Flaminia Talos

Functional inactivation of endogenous MDM2 and CHIP by HSP90 causes aberrant stabilization of mutant p53 in human cancer cells

The tight control of wild-type p53 by mainly MDM2 in normal cells is permanently lost in tumors harboring mutant p53, which exhibit dramatic constitutive p53 hyperstabilization that far exceeds that of wild-type p53 tumors. Importantly, mutant p53 hyperstabilization is critical for oncogenic gain of function of mutant p53 in vivo. Current insight into the mechanism of this dysregulation is fragmentary and largely derived from ectopically constructed cell systems. Importantly, mutant p53 knock-in mice established that normal mutant p53 tissues have sufficient enzymatic reserves in MDM2 and other E3 ligases to maintain full control of mutant p53. We find that in human cancer cells, endogenous mutant p53, despite its ability to interact with MDM2, suffers from a profound lack of ubiquitination as the root of its degradation defect. In contrast to wild-type p53, the many mutant p53 proteins which are conformationally aberrant are engaged in complexes with the HSP90 chaperone machinery to prevent its aggregation. In contrast to wild-type p53 cancer cells, we show that in mutant p53 cancer cells, this HSP90 interaction blocks the endogenous MDM2 and CHIP (carboxy-terminus of Hsp70-interacting protein) E3 ligase activity. Interference with HSP90 either by RNA interference against HSF1, the transcriptional regulator of the HSP90 pathway, or by direct knockdown of Hsp90 protein or by pharmacologic inhibition of Hsp90 activity with 17AAG (17-allylamino-17-demethoxygeldanamycin) destroys the complex, liberates mutant p53, and reactivates endogenous MDM2 and CHIP to degrade mutant p53. Of note, 17AAG induces a stronger viability loss in mutant p53 than in wild-type p53 cancer cells. Our data support the rationale that suppression of mutant p53 levels in vivo in established cancers might achieve clinically significant effects. Mol Cancer Res; 9(5); 577–88. ©2011 AACR.

p73 is an essential regulator of neural stem cell maintenance in embryonal and adult CNS neurogenesis

The p53 family member p73 is essential for brain development, but its precise role and scope remain unclear. Global p73 deficiency determines an overt and highly penetrant brain phenotype marked by cortical hypoplasia with ensuing hydrocephalus and hippocampal dysgenesis. The ΔNp73 isoform is known to function as a prosurvival factor of mature postmitotic neurons. In this study, we define a novel essential role of p73 in the regulation of the neural stem cell compartment. In both embryonic and adult neurogenesis, p73 has a critical role in maintaining an adequate neurogenic pool by promoting self-renewal and proliferation and inhibiting premature senescence of neural stem and early progenitor cells. Thus, products of the p73 gene locus are essential maintenance factors in the central nervous system, whose broad action stretches across the entire differentiation arch from stem cells to mature postmitotic neurons.

Mitochondrially Targeted p53 Has Tumor Suppressor Activities In vivo

Complex proapoptotic functions are essential for the tumor suppressor activity of p53. We recently described a novel transcription-independent mechanism that involves a rapid proapoptotic action of p53 at the mitochondria and executes the shortest known circuitry of p53 death signaling. Here, we examine if this p53-dependent mitochondrial program could be exploited for tumor suppression in vivo. To test this, we engage Emu-Myc transgenic mice, a well-established model of p53-dependent lymphomagenesis. We show that exclusive delivery of p53 to the outer mitochondrial membrane confers a significant growth disadvantage on Emu-Myc-transformed B-cells of p53-deficient or alternate reading frame-deficient genotypes, resulting in efficient induction of apoptosis and impinged proliferation. Conversely, normal cells from thymus, spleen, and bone marrow showed poor infectivity with these viruses. This proof-of-principle experiment shows that exclusive reliance on the direct mitochondrial program exerts a significant tumor suppressor activity in vivo. Our in vivo data on the direct mitochondrial apoptotic p53 program lays the groundwork to further investigate its efficacy and safety and to address its possible therapeutic value in the future.

The alpha/beta carboxy-terminal domains of p63 are required for skin and limb development. New insights from the Brdm2 mouse which is not a complete p63 knockout but expresses p63 gamma-like proteins.

p63, an ancestral transcription factor of the p53 family, has three C-terminal isoforms whose relative in vivo functions are elusive. The p63 gene is essential for skin and limb development, as vividly shown by two independent global knockout mouse models. Both strains, although constructed differently, have identical and severe phenotypes, characterized by absent epidermis and hindlimbs and only rudimentary forelimbs at birth. Here we show that mice from one model, Brdm2, express normal levels of truncated p63 proteins that contain the DNA binding and oligomerization domain but lack the long carboxy-terminal SAM (sterile α-motif) and post-SAM domains that are specific for the α and β isoforms. As such, transcriptionally active p63 proteins from Brdm2 mice resemble the naturally occurring p63γ isoforms, which of all the p63 isoforms most closely resemble p53. Thus, Brdm2 mice are p63α/β isoform-specific knockout mice, gaining unexpected new importance. Our studies identify that p63α/β but not p63γ are absolutely required for proper skin and limb development.

MIF loss impairs Myc-induced lymphomagenesis

Macrophage migration inhibitory factor (MIF) is a potent regulator of inflammation and cell growth. Using the Eμ-Myc lymphoma mouse model, we demonstrate that loss of MIF markedly delays the onset of B-cell lymphoma development in vivo. The molecular basis for this MIF-loss-induced phenotype is the perturbed DNA-binding activity of E2F factors and the concomitantly enhanced tumor suppressor activity of the p53 pathway. Accordingly, premalignant MIF-null Eμ-Myc B-cells are predisposed to delayed S-phase progression and increased apoptosis. MIF-deficient lymphomas that do arise under these conditions contain frequent ARF deletions and p53 inactivating mutations. Conversely, MIF expression is retained in tumors developed by wild-type Eμ-Myc animals, and the presence of one or both MIF alleles is sufficient to accelerate the development of Myc-induced lymphomas. Collectively, these results indicate that MIF promotes Myc-mediated tumorigenesis, at least in the B-lymphoid compartment, and implicate MIF as a mediator of malignant cell growth in vivo.

While p73 is essential, p63 is completely dispensable for the development of the central nervous system.

The ancient p53 paralogs p63 and p73 regulate specific tissue formation, cell survival and cell death via their TA and ΔN isoforms. Targeted disruption of the p73 locus leads to severe defects in the development of the central nervous system (CNS), and p73 has recently been shown to be an essential regulator of neural stem cell maintenance and differentiation in both embryonal and adult neurogenesis. In contrast, global p63-/- mice lack skin and limbs. Moreover, p63 is detectable in embryonic cortex. It has previously been proposed to also play critical pro-death and pro-survival roles in neural precursors of the developing sympathetic and central nervous system, respectively, based on experimental overexpression and siRNA-mediated knockdown of p63. Here we perform an extensive analysis of the developing central nervous system in global p63-/- mice and their wildtype littermates. Brain and spinal cord of embryos and newborn mice were assessed in vivo for neuroanatomy, histology, apoptosis, proliferation, stemness and differentiation, and in vitro for self-renewal and maturation in neurosphere assays. None of these analyses revealed a detectable phenotype in p63-/- mice. Hence, despite the profound impact of p63 on the development of stratified epithelia and limbs, p63 is completely dispensable for proper development of the central nervous system. Thus, despite their strong homology, the non-overlapping tissue specificity of p63 and p73 functions appears more pronounced than previously anticipated.

Role of the p53 family in stabilizing the genome and preventing polyploidization.

Cellular defects resulting in chromosomal instability and aneuploidy are the most common features of human cancers. As a major tumor suppressor and intrinsic part of several cellular checkpoints, p53 contributes to maintenance of the stability of the genetic material, both in quality (ensures faithful replication) and quantity (preservation of diploidy). Although the exact trigger of p53 in case of numerical chromosomal aberrations is unknown, the absence of p53 allows polyploid cells to proliferate and generate unstable aneuploid progeny. A more recent addition to the p53 family, p73, emerged as an important contributor to genomic integrity when p53 is inactivated. p73 loss in p53-null background leads to a rapid increase in polyploidy and aneuploidy, markedly exceeding that caused by p53 loss alone. Constitutive deregulation of Cyclin-Cdk and p27/Kip1 activities and excess failure of the G2/M DNA damage checkpoint are important deficiencies associated with p73 loss.

p63 is a prosurvival factor in the adult mammary gland during post-lactational involution, affecting PI-MECs and ErbB2 tumorigenesis

In embryogenesis, p63 is essential to develop mammary glands. In the adult mammary gland, p63 is highly expressed in the basal cell layer that comprises myoepithelial and interspersed stem/progenitor cells, and has limited expression in luminal epithelial cells. In adult skin, p63 has a crucial role in the maintenance of epithelial stem cells. However, it is unclear whether p63 also has an equivalent role as a stem/progenitor cell factor in adult mammary epithelium. We show that p63 is essential in vivo for the survival and maintenance of parity-identified mammary epithelial cells (PI-MECs), a pregnancy-induced heterogeneous population that survives post-lactational involution and contain multipotent progenitors that give rise to alveoli and ducts in subsequent pregnancies. p63+/− glands are normal in virgin, pregnant and lactating states. Importantly, however, during the apoptotic phase of post-lactational involution p63+/− glands show a threefold increase in epithelial cell death, concomitant with increased activation of the oncostatin M/Stat3 and p53 pro-apoptotic pathways, which are responsible for this phase. Thus, p63 is a physiologic antagonist of these pathways specifically in this regressive stage. After the restructuring phase when involution is complete, mammary glands of p63+/− mice again exhibit normal epithelial architecture by conventional histology. However, using RosaLSL-LacZ;WAP-Cre transgenics (LSL-LacZ, lox-stop-lox β-galactosidase), a genetic in vivo labeling system for PI-MECs, we find that p63+/− glands have a 30% reduction in the number of PI-MEC progenitors and their derivatives. Importantly, PI-MECs are also cellular targets of pregnancy-promoted ErbB2 tumorigenesis. Consistent with their PI-MEC pool reduction, one-time pregnant p63+/− ErbB2 mice are partially protected from breast tumorigenesis, exhibiting extended tumor-free and overall survival, and reduced tumor multiplicity compared with their p63+/+ ErbB2 littermates. Conversely, in virgin ErbB2 mice p63 heterozygosity provides no survival advantage. In sum, our data establish that p63 is an important survival factor for pregnancy-identified PI-MEC progenitors in breast tissue in vivo.

Brdm2—an aberrant hypomorphic p63 allele

In response to our recent report on the Brdm2 mouse model for p63 function1, Mikkola et al.2 raise concerns on three of our statements: 1) They maintain their initial claim that no functional p63 protein of any kind is synthesized in mice homozygous for the Brdm2 allele; 2) they report the occurrence of spontaneous wild-type allelic reversions in these mice, leading to patches of completely normal epidermis, and propose that these normal reverted skin patches are in fact the multilayered epithelia we described; 3) they insist that the Brdm2 allele is phenotypically indistinguishable from a global p63 knockout.

Rb function is required for E1A-induced S-phase checkpoint activation

It is widely accepted that adenoviral E1A exerts its influence on recipient cells through binding to the retinoblastoma (Rb) family proteins, followed by a global release of E2F factors from pocket-protein control. Our study challenges this simple paradigm by demonstrating previously unappreciated complexity. We show that E1A-expressing primary and transformed cells are characterized by the persistence of Rb–E2F1 complexes. We provide evidence that E1A causes Rb stabilization by interfering with its proteasomal degradation. Functional experiments supported by biochemical data reveal not only a dramatic increase in Rb and E2F1 protein levels in E1A-expressing cells but also demonstrate their activation throughout the cell cycle. We further show that E1A activates an Rb- and E2F1-dependent S-phase checkpoint that attenuates the growth of cells that became hyperploid through errors in mitosis and supports the fidelity DNA replication even in the absence of E2F complexes with other Rb family proteins, thereby functionally substituting for the loss of p53. Our results support the essential role of Rb and E2F1 in the regulation of genomic stability and DNA damage checkpoints.