Trp53 Mice Research Articles

Loss of p53 and genetic evolution in pancreatic cancer: Ordered chaos after the guardian is gone.

A recent Nature study delineates a stepwise genomic evolution during pancreatic cancer development, employing an engineered mutant Kras and heterozygous Trp53 mouse model that identifies cells undergoing Trp53 loss of heterozygosity (LOH). Genetic progression post-Trp53 LOH involves clonal deletions, then genome doubling and subsequent accumulation of subclonal gains and amplifications.

A17 TRP53 REGULATES ESLF-RENEWAL AND CELL DIFFERENTIATION IN KRT15+ INTESTINAL STEM CELLS

Abstract Background Different pools of stem cells assure the intestinal epithelium homeostasis and regeneration. We recently reported that Krt15+ intestinal cells harbor self-renewal, multipotent and regenerative capacities, characteristics consistent with a stem cell population. p53 has been associated with the development of various types of cancer, and recent studies suggested it also regulates adult stem cell behavior in some tissues. However, its role in the maintenance of intestinal stem cells has not been explicitly covered. Therefore, we hypothesize that Trp53 loss specifically in Krt15+ intestinal stem cells will perturb the epithelial homeostasis and differentiation. Aims Identify the role of Trp53 in the regulation of intestinal stem cell and intestinal homeostasis Methods To induce Trp53 loss specifically in Krt15+ cells, we generated Krt15 CrePR1;Trp53fl/fl ( Krt15△Trp53) mice and induced Cre recombination by injecting RU486 (PR agonist). Mice were euthanized after Cre recombination at different time points. Organoid cultures were established from control and experimental mice. Results Two-month post Cre recombination, we observed no major morphological changes in the intestinal epithelium. However, we observed that crypts isolated from Krt15△Trp53 mice died rapidly and had difficulties forming organoids. When hyperactivating the Wnt/beta-catenin pathway through CHIR treatment, we observed that budding capacity was maintained in experimental organoids while control organoids formed mainly cysts as expected. We also observed an increased in Paneth cell-specific genes, suggesting an enrichment in Paneth cells. Interestingly, the expression of EpHB2 receptor and its target genes was decreased in organoids derived from Krt15△Trp53 mice. Ephrin signaling plays a crucial role in intestinal homeostasis by regulating cell position and migration, and previous studies suggest that ephrin receptors are a target of p53. Indeed, twelve-month post Cre recombination, altered architecture of crypt and villi was noted in the small intestinal epithelium of Krt15△Trp53 mice. Differentiation towards secretory cell types, specifically Paneth, goblet, and tuft cells, are significantly increased in Krt15△Trp53 mice. Interestingly, we also observed crypt cells expressing both goblet and Paneth cell markers suggesting dysfunction in the cell fate decision. Finally, decreased Notch pathway activation was also observed supporting that Trp53 loss, specifically in Krt15+ cells, leads to a dysregulation of secretory cell fate. Conclusions Loss of Trp53 specifically in Krt15+ cells affects the small intestinal homeostasis and secretory cell differentiation. Decreased Notch and ephrin signaling support the possible role of Trp53 in cell fate decision and self-renewal. Funding Agencies None

A149 TRP53 LOSS IN KRT15+ INTESTINAL STEM CELLS SEVERELY AFFECTS SECRETORY CELL FATE AND SELF-RENEWAL

Abstract Background Intestinal epithelium homeostasis is maintained by two main populations of stem cells: Lgr5+ and reserve stem cells. Under injury, cell plasticity has been observed in progenitor and differentiated cells. We recently reported that Krt15+ cells are present in small intestinal and colon epithelia, and harbor self-renewal, multipotent and regenerative capacities. As in Lgr5+and reserve stem cells, hyperactivation of Wnt/b-catenin pathway in Krt15+ stem cells lead to tumor formation in the intestinal epithelium. While these intestinal stem cell populations can act as tumor-initiating cells in sporadic colon cancer, little is known about the cell-of-origin of colitis-associated colon cancer (CAC). TP53 alteration is reported as an early event in CAC. Therefore, we hypothesize that Trp53 loss specifically in Krt15+ stem cells will perturb the epithelial homeostasis and lead to tumor formation. Aims Identify if Krt15+ cells may act as the cell-of-origin in colitis-associated colorectal cancer Methods To induce Trp53 loss specifically in Krt15+ cells, we generated Krt15-CrePR1;Trp53fl/fl (Krt15△Trp53) mice, induced Cre recombination by injecting RU486 (PR agonist) and euthanized the mice at different time points following recombination. Results Results Twelve-month following Cre recombination, adenoma formation was observed in a small proportion of Krt15△Trp53 mice. Though, Trp53 loss in Krt15+ cells severely perturbed the small intestinal morphology in every mouse studied. Increased crypt length and villi width was observed in Krt15△Trp53 vs control mice without any changes in cell proliferation. We also observed an increased number of Tuft cells and goblet cells in the villi of experimental mice. In the crypt, higher number of Paneth cells and aberrant presence of goblet cells were noted in Krt15△Trp53mice. Interestingly, we also observed crypt cells expressing goblet and Paneth cell markers and decreased Notch pathway activation suggesting dysregulation of secretory cell fate. Krt15△Trp53 mice display higher number of fibroblasts in the villi and the submucosa, as well as thickening of the muscularis layer. Interestingly, similar observations (accumulation of secretory cells and fibrosis) have been reported in IBD patients, supporting the possible role of Krt15+ cells in CAC. Furthermore, crypts isolated from Krt15△Trp53 mice rapidly die when seeded as organoids vs crypts from control mice, suggesting that the alterations observed in vivo in Paneth cells might interfere with the stem cell niche and therefore reduce self-renewal of Krt15+ cells. Conclusions Trp53 loss specifically in Krt15+ cells impaired cell fate decision, induced secretory cell hyperplasia, affected self-renewal ability, and initiated adenoma formation supporting the possible role of Krt15+ cells in gut inflammation and cancer. Funding Agencies Canada Research Chair, Cancer Research Society, CFI

A31 ALTERED SMALL INTESTINAL EPITHELIAL HOMEOSTASIS AND CELL FATE DECISION UPON LOSS OF TRP53 IN KRT15+ INTESTINAL STEM CELLS

Abstract Background Intestinal homeostasis is mainly maintained by two groups of stem cells: Lgr5+ stem cells and reserve stem cells. Recently, we reported that Krt15+ cells are present in small intestinal and colon epithelia, and harbor self-renewal, multipotent and regenerative capacities. Initiation of sporadic colorectal cancer has been described in Krt15+ stem cells, Lgr5+ stem cells and reserve stem cells following the loss of Apc. While these intestinal stem cell (ISC) populations can act as tumor-initiating cells in sporadic colon cancer, little is known about the cell-of-origin of colitis-associated colon cancer. TP53 alteration is reported as an early event in colitis-associated colon cancer cases. Therefore, we hypothesize that Trp53 loss specifically in Krt15+ stem cells will perturb the epithelial homeostasis and lead to tumor formation Aims Identify if Krt15+ cells may act as the cell-of-origin in colitis-associated colorectal cancer. Methods To induce Trp53 loss specifically in Krt15+ cells, we generated Krt15-CrePR1;Trp53fl/fl (Krt15△Trp53) mice, induced Cre recombination by injecting RU486 (PR agonist) and euthanized the mice after 12 months following recombination. Histological analysis was performed on small intestinal tissues. Results Results: Trp53 loss in Krt15+ cells severely perturbed small intestinal morphology. Increased crypt (proliferative compartment) length correlated with no proliferation changes surprisingly but higher number of Paneth cells and abnormal presence of goblet cells. Interestingly, we also observed the presence of cells expressing both Paneth and goblet cells markers suggesting a deregulation of secretory cell fate decision. Decreased expression of Hes1 and increased β-catenin nuclear expression in the small intestinal crypt of Krt15△Trp53 mice suggest altered Notch and Wnt signaling. Furthermore, villi (differentiated compartment) were significantly wider and shorter, and showed accumulation of activated fibroblasts. Finally, we observed inflammatory lesions as well as adenomas in the small intestine of Krt15△Trp53 mice which remain to be further characterized Conclusions Conclusion: In summary, Trp53 loss specifically in Krt15+ cells impaired cell fate decision, induced inflammation and initiated tumor formation. Overall, these results suggest that Krt15+ cells could act as the cell-of-origin of colitis-associated colon cancer. Funding Agencies Cancer Research Society, CRC Tier 2

Role of smooth muscle cell p53 in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by remodeling and narrowing of the pulmonary arteries, which lead to elevation of right ventricular pressure, heart failure, and death. Proliferation of pulmonary artery smooth muscle cells (PASMCs) is thought to be central to the pathogenesis of PAH, although the underlying mechanisms are still being explored. The protein p53 is involved in cell cycle coordination, DNA repair, apoptosis, and cellular senescence, but its role in pulmonary hypertension (PH) is not fully known. We developed a mouse model of hypoxia-induced pulmonary hypertension (PH) and found significant reduction of p53 expression in the lungs. Our in vitro experiments with metabolomic analyses and the Seahorse XF extracellular flux analyzer indicated that suppression of p53 expression in PASMCs led to upregulation of glycolysis and downregulation of mitochondrial respiration, suggesting a proliferative phenotype resembling that of cancer cells. It was previously shown that systemic genetic depletion of p53 in a murine PH model led to more severe lung manifestations. Lack of information about the role of cell-specific p53 signaling promoted us to investigate it in our mouse PH model with the inducible Cre-loxP system. We generated a mouse model with SMC-specific gain or loss of p53 function by crossing Myh11-Cre/ERT2 mice with floxed Mdm4 mice or floxed Trp53 mice. After these animals were exposed to hypoxia for 4 weeks, we conducted hemodynamic and echocardiographic studies. Surprisingly, the severity of PH was similar in both groups of mice and there were no differences between the genotypes. Our findings in these mice indicate that activation or suppression of p53 signaling in SMCs has a minor role in the pathogenesis of PH and suggest that p53 signaling in other cells (endothelial cells, immune cells, or fibroblasts) may be involved in the progression of this condition.

Effect of Trp53 gene deficiency on brain injury after neonatal hypoxia-ischemia.

Hypoxia-ischemia (HI) can result in permanent life-long injuries such as motor and cognitive deficits. In response to cellular stressors such as hypoxia, tumor suppressor protein p53 is activated, potently initiating apoptosis and promoting Bax-dependent mitochondrial outer membrane permeabilization. The aim of this study was to investigate the effect of Trp53 genetic inhibition on injury development in the immature brain following HI. HI (50 min or 60 min) was induced at postnatal day 9 (PND9) in Trp53 heterozygote (het) and wild type (WT) mice. Utilizing Cre-LoxP technology, CaMK2α-Cre mice were bred with Trp53-Lox mice, resulting in knockdown of Trp53 in CaMK2α neurons. HI was induced at PND12 (50 min) and PND28 (40 min). Extent of brain injury was assessed 7 days following HI. Following 50 min HI at PND9, Trp53 het mice showed protection in the posterior hippocampus and thalamus. No difference was seen between WT or Trp53 het mice following a severe, 60 min HI. Cre-Lox mice that were subjected to HI at PND12 showed no difference in injury, however we determined that neuronal specific CaMK2α-Cre recombinase activity was strongly expressed by PND28. Concomitantly, Trp53 was reduced at 6 weeks of age in KO-Lox Trp53 mice. Cre-Lox mice subjected to HI at PND28 showed no significant difference in brain injury. These data suggest that p53 has a limited contribution to the development of injury in the immature/juvenile brain following HI. Further studies are required to determine the effect of p53 on downstream targets.

Hypothesis: The female excess in cranial neural tube defects reflects an epigenetic drag of the inactivating x chromosome on the molecular mechanisms of neural fold elevation

Females have long been known to be in excess among cranial neural tube defect (NTD) cases. Up to two thirds of human anencephalics and mouse exencephalics from various genetic causes are female, but the cause of this female excess is unknown. It appears not to be attributable to gonadal hormones, developmental delay in females, or preferential death of affected males. Recent studies of the Trp53 mouse mutant showed that exencephaly susceptibility depends on the presence of two X chromosomes, not the absence of the Y. Over a decade ago, we hypothesized that the relevant difference between female and male mammalian embryos at the time of cranial neural tube closure is the fact that females methylate most of the DNA in the large inactive X chromosome after every cell division, reducing the methylation available for other needs in female cells. Recently, the Whitelaw laboratory identified several proteins in mice (Momme D genes) involved in epigenetic silencing and methylation and shared in the silencing of transgenes, retrotransposons, and the inactive-X, and suggested that the inactive-X acts as a "sink" for epigenetic silencing proteins. The "inactive-X sink" hypothesis can be used to suggest expected changes in sex ratio in cranial NTDs in response to various genetic or environmental alterations. We recommend that observation of sex ratio become a standard component of all NTD studies. We suggest that the female excess among cranial NTDs is an epigenetic phenomenon whose molecular investigation will produce insight into the mechanisms underlying NTDs.

Abstract 1433: TC21/R-Ras2 is a critical mediator of the Nf1 oncogenic switch

Abstract The TC21/R-Ras2 protein is an oncogenic member of the Ras family. Ras proteins act as binary switches which are ‘active’ when bound to GTP and ‘inactive’ when bound to GDP. GTPase activating proteins (GAPs) accelerate the hydrolysis from Ras-GTP to Ras-GDP. This Ras-related GTPase TC21 has transforming capabilities similar to H, N and K-Ras. Activated alleles of TC21 transform epithelial and fibroblast cell lines and induce tumors in vivo. To explore the role of TC21 in cancer we used a model of Ras activation driven by loss of the NF1 tumor suppressor protein, a GAP for all Ras proteins including TC21. Thus loss of NF1 predicts increased levels of activated TC21/R-Ras2. NF1 loss in Schwann cells of the peripheral nervous system causes benign tumors known as neurofibromas. Neurofibromas can transform to sarcomas known as malignant peripheral nerve sheath tumors (MPNSTs). We found that TC21 loss delayed benign neurofibroma formation in Nf1fl/fl;DhhCre mice. Nf1 loss increased mRNA encoding the cytokine transforming growth factor-beta (TGF-β) and rendered Schwann cell progenitors insensitive to TGF-β; these phenotypes could be rescued by the Ras-related protein TC21/R-Ras2 and were mediated through TGF-β receptors. Conversely, growth of Nf1;Trp53 brain tumors and NF1−/− MPNST sarcomas were accelerated by TC21 loss. MPNST from Nf1;Trp53 mice and NF1−/− MPNST xenografts had increased levels of mRNA encoding TGF- ≤ ligands, and blocking TGF- ≤ decreased sarcoma size induced by shTC21. In benign and malignant tumors an AKT-dependent pathway regulated TGF- ≤ expression. Indicating relevance to human tumorigenesis, global gene expression analyses demonstrated increases in expression of TGF- ≤ ligands and decreases in TGF- ≤ receptors in human neurofibromas and MPNSTs. Thus, we identify critical roles for TC21 in regulation of TGF- ≤ expression. The results are important because TGF- ≤ acts as a tumor suppressor in numerous types of benign tumors and is also known for its oncogenic role in cellular transformation and tumor progression. While it has been known that Ras proteins are involved in TGF- ≤ mediated tumor suppression and oncogenesis, integration between the pathways is incompletely understood, especially in vivo. The study demonstrates that TC21 can be a major regulator of the duality of TGF- ≤ effects on tumorigenesis in vivo. This work was supported by a grant to NR (NIH P50NS057531-03). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1433. doi:1538-7445.AM2012-1433

FAK Deletion Promotes Increased Radioresistance in a Wildtype Trp53 Mouse Squamous Cell Carcinoma Cell Line by p53 Mediated Induction of p21

FAK Deletion Promotes Increased Radioresistance in a Wildtype Trp53 Mouse Squamous Cell Carcinoma Cell Line by p53 Mediated Induction of p21

Enrichment of hematopoietic stem cells with SLAM and LSK markers for the detection of hematopoietic stem cell function in normal and Trp53 null mice

To test function of hematopoietic stem cells (HSCs) in vivo in C57BL/6 (B6) and Trp53-deficient (Trp53 null) mice by using two HSC enrichment schemes. Bone marrow (BM) Lin-CD41-CD48-CD150+ (signaling lymphocyte activation molecules [SLAM]), Lin-CD41-CD48-CD150- (SLAM-) and Lin-Sca1+CD117+ (LSK) cells were defined by fluorescence-activated cell staining (FACS). Cellular reactive oxygen species (ROS) level was also analyzed by FACS. Sorted SLAM, SLAM-, and LSK cells were tested in vivo in the competitive repopulation (CR) and serial transplantation assays. SLAM cell fraction was 0.0078%+/-0.0010% and 0.0135%+/-0.0010% of total BM cells in B6 and Trp53 null mice, and was highly correlated (R2=0.7116) with LSK cells. CD150+ BM cells also contained more ROSlow cells than did CD150- cells. B6 SLAM cells repopulated recipients much better than B6 SLAM- cells, showing high HSC enrichment. B6 SLAM cells also engrafted recipients better than Trp53 null SLAM cells in the CR and the follow-up serial transplantation assays. Similarly, LSK cells from B6 donors also had higher repopulating ability than those from Trp53 null donors. However, whole BM cells from the same B6 and Trp53 null donors showed the opposite functional trend in recipient engraftment. Both SLAM and LSK marker sets can enrich HSCs from B6 and Trp53 mice. Deficiency of Trp53 upregulates HSC self-renewal but causes no gain of HSC function.

SnapShot: Genetic Mouse Models of Cancer

p 53 T um o r S up p re ss o r Trp53 mouse germline knockout. Trp53−/− homozygous: 100% tumor penetrance at ?4.5 months. Typical tumors: T cell lymphoma (>60%); soft tissue sarcoma (?25%); osteosarcoma, brain tumors, teratoma (together <15%); carcinomas rarely observed. Trp53+/− heterozygous: 50% tumor penetrance at 17 months. Typical tumors: T cell lymphoma (?30%); soft tissue sarcoma (?30%); osteosarcoma (?30%); more carcinomas than Trp53−/− mice. Oncogenic cooperativity observed between Trp53−/− and other lesions such as Rb−/− or Eμ-Myc. Carcinogenesis induced by different genotoxic agents or irradiation is accelerated in Trp53-deficient mice. Mutations in TP53 found in more than 50% of all human tumors.

Chromosome 11 loss from thymic lymphomas induced in heterozygous Trp53 mice by phenolphthalein.

C57BL/6 p53 (+/-) N5 mice heterozygous for a null p53 allele were given phenolphthalein to learn more about mechanisms of carcinogenesis and to evaluate the p53-deficient mouse as a tool for identifying potential human carcinogens. DNA samples isolated from 10 phenolphthalein-induced thymic lymphomas were analyzed for loss of heterozygosity (LOH) at the Trp53 locus and simple sequence length polymorphic (SSLP) loci. The initial screening revealed remarkable results from only chromosome 11. Allelotyping at approximately five centiMorgan intervals, we found SSLP heterozygosity for C57BL/6 and 129Sv over much of chromosome 11. In the tumors, treatment-related LOH was apparent on chromosome 11 at each of the 28 informative loci examined. The strain-specific polymorphism lost from individual tumors allowed us to deduce the distribution of alleles along the length of the maternal and paternal chromosomes 11. The allelic patterns indicate that mitotic homologous recombination occurred during embryogenesis if breeding protocols were carried out as described. The mitotic recombination observed may be attributable to p53 haploinsufficiency for normal suppression of mitotic recombination.