RESUMEN
KRAS is the most frequently mutated oncogene. The incidence of specific KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specific mutant KRAS proteins. We used a cross-disciplinary approach to compare KRASG12D, a common mutant form, and KRASA146T, a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRASA146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor-induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRASG12D and KRASA146T exhibit distinct tissue-specific effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specific phenotypes result from allele-specific signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. SIGNIFICANCE: Although epidemiologic and clinical studies have suggested allele-specific behaviors for KRAS, experimental evidence for allele-specific biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties.See related commentary by Hobbs and Der, p. 696.This article is highlighted in the In This Issue feature, p. 681.
Asunto(s)
Alelos , Mutación , Oncogenes , Proteínas Proto-Oncogénicas p21(ras)/genética , Transformación Celular Neoplásica/genética , Humanos , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Especificidad de Órganos , Fenotipo , Conformación Proteica , Proteoma , Proteómica/métodos , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Relación Estructura-ActividadRESUMEN
Inflammatory bowel disease (IBD) is a chronic condition driven by loss of homeostasis between the mucosal immune system, the commensal gut microbiota, and the intestinal epithelium. Our goal is to understand how these components of the intestinal ecosystem cooperate to control homeostasis. By combining quantitative measures of epithelial hyperplasia and immune infiltration with multivariate analysis of inter- and intracellular signaling, we identified epithelial mammalian target of rapamycin (mTOR) signaling as a potential driver of inflammation in a mouse model of colitis. A kinetic analysis of mTOR inhibition revealed that the pathway regulates epithelial differentiation, which in turn controls the cytokine milieu of the colon. Consistent with our in vivo analysis, we found that cytokine expression of organoids grown ex vivo, in the absence of bacteria and immune cells, was dependent on differentiation state. Our study suggests that proper differentiation of epithelial cells is an important feature of colonic homeostasis because of its effect on the secretion of inflammatory cytokines.
Asunto(s)
Colitis/metabolismo , Colon/inmunología , Citocinas/metabolismo , Animales , Autofagia , Comunicación Celular , Diferenciación Celular , Colon/metabolismo , Colon/patología , Epitelio/inmunología , Epitelio/metabolismo , Microbioma Gastrointestinal , Homeostasis , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Cinética , Ratones , Análisis Multivariante , Fosforilación , Análisis de Componente Principal , Transducción de Señal , Sirolimus/farmacología , Biología de Sistemas , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
UNLABELLED: Disruption of the gene encoding Protein Tyrosine Kinase 6 (Ptk6) delayed differentiation and increased growth in the mouse intestine. However, Ptk6-null mice were also resistant to azoxymethane-induced colon tumorigenesis. To further explore functions of PTK6 in colon cancer, expression of epithelial and mesenchymal markers, as well as proliferation, migration, and xenograft tumor growth, was examined in human colon tumor cell lines with knockdown or overexpression of PTK6. PTK6 protein, transcript, and activation were also examined in a human colon tumor tissue array, using immunohistochemistry and qRT-PCR. Knockdown of PTK6 led to the epithelial-mesenchymal transition (EMT) in SW480 and HCT116 cells, whereas overexpression of PTK6 in SW620 cells restored an epithelial phenotype in a kinase-independent manner. PTK6 knockdown also increased xenograft tumor growth of SW480 cells, suggesting tumor suppressor functions. In clinical specimens, PTK6 expression was highest in normal differentiated epithelial cells and reduced in tumors. In contrast, overexpression of constitutively active PTK6 promoted STAT3 and ERK5 activation in colon cancer cells, and endogenous PTK6 promoted cell survival and oncogenic signaling in response to DNA-damaging treatments. These data indicate that PTK6 has complex, context-specific functions in colon cancer; PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner, whereas activation of PTK6 promotes oncogenic signaling. IMPLICATIONS: Understanding context-specific functions of PTK6 is important, because although it promotes cell survival and oncogenic signaling after DNA damage, expression of PTK6 in established tumors may maintain the epithelial phenotype, preventing tumor progression. Mol Cancer Res; 14(6); 563-73. ©2016 AACR.
Asunto(s)
Neoplasias del Colon/enzimología , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Femenino , Células HCT116 , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Proteínas Tirosina Quinasas/genética , Transducción de SeñalRESUMEN
Individual signaling pathways operate in the context of the broader signaling network. Thus, the response of a cell to signals from the environment is affected by the state of the signaling network, such as the clinically relevant example of whether some components in the network are inhibited. The cytokine tumor necrosis factor-α (TNF-α) promotes opposing cellular behaviors under different conditions; the outcome is influenced by the state of the network. For example, in the mouse intestinal epithelium, inhibition of the mitogen-activated protein kinase (MAPK) kinase MEK alters the timing of TNF-α-induced apoptosis. We investigated whether MAPK signaling directly influences TNF-α-induced apoptosis or whether network-level effects secondary to inhibition of the MAPK pathway alter the cellular response. We found that inhibitors of the MAPK kinase kinase Raf, MEK, or extracellular signal-regulated kinase (ERK) exerted distinct effects on the timing and magnitude of TNF-α-induced apoptosis in the mouse intestine. Furthermore, even different MEK inhibitors exerted distinct effects; one, CH5126766, potentiated TNF-α-induced apoptosis, and the others reduced cell death. Computational modeling and experimental perturbation identified the kinase Akt as the primary signaling node that enhanced apoptosis in the context of TNF-α signaling in the presence of CH5126766. Our work emphasizes the importance of integrated network signaling in specifying cellular behavior in response to experimental or therapeutic manipulation. More broadly, this study highlighted the importance of considering the network-level effects of pathway inhibitors and showed the distinct effects of inhibitors that share the same target.
Asunto(s)
Apoptosis/efectos de los fármacos , Mucosa Intestinal/metabolismo , Modelos Biológicos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Mucosa Intestinal/citología , Masculino , Ratones , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidoresRESUMEN
The retinoblastoma tumor suppressor (pRb) protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb loss are largely unexplored. We acutely ablated Rb in adult mice and conducted a quantitative analysis of RNA and proteomic changes in the colon and lungs, where Rb(KO) was sufficient or insufficient to induce ectopic proliferation, respectively. As expected, Rb(KO) caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but, unexpectedly, their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb loss are coupled with proliferation but uncoupled from transcription. The proteomic changes in common between Rb(KO) tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and oxidative phosphorylation (OXPHOS) function. RB(KO) cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb loss altered mitochondrial pyruvate oxidation from (13)C-glucose through the TCA cycle in mouse tissues and cultured cells. Consequently, RB(KO) cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.
Asunto(s)
Mitocondrias/metabolismo , Fosforilación Oxidativa , Proteína de Retinoblastoma/genética , Animales , Células Cultivadas , Colon/fisiopatología , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Pulmón/fisiopatología , Ratones , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteómica , Proteína de Retinoblastoma/metabolismo , Estrés Fisiológico/genética , TranscriptomaRESUMEN
K-Ras is a monomeric GTPase that controls cellular and tissue homeostasis. Prior studies demonstrated that mutationally activated K-Ras (K-Ras(G12D)) signals through MEK to promote expansion and hyperproliferation of the highly mitotically active transit-amplifying cells (TACs) in the intestinal crypt. Its effect on normally quiescent stem cells was unknown, however. Here, we have used an H2B-Egfp transgenic system to demonstrate that K-Ras(G12D) accelerates the proliferative kinetics of quiescent intestinal stem cells. As in the TAC compartment, the effect of mutant K-Ras on the quiescent stem cell is dependent upon activation of MEK. Mutant K-Ras is also able to increase self-renewal potential of intestinal stem cells following damage. These results demonstrate that mutant K-Ras can influence intestinal homeostasis on multiple levels.
Asunto(s)
Intestinos/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Células Madre/patología , Animales , Proliferación Celular , Sulfato de Dextran , Proteínas Fluorescentes Verdes/metabolismo , Mucosa Intestinal/metabolismo , Ratones Transgénicos , Células Madre/metabolismoRESUMEN
The two products of the KRAS locus, K-Ras4A and K-Ras4B, are encoded by alternative fourth exons and therefore, possess distinct membrane-targeting sequences. The common activating mutations occur in exons 1 or 2 and therefore, render both splice variants oncogenic. K-Ras4A has been understudied, because it has been considered a minor splice variant. By priming off of the splice junction, we developed a quantitative RT-PCR assay for K-Ras4A and K-Ras4B message capable of measuring absolute amounts of the two transcripts. We found that K-Ras4A was widely expressed in 30 of 30 human cancer cell lines and amounts equal to K-Ras4B in 17 human colorectal tumors. Using splice variant-specific antibodies, we detected K-Ras4A protein in several tumor cell lines at a level equal to or greater than that of K-Ras4B. In addition to the CAAX motif, the C terminus of K-Ras4A contains a site of palmitoylation as well as a bipartite polybasic region. Although both were required for maximal efficiency, each of these could independently deliver K-Ras4A to the plasma membrane. Thus, among four Ras proteins, K-Ras4A is unique in possessing a dual membrane-targeting motif. We also found that, unlike K-Ras4B, K-Ras4A does not bind to the cytosolic chaperone δ-subunit of cGMP phosphodiesterase type 6 (PDE6δ). We conclude that efforts to develop anti-K-Ras drugs that interfere with membrane trafficking will have to take into account the distinct modes of targeting of the two K-Ras splice variants.
Asunto(s)
Genes ras , Neoplasias/genética , Empalme del ARN , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
The laboratory mouse is an ideal model organism for studying disease because it is physiologically similar to human and also because its genome is readily manipulated. Genetic engineering allows researchers to introduce specific loss-of-function or gain-of-function mutations into genes and then to study the resulting phenotypes in an in vivo context. One drawback of using traditional transgenic and knockout mice to study human diseases is that many mutations passed through the germline can profoundly affect development, thus impeding the study of disease phenotypes in adults. New technology has made it possible to generate conditional mutations that can be introduced in a spatially and/or temporally restricted manner. Mouse strains carrying conditional mutations represent valuable experimental models for the study of human diseases and they can be used to develop strategies for prevention and treatment of these diseases. In this article, we will describe the most widely used DNA recombinase systems used to achieve conditional gene mutation in mouse models and discuss how these systems can be employed in vivo.
Asunto(s)
Animales Modificados Genéticamente , Regulación de la Expresión Génica , Mutación , Animales , Ratones , Recombinación GenéticaRESUMEN
Although the development of improved mouse models, including conditional deletions, marks an exciting time in mouse genetics, it is important to characterize and validate these models. Cre reporter strains allow researchers to assess the recombinase expression profile and function in individual Cre mouse lines. These strains are engineered to express a reporter gene (usually LacZ) following the removal of a floxed STOP cassette, thus marking cell lineages that can be targeted with a given Cre line. This protocol provides a detailed method for the histochemical detection of ß-galactosidase activity in Cre mouse strains.
Asunto(s)
Animales Modificados Genéticamente , Galactósidos/metabolismo , Genes Reporteros , Indoles/metabolismo , Coloración y Etiquetado/métodos , beta-Galactosidasa/análisis , Animales , RatonesRESUMEN
Lentiviral vectors offer versatility as vehicles for gene delivery. They can transduce a wide range of cell types and integrate into the host genome, which results in long-term expression of the transgene (Cre) both in vitro and in vivo. This protocol describes how lentiviral particles are produced, purified, and concentrated.
Asunto(s)
Vectores Genéticos , Infecciones/terapia , Integrasas/genética , Lentivirus/genética , Animales , RatonesRESUMEN
Lung cancer remains the leading cause of cancer deaths among both men and women, with a lower rate of survival than both breast and prostate cancer. Development of the Cre/lox system and improved mouse models have allowed researchers to gain a better understanding of human disease, including lung cancer. Through the viral delivery of Cre, gene function in adult mice can be precisely studied at a specific developmental stage or in a specific cell/tissue type of choice. This protocol describes how to produce adenovirus-Cre precipitate. Using this adeno-Cre (or lentivirus-Cre), Cre can be expressed in mouse lungs. The virus is delivered by intranasal instillation.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos/administración & dosificación , Lentivirus/genética , Administración Intranasal , Animales , Integrasas/genética , RatonesRESUMEN
Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that has distinct functions in normal epithelia and cancer. It is expressed primarily in nondividing epithelial cells in the normal intestine, where it promotes differentiation. However, after DNA damage, PTK6 is induced in proliferating progenitor cells, where it contributes to apoptosis. We examined links between PTK6 and the tumor suppressor p53 in the isogenic p53(+/+) and p53(-/-) HCT116 colon tumor cell lines. We found that p53 promotes expression of PTK6 in HCT116 cells, and short hairpin RNA-mediated knockdown of PTK6 leads to reduced induction of the cyclin-dependent kinase inhibitor p21. Knockdown of PTK6 enhances apoptosis in HCT116 cells with wild-type p53, following treatment of cells with γ-radiation, doxorubicin, or 5-fluorouracil. No differences in the activation of AKT, ERK1/2, or ERK5, known PTK6-regulated prosurvival signaling proteins, were detected. However, activity of STAT3, a PTK6 substrate, was impaired in cells with knockdown of PTK6 following DNA damage. In contrast to its role in the normal epithelium following DNA damage, PTK6 promotes survival of cancer cells with wild-type p53 by promoting p21 expression and STAT3 activation. Targeting PTK6 in combination with use of chemotherapeutic drugs or radiation may enhance death of colon tumor cells with wild-type p53.
Asunto(s)
Apoptosis , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Daño del ADN , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Rayos gamma , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Ratones , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Disruption of the gene encoding protein tyrosine kinase 6 (PTK6) leads to increased growth, impaired enterocyte differentiation and higher levels of nuclear beta-catenin in the mouse small intestine. Here, we demonstrate that PTK6 associates with nuclear and cytoplasmic beta-catenin and inhibits beta-catenin- and T-cell factor (TCF)-mediated transcription. PTK6 directly phosphorylates beta-catenin on Tyr64, Tyr142, Tyr331 and/or Tyr333, with the predominant site being Tyr64. However, mutation of these sites does not abrogate the ability of PTK6 to inhibit beta-catenin transcriptional activity. Outcomes of PTK6-mediated regulation appear to be dependent on its intracellular localization. In the SW620 colorectal adenocarcinoma cell line, nuclear-targeted PTK6 negatively regulates endogenous beta-catenin/TCF transcriptional activity, whereas membrane-targeted PTK6 enhances beta-catenin/TCF regulated transcription. Levels of TCF4 and the transcriptional co-repressor TLE/Groucho increase in SW620 cells expressing nuclear-targeted PTK6. Knockdown of PTK6 in SW620 cells leads to increased beta-catenin/TCF transcriptional activity and increased expression of beta-catenin/TCF target genes Myc and Survivin. Ptk6-null BAT-GAL mice, containing a beta-catenin-activated LacZ reporter transgene, have increased levels of beta-galactosidase expression in the gastrointestinal tract. The ability of PTK6 to negatively regulate beta-catenin/TCF transcription by modulating levels of TCF4 and TLE/Groucho could contribute to its growth-inhibitory activities in vivo.
Asunto(s)
Espacio Intracelular/enzimología , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas/metabolismo , beta Catenina/metabolismo , Familia-src Quinasas/metabolismo , Animales , Línea Celular Tumoral , Regulación hacia Abajo/genética , Activación Enzimática , Genes Reporteros , Humanos , Intestinos/enzimología , Intestinos/patología , Ratones , Ratones Transgénicos , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Especificidad por Sustrato , Factores de Transcripción TCF/metabolismo , Transcripción Genética , beta Catenina/genéticaRESUMEN
BACKGROUND & AIMS: Protein tyrosine kinase 6 (PTK6) is expressed in epithelial linings of the gastrointestinal tract. PTK6 sensitizes the nontransformed Rat1a fibroblast cell line to apoptotic stimuli. The aim of this study was to determine if PTK6 regulates apoptosis in vivo after DNA damage in the small intestine. METHODS: Wild-type and Ptk6(-/-) mice were subjected to gamma-irradiation; intestinal tissues were collected, protein was isolated, and samples were fixed for immunohistochemical analyses at 0, 6, and 72 hours after the mice were irradiated. Expression of PTK6 was examined in the small intestine before and after irradiation. Apoptosis and proliferation were compared between wild-type and Ptk6(-/-) mice. Expression and activation of prosurvival signaling proteins were assessed. RESULTS: Irradiation induced PTK6 in crypt epithelial cells of the small intestine in wild-type mice. Induction of PTK6 corresponded with DNA damage-induced apoptosis in the wild-type small intestine. Following irradiation, the apoptotic response was impaired in the intestinal crypts of Ptk6(-/-) mice. Increased activation of AKT and extracellular signal-regulated kinase (ERK)1/2 and increased inhibitory phosphorylation of the proapoptotic protein BAD were detected in Ptk6(-/-) mice after irradiation. In response to the induction of apoptosis, compensatory proliferation increased in the small intestines of wild-type mice but not in Ptk6(-/-) mice at 6 hours after irradiation. CONCLUSIONS: PTK6 is a stress-induced kinase that promotes apoptosis by inhibiting prosurvival signaling. After DNA damage, induction of PTK6 is required for efficient apoptosis and inhibition of AKT and ERK1/2.