Alice Nemajerova

WT p53, but Not Tumor-derived Mutants, Bind to Bcl2 via the DNA Binding Domain and Induce Mitochondrial Permeabilization

The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the anti-apoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component KD 535 ± 24 nm. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.

A Tautomerase-Null Macrophage Migration-Inhibitory Factor (MIF) Gene Knock-In Mouse Model Reveals That Protein Interactions and Not Enzymatic Activity Mediate MIF-Dependent Growth Regulation

ABSTRACT Macrophage migration-inhibitory factor (MIF) is an upstream regulator of innate immunity and a potential molecular link between inflammation and cancer. The unusual structural homology between MIF and certain tautomerases, which includes both a conserved substrate-binding pocket and a catalytic N-terminal proline (Pro1), has fueled speculation that an enzymatic reaction underlies MIFs biologic function. To address the functional role of the MIF tautomerase activity in vivo, we created a knock-in mouse in which the endogenous mif gene was replaced by one encoding a tautomerase-null, Pro1→Gly1 MIF protein (P1G-MIF). While P1G-MIF is completely inactive catalytically, it maintains significant, albeit reduced, binding to its cell surface receptor (CD74) and to the intracellular binding protein JAB1/CSN5. P1G-MIF knock-in mice (mifP1G/P1G) and cells derived from these mice show a phenotype in assays of growth control and tumor induction that is intermediate between those of the wild type (mif+/+) and complete MIF deficiency (mif−/−). These data provide genetic evidence that MIFs intrinsic tautomerase activity is dispensable for this cytokines growth-regulatory properties and support a role for the N-terminal region in protein-protein interactions.

Direct reprogramming by oncogenic Ras and Myc

Genetically or epigenetically defined reprogramming is a hallmark of cancer cells. However, a causal association between genome reprogramming and cancer has not yet been conclusively established. In particular, little is known about the mechanisms that underlie metastasis of cancer, and even less is known about the identity of metastasizing cancer cells. In this study, we used a model of conditional expression of oncogenic KrasG12D allele in primary mouse cells to show that reprogramming and dedifferentiation is a fundamental early step in malignant transformation and cancer initiation. Our data indicate that stable expression of activated KrasG12D confers on cells a large degree of phenotypic plasticity that predisposes them to neoplastic transformation and acquisition of stem cell characteristics. We have developed a genetically tractable model system to investigate the origins and evolution of metastatic pancreatic cancer cells. We show that metastatic conversion of KrasG12D-expressing cells that exhibit different degrees of differentiation and malignancy can be reconstructed in cell culture, and that the proto-oncogene c-Myc controls the generation of self-renewing metastatic cancer cells. Collectively, our results support a model wherein non-stem cancer cells have the potential to dedifferentiate and acquire stem cell properties as a direct consequence of oncogene-induced plasticity. Moreover, the disturbance in the normally existing dynamic equilibrium between cancer stem cells and non-stem cancer cells allows the formation of cancer stem cells with high metastatic capacity at any time during cancer progression.

Impaired DNA damage checkpoint response in MIF-deficient mice

Recent studies demonstrated that proinflammatory migration inhibitory factor(MIF) blocks p53‐dependent apoptosis and interferes with the tumor suppressor activity of p53. To explore the mechanism underlying this MIF‐p53 relationship, we studied spontaneous tumorigenesis in genetically matched p53−/− and MIF−/−p53−/− mice. We show that the loss of MIF expression aggravates the tumor‐prone phenotype of p53−/− mice and predisposes them to a broader tumor spectrum, including B‐cell lymphomas and carcinomas. Impaired DNA damage response is at the root of tumor predisposition of MIF−/−p53−/− mice. We provide evidence that MIF plays a role in regulating the activity of Cul1‐containing SCF ubiquitin ligases. The loss of MIF expression uncouples Chk1/Chk2‐responsive DNA damage checkpoints from SCF‐dependent degradation of key cell‐cycle regulators such as Cdc25A, E2F1 and DP1, creating conditions for the genetic instability of cells. These MIF effects depend on its association with the Jab1/CSN5 subunit of the COP9/CSN signalosome. Given that CSN plays a central role in the assembly of SCF complexes in vivo, regulation of Jab1/CSN5 by MIF is required to sustain optimal composition and function of the SCF complex.

The post-translational phosphorylation and acetylation modification profile is not the determining factor in targeting endogenous stress-induced p53 to mitochondria

The post-translational phosphorylation and acetylation modification profile is not the determining factor in targeting endogenous stress-induced p53 to mitochondria

p53Ψ is a transcriptionally inactive p53 isoform able to reprogram cells toward a metastatic-like state.

Significance p53 is one of the most intensively studied tumor-suppressor genes. We identified a naturally occurring p53 isoform, generated by an alternative-splicing event, that, although lacking transcriptional activity and canonical tumor suppressor functions, is able to reprogram cells toward the acquisition of metastatic features via a cyclophilin D interaction in the mitochondria matrix. Interestingly, this isoform is expressed on tissue injury and in tumors characterized by increased metastatic spread. In some of these tumors, p53-like isoforms are generated by intron 6 mutations. This suggests a possible physiological origin of certain p53 mutations and indicates that mutations resulting in the generation of truncated p53Ψ-like proteins do more than create a 53-null state. Although much is known about the underlying mechanisms of p53 activity and regulation, the factors that influence the diversity and duration of p53 responses are not well understood. Here we describe a unique mode of p53 regulation involving alternative splicing of the TP53 gene. We found that the use of an alternative 3′ splice site in intron 6 generates a unique p53 isoform, dubbed p53Ψ. At the molecular level, p53Ψ is unable to bind to DNA and does not transactivate canonical p53 target genes. However, like certain p53 gain-of-function mutants, p53Ψ attenuates the expression of E-cadherin, induces expression of markers of the epithelial-mesenchymal transition, and enhances the motility and invasive capacity of cells through a unique mechanism involving the regulation of cyclophilin D activity, a component of the mitochondrial inner pore permeability. Hence, we propose that p53Ψ encodes a separation-of-function isoform that, although lacking canonical p53 tumor suppressor/transcriptional activities, is able to induce a prometastatic program in a transcriptionally independent manner.

Macrophage migration inhibitory factor coordinates DNA damage response with the proteasomal control of the cell cycle.

Proper repair of DNA damage is critical for protecting genomic stability, cellular viability and suppression of tumorigenesis. Both p53-dependent and p53-independent pathways have evolved to coordinate the cellular response following DNA damage. In this review, we highlight the importance of the ubiquitously expressed protein macrophage migration inhibitory factor (MIF) for an appropriate response to DNA damage. We discuss the mechanisms by which MIF affects the activity of the ubiquitin-proteasome system, and how this impacts on the integrity of the genome and on cancer.

MDM2 Associates with Polycomb Repressor Complex 2 and Enhances Stemness-Promoting Chromatin Modifications Independent of p53

The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of lineage-specific genes, independent of p53.

TAp73 is a central transcriptional regulator of airway multiciliogenesis

Motile multiciliated cells (MCCs) have critical roles in respiratory health and disease and are essential for cleaning inhaled pollutants and pathogens from airways. Despite their significance for human disease, the transcriptional control that governs multiciliogenesis remains poorly understood. Here we identify TP73, a p53 homolog, as governing the program for airway multiciliogenesis. Mice with TP73 deficiency suffer from chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance. Organotypic airway cultures pinpoint TAp73 as necessary and sufficient for basal body docking, axonemal extension, and motility during the differentiation of MCC progenitors. Mechanistically, cross-species genomic analyses and complete ciliary rescue of knockout MCCs identify TAp73 as the conserved central transcriptional integrator of multiciliogenesis. TAp73 directly activates the key regulators FoxJ1, Rfx2, Rfx3, and miR34bc plus nearly 50 structural and functional ciliary genes, some of which are associated with human ciliopathies. Our results position TAp73 as a novel central regulator of MCC differentiation.

TAp73 is essential for germ cell adhesion and maturation in testis

The p53 family member TAp73 is required for sperm maturation through promotion of adhesion between developing germ cells and Sertoli nurse cells.