ABSTRACT
Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf-/- mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 (Trp53) in 54% of tumors and transposon-mediated gain-of-function alterations in B-cell lymphoma-extra large (Bcl-xL), Mdm4, and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.
Subject(s)
DNA Transposable Elements , Drug Resistance, Neoplasm/genetics , Genetic Vectors/genetics , Mutagenesis, Insertional , Proto-Oncogene Proteins c-mdm2/genetics , Tumor Suppressor Protein p53/genetics , Allografts , Animals , Antineoplastic Agents/pharmacology , Biomarkers, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Disease Models, Animal , Genetic Drift , Humans , Kaplan-Meier Estimate , Mice , Mice, Knockout , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/mortality , Neoplasms/pathology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , bcl-X Protein/genetics , bcl-X Protein/metabolismABSTRACT
Hypoxia is a prominent feature in the maintenance of hematopoietic stem cell (HSC) quiescence and multipotency. Hypoxia-inducible factor (HIF) prolyl hydroxylase domain proteins (PHDs) serve as oxygen sensors and may therefore regulate this system. Here, we describe a mouse line with conditional loss of HIF prolyl hydroxylase 2 (PHD2) in very early hematopoietic precursors that results in self-renewal of multipotent progenitors under steady-state conditions in a HIF1α- and SMAD7-dependent manner. Competitive bone marrow (BM) transplantations show decreased peripheral and central chimerism of PHD2-deficient cells but not of the most primitive progenitors. Conversely, in whole BM transfer, PHD2-deficient HSCs replenish the entire hematopoietic system and display an enhanced self-renewal capacity reliant on HIF1α. Taken together, our results demonstrate that loss of PHD2 controls the maintenance of the HSC compartment under physiological conditions and causes the outcompetition of PHD2-deficient hematopoietic cells by their wild-type counterparts during stress while promoting the self-renewal of very early hematopoietic progenitors.
Subject(s)
Hematopoietic Stem Cells/cytology , Hypoxia/physiopathology , Multipotent Stem Cells/cytology , Procollagen-Proline Dioxygenase/physiology , Stress, Physiological , Animals , Bone Marrow Transplantation , Cell Cycle , Cell Differentiation , Hematopoietic Stem Cells/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit , Hypoxia-Inducible Factor-Proline Dioxygenases , Integrases/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Multipotent Stem Cells/metabolism , Smad7 Protein/metabolismABSTRACT
The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3-4 weeks by using Gateway cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.
Subject(s)
Cloning, Molecular/methods , Embryonic Stem Cells/metabolism , Gene Targeting/methods , Genetic Vectors , Mice, Transgenic , Proteins/genetics , Alleles , Animals , Cell Line , Diploidy , Hybrid Cells , Mice , RNA, Untranslated , Recombination, Genetic , TransgenesABSTRACT
The inactivation of the p53 tumor suppressor pathway, which often occurs through mutations in TP53 (encoding tumor protein 53) is a common step in human cancer. However, in melanoma-a highly chemotherapy-resistant disease-TP53 mutations are rare, raising the possibility that this cancer uses alternative ways to overcome p53-mediated tumor suppression. Here we show that Mdm4 p53 binding protein homolog (MDM4), a negative regulator of p53, is upregulated in a substantial proportion (â¼65%) of stage I-IV human melanomas and that melanocyte-specific Mdm4 overexpression enhanced tumorigenesis in a mouse model of melanoma induced by the oncogene Nras. MDM4 promotes the survival of human metastatic melanoma by antagonizing p53 proapoptotic function. Notably, inhibition of the MDM4-p53 interaction restored p53 function in melanoma cells, resulting in increased sensitivity to cytotoxic chemotherapy and to inhibitors of the BRAF (V600E) oncogene. Our results identify MDM4 as a key determinant of impaired p53 function in human melanoma and designate MDM4 as a promising target for antimelanoma combination therapy.
Subject(s)
Melanoma/chemistry , Neoplasm Proteins/physiology , Nuclear Proteins/physiology , Proto-Oncogene Proteins/physiology , Skin Neoplasms/chemistry , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Cycle Proteins , Cell Line, Tumor/metabolism , Cell Line, Tumor/transplantation , Cell-Penetrating Peptides/pharmacology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/physiology , Female , GTP Phosphohydrolases/genetics , Gene Expression Regulation, Neoplastic , Humans , Keratinocytes/metabolism , Male , Melanocytes/metabolism , Melanoma/pathology , Melanoma/secondary , Melanoma, Experimental/etiology , Melanoma, Experimental/genetics , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Nude , Mice, Transgenic , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/biosynthesis , Proto-Oncogene Proteins c-mdm2/genetics , Recombinant Fusion Proteins/physiology , Signal Transduction/physiology , Tumor Stem Cell Assay , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/physiology , Up-Regulation , Xenograft Model Antitumor AssaysABSTRACT
Mdm2 and Mdm4 are critical negative regulators of p53. A large body of evidence indicates that elevated expression of either Mdm2 or Mdm4 may favor tumor formation by inhibiting p53 tumor suppression function. To explore this possibility in vivo, we generated conditional Mdm2 and Mdm4 transgenic mice. We show that although both transgenes are designed to be expressed ubiquitously and at comparable levels, only the Mdm4 transgenic protein is produced at high levels in vivo. In contrast, exogenous Mdm2 is constitutively degraded in a proteasome-dependent manner, indicating that cells are equipped with efficient mechanisms that prevent Mdm2 accumulation in vivo. Mice that are homozygous for the Mdm4 transgene die during embryogenesis owing to severe vascular maturation defects. Importantly, this lethality is not rescued on a p53-null background, indicating that high levels of Mdm4 impact on a pathway(s) other than p53 that controls vascular and embryonic development. Mice expressing a single copy of the Mdm4 transgene are viable and, surprisingly, are not prone to spontaneous, radiation-induced or Eµ-myc-induced tumor formation. The findings have clear implications for cancer etiology as well as for cancer therapy.
Subject(s)
Epitopes , Neoplasms/metabolism , Neoplasms/pathology , Proto-Oncogene Proteins c-mdm2/metabolism , Proto-Oncogene Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Embryo, Mammalian/anatomy & histology , Embryo, Mammalian/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Kaplan-Meier Estimate , Lymphoma/genetics , Lymphoma/metabolism , Lymphoma/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neoplasms/etiology , Neoplasms/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-mdm2/genetics , Tissue Distribution , Transgenes , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/geneticsABSTRACT
MAPK phosphatases (MKPs) are dual specificity phosphatases that dephosphorylate and thereby inactivate MAPKs. In the present study, we provide evidence that platelet-derived growth factor BB (PDGF-BB) regulates MKP3 (DUSP6), which is considered to be a phosphatase highly selective for Erk. Intriguingly, we observed that Mek is positively regulated by MKP3, whereas Erk itself is negatively regulated. In addition, we found that activation of PDGF receptor alpha or beta leads to a rapid proteasomal degradation of MKP3 in a manner that requires Mek activation; this feed-forward mechanism was found to be essential for efficient Erk phosphorylation. We could also demonstrate that PDGF-BB stimulation induces phosphorylation of MKP3 at Ser-174 and Ser-300; phosphorylation of Ser-174 is involved in PDGF-induced MKP3 degradation, since mutation of this site stabilized MKP3. Moreover, activated Erk induces mkp3 expression, leading to restoration of MKP3 levels after 1-2 h and a concomitant dephosphorylation of Erk in cells with activated PDGFRalpha. Reducing the MKP3 level by small interfering RNA leads to an increased Erk activation and mitogenic response to PDGF-BB. In conclusion, MKP3 is an important regulator of PDGF-induced Erk phosphorylation acting in both a rapid positive feed-forward and a later negative feed-back loop.