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1.
Mol Cell ; 81(11): 2290-2302.e7, 2021 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-33831358

RESUMEN

Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis.


Asunto(s)
Carcinoma Ductal Pancreático/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Neoplasias Pancreáticas/genética , Células Estrelladas Pancreáticas/metabolismo , Escape del Tumor/efectos de los fármacos , Aloinjertos , Animales , Antineoplásicos/farmacología , Carbono/inmunología , Carbono/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/mortalidad , Línea Celular Tumoral , Formiatos/inmunología , Formiatos/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Redes y Vías Metabólicas/efectos de los fármacos , Redes y Vías Metabólicas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Oximas/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/mortalidad , Células Estrelladas Pancreáticas/efectos de los fármacos , Células Estrelladas Pancreáticas/inmunología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/inmunología , Serina/inmunología , Serina/metabolismo , Serina/farmacología , Transducción de Señal , Sulfonamidas/farmacología , Triptófano/inmunología , Triptófano/metabolismo , Triptófano/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunología
2.
Nature ; 544(7650): 372-376, 2017 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-28425994

RESUMEN

The non-essential amino acids serine and glycine are used in multiple anabolic processes that support cancer cell growth and proliferation (reviewed in ref. 1). While some cancer cells upregulate de novo serine synthesis, many others rely on exogenous serine for optimal growth. Restriction of dietary serine and glycine can reduce tumour growth in xenograft and allograft models. Here we show that this observation translates into more clinically relevant autochthonous tumours in genetically engineered mouse models of intestinal cancer (driven by Apc inactivation) or lymphoma (driven by Myc activation). The increased survival following dietary restriction of serine and glycine in these models was further improved by antagonizing the anti-oxidant response. Disruption of mitochondrial oxidative phosphorylation (using biguanides) led to a complex response that could improve or impede the anti-tumour effect of serine and glycine starvation. Notably, Kras-driven mouse models of pancreatic and intestinal cancers were less responsive to depletion of serine and glycine, reflecting an ability of activated Kras to increase the expression of enzymes that are part of the serine synthesis pathway and thus promote de novo serine synthesis.


Asunto(s)
Glicina/deficiencia , Neoplasias Intestinales/dietoterapia , Neoplasias Intestinales/metabolismo , Linfoma/dietoterapia , Linfoma/metabolismo , Serina/deficiencia , Animales , Antioxidantes/metabolismo , Biguanidas/farmacología , Línea Celular Tumoral , Dieta , Modelos Animales de Enfermedad , Femenino , Privación de Alimentos , Glicina/metabolismo , Humanos , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Linfoma/patología , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estado Nutricional , Fosforilación Oxidativa/efectos de los fármacos , Neoplasias Pancreáticas/dietoterapia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Serina/biosíntesis , Serina/metabolismo , Serina/farmacología , Tasa de Supervivencia
4.
Mol Cell ; 57(5): 860-872, 2015 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-25702873

RESUMEN

During apoptosis, the mitochondrial outer membrane is permeabilized, leading to the release of cytochrome c that activates downstream caspases. Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis. Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event. Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term "minority MOMP." Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death. Instead, this caspase activity leads to DNA damage that, in turn, promotes genomic instability, cellular transformation, and tumorigenesis. Our data demonstrate that, in contrast to its well-established tumor suppressor function, apoptosis also has oncogenic potential that is regulated by the extent of MOMP. These findings have important implications for oncogenesis following either physiological or therapeutic engagement of apoptosis.


Asunto(s)
Apoptosis/fisiología , Daño del ADN , Inestabilidad Genómica , Membranas Mitocondriales/fisiología , Animales , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Western Blotting , Caspasas/metabolismo , Línea Celular Tumoral , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/genética , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HCT116 , Células HeLa , Histonas/metabolismo , Humanos , Células MCF-7 , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Nitrofenoles/farmacología , Permeabilidad , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Estaurosporina/farmacología , Sulfonamidas/farmacología , Factores de Tiempo
5.
Br J Cancer ; 127(10): 1773-1786, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36115879

RESUMEN

BACKGROUND: Cellular metabolism is an integral component of cellular adaptation to stress, playing a pivotal role in the resistance of cancer cells to various treatment modalities, including radiotherapy. In response to radiotherapy, cancer cells engage antioxidant and DNA repair mechanisms which mitigate and remove DNA damage, facilitating cancer cell survival. Given the reliance of these resistance mechanisms on amino acid metabolism, we hypothesised that controlling the exogenous availability of the non-essential amino acids serine and glycine would radiosensitise cancer cells. METHODS: We exposed colorectal, breast and pancreatic cancer cell lines/organoids to radiation in vitro and in vivo in the presence and absence of exogenous serine and glycine. We performed phenotypic assays for DNA damage, cell cycle, ROS levels and cell death, combined with a high-resolution untargeted LCMS metabolomics and RNA-Seq. RESULTS: Serine and glycine restriction sensitised a range of cancer cell lines, patient-derived organoids and syngeneic mouse tumour models to radiotherapy. Comprehensive metabolomic and transcriptomic analysis of central carbon metabolism revealed that amino acid restriction impacted not only antioxidant response and nucleotide synthesis but had a marked inhibitory effect on the TCA cycle. CONCLUSION: Dietary restriction of serine and glycine is a viable radio-sensitisation strategy in cancer.


Asunto(s)
Neoplasias Pancreáticas , Serina , Ratones , Animales , Serina/metabolismo , Glicina/farmacología , Antioxidantes/metabolismo , Aminoácidos
6.
EMBO J ; 36(16): 2373-2389, 2017 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-28694244

RESUMEN

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates ß-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis.


Asunto(s)
Comunicación Celular , Células Endoteliales/fisiología , Melanocitos/fisiología , Cadherinas/análisis , Línea Celular , Proteína 61 Rica en Cisteína/análisis , Regulación de la Expresión Génica , Humanos , Espectrometría de Masas , beta Catenina/análisis
7.
Haematologica ; 105(2): 375-386, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31097632

RESUMEN

RAS-signaling mutations induce the myelomonocytic differentiation and proliferation of hematopoietic stem and progenitor cells. Moreover, they are important players in the development of myeloid neoplasias. RAF kinase inhibitor protein (RKIP) is a negative regulator of RAS-signaling. As RKIP loss has recently been described in RAS-mutated myelomonocytic acute myeloid leukemia, we now aimed to analyze its role in myelomonocytic differentiation and RAS-driven leukemogenesis. Therefore, we initially analyzed RKIP expression during human and murine hematopoietic differentiation and observed that it is high in hematopoietic stem and progenitor cells and lymphoid cells but decreases in cells belonging to the myeloid lineage. By employing short hairpin RNA knockdown experiments in CD34+ umbilical cord blood cells and the undifferentiated acute myeloid leukemia cell line HL-60, we show that RKIP loss is indeed functionally involved in myelomonocytic lineage commitment and drives the myelomonocytic differentiation of hematopoietic stem and progenitor cells. These results could be confirmed in vivo, where Rkip deletion induced a myelomonocytic differentiation bias in mice by amplifying the effects of granulocyte macrophage-colony-stimulating factor. We further show that RKIP is of relevance for RAS-driven myelomonocytic leukemogenesis by demonstrating that Rkip deletion aggravates the development of a myeloproliferative disease in NrasG12D -mutated mice. Mechanistically, we demonstrate that RKIP loss increases the activity of the RAS-MAPK/ERK signaling module. Finally, we prove the clinical relevance of these findings by showing that RKIP loss is a frequent event in chronic myelomonocytic leukemia, and that it co-occurs with RAS-signaling mutations. Taken together, these data establish RKIP as novel player in RAS-driven myeloid leukemogenesis.


Asunto(s)
Leucemia Mieloide Aguda , Proteínas de Unión a Fosfatidiletanolamina , Animales , Diferenciación Celular , Leucemia Mieloide Aguda/genética , Ratones , Monocitos/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Transducción de Señal
8.
Int J Mol Sci ; 20(14)2019 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-31336867

RESUMEN

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. METHODS: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. RESULTS: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. CONCLUSIONS: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation.


Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Neoplasias Cutáneas/patología , Animales , Biomarcadores de Tumor , Biopsia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Masculino , Mutación , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Gut ; 66(7): 1268-1277, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-27618837

RESUMEN

OBJECTIVE: Transcribed-ultraconserved regions (T-UCR) are long non-coding RNAs which are conserved across species and are involved in carcinogenesis. We studied T-UCRs downstream of the Wnt/ß-catenin pathway in liver cancer. DESIGN: Hypomorphic Apc mice (Apcfl/fl) and thiocetamide (TAA)-treated rats developed Wnt/ß-catenin dependent hepatocarcinoma (HCC) and cholangiocarcinoma (CCA), respectively. T-UCR expression was assessed by microarray, real-time PCR and in situ hybridisation. RESULTS: Overexpression of the T-UCR uc.158- could differentiate Wnt/ß-catenin dependent HCC from normal liver and from ß-catenin negative diethylnitrosamine (DEN)-induced HCC. uc.158- was overexpressed in human HepG2 versus Huh7 cells in line with activation of the Wnt pathway. In vitro modulation of ß-catenin altered uc.158- expression in human malignant hepatocytes. uc.158- expression was increased in CTNNB1-mutated human HCCs compared with non-mutated human HCCs, and in human HCC with nuclear localisation of ß-catenin. uc.158- was increased in TAA rat CCA and reduced after treatment with Wnt/ß-catenin inhibitors. uc.158- expression was negative in human normal liver and biliary epithelia, while it was increased in human CCA in two different cohorts. Locked nucleic acid-mediated inhibition of uc.158- reduced anchorage cell growth, 3D-spheroid formation and spheroid-based cell migration, and increased apoptosis in HepG2 and SW1 cells. miR-193b was predicted to have binding sites within the uc.158- sequence. Modulation of uc.158- changed miR-193b expression in human malignant hepatocytes. Co-transfection of uc.158- inhibitor and anti-miR-193b rescued the effect of uc.158- inhibition on cell viability. CONCLUSIONS: We showed that uc.158- is activated by the Wnt pathway in liver cancers and drives their growth. Thus, it may represent a promising target for the development of novel therapeutics.


Asunto(s)
Neoplasias de los Conductos Biliares/metabolismo , Carcinoma Hepatocelular/metabolismo , Colangiocarcinoma/metabolismo , Secuencia Conservada/genética , Neoplasias Hepáticas/metabolismo , ARN no Traducido/genética , Vía de Señalización Wnt , Animales , Neoplasias de los Conductos Biliares/genética , Carcinoma Hepatocelular/genética , Colangiocarcinoma/genética , Regulación Neoplásica de la Expresión Génica/genética , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas/genética , Ratones Noqueados , MicroARNs/metabolismo , Neoplasias Experimentales , Transfección , beta Catenina/genética , beta Catenina/metabolismo
10.
J Proteome Res ; 15(7): 2187-97, 2016 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-27246970

RESUMEN

Endothelial cells (ECs) form the inner layer of blood vessels and physically separate the blood from the surrounding tissue. To support tissues with nutrients and oxygen, the endothelial monolayer is semipermeable. When EC permeability is altered, blood vessels are not functional, and this is associated with disease. A comprehensive knowledge of the mechanisms regulating EC permeability is key in developing strategies to target this mechanism in pathologies. Here we have used an in vitro model of human umbilical vein endothelial cells mimicking the formation of a physiologically permeable vessel and performed time-resolved in-depth molecular profiling using stable isotope labeling by amino acids in cell culture mass spectrometry (MS)-proteomics. Autophagy is induced when ECs are assembled into a physiologically permeable monolayer. By using siRNA and drug treatment to block autophagy in combination with functional assays and MS proteomics, we show that ECs require autophagy flux to maintain intracellular reactive oxygen species levels, and this is required to maintain the physiological permeability of the cells.


Asunto(s)
Autofagia/fisiología , Especies Reactivas de Oxígeno/metabolismo , Células Cultivadas , Endotelio Vascular/fisiología , Humanos , Espectrometría de Masas , Modelos Biológicos , Permeabilidad , Proteómica/métodos , Especies Reactivas de Oxígeno/análisis
11.
Sci Signal ; 17(827): eade0580, 2024 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-38470957

RESUMEN

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we showed that CAFs with a myofibroblast phenotype released extracellular vesicles that transferred proteins to endothelial cells (ECs) that affected their interaction with immune cells. Mass spectrometry-based proteomics identified proteins transferred from CAFs to ECs, which included plasma membrane receptors. Using THY1 as an example of a transferred plasma membrane-bound protein, we showed that CAF-derived proteins increased the adhesion of a monocyte cell line to ECs. CAFs produced high amounts of matrix-bound EVs, which were the primary vehicles of protein transfer. Hence, our work paves the way for future studies that investigate how CAF-derived matrix-bound EVs influence tumor pathology by regulating the function of neighboring cancer, stromal, and immune cells.


Asunto(s)
Fibroblastos Asociados al Cáncer , Neoplasias , Humanos , Fibroblastos Asociados al Cáncer/metabolismo , Células Endoteliales , Neoplasias/metabolismo , Membrana Celular , Línea Celular , Fibroblastos/metabolismo , Microambiente Tumoral , Línea Celular Tumoral
12.
Nature ; 446(7136): 676-9, 2007 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-17377531

RESUMEN

The APC gene encodes the adenomatous polyposis coli tumour suppressor protein, germline mutation of which characterizes familial adenomatous polyposis (FAP), an autosomal intestinal cancer syndrome. Inactivation of APC is also recognized as the key early event in the development of sporadic colorectal cancers, and its loss results in constitutive activity of the beta-catenin-Tcf4 transcription complex. The proto-oncogene c-MYC has been identified as a target of the Wnt pathway in colorectal cancer cells in vitro, in normal crypts in vivo and in intestinal epithelial cells acutely transformed on in vivo deletion of the APC gene; however, the significance of this is unclear. Therefore, to elucidate the role Myc has in the intestine after Apc loss, we have simultaneously deleted both Apc and Myc in the adult murine small intestine. Here we show that loss of Myc rescued the phenotypes of perturbed differentiation, migration, proliferation and apoptosis, which occur on deletion of Apc. Remarkably, this rescue occurred in the presence of high levels of nuclear beta-catenin. Array analysis revealed that Myc is required for the majority of Wnt target gene activation following Apc loss. These data establish Myc as the critical mediator of the early stages of neoplasia following Apc loss.


Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/deficiencia , Proteína de la Poliposis Adenomatosa del Colon/genética , Eliminación de Gen , Genes APC , Genes myc/genética , Intestino Delgado/metabolismo , Proteínas Proto-Oncogénicas c-myc/deficiencia , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Animales , Enterocitos/metabolismo , Intestino Delgado/citología , Masculino , Ratones , Fenotipo , Proteínas Proto-Oncogénicas c-myc/genética
13.
Proc Natl Acad Sci U S A ; 107(1): 246-51, 2010 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-20018721

RESUMEN

TP53 mutation occurs in 50-75% of human pancreatic ductal adenocarcinomas (PDAC) following an initiating activating mutation in the KRAS gene. These p53 mutations frequently result in expression of a stable protein, p53(R175H), rather than complete loss of protein expression. In this study we elucidate the functions of mutant p53 (Trp53(R172H)), compared to knockout p53 (Trp53(fl)), in a mouse model of PDAC. First we find that although Kras(G12D) is one of the major oncogenic drivers of PDAC, most Kras(G12D)-expressing pancreatic cells are selectively lost from the tissue, and those that remain form premalignant lesions. Loss, or mutation, of Trp53 allows retention of the Kras(G12D)-expressing cells and drives rapid progression of these premalignant lesions to PDAC. This progression is consistent with failed growth arrest and/or senescence of premalignant lesions, since a mutant of p53, p53(R172P), which can still induce p21 and cell cycle arrest, is resistant to PDAC formation. Second, we find that despite similar kinetics of primary tumor formation, mutant p53(R172H), as compared with genetic loss of p53, specifically promotes metastasis. Moreover, only mutant p53(R172H)-expressing tumor cells exhibit invasive activity in an in vitro assay. Importantly, in human PDAC, p53 accumulation significantly correlates with lymph node metastasis. In summary, by using 'knock-in' mutations of Trp53 we have identified two critical acquired functions of a stably expressed mutant form of p53 that drive PDAC; first, an escape from Kras(G12D)-induced senescence/growth arrest and second, the promotion of metastasis.


Asunto(s)
Carcinoma Ductal Pancreático , Senescencia Celular/genética , Mutación , Metástasis de la Neoplasia/genética , Proteína p53 Supresora de Tumor/genética , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Ciclo Celular/fisiología , Genes Reporteros , Humanos , Ratones , Análisis por Micromatrices , Metástasis de la Neoplasia/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
14.
PLoS Genet ; 6(1): e1000816, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-20084116

RESUMEN

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/beta-catenin pathway, we challenged the allele combinations by genetically restricting intracellular beta-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/beta-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/beta-catenin signaling capacity similar to that in the germline of the Apc(min) mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc(min) mice arise independently of intestinal tumorigenesis. Together, the present genotype-phenotype analysis suggests tissue-specific response levels for the Wnt/beta-catenin pathway that regulate both physiological and pathophysiological conditions.


Asunto(s)
Ratones/genética , Ratones/metabolismo , Transducción de Señal , beta Catenina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos , Femenino , Fibroblastos/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/embriología , Intestinos/crecimiento & desarrollo , Hígado/embriología , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Masculino , Ratones/embriología , Ratones/crecimiento & desarrollo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Wnt , Proteína Wnt3 , beta Catenina/genética
15.
Nat Metab ; 5(12): 2148-2168, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38066114

RESUMEN

Serine is a vital amino acid in tumorigenesis. While cells can perform de novo serine synthesis, most transformed cells rely on serine uptake to meet their increased biosynthetic requirements. Solute carriers (SLCs), a family of transmembrane nutrient transport proteins, are the gatekeepers of amino acid acquisition and exchange in mammalian cells and are emerging as anticancer therapeutic targets; however, the SLCs that mediate serine transport in cancer cells remain unknown. Here we perform an arrayed RNAi screen of SLC-encoding genes while monitoring amino acid consumption and cell proliferation in colorectal cancer cells using metabolomics and high-throughput imaging. We identify SLC6A14 and SLC25A15 as major cytoplasmic and mitochondrial serine transporters, respectively. We also observe that SLC12A4 facilitates serine uptake. Dual targeting of SLC6A14 and either SLC25A15 or SLC12A4 diminishes serine uptake and growth of colorectal cancer cells in vitro and in vivo, particularly in cells with compromised de novo serine biosynthesis. Our results provide insight into the mechanisms that contribute to serine uptake and intracellular handling.


Asunto(s)
Neoplasias Colorrectales , Proteínas de Transporte de Membrana , Animales , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico , Aminoácidos/metabolismo , Serina/metabolismo , Neoplasias Colorrectales/genética , Mamíferos/metabolismo
16.
Nat Metab ; 5(8): 1303-1318, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37580540

RESUMEN

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC.


Asunto(s)
Neoplasias Colorrectales , Animales , Humanos , Ratones , Adenosilhomocisteinasa/genética , Adenosilhomocisteinasa/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Metabolómica , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética
17.
Nat Metab ; 4(6): 693-710, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35760868

RESUMEN

Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production.


Asunto(s)
Neoplasias de la Mama , Fibroblastos Asociados al Cáncer , Pirrolina Carboxilato Reductasas/metabolismo , Neoplasias de la Mama/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinogénesis/metabolismo , Carcinogénesis/patología , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Femenino , Glutamina/metabolismo , Humanos , Prolina , delta-1-Pirrolina-5-Carboxilato Reductasa
18.
Gastroenterology ; 139(1): 292-303, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20303350

RESUMEN

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive and metastatic disease for which conventional treatments are of limited efficacy. A number of agents in development are potential anti-invasive and antimetastatic agents, including the Src kinase inhibitor dasatinib. The aim of this study was to assess the importance of Src in human PDAC and to use a genetically engineered mouse model of PDAC to determine the effects of dasatinib on PDAC progression. METHODS: Src expression and activity was measured by immunohistochemistry in 114 human PDACs. Targeting expression of Trp53(R172H) and Kras(G12D) to the mouse pancreas results in the formation of invasive and metastatic PDAC. These mice were treated with dasatinib, and disease progression monitored. Cell lines were derived from mouse PDACs, and in vitro effects of dasatinib assessed. RESULTS: Src expression and activity were up-regulated in human PDAC and this correlated with reduced survival. Dasatinib inhibited the migration and invasion of PDAC cell lines, although no effects on proliferation were seen at concentrations that inhibited Src kinase activity. In addition, dasatinib significantly inhibited the development of metastases in Pdx1-Cre, Z/EGFP, LSL-Kras(G12D/+), LSL-Trp53(R172H/+) mice. However, there was no survival advantage in the dasatinib-treated animals owing to continued growth of the primary tumor. CONCLUSIONS: This study confirms the importance of Src in human PDAC and shows the usefulness of a genetically engineered mouse model of PDAC for assessing the activity of potential antimetastatic agents and suggests that dasatinib should be evaluated further as monotherapy after resection of localized invasive PDAC.


Asunto(s)
Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Tiazoles/uso terapéutico , Familia-src Quinasas/antagonistas & inhibidores , Animales , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Dasatinib , Modelos Animales de Enfermedad , Femenino , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Familia-src Quinasas/metabolismo
19.
Gastroenterology ; 139(2): 586-97, 597.e1-6, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20452353

RESUMEN

BACKGROUND & AIMS: Patients carrying germline mutations of LKB1 have an increased risk of pancreatic cancer; however, it is unclear whether down-regulation of LKB1 is an important event in sporadic pancreatic cancer. In this study, we aimed to investigate the impact of LKB1 down-regulation for pancreatic cancer in mouse and human and to elucidate the mechanism by which Lkb1 deregulation contributes to this disease. METHODS: We first investigated the consequences of Lkb1 deficiency in a genetically modified mouse model of pancreatic cancer, both in terms of disease progression and at the molecular level. To test the relevance of our findings to human pancreatic cancer, we investigated levels of LKB1 and its potential targets in human pancreatic cancer. RESULTS: We definitively show that Lkb1 haploinsufficiency can cooperate with oncogenic KrasG12D to cause pancreatic ductal adenocarcinoma (PDAC) in the mouse. Mechanistically, this was associated with decreased p53/p21-dependent growth arrest. Haploinsufficiency for p21 (Cdkn1a) also synergizes with KrasG12D to drive PDAC in the mouse. We also found that levels of LKB1 expression were decreased in around 20% of human PDAC and significantly correlated with low levels of p21 and a poor prognosis. Remarkably, all tumors that had low levels of LKB1 had low levels of p21, and these tumors did not express mutant p53. CONCLUSIONS: We have identified a novel LKB1-p21 axis that suppresses PDAC following Kras mutation in vivo. Down-regulation of LKB1 may therefore serve as an alternative to p53 mutation to drive pancreatic cancer in vivo.


Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Transformación Celular Neoplásica/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Progresión de la Enfermedad , Genes Supresores de Tumor , Genotipo , Haplotipos , Heterocigoto , Proteínas de Homeodominio/genética , Homocigoto , Humanos , Ratones , Ratones Noqueados , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Fenotipo , Pronóstico , Regiones Promotoras Genéticas , Modelos de Riesgos Proporcionales , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras)/deficiencia , Proteínas Proto-Oncogénicas p21(ras)/genética , Medición de Riesgo , Factores de Tiempo , Transactivadores/genética , Proteína p53 Supresora de Tumor/metabolismo
20.
Proc Natl Acad Sci U S A ; 105(48): 18919-23, 2008 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-19033191

RESUMEN

Dysregulated Wnt signaling is seen in approximately 30% of hepatocellular carcinomas; thus, finding pathways downstream of the activation of Wnt signaling is key. Here, using cre-lox technology, we deleted the Apc gene in the adult mouse liver and observed a rapid increase in nuclear beta-catenin and c-Myc, which is associated with an induction of proliferation that led to hepatomegaly within 4 days of gene deletion. To investigate the downstream pathways responsible for these phenotypes, we analyzed the impact of inactivating APC in the context of deficiency of the potentially key effectors beta-catenin and c-Myc. beta-catenin loss rescues both the proliferation and hepatomegaly phenotypes after APC loss. However, c-Myc deletion, which rescues the phenotypes of APC loss in the intestine, had no effect on the phenotypes of APC loss in the liver. The consequences of the deregulation of the Wnt pathway within the liver are therefore strikingly different from those observed within the intestine, with the vast majority of Wnt targets being beta-catenin-dependent but c-Myc-independent in the liver.


Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Hígado/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , beta Catenina/deficiencia , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Eliminación de Gen , Hígado/citología , Hígado/patología , Masculino , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Fenotipo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Wnt/genética , beta Catenina/genética
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