Project description:RNA-sequencing was performed on p53-inactivated human triple negative breat cancer cells to evaluate gene expression changes following MDM2 loss. Genes and pathways that are regulated by p53/p73 were induced with MDM2 loss.
Project description:The pulmonary alveolar epithelium mainly composed of two types of epithelial cells: alveolar type I (AT1) and type II (AT2) cells. AT2 cells are the alveolar stem cells, and can differentiate into AT1 cells post-pneumonectomy (PNX). Here, we found that, compared with control mice (Sftpc-CreER; Cdc42flox/+; Rosa26-mTmG) at post-PNX day 21, Cdc42 AT2 null mice (Sftpc-CreER; Cdc42flox/-; Rosa26-mTmG) at post-PNX day 21 undergone fibrotic change. By using 10X genomics “Chromium Single Cell” technology, we performed single-cell RNA-seq analyses of AT2 cells of sham treated control mice (C0), AT2 cells of control mice at post PNX day 21 (C21) , AT2 cells of sham treated Cdc42 AT2 null mice (N0), and AT2 cells of Cdc42 AT2 null mice at post PNX day 21 (N21). The study identified a specific gene signature in AT2 cells of Cdc42 AT2 null mice at post PNX day 21 which is related to the fibrosis phenotype of Cdc42 AT2 null mice.
Project description:Carnitine palmitoyltransferase 1a (CPT1a) is the key regulator of mitochondrial long-chain fatty acid beta-oxidation (LCFAO). However, the functional significance of AT2 cell-specific LCFAO at baseline and during acute lung injury (ALI) is not fully understood. In this study, Murine models of AT2 cell-specific Cpt1a deletion were generated for investigating the role of CPT1a in regulating AT2 cell function and the severity of lipopolysaccharide-induced murine ALI models.
Project description:AT2 cells are the resident progenitor cells in alveoli, capable of self-proliferation and differentiation into alveolar type I cells during homeostatic maintenance and tissue regeneration. The AT2 cell population is heterogenous. We identified a small subpopulation of AT2 cells that express high levels of CD44 (CD44hi) and display progenitor functions during alveoli homeostasis. To further analyze the heterogeneity of the AT2 cell population and characterize CD44hi AT2 cells, we performed single cell RNA-seq on the total AT2 cell population and CD44hi AT2 cells.
Project description:Alveolar type II (AT2) cell dysfunction contributes to a number of significant human pathologies including respiratory distress syndrome, lung adenocarcinoma, and debilitating fibrotic diseases, but the critical transcription factors that maintain AT2 cell identity are unknown. Here we show that the E26 transformation-specific (ETS) family transcription factor Etv5 is essential tomaintain AT2 cell identity. Deletion of Etv5 from AT2 cells produced gene and protein signatures characteristic of differentiated alveolar type I (AT1) cells. Consistent with a defect in the AT2 stem cell population, Etv5 deficiency markedly reduced recovery following bleomycin-induced lung injury. Lung tumorigenesis driven by mutant KrasG12D was also compromised by Etv5 deficiency. ERK activation downstream of Ras was found to stabilize Etv5 through inactivation of the cullin- RING ubiquitin ligase CRL4COP1/DET1 that targets Etv5 for proteasomal degradation. These findings identify Etv5 as a critical output of Ras signaling in AT2 cells, contributing to both lung homeostasis and tumor initiation.
Project description:Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. We screened several MRT cell lines each with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered MDM2 and MDM4, the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin and ATSP-7041, we show that MRT cells are more sensitive than other p53 wild-type cancer cell lines to MDM2 and dual MDM2/4 inhibition in vitro. These compounds cause significant upregulation of the p53 pathway in MRT cells, and sensitivity is ablated by CRISPR-Cas9-mediated inactivation of TP53. We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRT, sensitizes cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth in vivo, with a five-day idasanutlin pulse causing marked regression of all xenografts including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene p53, and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer.
Project description:Adult cardiomyocytes (CM) are terminally differentiated cells with minimal regenerative capacity, making cardiac tissue particularly vulnerable to injury. Thus, defining the roadblocks responsible for adult CM cell cycle arrest lies at the core of developing therapies to regenerate myocyte loss following injurious events such as myocardial infarction. We have previously shown that inactivating the p53/Mdm2 tumor suppressor circuitry, specifically in the heart (using the Cre-loxP recombination system of bacteriophage P1), can allow differentiated CMs to regain proliferative capacity, through an upregulation of factors involved in cell cycle re-entry. These factors are repressed in quiescent CMs, in part through the action of microRNAs (miRNAs). Notably, knockout of either p53 or Mdm2 individually was insufficient to promote CM proliferation. Therefore, we hypothesized that inactivation of p53/Mdm2-regulated miRNAs could promote the expression of cell cycle activators and induce proliferation of adult murine CMs. To identify miRNAs regulated by both p53 and Mdm2, total miRNA expression profiles from cardiac specific p53/Mdm2 double knockout (DKO) mouse hearts were compared with those from cardiac-specific single knockouts (p53KO and Mdm2KO), and vehicle-injected controls using the Nanostring nCounter mouse miRNA expression assay. This revealed a profile of 11 significantly downregulated miRNAs in the proliferative DKO hearts (versus vehicle-injected control), that were enriched for mRNA targets involved in cell cycle regulation. In vitro studies have demonstrated that knockdown of these 11 miRNAs in neonatal rat cardiomyocytes can increase the occurrence of cytokinetic events. Ultimately, we aim to inject antagomirs targeting these miRNAs into animals post-myocardial infarction to determine the effect of p53/Mdm2-regulated miRNAs on heart function and CM proliferation in vivo.
Project description:FMTg (Mdm2FM/- Cag-CreERTg) and Tg (Mdm2+/- Cag-CreERTg) mice were administered 6mg/40g of Tamoxifen dissolved in corn-oil and sacrificed 24 hours after injection. Single cells were isolated from fresh small intestine samples to compare single cell transcriptomes between intestine with high or control p53 activity