunit 3 (RPA3). We generated transgenic mice with TRE-driven Rpa3 shRNAs whose expression enforced a reversible cell cycle arrest. In adult mice, the block in cell proliferation caused rapid atrophy of the intestinal epithelium which led to weight loss and lethality within 8-11 d of shRNA induction. Upon shRNA withdrawal, villus atrophy and weight loss were fully reversible. Thus, shRpa3 transgenic mice provide an interesting tool to study tissue maintenance and regeneration. Overall, we have established a robust system that serves the purpose of temperature-sensitive alleles in other model organisms, enabling inducible and reversible suppression of essential genes in a mammalian system.


Assuntos
Alelos , Ciclo Celular/fisiologia , Replicação do DNA/fisiologia , Interferência de RNA , Elementos de Resposta/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Proteína de Replicação A/metabolismo
8.
Nat Cancer ; 5(2): 315-329, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38177458

RESUMO

Metastatic gastric carcinoma is a highly lethal cancer that responds poorly to conventional and molecularly targeted therapies. Despite its clinical relevance, the mechanisms underlying the behavior and therapeutic response of this disease are poorly understood owing, in part, to a paucity of tractable models. Here we developed methods to somatically introduce different oncogenic lesions directly into the murine gastric epithelium. Genotypic configurations observed in patients produced metastatic gastric cancers that recapitulated the histological, molecular and clinical features of all nonviral molecular subtypes of the human disease. Applying this platform to both wild-type and immunodeficient mice revealed previously unappreciated links between the genotype, organotropism and immune surveillance of metastatic cells, which produced distinct patterns of metastasis that were mirrored in patients. Our results establish a highly portable platform for generating autochthonous cancer models with flexible genotypes and host backgrounds, which can unravel mechanisms of gastric tumorigenesis or test new therapeutic concepts.


Assuntos
Neoplasias Gástricas , Humanos , Camundongos , Animais , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Modelos Animais de Doenças , Mucosa Gástrica/patologia , Genótipo
9.
Nat Biotechnol ; 42(3): 437-447, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37563300

RESUMO

Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo. Combined with plasmid-based or synthetic guide RNAs, iBE drives efficient engineering of individual or multiple SNVs in intestinal, lung and pancreatic organoids. Temporal regulation of base editor activity allows controlled sequential genome editing ex vivo and in vivo, and delivery of sgRNAs directly to target tissues facilitates generation of in situ preclinical cancer models.


Assuntos
Edição de Genes , Neoplasias , Camundongos , Animais , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas , Neoplasias/genética , Neoplasias/terapia , Pulmão
10.
bioRxiv ; 2024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-38562717

RESUMO

Driver gene mutations can increase the metastatic potential of the primary tumor1-3, but their role in sustaining tumor growth at metastatic sites is poorly understood. A paradigm of such mutations is inactivation of SMAD4 - a transcriptional effector of TGFß signaling - which is a hallmark of multiple gastrointestinal malignancies4,5. SMAD4 inactivation mediates TGFß's remarkable anti- to pro-tumorigenic switch during cancer progression and can thus influence both tumor initiation and metastasis6-14. To determine whether metastatic tumors remain dependent on SMAD4 inactivation, we developed a mouse model of pancreatic ductal adenocarcinoma (PDAC) that enables Smad4 depletion in the pre-malignant pancreas and subsequent Smad4 reactivation in established metastases. As expected, Smad4 inactivation facilitated the formation of primary tumors that eventually colonized the liver and lungs. By contrast, Smad4 reactivation in metastatic disease had strikingly opposite effects depending on the tumor's organ of residence: suppression of liver metastases and promotion of lung metastases. Integrative multiomic analysis revealed organ-specific differences in the tumor cells' epigenomic state, whereby the liver and lungs harbored chromatin programs respectively dominated by the KLF and RUNX developmental transcription factors, with Klf4 depletion being sufficient to reverse Smad4's tumor-suppressive activity in liver metastases. Our results show how epigenetic states favored by the organ of residence can influence the function of driver genes in metastatic tumors. This organ-specific gene-chromatin interplay invites consideration of anatomical site in the interpretation of tumor genetics, with implications for the therapeutic targeting of metastatic disease.

11.
Nat Cancer ; 4(6): 872-892, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37142692

RESUMO

Immunotherapies that produce durable responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of the senescence-associated secretory phenotype (SASP) can be an effective approach to activate anti-tumor natural killer (NK) cell and T cell immunity. In the present study, we found that the pancreas tumor microenvironment suppresses NK cell and T cell surveillance after therapy-induced senescence through enhancer of zeste homolog 2 (EZH2)-mediated epigenetic repression of proinflammatory SASP genes. EZH2 blockade stimulated production of SASP chemokines CCL2 and CXCL9/10, leading to enhanced NK cell and T cell infiltration and PDAC eradication in mouse models. EZH2 activity was also associated with suppression of chemokine signaling and cytotoxic lymphocytes and reduced survival in patients with PDAC. These results demonstrate that EZH2 represses the proinflammatory SASP and that EZH2 inhibition combined with senescence-inducing therapy could be a powerful means to achieve immune-mediated tumor control in PDAC.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Camundongos , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Fenótipo Secretor Associado à Senescência , Microambiente Tumoral/genética
12.
Nat Cancer ; 3(11): 1367-1385, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36344707

RESUMO

The most prominent homozygous deletions in cancer affect chromosome 9p21.3 and eliminate CDKN2A/B tumor suppressors, disabling a cell-intrinsic barrier to tumorigenesis. Half of 9p21.3 deletions, however, also encompass a type I interferon (IFN) gene cluster; the consequences of this co-deletion remain unexplored. To functionally dissect 9p21.3 and other large genomic deletions, we developed a flexible deletion engineering strategy, MACHETE (molecular alteration of chromosomes with engineered tandem elements). Applying MACHETE to a syngeneic mouse model of pancreatic cancer, we found that co-deletion of the IFN cluster promoted immune evasion, metastasis and immunotherapy resistance. Mechanistically, IFN co-deletion disrupted type I IFN signaling in the tumor microenvironment, leading to marked changes in infiltrating immune cells and escape from CD8+ T-cell surveillance, effects largely driven by the poorly understood interferon epsilon. These results reveal a chromosomal deletion that disables both cell-intrinsic and cell-extrinsic tumor suppression and provide a framework for interrogating large deletions in cancer and beyond.


Assuntos
Interferons , Neoplasias , Animais , Camundongos , Deleção Cromossômica , Cromossomos , Evasão da Resposta Imune , Microambiente Tumoral/genética , Sequências de Repetição em Tandem
13.
Nat Biotechnol ; 40(6): 862-873, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35165384

RESUMO

Base editing can be applied to characterize single nucleotide variants of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular base-editing-activity 'sensors' that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor-sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated single nucleotide variants in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput base editing screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.


Assuntos
Edição de Genes , Neoplasias , Animais , Sistemas CRISPR-Cas/genética , Neoplasias/genética , Nucleotídeos , RNA Guia de Cinetoplastídeos/genética
14.
Nat Biotechnol ; 35(6): 577-582, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28459450

RESUMO

Colorectal cancer (CRC) is a leading cause of death in the developed world, yet facile preclinical models that mimic the natural stages of CRC progression are lacking. Through the orthotopic engraftment of colon organoids we describe a broadly usable immunocompetent CRC model that recapitulates the entire adenoma-adenocarcinoma-metastasis axis in vivo. The engraftment procedure takes less than 5 minutes, shows efficient tumor engraftment in two-thirds of mice, and can be achieved using organoids derived from genetically engineered mouse models (GEMMs), wild-type organoids engineered ex vivo, or from patient-derived human CRC organoids. In this model, we describe the genotype and time-dependent progression of CRCs from adenocarcinoma (6 weeks), to local disseminated disease (11-12 weeks), and spontaneous metastasis (>20 weeks). Further, we use the system to show that loss of dysregulated Wnt signaling is critical for the progression of disseminated CRCs. Thus, our approach provides a fast and flexible means to produce tailored CRC mouse models for genetic studies and pre-clinical investigation.


Assuntos
Neoplasias Colorretais/genética , Modelos Animais de Doenças , Edição de Genes/métodos , Genes Neoplásicos/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Transplante de Órgãos/métodos , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Metástase Neoplásica
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