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1.
Cell ; 160(1-2): 324-38, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25557080

RESUMO

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.


Assuntos
Carcinoma Ductal Pancreático/patologia , Modelos Biológicos , Técnicas de Cultura de Órgãos , Organoides/patologia , Neoplasias Pancreáticas/patologia , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Pâncreas/metabolismo , Pâncreas/patologia
2.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34021083

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4BG12D), and nontransforming cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRASG12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.


Assuntos
Carcinoma Ductal Pancreático/genética , Neurofibromina 1/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Camundongos , Mutação , Pâncreas/patologia , Proteínas Repressoras/genética , Transdução de Sinais/genética
3.
Nature ; 487(7408): 510-3, 2012 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-22763454

RESUMO

Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. Although these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as a candidate gene enriched in CTCs. Expression of WNT2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. This effect is correlated with fibronectin upregulation and suppressed by inhibition of MAP3K7 (also known as TAK1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple WNT genes, and pancreatic CTCs revealed enrichment for WNT signalling in 5 out of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Metástase Neoplásica/genética , Células Neoplásicas Circulantes/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , Animais , Sobrevivência Celular , Inibição de Contato , Modelos Animais de Doenças , Genes Neoplásicos/genética , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Camundongos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Análise de Sequência de RNA , Proteínas Wnt/genética , Proteína Wnt2/genética , Proteína Wnt2/metabolismo
4.
Nature ; 468(7324): 659-63, 2010 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-21124451

RESUMO

Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. Here we show that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint.


Assuntos
Metabolismo Energético , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Autofagia , Medula Óssea/metabolismo , Medula Óssea/patologia , Ciclo Celular , Proliferação de Células , Sobrevivência Celular , Ativação Enzimática , Feminino , Hematopoese , Células-Tronco Hematopoéticas/patologia , Homeostase , Metabolismo dos Lipídeos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Complexos Multiproteicos , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
5.
Cancer Discov ; 10(10): 1566-1589, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32703770

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common malignancy, with little improvement in patient outcomes over the past decades. Recently, subtypes of pancreatic cancer with different prognoses have been elaborated; however, the inability to model these subtypes has precluded mechanistic investigation of their origins. Here, we present a xenotransplantation model of PDAC in which neoplasms originate from patient-derived organoids injected directly into murine pancreatic ducts. Our model enables distinction of the two main PDAC subtypes: intraepithelial neoplasms from this model progress in an indolent or invasive manner representing the classical or basal-like subtypes of PDAC, respectively. Parameters that influence PDAC subtype specification in this intraductal model include cell plasticity and hyperactivation of the RAS pathway. Finally, through intratumoral dissection and the direct manipulation of RAS gene dosage, we identify a suite of RAS-regulated secreted and membrane-bound proteins that may represent potential candidates for therapeutic intervention in patients with PDAC. SIGNIFICANCE: Accurate modeling of the molecular subtypes of pancreatic cancer is crucial to facilitate the generation of effective therapies. We report the development of an intraductal organoid transplantation model of pancreatic cancer that models the progressive switching of subtypes, and identify stochastic and RAS-driven mechanisms that determine subtype specification.See related commentary by Pickering and Morton, p. 1448.This article is highlighted in the In This Issue feature, p. 1426.


Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica/genética , Ductos Pancreáticos/transplante , Animais , Carcinoma Ductal Pancreático , Modelos Animais de Doenças , Humanos , Camundongos , Prognóstico
6.
Cancer Discov ; 9(9): 1268-1287, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31263025

RESUMO

Activating KRAS mutations are found in nearly all cases of pancreatic ductal adenocarcinoma (PDAC), yet effective clinical targeting of oncogenic KRAS remains elusive. Understanding of KRAS-dependent PDAC-promoting pathways could lead to the identification of vulnerabilities and the development of new treatments. We show that oncogenic KRAS induces BNIP3L/NIX expression and a selective mitophagy program that restricts glucose flux to the mitochondria and enhances redox capacity. Loss of Nix restores functional mitochondria to cells, increasing demands for NADPH reducing power and decreasing proliferation in glucose-limited conditions. Nix deletion markedly delays progression of pancreatic cancer and improves survival in a murine (KPC) model of PDAC. Although conditional Nix ablation in vivo initially results in the accumulation of mitochondria, mitochondrial content eventually normalizes via increased mitochondrial clearance programs, and pancreatic intraepithelial neoplasia (PanIN) lesions progress to PDAC. We identify the KRAS-NIX mitophagy program as a novel driver of glycolysis, redox robustness, and disease progression in PDAC. SIGNIFICANCE: NIX-mediated mitophagy is a new oncogenic KRAS effector pathway that suppresses functional mitochondrial content to stimulate cell proliferation and augment redox homeostasis. This pathway promotes the progression of PanIN to PDAC and represents a new dependency in pancreatic cancer.This article is highlighted in the In This Issue feature, p. 1143.


Assuntos
Carcinoma Ductal Pancreático/patologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glicólise , Humanos , Proteínas de Membrana/genética , Camundongos , Mitofagia , Mutação , NADP/metabolismo , Transplante de Neoplasias , Oxirredução , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Supressoras de Tumor/genética
7.
Science ; 364(6446): 1156-1162, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31221853

RESUMO

Glycosylation alterations are indicative of tissue inflammation and neoplasia, but whether these alterations contribute to disease pathogenesis is largely unknown. To study the role of glycan changes in pancreatic disease, we inducibly expressed human fucosyltransferase 3 and ß1,3-galactosyltransferase 5 in mice, reconstituting the glycan sialyl-Lewisa, also known as carbohydrate antigen 19-9 (CA19-9). Notably, CA19-9 expression in mice resulted in rapid and severe pancreatitis with hyperactivation of epidermal growth factor receptor (EGFR) signaling. Mechanistically, CA19-9 modification of the matricellular protein fibulin-3 increased its interaction with EGFR, and blockade of fibulin-3, EGFR ligands, or CA19-9 prevented EGFR hyperactivation in organoids. CA19-9-mediated pancreatitis was reversible and could be suppressed with CA19-9 antibodies. CA19-9 also cooperated with the KrasG12D oncogene to produce aggressive pancreatic cancer. These findings implicate CA19-9 in the etiology of pancreatitis and pancreatic cancer and nominate CA19-9 as a therapeutic target.


Assuntos
Antígeno CA-19-9/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pancreáticas/metabolismo , Pancreatite/metabolismo , Doença Aguda , Animais , Antígeno CA-19-9/imunologia , Carcinogênese/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Doença Crônica , Proteínas da Matriz Extracelular/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Glicosilação , Humanos , Camundongos , Terapia de Alvo Molecular/métodos , Neoplasias Pancreáticas/patologia , Pancreatite/patologia
8.
Clin Cancer Res ; 21(2): 396-404, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25348516

RESUMO

PURPOSE: Improved therapeutic approaches are needed for the treatment of pancreatic ductal adenocarcinoma (PDAC). As dual MEK and PI3K inhibition is presently being used in clinical trials for patients with PDAC, we sought to test the efficacy of combined targeting of these pathways in PDAC using both in vitro drug screens and genetically engineered mouse models (GEMM). EXPERIMENTAL DESIGN: We performed high-throughput screening of >500 human cancer cell lines (including 46 PDAC lines), for sensitivity to 50 clinically relevant compounds, including MEK and PI3K inhibitors. We tested the top hit in the screen, the MEK1/2 inhibitor, AZD6244, for efficacy alone or in combination with the PI3K inhibitors, BKM120 or GDC-0941, in a Kras(G12D)-driven GEMM that recapitulates the histopathogenesis of human PDAC. RESULTS: In vitro screens revealed that PDAC cell lines are relatively resistant to single-agent therapies. The response profile to the MEK1/2 inhibitor, AZD6244, was an outlier, showing the highest selective efficacy in PDAC. Although MEK inhibition alone was mainly cytostatic, apoptosis was induced when combined with PI3K inhibitors (BKM120 or GDC-0941). When tested in a PDAC GEMM and compared with the single agents or vehicle controls, the combination delayed tumor formation in the setting of prevention and extended survival when used to treat advanced tumors, although no durable responses were observed. CONCLUSIONS: Our studies point to important contributions of MEK and PI3K signaling to PDAC pathogenesis and suggest that dual targeting of these pathways may provide benefit in some patients with PDAC. Clin Cancer Res; 21(2); 396-404. ©2014 AACR.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Aminopiridinas , Animais , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Cloridrato de Erlotinib , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Camundongos Transgênicos , Morfolinas , Inibidores de Fosfoinositídeo-3 Quinase , Quinazolinas/farmacologia
9.
Cancer Res ; 72(18): 4840-5, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22787119

RESUMO

The TGF-ß pathway is under active consideration as a cancer drug target based on its capacity to promote cancer cell invasion and to create a protumorigenic microenvironment. However, the clinical application of TGF-ß inhibitors remains uncertain as genetic studies show a tumor suppressor function of TGF-ß in pancreatic cancer and other epithelial malignancies. Here, we used genetically engineered mouse models to investigate the therapeutic impact of global TGF-ß inhibition in pancreatic cancer in relation to tumor stage, genetic profile, and concurrent chemotherapy. We found that αvß6 integrin acted as a key upstream activator of TGF-ß in evolving pancreatic cancers. In addition, TGF-ß or αvß6 blockade increased tumor cell proliferation and accelerated both early and later disease stages. These effects were dependent on the presence of Smad4, a central mediator of TGF-ß signaling. Therefore, our findings indicate that αvß6 and TGF-ß act in a common tumor suppressor pathway whose pharmacologic inactivation promotes pancreatic cancer progression.


Assuntos
Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Modelos Animais de Doenças , Progressão da Doença , Imuno-Histoquímica , Camundongos
10.
Sci Transl Med ; 4(126): 126ra34, 2012 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-22440736

RESUMO

Testosterone is necessary for the development of male pattern baldness, known as androgenetic alopecia (AGA); yet, the mechanisms for decreased hair growth in this disorder are unclear. We show that prostaglandin D(2) synthase (PTGDS) is elevated at the mRNA and protein levels in bald scalp compared to haired scalp of men with AGA. The product of PTGDS enzyme activity, prostaglandin D(2) (PGD(2)), is similarly elevated in bald scalp. During normal follicle cycling in mice, Ptgds and PGD(2) levels increase immediately preceding the regression phase, suggesting an inhibitory effect on hair growth. We show that PGD(2) inhibits hair growth in explanted human hair follicles and when applied topically to mice. Hair growth inhibition requires the PGD(2) receptor G protein (heterotrimeric guanine nucleotide)-coupled receptor 44 (GPR44), but not the PGD(2) receptor 1 (PTGDR). Furthermore, we find that a transgenic mouse, K14-Ptgs2, which targets prostaglandin-endoperoxide synthase 2 expression to the skin, demonstrates elevated levels of PGD(2) in the skin and develops alopecia, follicular miniaturization, and sebaceous gland hyperplasia, which are all hallmarks of human AGA. These results define PGD(2) as an inhibitor of hair growth in AGA and suggest the PGD(2)-GPR44 pathway as a potential target for treatment.


Assuntos
Alopecia/metabolismo , Cabelo/efeitos dos fármacos , Cabelo/crescimento & desenvolvimento , Prostaglandina D2/metabolismo , Couro Cabeludo/metabolismo , Alopecia/enzimologia , Animais , Epiderme/efeitos dos fármacos , Epiderme/enzimologia , Feminino , Perfilação da Expressão Gênica , Cabelo/enzimologia , Folículo Piloso/efeitos dos fármacos , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Prostaglandina D2/análogos & derivados , Prostaglandina D2/análise , Prostaglandina D2/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Couro Cabeludo/efeitos dos fármacos , Couro Cabeludo/enzimologia
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