Transformation of the intestinal epithelium by the MSI2 RNA binding protein
ABSTRACT: The MSI2 RNA binding protein has recently emerged as a potent oncogene playing key roles in hematopoietic stem cell homeostasis and malignant hematopoiesis. Here we demonstrate that MSI2 is expressed in the intestinal stem cell compartment, that its expression is elevated in colorectal adenocarcinomas, and that MSI2 loss of function abrogates colorectal cancer cell growth. We thus examined the oncogenic consequences of MSI2 gain of function in the intestinal epithelium with a drug inducible mouse model. Strikingly, MSI2 induction alone was sufficient to phenocopy the majority of morphological and molecular consequences of acute loss of the APC tumor suppressor in the intestinal epithelium. We demonstrate that this phenotype is independent of both the activation of the other oncogenic Musashi family member, Msi1, and of canonical Wnt pathway activation. Transcriptome-wide RNA-binding analysis indicates that MSI2 acts as a pleiotropic inhibitor of known intestinal tumor suppressors including Lrig1, Bmpr1a, Cdkn1a, and Pten. Finally, we demonstrate that inhibition of the PDK-AKT-mTORC1 axis downstream of Pten rescues oncogenic consequences of MSI2 induction. Taken together, our findings identify MSI2 as a central component in an unappreciated oncogenic pathway promoting intestinal transformation. 2 wild-type samples, 2 TRE-Msi2 samples
Project description:Loss of the APC tumor suppressor in the intestinal epithelium initiates the majority of human colorectal adenocarcinomas. Constitutive β-catenin activation is thought to underlie tumorigenesis induced by loss of APC, however β-catenin activation alone does not recapitulate all APC-loss phenotypes, suggesting that additional pathways are required. We demonstrate that aberrant activation of the Msi1 RNA binding protein occurs upon APC loss and that constitutive Msi1 activation alone is sufficient to phenocopy APC loss in the intestinal epithelium. Msi1 elicits these effects through binding of mRNAs encoding pleiotropic tumor suppressors resulting in promiscuous activation of quiescent intestinal stem cells, proliferative expansion of the stem cell compartment, crypt fission, and blocked differentiation. Further, we find these phenotypes to be largely dependent on mTORC1 activity, and demonstrate that loss of Msi activity is sufficient to abrogate tumorigenesis in mouse and human systems. Our findings implicate Msi1 as a central coordinator of APC loss-induced intestinal stem cell transformation and adenocarcinoma progression. 2 wild-type, 2 transgenic samples
Project description:We demonstrate that Msi2 is the predominant form expressed in hematopoietic stem cells (HSC), and its knockdown leads to reduced engraftment and depletion of HSCs in vivo. Overexpression of Msi2 in a mouse model increases HSC cell cycle progression and cooperates with BCR-ABL1 to induce an aggressive leukemia. MSI2 is over-expressed in human myeloid leukemia, and expression levels directly correlate with decreased patient survival, thereby defining MSI2 expression as a novel prognostic marker in acute myeloid leukemia (AML). Depletion of MSI2 in human myeloid leukemia cells leads to decreased proliferation and apoptosis. These data implicate the MSI2 RNA binding protein in myeloid leukemogenesis and identify a novel potential target for therapy in AML. Hematopoietic stem cells and progenitor cells (LineageLow, Sca1-, Kit+; LK) from control and transgenic MSI2-inducible mice were isolated and RNA was extracted using Qiagen RNeasy Micro Kit according to manufacturers instruction. cDNA was fragmented and biotinylated before hybridization onto Affymetrix Mouse Expression Array 430 2.0.
Project description:Recent studies have shown that the RNA binding protein Musashi 2 (Msi2) plays prominent roles during development and leukemia. Additionally, in embryonic stem cells (ESC) undergoing the early stages of differentiation, Msi2 has been shown to associate with Sox2, which is required for the self-renewal of ESC. These findings led us to examine the effects of Msi2 on the behavior of ESC. Using an shRNA sequence that targets Msi2 and a scrambled shRNA sequence, we determined that knockdown of Msi2 disrupts the self-renewal of ESC and promotes their differentiation. Collectively, our findings argue that Msi2 is required to support the self-renewal and pluripotency of ESC. We used microarrays to better understand global changes in ESC gene expression following the knockdown of the RNA-binding protein Msi2 as compared to control ESC expressing a scrambled shRNA. Mouse embryonic stem cells (D3) were treated with lentivirus engineered for the expression of a Msi2 targeting shRNA sequence or scrambled (control) shRNA sequence. Following selection for infected cells with puromycin, cells were subcultured at low density and allowed to grow for 4 days (see treatment protocol) before RNA was extracted. RNA was collected and analyzed one time for each of the two samples.
Project description:Comparison of gene expression in intestinal epithelial cells in the presence or absence of ectopic induction of MSI2 in vivo Total RNA was isolated from preparations of total intestinal epithelial cells taken from the jejunum from 3 control (R26-M2rtTA +doxycycline for 24 hrs) and 3 experimental (TRE-MSI2::R26-M2rtTA +doxycycline for 24 hrs) and subjected to profiling on affymetrix Gene 1.0ST arrays
Project description:Msi2 is a critical regulatior of myeoid leukemia, and these data identify genes that are changed following Msi2 deletion in bcCML and de novo AML stem cells. Leukemic stem cells were extracted from wild-type and Msi2 mutant MLL-AF9 driven AML and BCR-ABL/NUP98-HOXA9 driven bcCML, in triplicate. RNA was extracted and hybridized on Affymetrix microarrays. The mouse strain was a genetic trap Msi2 mutant made by Center for Animal Resources and Development (CARD) of Kumamoto University. They call their strain B6;CB-Msi2Gt(pU-21T)2Imeg, see also http://cardb.cc.kumamoto-u.ac.jp/transgenic/strainsDetailAction.do?strainId=834
Project description:This SuperSeries is composed of the following subset Series: GSE16432: MSI2 regulates hematopoiesis and accelerates leukemogenesis GSE22773: Musashi 2 regulates normal hematopoiesis and accelerates leukemogenesis (LK and MS12-inducible) GSE22774: Musashi 2 regulates normal hematopoiesis and accelerates leukemogenesis (LSK and LK) GSE22775: Musashi 2 regulates normal hematopoiesis and accelerates leukemogenesis (Leukemia cell lines) Refer to individual Series
Project description:Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. Genetic and epigenetic alterations cause a dysregulated developmental program in leukemia. The MSI2 RNA binding protein has been previously shown to predict poor survival in leukemia. We demonstrate that the conditional deletion of Msi2 results in delayed leukemogenesis, reduced disease burden and a loss of LSC function. Gene expression profiling of the Msi2 ablated LSCs demonstrates a loss of the HSC/LSC and an increase in the differentiation program. The gene signature from the Msi2 deleted LSCs correlates with survival in AML patients. MSI2’s maintains the MLL self-renewal program by interacting with and retaining efficient translation of Hoxa9, Myc and Ikzf2. We further demonstrate that shRNA depletion of the MLL target gene Ikzf2 also contributes to MLL leukemia cell survival. Our data provides evidence that MSI2 controls efficient translation of the oncogenic LSC self-renewal program and a rationale for clinically targeting MSI2 in myeloid leukemia. RNA-Seq was performed on sorted c-Kit high leukemic cells from 2 Msi2 -/- and 2 Msi2 f/f mice.
Project description:Comparison of gene expression in intestinal epithelial cells in the presence or absence of ectopic induction of MSI2 in vivo Overall design: Total RNA was isolated from preparations of total intestinal epithelial cells taken from the jejunum from 3 control (R26-M2rtTA +doxycycline for 24 hrs) and 3 experimental (TRE-MSI2::R26-M2rtTA +doxycycline for 24 hrs) and subjected to profiling on affymetrix Gene 1.0ST arrays
Project description:BACKGROUND AND AIMS: Loss of epithelial cell homeostasis and apoptosis highly con-tribute to intestinal inflammation. While endoplasmic reticulum unfolded protein response (UPR) has been implicated in chronic intestinal inflammation, functional correlation between UPR-related C/EBP homologous protein (CHOP) expression and CHOP-mediated programming towards inflammation-related disease susceptibility remains unclear. In this study, we generated the new mouse model ChopIEC Tg/Tg to investigate consequences of intestinal epithelial cell (IEC)-specific CHOP overexpression. Transcriptional profiling of transgenic mice identified a set of CHOP-dependent target genes related to inflammatory and microbial defense program in the intestinal epithelium. Effect of CHOP overexpression in intestial epithelial cells was investigated on epithelial homeostasis using transgenic mice Disease-free mice do not show enhanced apoptotic signaling Intestinal epithelial cells were isolated from 12 week old females
Project description:In an effort to identify genes whose expression is regulated by activated PI3K signaling, we performed microarray analysis and subsequent qRT-PCR on an isogenic set of PTEN gene-targeted human cancer cells. Numerous p53 effectors were upregulated following PTEN deletion, including p21, GDF15, PIG3, NOXA, and PLK2. Stable depletion of p53 led to reversion of the gene expression program. Western blots revealed that p53 was stabilized in HCT116 PTEN-/- cells via an Akt1-dependent and p14ARF-independent mechanism. Stable depletion of PTEN in untransformed human fibroblasts and epithelial cells also led to upregulation of p53 and senescent-like growth arrest. Simultaneous depletion of p53 rescued this phenotype, enabling PTEN-depleted cells to continue proliferating. Next, we tested whether oncogenic PIK3CA, like inactivated PTEN, could activate p53. Retroviral expression of oncogenic human PIK3CA in MCF10A cells led to activation of p53 and upregulation of p53-regulated genes. Stable depletion of p53 reversed these PIK3CA-induced expression changes and synergized with oncogenic PIK3CA in inducing anchorage-independent growth. Finally, targeted deletion of an endogenous allele of oncogenic but not wild-type PIK3CA in a human cancer cell line led to a reduction in p53 levels and a decrease in the expression of p53-regulated genes. These studies demonstrate that activation of PI3K signaling by mutations in PTEN or PIK3CA can lead to activation of p53-mediated growth suppression in human cells, indicating that p53 can function as a brake on PIP3-induced mitogenesis during human cancer pathogenesis. Experiment Overall Design: Two HCT116 PTEN+/+ cell lines (parental cells and a clone with random integration of the targeting vector) and three independently-derived HCT116 PTEN-/- cell lines were studied