RESUMEN
Many epithelial stem cell populations follow a pattern of stochastic stem cell divisions called 'neutral drift'. It is hypothesised that neutral competition between stem cells protects against the acquisition of deleterious mutations. Here we use a Porcupine inhibitor to reduce Wnt secretion at a dose where intestinal homoeostasis is maintained despite a reduction of Lgr5+ stem cells. Functionally, there is a marked acceleration in monoclonal conversion, so that crypts become rapidly derived from a single stem cell. Stem cells located further from the base are lost and the pool of competing stem cells is reduced. We tested whether this loss of stem cell competition would modify tumorigenesis. Reduction of Wnt ligand secretion accelerates fixation of Apc-deficient cells within the crypt leading to accelerated tumorigenesis. Therefore, ligand-based Wnt signalling influences the number of stem cells, fixation speed of Apc mutations and the speed and likelihood of adenoma formation.
Asunto(s)
Carcinogénesis/metabolismo , Transformación Celular Neoplásica/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Células Madre/citología , Células Madre/metabolismo , Vía de Señalización Wnt , Aciltransferasas/antagonistas & inhibidores , Adenoma/etiología , Adenoma/metabolismo , Adenoma/patología , Proteína de la Poliposis Adenomatosa del Colon/deficiencia , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Inhibidores Enzimáticos/farmacología , Mucosa Intestinal/efectos de los fármacos , Ligandos , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pirazinas/farmacología , Piridinas/farmacología , Células Madre/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.
Asunto(s)
Apoptosis/genética , Enterocitos/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Enterocitos/efectos de los fármacos , Enterocitos/efectos de la radiación , Humanos , Ratones , Proteínas Proto-Oncogénicas c-myc/genética , Radiación IonizanteRESUMEN
A role for WNT signalling in gastric carcinogenesis has been suggested due to two major observations. First, patients with germline mutations in adenomatous polyposis coli (APC) are susceptible to stomach polyps and second, in gastric cancer, WNT activation confers a poor prognosis. However, the functional significance of deregulated WNT signalling in gastric homoeostasis and cancer is still unclear. In this study we have addressed this by investigating the immediate effects of WNT signalling activation within the stomach epithelium. We have specifically activated the WNT signalling pathway within the mouse adult gastric epithelium via deletion of either glycogen synthase kinase 3 (GSK3) or APC or via expression of a constitutively active ß-catenin protein. WNT pathway deregulation dramatically affects stomach homoeostasis at very short latencies. In the corpus, there is rapid loss of parietal cells with fundic gland polyp (FGP) formation and adenomatous change, which are similar to those observed in familial adenomatous polyposis. In the antrum, adenomas occur from 4 days post-WNT activation. Taken together, these data show a pivotal role for WNT signalling in gastric homoeostasis, FGP formation and adenomagenesis. Loss of the parietal cell population and corresponding FGP formation, an early event in gastric carcinogenesis, as well as antral adenoma formation are immediate effects of nuclear ß-catenin translocation and WNT target gene expression. Furthermore, our inducible murine model will permit a better understanding of the molecular changes required to drive tumourigenesis in the stomach.
Asunto(s)
Poliposis Adenomatosa del Colon/metabolismo , Transformación Celular Neoplásica/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Wnt/metabolismo , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Animales , Diferenciación Celular/fisiología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Transgenes , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Although deregulation of the Wnt signalling pathway has been implicated in urothelial cell carcinoma (UCC), the functional significance is unknown. To test its importance, we have targeted expression of an activated form of ß-catenin to the urothelium of transgenic mice using Cre-Lox technology (UroIICRE(+) ß-catenin(exon3/+)). Expression of this activated form of ß-catenin led to the formation of localized hyperproliferative lesions by 3 months, which did not progress to malignancy. These lesions were characterized by a marked increase of the phosphatase and tensin homologue (PTEN) tumour suppressor protein. This appears to be a direct consequence of activating Wnt signalling in the bladder as conditional deletion of the adenomatous polyposis coli (Apc) gene within the adult bladder led rapidly to coincident ß-catenin and PTEN expression. This PTEN expression blocked proliferation. Next, we combined PTEN deficiency with ß-catenin activation and found that this caused papillary UCC. These tumours had increased pAKT signalling and were dependent on mammalian target of rapamycin (mTOR). Importantly, in human UCC, there was a significant correlation between high levels of ß-catenin and pAKT (and low levels of PTEN). Taken together these data show that deregulated Wnt signalling has a critical role in promoting UCC, and suggests that human UCC that have high levels of Wnt and PI3 kinase signalling may be responsive to mTOR inhibition.
Asunto(s)
Carcinoma Papilar/metabolismo , Transformación Celular Neoplásica/metabolismo , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Vejiga Urinaria/inducido químicamente , beta Catenina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Proliferación Celular , Estudios de Cohortes , Femenino , Humanos , Hiperplasia/inducido químicamente , Masculino , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Eliminación de Secuencia , Serina-Treonina Quinasas TOR/metabolismo , Vejiga Urinaria/metabolismo , Urotelio/metabolismo , Proteínas Wnt/metabolismoRESUMEN
Mutations in the adenomatous polyposis coli (APC) tumour suppressor are the key initiating event of colorectal cancer. Although the control of WNT signalling is well established as a central tumour-suppressive function, the significance of APC in regulating chromosome instability is less well established. In this study, we test whether APC-deficient cells have a functional spindle assembly checkpoint (SAC) in vivo by examining the response of these cells to Taxol and Vinorelbine. We also show for the first time that APC deficiency compromises the arrest response to Taxol in vivo. This effect is independent of the role that APC has in WNT signalling. At higher levels of Taxol, APC-deficient cells arrest as efficiently as wild-type cells. Importantly, this dose of Taxol strongly suppresses intestinal tumourigenesis in models of benign (APC(Min/+) mouse) and invasive (AhCreER(+)APC(fl/+)PTEN(fl/fl)) cancer. In contrast to intestinal enterocytes with a general SAC defect because of Bub1 (budding uninhibited by benzimidazole 1) deletion, APC-deficient enterocytes arrest equivalently to wild type when treated with Vinorelbine. This suggests that the failed arrest in response to Taxol is because of a specific defect in microtubule stabilization following Taxol treatment rather than a general role of the APC protein in the mitotic spindle checkpoint. In summary, this study clarifies the role of APC as a mitotic spindle checkpoint protein in vivo and shows that APC-deficient cells have a compromised response to Taxol.