RHOA inactivation enhances Wnt signaling and promotes colorectal cancer
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ABSTRACT: Microarray transcriptomic analysis of formalin-fixed, paraffin-embedded small intestinal tumors from control (Apcmin/+;Vil-Cre-/-;RhoADN/-) and DN-RhoA (Apcmin/+;Vil-CreTG/-;RhoADN/-) mice.
Project description:To analyse roles of HAI-1/Spint1 in intestinal tumorigenesis, we examined the effect of intestine-specific deletion of Spint1 gene on Apc(Min/+) mice. The loss of Hai-1/Spint1 significantly accelerated tumor formation in ApcMin/+ mice and shortened their survival periods. Mouse small intestine tumor tissue or background mucosa lacking macroscopically visible tumors were proceeded to RNA extraction and hybridization on microarrays (Affymetrix Mouse Genome 430 2.0 Array). Non-tumor or tumor intestinal mucosa tissues of Apc (Min/+)/Spint1 (flox/flox) mice and non-tumor or tumor intestinal mucosa tissues of Apc (Min/+)/Spint1 (flox/flox)/Vil-Cre mice were analysed. The experiment was repeated respectively.
Project description:Expression profiles of normal colons in ApcMin mice that were fed methyl deficient diet for 11 weeks vs controls Expression profiles of normal colons in ApcMin mice that were fed methyl deficient diet for 11 weeks vs controls fed methyl donor sufficient diets
Project description:Crypts were isolated from either control or YY1f/f; Vil-Cre-ERT2 mice treated with tamoxifen for 4 days to induce knockout Jejunal crypt epithelia were isolated and processed for microarray
Project description:We performed RNAseq on intestinal polyps from diptheria toxin-treated ApcMin;Lgr5DTR mice to investigate the effect of an acute selective pressure on stem cell populations in intestinal lesions. Lgr5+ cells in the ApcMin;Lgr5DTR mice were ablated with a single intraperitoneal dose of diphtheria toxin in saline (50 μg/kg), and samples were collected after 24 hours and after 5 days. Untreated ApcMin mice were used as control. Intestinal polyps were excised and collected for RNA sequencing.
Project description:The transcription factor Zinc finger protein 148 (Zfp148) interacts physically with the tumor suppressor p53, but the siginficance of this interaction is not known. We recently showed that knockout of Zfp148 in mice leads to ectopic activation of p53 in tissues and cultured fibroblasts, suggesting that Zfp148 represses p53 activity. Here we hypothesized that targeting Zfp148 would unleash p53 activity and protect against cancer development, and test this idea in the APCMin/+ mouse model of intestinal adenomas. Crypt-enriched tissues were isolated by laser microdissection (PALM) from the small intestines (proximal) of Zfp148gt/+APCMin/+ and Zfp148+/+APCMin/+ mice for RNA extraction and hybridization to Affymetrix microarrays.
Project description:In this study, we investigated the role of LIN28 in intestinal tumor initiation and invasive progression. We generated animal models with just intestinal LIN28B overexpression, or in combination with Apcmin/+ background. The animals develop intestinal and colorectal tumors with histology ranging from adenoma to adenocarcinoma. total RNA isolated from mouse small intestinal tumors with LIN28B overexpression, or duodenum and colon Apcmin tumors and LIN28B;Apcmin tumors
Project description:Apc, a negative regulator of the canonical Wnt signaling pathway, is a bona-fide tumor suppressor whose loss of function results in intestinal polyposis. APC is located in a commonly deleted region on human chromosome 5q, associated with myelodysplastic syndrome (MDS) suggesting that haploinsufficiency of APC contributes to the MDS phenotype. Analysis of the hematopoietic system of mice with the Apcmin allele that results in a premature stop codon and loss of function, showed no abnormality in steady state hematopoiesis. Bone marrow derived from Apcmin mice showed enhanced repopulation potential, indicating of a cell intrinsic gain of function in the long-term hematopoietic stem cell (HSC) population. However, Apcmin bone marrow was unable to repopulate secondary recipients due to loss of the quiescent HSC population. Apcmin mice developed a myelodysplastic/ myeloproliferative phenotype. Our data indicate that Wnt activation through haploinsufficiency of Apc causes insidious loss of HSC function that is only evident in serial transplantation strategies. These data provide a cautionary note for HSC expansion strategies through Wnt pathway activation, provide evidence that cell extrinsic factors can contribute to the development of myeloid disease and indicate that loss of function of APC may contribute to the phenotype observed in patients with MDS and del(5q). LKS+ cells were isolated from Apcmin or WT mice using high-speed multiparameter flow cytometry. At least 2x104 cells per mouse were isolated with confirmed purity in excess of 90%. The cells were treated with RLT lysis buffer (Qiagen) containing beta-mercaptoethanol to stabilize RNA. RNA was extracted using Qiagen RNeasy Micro Kit according to manufacturers instruction. The RNA was amplified using a linear amplification protocol (Nugen Ovation V2 amplification system). cDNA was fragmented and biotinylated before hybridization onto Affymetrix mouse genome 430 2.0 Array chips
Project description:To uncover molecular mechanisms specifically involved in the pathogenesis of colitis-associated colon cancer (CAC), we studied tumorigenesis in experimental models of CAC and sporadic CRC that mimic characteristics of human CRC. Using comparative whole genome expression profiling, we observed differential expression of epiregulin (Ereg) in mouse models of colitis-associated, but not sporadic colorectal cancer. Similarly, highly significant upregulation of Ereg expression was found in cohorts of patients with colitis-associated cancer in inflammatory bowel disease but not in sporadic colorectal cancer. Furthermore, tumor-associated fibroblasts were identified as major source of Ereg in colitis-associated neoplasias. Functional studies showed that Ereg-deficient mice, although more prone to colitis, are strongly protected from colitis-associated tumors, and data from serial endoscopic studies revealed that Ereg promotes growth rather than initiation of tumors. 4 samples of individual distal colitis-associated tumors (CAC) from 4 mice, 2 samples of tumor-free distal colon epithelium with a pool of 5 mice per sample (CAC contr), 5 samples of individual Apcmin/+ tumors from the distal colorectum of 5 mice (sporCRC) and 3 samples of tumor-free distal colon epithelium (pool of 4 mice per sample) (sporCRC contr). Colitis-associated tumorigenesis was performed by intraperitoneal injection of Azoxymethane (10mg/kg) (Sigma) into C57BL/6J wildtype mice followed by 3 cycles of Dextran Sodium Sulfate (DSS) in drinking water. Each DSS-cycle was composed of DSS (2.5% (w/v) (MP Biomedicals) in drinking water for 7 days, followed by a recovery phase with regular drinking water for 14 days. Sporadic tumors were from C57BL/6J-ApcMin/+/J mice. All tumors were obtained from the from the lower 6th of the large intestine and they had the same size covering between ¼ and up to ½ of the colonic circumferenc as evaluated by mini-endoscopy.
Project description:This experiment was done to analyze the p53 status in gastrointestinal tumors in the colon and small intestine in APC mice carrying either a wild type p53 or a mutant p53 (R172H) allele and compare colon and small intestine tumors.