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1.
Cell ; 155(2): 397-409, 2013 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-24120138

RESUMEN

The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.


Asunto(s)
Neoplasias de la Mama/metabolismo , Eliminación de Gen , Neoplasias Mamarias Experimentales/metabolismo , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Animales , Secuencia de Bases , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Exones , Femenino , Técnicas de Inactivación de Genes , Xenoinjertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Mutación , Metástasis de la Neoplasia , Trasplante de Neoplasias , Empalme del ARN
2.
Genes Dev ; 30(9): 1020-33, 2016 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-27125672

RESUMEN

Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of pyruvate kinase (PK), and PKM2 expression is closely linked to embryogenesis, tissue regeneration, and cancer. To interrogate the functional requirement for PKM2 during development and tissue homeostasis, we generated germline PKM2-null mice (Pkm2(-/-)). Unexpectedly, despite being the primary isoform expressed in most wild-type adult tissues, we found that Pkm2(-/-) mice are viable and fertile. Thus, PKM2 is not required for embryonic or postnatal development. Loss of PKM2 leads to compensatory expression of PKM1 in the tissues that normally express PKM2. Strikingly, PKM2 loss leads to spontaneous development of hepatocellular carcinoma (HCC) with high penetrance that is accompanied by progressive changes in systemic metabolism characterized by altered systemic glucose homeostasis, inflammation, and hepatic steatosis. Therefore, in addition to its role in cancer metabolism, PKM2 plays a role in controlling systemic metabolic homeostasis and inflammation, thereby preventing HCC by a non-cell-autonomous mechanism.


Asunto(s)
Carcinoma Hepatocelular/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Metabolismo Energético/genética , Neoplasias Hepáticas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Hormonas Tiroideas/genética , Hormonas Tiroideas/metabolismo , Animales , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/fisiopatología , Proliferación Celular/genética , Dieta Alta en Grasa , Embrión de Mamíferos , Desarrollo Embrionario/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Mutación de Línea Germinal , Crecimiento y Desarrollo/genética , Hepatocitos/citología , Homeostasis/genética , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/fisiopatología , Masculino , Ratones , Isoformas de Proteínas , Proteínas de Unión a Hormona Tiroide
3.
Mol Cell ; 57(1): 95-107, 2015 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-25482511

RESUMEN

Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2 deletion affects proliferation and metabolism in nontransformed, nonimmortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis.


Asunto(s)
Fibroblastos/metabolismo , Metaboloma/genética , Nucleótidos/metabolismo , Piruvato Quinasa/genética , Animales , Ciclo Celular/genética , Proliferación Celular , ADN/biosíntesis , Embrión de Mamíferos , Fibroblastos/citología , Regulación de la Expresión Génica , Redes y Vías Metabólicas/genética , Ratones , Ratones Noqueados , Cultivo Primario de Células , Piruvato Quinasa/deficiencia , Transducción de Señal
4.
Genes Dev ; 27(14): 1557-67, 2013 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-23873940

RESUMEN

Metastasis accounts for the vast majority of cancer-related deaths, yet the molecular mechanisms that drive metastatic spread remain poorly understood. Here we report that Tks5, which has been linked to the formation of proteolytic cellular protrusions known as invadopodia, undergoes an isoform switch during metastatic progression in a genetically engineered mouse model of lung adenocarcinoma. Nonmetastatic primary tumor-derived cells predominantly expressed a short isoform, Tks5short, while metastatic primary tumor- and metastasis-derived cells acquired increased expression of the full-length isoform Tks5long. This elevation of Tks5long to Tks5short ratio correlated with a commensurate increase in invadopodia activity in metastatic cells compared with nonmetastatic cells. Further characterization of these isoforms by knockdown and overexpression experiments demonstrated that Tks5long promoted invadopodia in vitro and increased metastasis in transplant models and an autochthonous model of lung adenocarcinoma. Conversely, Tks5short decreased invadopodia stability and proteolysis, acting as a natural dominant-negative inhibitor to Tks5long. Importantly, high Tks5long and low Tks5short expressions in human lung adenocarcinomas correlated with metastatic disease and predicted worse survival of early stage patients. These data indicate that tipping the Tks5 isoform balance to a high Tks5long to Tks5short ratio promotes invadopodia-mediated invasion and metastasis.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Fosfoproteínas/genética , Adenocarcinoma/mortalidad , Adenocarcinoma del Pulmón , Animales , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/mortalidad , Ratones , Ratones Desnudos , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Proteínas de Unión a Fosfato , Isoformas de Proteínas , Análisis de Supervivencia
5.
Proc Natl Acad Sci U S A ; 114(28): E5625-E5634, 2017 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-28652369

RESUMEN

The extracellular microenvironment is an integral component of normal and diseased tissues that is poorly understood owing to its complexity. To investigate the contribution of the microenvironment to lung fibrosis and adenocarcinoma progression, two pathologies characterized by excessive stromal expansion, we used mouse models to characterize the extracellular matrix (ECM) composition of normal lung, fibrotic lung, lung tumors, and metastases. Using quantitative proteomics, we identified and assayed the abundance of 113 ECM proteins, which revealed robust ECM protein signatures unique to fibrosis, primary tumors, or metastases. These analyses indicated significantly increased abundance of several S100 proteins, including Fibronectin and Tenascin-C (Tnc), in primary lung tumors and associated lymph node metastases compared with normal tissue. We further showed that Tnc expression is repressed by the transcription factor Nkx2-1, a well-established suppressor of metastatic progression. We found that increasing the levels of Tnc, via CRISPR-mediated transcriptional activation of the endogenous gene, enhanced the metastatic dissemination of lung adenocarcinoma cells. Interrogation of human cancer gene expression data revealed that high TNC expression correlates with worse prognosis for lung adenocarcinoma, and that a three-gene expression signature comprising TNC, S100A10, and S100A11 is a robust predictor of patient survival independent of age, sex, smoking history, and mutational load. Our findings suggest that the poorly understood ECM composition of the fibrotic and tumor microenvironment is an underexplored source of diagnostic markers and potential therapeutic targets for cancer patients.


Asunto(s)
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Proteómica/métodos , Tenascina/fisiología , Adenocarcinoma/metabolismo , Animales , Anexina A2/metabolismo , Sistemas CRISPR-Cas , Progresión de la Enfermedad , Matriz Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis Multivariante , Metástasis de la Neoplasia , Pronóstico , Proteínas S100/metabolismo , Factor Nuclear Tiroideo 1/metabolismo , Resultado del Tratamiento , Microambiente Tumoral
6.
Genes Dev ; 25(14): 1470-5, 2011 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-21764851

RESUMEN

Small cell lung cancer (SCLC) is an aggressive cancer often diagnosed after it has metastasized. Despite the need to better understand this disease, SCLC remains poorly characterized at the molecular and genomic levels. Using a genetically engineered mouse model of SCLC driven by conditional deletion of Trp53 and Rb1 in the lung, we identified several frequent, high-magnitude focal DNA copy number alterations in SCLC. We uncovered amplification of a novel, oncogenic transcription factor, Nuclear factor I/B (Nfib), in the mouse SCLC model and in human SCLC. Functional studies indicate that NFIB regulates cell viability and proliferation during transformation.


Asunto(s)
Factores de Transcripción NFI/genética , Factores de Transcripción NFI/metabolismo , Oncogenes/fisiología , Carcinoma Pulmonar de Células Pequeñas/genética , Animales , Animales Modificados Genéticamente , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Oncogenes/genética
7.
EMBO Rep ; 17(12): 1721-1730, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27856534

RESUMEN

A major metabolic aberration associated with cancer is a change in glucose metabolism. Isoform selection of the glycolytic enzyme pyruvate kinase has been implicated in the metabolic phenotype of cancer cells, and specific pyruvate kinase isoforms have been suggested to support divergent energetic and biosynthetic requirements of cells in tumors and normal tissues. PKM2 isoform expression has been closely linked to embryogenesis, tissue repair, and cancer. In contrast, forced expression of the PKM1 isoform has been associated with reduced tumor cell proliferation. Here, we discuss the role that PKM2 plays in cells and provide a historical perspective for how the study of PKM2 has contributed to understanding cancer metabolism. We also review recent studies that raise important questions with regard to the role of PKM2 in both normal and cancer cell metabolism.


Asunto(s)
Proteínas Portadoras/metabolismo , Glucólisis , Proteínas de la Membrana/metabolismo , Neoplasias/metabolismo , Hormonas Tiroideas/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Proliferación Celular , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de la Membrana/genética , Redes y Vías Metabólicas , Ratones , Neoplasias/genética , Piruvato Quinasa/metabolismo , Hormonas Tiroideas/genética , Proteínas de Unión a Hormona Tiroide
8.
Nature ; 473(7345): 101-4, 2011 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-21471965

RESUMEN

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of Kras(LSL-G12D/+);p53(flox/flox) mice initiates lung adenocarcinoma development. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/fisiopatología , Adenocarcinoma del Pulmón , Animales , Diferenciación Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación hacia Abajo , Proteína HMGA2/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatología , Ratones , Factor Nuclear Tiroideo 1
9.
Gigascience ; 132024 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-38451475

RESUMEN

BACKGROUND: Organoids are 3-dimensional experimental models that summarize the anatomical and functional structure of an organ. Although a promising experimental model for precision medicine, patient-derived tumor organoids (PDTOs) have currently been developed only for a fraction of tumor types. RESULTS: We have generated the first multi-omic dataset (whole-genome sequencing [WGS] and RNA-sequencing [RNA-seq]) of PDTOs from the rare and understudied pulmonary neuroendocrine tumors (n = 12; 6 grade 1, 6 grade 2) and provide data from other rare neuroendocrine neoplasms: small intestine (ileal) neuroendocrine tumors (n = 6; 2 grade 1 and 4 grade 2) and large-cell neuroendocrine carcinoma (n = 5; 1 pancreatic and 4 pulmonary). This dataset includes a matched sample from the parental sample (primary tumor or metastasis) for a majority of samples (21/23) and longitudinal sampling of the PDTOs (1 to 2 time points), for a total of n = 47 RNA-seq and n = 33 WGS. We here provide quality control for each technique and the raw and processed data as well as all scripts for genomic analyses to ensure an optimal reuse of the data. In addition, we report gene expression data and somatic small variant calls and describe how they were generated, in particular how we used WGS somatic calls to train a random forest classifier to detect variants in tumor-only RNA-seq. We also report all histopathological images used for medical diagnosis: hematoxylin and eosin-stained slides, brightfield images, and immunohistochemistry images of protein markers of clinical relevance. CONCLUSIONS: This dataset will be critical to future studies relying on this PDTO biobank, such as drug screens for novel therapies and experiments investigating the mechanisms of carcinogenesis in these understudied diseases.


Asunto(s)
Multiómica , Tumores Neuroendocrinos , Humanos , Tumores Neuroendocrinos/genética , Eosina Amarillenta-(YS) , Genómica
10.
Cell Rep ; 42(4): 112335, 2023 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-37027294

RESUMEN

Neurons require large amounts of energy, but whether they can perform glycolysis or require glycolysis to maintain energy remains unclear. Using metabolomics, we show that human neurons do metabolize glucose through glycolysis and can rely on glycolysis to supply tricarboxylic acid (TCA) cycle metabolites. To investigate the requirement for glycolysis, we generated mice with postnatal deletion of either the dominant neuronal glucose transporter (GLUT3cKO) or the neuronal-enriched pyruvate kinase isoform (PKM1cKO) in CA1 and other hippocampal neurons. GLUT3cKO and PKM1cKO mice show age-dependent learning and memory deficits. Hyperpolarized magnetic resonance spectroscopic (MRS) imaging shows that female PKM1cKO mice have increased pyruvate-to-lactate conversion, whereas female GLUT3cKO mice have decreased conversion, body weight, and brain volume. GLUT3KO neurons also have decreased cytosolic glucose and ATP at nerve terminals, with spatial genomics and metabolomics revealing compensatory changes in mitochondrial bioenergetics and galactose metabolism. Therefore, neurons metabolize glucose through glycolysis in vivo and require glycolysis for normal function.


Asunto(s)
Metabolismo Energético , Glucólisis , Humanos , Femenino , Ratones , Animales , Glucólisis/fisiología , Imagen por Resonancia Magnética , Neuronas/metabolismo , Glucosa/metabolismo
11.
Cancer Cell ; 41(12): 2083-2099.e9, 2023 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-38086335

RESUMEN

Neuroendocrine neoplasms (NENs) comprise well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). Treatment options for patients with NENs are limited, in part due to lack of accurate models. We establish patient-derived tumor organoids (PDTOs) from pulmonary NETs and derive PDTOs from an understudied subtype of NEC, large cell neuroendocrine carcinoma (LCNEC), arising from multiple body sites. PDTOs maintain the gene expression patterns, intra-tumoral heterogeneity, and evolutionary processes of parental tumors. Through hypothesis-driven drug sensitivity analyses, we identify ASCL1 as a potential biomarker for response of LCNEC to treatment with BCL-2 inhibitors. Additionally, we discover a dependency on EGF in pulmonary NET PDTOs. Consistent with these findings, we find that, in an independent cohort, approximately 50% of pulmonary NETs express EGFR. This study identifies an actionable vulnerability for a subset of pulmonary NETs, emphasizing the utility of these PDTO models.


Asunto(s)
Carcinoma Neuroendocrino , Neoplasias Pulmonares , Tumores Neuroendocrinos , Neoplasias Pancreáticas , Humanos , Tumores Neuroendocrinos/tratamiento farmacológico , Tumores Neuroendocrinos/genética , Tumores Neuroendocrinos/metabolismo , Carcinoma Neuroendocrino/tratamiento farmacológico , Carcinoma Neuroendocrino/genética , Carcinoma Neuroendocrino/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pancreáticas/patología
13.
Cancer Metab ; 6: 17, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30386596

RESUMEN

BACKGROUND: While most cancer cells preferentially express the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2), PKM2 is dispensable for tumor development in several mouse cancer models. PKM2 is expressed in human pancreatic cancer, and there have been conflicting reports on the association of PKM2 expression and pancreatic cancer patient survival, but whether PKM2 is required for pancreatic cancer progression is unknown. To investigate the role of PKM2 in pancreatic cancer, we used a conditional allele to delete PKM2 in a mouse model of pancreatic ductal adenocarcinoma (PDAC). RESULTS: PDAC tumors were initiated in LSL-Kras G12D/+ ;Trp53 flox/flox ;Pdx-1-Cre (KP-/-C) mice harboring a conditional Pkm2 allele. Immunohistochemical analysis showed PKM2 expression in wild-type tumors and loss of PKM2 expression in tumors from Pkm2 conditional mice. PKM2 deletion had no effect on overall survival or tumor size. Loss of PKM2 resulted in pyruvate kinase M1 (PKM1) expression, but did not affect the number of proliferating cells. These findings are consistent with results in other cancer models. CONCLUSIONS: PKM2 is not required for initiation or growth of PDAC tumors arising in the KP-/-C pancreatic cancer model. These findings suggest that, in this mouse PDAC model, PKM2 expression is not required for pancreatic tumor formation or progression.

14.
Cancer Metab ; 6: 6, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29854399

RESUMEN

BACKGROUND: Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of the glycolytic enzyme pyruvate kinase. PKM2 expression is associated with embryogenesis, tissue regeneration, and cancer. PKM2 is also the pyruvate kinase isoform expressed in most wild-type adult tissues, with PKM1 restricted primarily to skeletal muscle, heart, and brain. To interrogate the functional requirement for PKM2 during tumor initiation in an autochthonous mouse model for soft tissue sarcoma (STS), we used a conditional Pkm2 allele (Pkm2fl ) to abolish PKM2 expression. RESULTS: PKM2 deletion slowed tumor onset but did not abrogate eventual tumor outgrowth. PKM2-null sarcoma cells expressed PKM1 with tumors containing a high number of infiltrating PKM2 expressing stromal cells. End-stage PKM2-null tumors showed increased proliferation compared to tumors with a wild-type Pkm2 allele, and tumor metabolite analysis revealed metabolic changes associated with PKM2 loss. CONCLUSIONS: While PKM2 is not required for soft tissue sarcoma growth, PKM2 expression may facilitate initiation of this tumor type. Because these data differ from what has been observed in other cancer models where PKM2 has been deleted, they argue that the consequences of PKM2 loss during tumor initiation are dependent on the tumor type.

15.
Cell Res ; 27(7): 851-852, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28429766

RESUMEN

Single Lgr5 intestinal stem cells (ISCs) can be expanded in vitro into epithelial organoids or "mini-guts", self-organizing cellular structures that recreate the intestinal differentiation program; Paneth cells, which constitute the intestinal stem cell niche, secrete stem cell growth signals, and are thus essential for this process. In a recent paper published in Nature, Rodríguez-Colman et al. describe how Paneth cells may be supporting the metabolic state of ISCs.


Asunto(s)
Células de Paneth , Células Madre , Intestinos , Transducción de Señal , Nicho de Células Madre
16.
Cancer Metab ; 5: 10, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29214019

RESUMEN

BACKGROUND: Cancer cells express the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2). PKM2 expression is not required for some cancers, and PKM2 loss can promote cancer progression; however, PKM2 has been reported to be essential in other tumor contexts, including a proposed non-metabolic role in ß-catenin nuclear translocation. PKM2 is expressed in colon cancers where loss of the Apc tumor suppressor results in ß-catenin nuclear translocation and aberrant activation of the canonical Wnt signaling pathway. Whether PKM2 is required in this colon cancer context has not been investigated. RESULTS: Colon tumorigenesis was induced in mice harboring conditional Apc and Pkm2 alleles, and tumor progression was monitored by serial colonoscopy. PKM2 deletion had no effect on overall survival, the number of mice that developed tumors, or the number of tumors that developed per animal. Immunohistochemical analysis demonstrated PKM2 expression in wild-type tumors and the expected loss of PKM2 expression in tumors from Pkm2 conditional mice. Loss of PKM2 resulted in pyruvate kinase M1 expression but had no effect on nuclear ß-catenin staining. These findings are consistent with tumor growth and activated Wnt signaling despite PKM2 loss in this model. We also found a large fraction of human colon cancers had very low or undetectable levels of PKM2 expression. CONCLUSIONS: PKM2 is not required for Apc-deficient colon cancer or for nuclear translocation of ß-catenin in Apc-null tumor cells. These findings suggest that PKM2 expression is not required for colon tumor formation or progression.

18.
Nat Med ; 20(10): 1193-1198, 2014 10.
Artículo en Inglés | MEDLINE | ID: mdl-25261994

RESUMEN

Most patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed with advanced disease and survive less than 12 months. PDAC has been linked with obesity and glucose intolerance, but whether changes in circulating metabolites are associated with early cancer progression is unknown. To better understand metabolic derangements associated with early disease, we profiled metabolites in prediagnostic plasma from individuals with pancreatic cancer (cases) and matched controls from four prospective cohort studies. We find that elevated plasma levels of branched-chain amino acids (BCAAs) are associated with a greater than twofold increased risk of future pancreatic cancer diagnosis. This elevated risk was independent of known predisposing factors, with the strongest association observed among subjects with samples collected 2 to 5 years before diagnosis, when occult disease is probably present. We show that plasma BCAAs are also elevated in mice with early-stage pancreatic cancers driven by mutant Kras expression but not in mice with Kras-driven tumors in other tissues, and that breakdown of tissue protein accounts for the increase in plasma BCAAs that accompanies early-stage disease. Together, these findings suggest that increased whole-body protein breakdown is an early event in development of PDAC.


Asunto(s)
Aminoácidos de Cadena Ramificada/sangre , Carcinoma Ductal Pancreático/sangre , Neoplasias Pancreáticas/sangre , Adulto , Anciano , Anciano de 80 o más Años , Animales , Carcinoma Ductal Pancreático/etiología , Estudios de Casos y Controles , Estudios de Cohortes , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Neoplasias Pancreáticas/etiología , Estudios Prospectivos , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de Riesgo , Factores de Tiempo
19.
Langmuir ; 23(8): 4432-7, 2007 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-17352500

RESUMEN

This work describes the immobilization of beta-galactosidase onto polyelectrolyte multilayer assemblies of the polyanion poly[1-[4-(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido]-1,2-ethanediyl, sodium salt] (PAZO) and the polycation poly(ethylenimine) (PEI) constructed by electrostatic self-assembly (ESA). A single layer of beta-galactosidase was deposited over a precursor film comprising up to five bilayers of the PEI/PAZO polyelectrolyte pair. The enzyme was deposited on both the polycationic (PEI) and the polyanionic (PAZO) surfaces. Quartz crystal microbalance with dissipation monitoring (QCM-D), single-wavelength ellipsometry, and UV-visible absorption spectroscopy revealed differences in both the amount of beta-galactosidase incorporated in each of the multilayer assemblies and the resulting enzyme packing density in the films. The enzymatic films were immersed in a reaction solution containing o-nitrophenyl-beta-d-galactopyranoside (ONPG), and absorbance measurements were used to monitor the concentration of o-nitrophenyl (ONP), the product of the beta-galactosidase catalyzed by hydrolysis of ONPG. Although our data indicate that comparable amounts of beta-galactosidase are incorporated onto both surfaces, enzymatic activity is substantially inhibited when the beta-galactosidase is immobilized on the polyanionic surface compared to the enzyme on the polycationic surface. The difference in catalytic activities reflects the different abilities of the two polyelectrolytes to screen the protein's active site from the substrate environment. In both assemblies, the protein interpenetrated the PEI/PAZO multilayer, disrupting the J-aggregated state of the PAZO chromophores. This work demonstrates that the charge, conformation, and composition of underlying polyelectrolyte cushions have a significant effect on the structure and function of an immobilized protein within functional nanoassemblies.


Asunto(s)
Química Física/métodos , Electroquímica/métodos , Iones/química , beta-Galactosidasa/química , Adsorción , Aniones , Catálisis , Electrólitos , Enzimas Inmovilizadas/química , Hidrólisis , Modelos Químicos , Nanopartículas/química , Espectrofotometría Ultravioleta , Electricidad Estática , Propiedades de Superficie
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