Vincenzo De Laurenzi

p73: Friend or foe in tumorigenesis

As p53 and its homologue p73 have significant sequence and functional similarities, p73 might also be expected to act as a tumour suppressor. However, p73 is activated after DNA damage in a way that is distinct from that of p53. The existence of ΔNp73 — an isoform of p73 that is encoded by a distinct promoter and that lacks the transactivation domain — further complicates matters. It seems to function as an oncogene by inhibiting both p73- and p53-induced apoptosis. So how can these opposing functions be reconciled in human tumours?

Gene Disruption of Tissue Transglutaminase

ABSTRACT Transglutaminase 2 (TGase 2), or tissue transglutaminase, catalyzes either ɛ-(γ-glutamyl)lysine orN 1,N 8-(γ-glutamyl)spermidine isopeptide bonds. TGase 2 expression has been associated with apoptosis, and it has been proposed that its activation should lead to the irreversible assembly of a cross-linked protein scaffold in dead cells. Thus, TGase 2-catalyzed protein polymerization contributes to the ultrastructural changes typical of dying apoptotic cells; it stabilizes the integrity of the apoptotic cells, preventing the release of harmful intracellular components into the extracellular space and, consequently, inflammation and scar formation. In order to perform a targeted disruption of the enzyme, we prepared a construct deleting part of exons 5 and 6, containing the active site, and intron 5. Complete absence of TGase 2 was demonstrated by reverse transcription-PCR and Western blot analysis. TGase activity measured on liver and thymus extracts showed, however, a minimal residual activity in TGase 2−/− mice. PCR analysis of mRNA extracted from the same tissues demonstrated that at least TGase 1 (normally present in the skin) is also expressed in these tissues and contributes to this residual activity. TGase 2−/− mice showed no major developmental abnormalities, and histological examination of the major organs appeared normal. Induction of apoptosis ex vivo in TGase 2−/− thymocytes (by CD95, dexamethasone, etoposide, and H2O2) and in vitro on TGase 2−/−mouse embryonal fibroblasts (by retinoids, UV, and H2O2) showed no significant differences. A reduction in cross-linked apoptotic bodies with a modestly increased release of lactate dehydrogenase has been detected in some cases. Together our results show that TGase 2 is not a crucial component of the main pathway of the apoptotic program. It is possible that the residual enzymatic activity, due to TGase 1 or redundancy of other still-unidentified TGases, can compensate for the lack of TGase 2.

The ubiquitin-protein ligase Itch regulates p73 stability

p73, a member of the p53 family of transcription factors, is upregulated in response to DNA damage, inducing cell cycle arrest and apoptosis. Besides indications that this p73 response is post‐transcriptional, little is known about the underlying molecular mechanisms of p73 protein degradation. Ubiquitination and proteasomal‐dependent degradation of p53 are regulated by its transcriptional target MDM2. However, unlike p53, p73 binds to, but is not degraded by, MDM2. Here we describe the binding of p73 to Itch, a Hect ubiquitin–protein ligase. Itch selectively binds and ubiquitinates p73 but not p53; this results in the rapid proteasome‐dependent degradation of p73. Upon DNA damage Itch itself is downregulated, allowing p73 protein levels to rise and thus interfere with p73 function. In conclusion, we have identified a key mechanism in the control of p73 protein levels both in normal as well as in stress conditions.

Role of transglutaminase 2 in glucose tolerance: knockout mice studies and a putative mutation in a MODY patient

Transglutaminase 2 (TGase 2) is a Ca+2‐ dependent enzyme that catalyzes both intracellular and extracellular cross‐linking reactions by transamidation of specific glutamine residues. TGase 2 is known to be involved in the membrane‐mediated events required for glucose‐stimulated insulin release from the pancreatic β cells. Here we show that targeted disruption of TGase 2 impairs glucose‐stimulated insulin secretion. TGase 2‐/‐mice show glucose intolerance after intraperitoneal glucose loading. TGase 2‐/‐mice manifest a tendency to develop hypoglycemia after administration of exogenous insulin as a consequence of enhanced insulin receptor substrate 2 (IRS‐2) phosphorylation. We suggest that the increased peripheral sensitivity to insulin partially compensates for the defective secretion in this animal model. TGase 2‐/‐mouse phenotype resembles that of the maturity‐onset diabetes of young (MODY) patients. In the course of screening for human TGase 2 gene in Italian subjects with the clinical features of MODY, we detected a missense mutation (N333S) in the active site of the enzyme. Collectively, these results identify TGase 2 as a potential candidate gene in type 2 diabetes.—Bernassola, F., Federici, M., Corazzari, M., Terrinoni, A., Hribal, M. L., De Laurenzi, V., Ranalli, M., Massa, O., Sesti, G., Mclean, W. H. I., Citro, G., Barbetti, F., Melino, G. Role of transglutaminase 2 in glucose tolerance: knockout mice studies and a putative mutation in a MODY patient. FASEB J. 16, 1371–1378 (2002)

Evolution of Functions within the p53/p63/p73 Family

Abstract: Even though the tumor suppressor gene p53 is highly important in human cancer, as indicated by the fact that it is mutated in about 50% of cases, up to a few years ago no similar proteins had been identified. Recently, two p53 homologues have been identified, p73 and p63, with high aminoacid identity suggesting similar functions. Indeed, like p53, p73 as well (i) can bind mdmx, mdm2, p300/CAF and adenovirus E4‐ORF6 proteins, (ii) can trigger several promoters including p21, bax, mdm2, gadd45, cyclin G, IGFBP3, 14–3–3σ, (iii) is able to trigger cell death, (iv) is involved in the DNA damage response, although through a different pathway. Here we analyze the data present in the literature in search of diverging pathways among the p53, p63, p73 family. Both p63 and p73 present two significant structural peculiarities: the presence of an extended non‐conserved C‐terminus containing a sterile alpha motive (SAM), typical of developmental proteins, and the presence of number of different splicing isoforms differing in the N‐terminus or in the absence of the transactivation domain (ΔN forms), acting as dominant negative. The mouse knockout of p63 and p73, unlike the ones for p53, shows developmental abnormalities; p63 and p73 are rarely mutated in human cancers; both genes are regulated in different differentiation models. This strongly suggests the involvement of p63 and p73 in development. A picture is emerging showing a gradient of function among p53, p73, p63 ranging from tumor suppression to development.

BAG3 protein is overexpressed in human glioblastoma and is a potential target for therapy.

Glioblastoma multiforme, which represents 80% of malignant gliomas, is characterized by aggressiveness and high recurrence rates. Despite therapeutic advances, patients with glioblastoma multiforme show a poor survival, and identification of novel markers and molecular targets for therapy is needed. A role for BAG3, a member of the BAG family of HSC/HSP70 co-chaperones, in promoting tumor cell growth in vivo has recently been described. We analyzed BAG3 levels by IHC in specimens from patients affected by brain tumors and we found that BAG3, although negative in normal brain tissues, was highly expressed in astrocytic tumors and increasingly expressed in more aggressive types of cancer; it was particularly high in glioblastomas. Down-regulating BAG3 both in vitro and in vivo in a rat glioblastoma model resulted in increased sensitivity to apoptosis, suggesting that BAG3 is a potential target for novel therapies. Finally, we determined that the underlying molecular mechanism requires the formation of a complex of BAG3, HSP70, and BAX that prevents BAX translocation to mitochondria, thus protecting tumor cells from apoptosis. Our data identify BAG3 as a potential marker of glial brain tumor sensitivity to therapy and thus also an attractive candidate for new molecular therapies.

PIAS-1 is a checkpoint regulator which affects exit from G1 and G2 by sumoylation of p73

ABSTRACT p73 is a recently described member of the p53 family, and, like p53, it undergoes a number of posttranslational modifications. Here we show, by yeast two-hybrid screening, pull-down assays, and coimmunoprecipitation, that p73α, -β, and -γ bind to the protein inhibitor of activated STAT-1 (PIAS-1) and that this binding stabilizes p73. PIAS-1 also sumoylates p73α, although not the C-terminally truncated isoforms p73β and -γ, and this requires the RING finger domain of PIAS-1. The ΔNp73α isoform can also bind, and be sumoylated by, PIAS-1. PIAS-1-mediated sumoylation decreases p73 transcriptional activity on several target promoters, such as Bax. p73 is colocalized in the nucleus with PIAS-1, and sumoylated p73 is located exclusively in the nuclear matrix. PIAS-1 is expressed predominantly during S phase, and PIAS-1 overexpression reduces p73-mediated transcription of p21, with a reduction of cells in G1 and cell cycle reentry. Inhibition of endogenous PIAS-1 by RNA interference reduces the proportion of cells in S phase and induces G2 arrest. These data suggest that PIAS-1, acting partly through binding and sumoylation of p73, is an important component of the cell cycle machinery.

FLASH and NPAT positive but not Coilin positive Cajal Bodies correlate with cell ploidy

Cajal Bodies are one of many specialised organelles contained in the eukaryotic cell nucleus, and are involved in a number of functions, including regulation of replication-dependent histone gene transcription. In normal diploid cells their number varies between 0 and 4 depending on the cell cycle phase, although in cancer cell lines their number is extremely variable and it has been suggested that it correlates with cell ploidy. Here we show that in mammals cells, as in Drosophila, two distinct though functionally related bodies exist: a histone gene locus body and a Cajal body. The first one can be detected using FLASH or NPAT as markers while the second is labelled using antibodies against Coilin.Only the number of FLASH/NPAT histone gene locus bodies correlates with ploidy and only these organelles appear to be regulated during the cell cycle. Finally, we show that the two organelles completely co-localize during the S phase of the cell cycle.

Role of p63 in cancer development

Since their initial identification p53 homologues p63 and p73 have been expected to play a role in cancer development due to their close homology to p53, notoriously one of the most mutated genes in cancer. However soon after their discovery the awareness that these genes were rarely mutated in cancer seemed to indicate that they did not play a role in its development. However a large number of data collected in the following years indicated that altered expression rather than mutation could be found in different neoplasia and play a role in its biology. In particular p63 due to its fundamental role in epithelial development seems to play a role in a number of tumors of epithelial origin. In this review we summarize some of the evidence linking p63 to carcinogenesis.

Methionine sulfoxide reductase A down-regulation in human breast cancer cells results in a more aggressive phenotype

Breast cancer is one of the most frequent of human malignacies, and it is therefore fundamental to identify the underlying molecular mechanisms leading to cancer transformation. Among other causative agents in the development of breast cancers, an important role for reactive oxygen species (ROS) has emerged. However, most studies on the role of ROS in cancer have not reached specific conclusions, and many issues remain controversial. In the present study, we show that methionine sulfoxide reductase A (MsrA), which is known to protect proteins from oxidation and which acts as a ROS scavenger, is down-regulated in a number of breast cancers. Moreover, levels of MsrA correlate with advanced tumor grade. We therefore investigated the functional role of MsrA in breast cancer cells. Our data show that reduction of MsrA levels results in increased cell proliferation and extracellular matrix degradation, and consequently in a more aggressive cellular phenotype, both in vivo and in vitro. We also show that the underlying molecular mechanisms involve increased ROS levels, resulting in reduction of phosphatase and tensin homolog deleted on chromosome ten protein (PTEN), and activation of the phosphoinositide 3-kinase pathway. In addition, MsrA down-regulation results in up-regulation of VEGF, providing additional support for tumor growth in vivo.