Giovanni Blandino

Interaction of c-Abl and p73α and their collaboration to induce apoptosis

c-Abl, a non-receptor tyrosine kinase, is activated by agents that damage DNA. This activation results in either arrest of the cell cycle in phase G1 or apoptotic cell death, both of which are dependent on the kinase activity of c-Abl. p73, a member of the p53 family of tumour-suppressor proteins,, can also induce apoptosis. Here we show that the apoptotic activity of p73α requires the presence of functional, kinase-competent c-Abl. Furthermore, p73 and c-Abl can associate with each other, and this binding is mediated by a PxxP motif in p73 and the SH3 domain of c-Abl. We find that p73 is a substrate of the c-Abl kinase and that the ability of c-Abl tophosphorylate p73 is markedly increased by γ-irradiation. Moreover, p73 is phosphorylated in vivo in response to ionizing radiation. These findings define a pro-apoptotic signalling pathway involving p73 and c-Abl.

Mutant p53 gain of function: Differential effects of different p53 mutants on resistance of cultured cells to chemotherapy

Many tumors overexpress mutant forms of p53. A growing number of studies suggest that the nature of a p53 mutation in a cell can impact upon cellular properties, clinical responses to therapy and prognosis of a tumor. To explore the cellular basis of these observations, experiments were designed to compare the properties of cells with and without p53 mutations within the same cell population. To that end, various tumor-derived human p53 mutants were introduced into p53-null H1299 lung adenocarcinoma cells. Clonogenic survival assays revealed that cells overexpressing the p53His175 mutant, but not the p53His273 mutant, recover preferentially from etoposide treatment. Moreover, p53His175 as well as p53His179 reduced substantially the rate of etoposide-induced apoptosis, whereas p53His273 and p53Trp248 had a much milder protective effect. In contrast, p53His175 and p53His273 exerted very similar effects on the cellular response to cisplatin; both conferred increased resistance to low concentrations of the drug (2.5 μg/ml), but did not protect at all against high concentrations (10 μg/ml). Hence particular p53 mutants may confer upon tumor cells a selective survival advantage during chemotherapy. These findings define a new type of mutant p53 selective gain of function, which may compromise the efficacy of cancer chemotherapy.

DNA Damage-Dependent Acetylation of p73 Dictates the Selective Activation of Apoptotic Target Genes

The tumor suppressor p53 and its close relative p73 are activated in response to DNA damage resulting in either cell cycle arrest or apoptosis. Here, we show that DNA damage induces the acetylation of p73 by the acetyltransferase p300. Inhibiting the enzymatic activity of p300 hampers apoptosis in a p53(-/-) background. Furthermore, a nonacetylatable p73 is defective in activating transcription of the proapoptotic p53AIP1 gene but retains an intact ability to regulate other targets such as p21. Finally, p300-mediated acetylation of p73 requires the protooncogene c-abl. Our results suggest that DNA damage-induced acetylation potentiates the apoptotic function of p73 by enhancing the ability of p73 to selectively activate the transcription of proapoptotic target genes.

Physical and Functional Interaction between p53 Mutants and Different Isoforms of p73

p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro andin vivo with p73α, β, γ, and δ. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage.

Mutant p53: an oncogenic transcription factor.

Inactivation of tumor-suppressor genes is one of the key hallmarks of a tumor. Unlike other tumor-suppressor genes, p53 is inactivated by missense mutations in half of all human cancers. It has become increasingly clear that the resulting mutant p53 proteins do not represent only the mere loss of wild-type p53 tumor suppressor activity, but gain new oncogenic properties favoring the insurgence, the maintenance, the spreading and the chemoresistance of malignant tumors. The actual challenge is the fine deciphering of the molecular mechanisms underlying the gain of function of mutant p53 proteins. In this review, we will focus mainly on the transcriptional activity of mutant p53 proteins as one of the potential molecular mechanisms. To date, the related knowledge is still quite scarce and many of the raised questions of this review are yet unanswered.

Mutant p53 gain of function: reduction of tumor malignancy of human cancer cell lines through abrogation of mutant p53 expression

Mutations in the TP53 tumor suppressor gene are the most frequent genetic alteration in human cancers. These alterations are mostly missense point mutations that cluster in the DNA binding domain. There is growing evidence that many of these mutations generate mutant p53 proteins that have acquired new biochemical and biological properties. Through this gain of function activity, mutant p53 is believed to contribute to tumor malignancy. The purpose of our study was to explore mutant p53 as a target for novel anticancer treatments. To this aim, we inhibited mutant p53 expression by RNA interference in three different cancer cell lines endogenously expressing mutant p53 proteins, and evaluated the effects on the biological activities through which mutant p53 exerts gain of function. We found that depletion of mutant p53 reduces cell proliferation, in vitro and in vivo tumorigenicity, and resistance to anticancer drugs. Our results demonstrate that mutant p53 knocking down weakens the aggressiveness of human cancer cells, and provides further insight into the comprehension of mutant p53 gain of function activity in human tumor.

PML, YAP, and p73 Are Components of a Proapoptotic Autoregulatory Feedback Loop

p73 has been identified as a structural and functional homolog of the tumor suppressor p53. The transcriptional coactivator Yes-associated protein (YAP) has been demonstrated to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here, we show the existence of a proapoptotic autoregulatory feedback loop between p73, YAP, and the promyelocytic leukemia (PML) tumor suppressor gene. We demonstrate that PML is a direct transcriptional target of p73/YAP, and we show that PML transcriptional activation by p73/YAP is under the negative control of the proto-oncogenic Akt/PKB kinase. Importantly, we find that PML and YAP physically interact through their PVPVY and WW domains, respectively, causing PML-mediated sumoylation and stabilization of YAP. Hence, we determine a mechanistic pathway in response to DNA damage that could have relevant implications for the treatment of human cancer.

Modulation of the Vitamin D3 Response by Cancer-Associated Mutant p53

The p53 gene is mutated in many human tumors. Cells of such tumors often contain abundant mutant p53 (mutp53) protein, which may contribute actively to tumor progression via a gain-of-function mechanism. We applied ChIP-on-chip analysis and identified the vitamin D receptor (VDR) response element as overrepresented in promoter sequences bound by mutp53. We report that mutp53 can interact functionally and physically with VDR. Mutp53 is recruited to VDR-regulated genes and modulates their expression, augmenting the transactivation of some genes and relieving the repression of others. Furthermore, mutp53 increases the nuclear accumulation of VDR. Importantly, mutp53 converts vitamin D into an antiapoptotic agent. Thus, p53 status can determine the biological impact of vitamin D on tumor cells.

The execution of the transcriptional axis mutant p53, E2F1 and ID4 promotes tumor neo-angiogenesis

ID4 (inhibitor of DNA binding 4) is a member of a family of proteins that function as dominant-negative regulators of basic helix-loop-helix transcription factors. Growing evidence links ID proteins to cell proliferation, differentiation and tumorigenesis. Here we identify ID4 as a transcriptional target of gain-of-function p53 mutants R175H, R273H and R280K. Depletion of mutant p53 protein severely impairs ID4 expression in proliferating tumor cells. The protein complex mutant p53–E2F1 assembles on specific regions of the ID4 promoter and positively controls ID4 expression. The ID4 protein binds to and stabilizes mRNAs encoding pro-angiogenic factors IL8 and GRO-α. This results in the increase of the angiogenic potential of cancer cells expressing mutant p53. These findings highlight the transcriptional axis mutant p53, E2F1 and ID4 as a still undefined molecular mechanism contributing to tumor neo-angiogenesis.

Induced p21waf expression in H1299 cell line promotes cell senescence and protects against cytotoxic effect of radiation and doxorubicin

The CDK inhibitor p21waf is a principal mediator of p53 function but can also be transactivated by many p53-independent stimuli leading to cell growth arrest or differentiation. In order to study the function of p21waf in a p53-deficient environment, we established an inducible expression of p21waf in the p53-null lung cancer cell line H1299, based on the muristerone-regulated system. Overexpression of p21waf led cells to growth arrest which after several days became irreversible and the arrested cells acquired a senescent phenotype as judged by cell shape, the senescence-associated β-gal marker and inhibition of colony formation. The effect of p21waf overexpression, in the absence of p53, on the cytotoxicity caused by irradiation, doxorubicin and taxol was studied. Expression of p21waf provided protection against the cytotoxic effect of radiation and doxorubicin but not of taxol. These results are relevant to treatment of cancer when p53 is inactive.