Mutant p53 gains new function in promoting inflammatory signals by repression of the secreted interleukin-1 receptor antagonist.

The TP53 tumor-suppressor gene is frequently mutated in human cancer. Missense mutations can add novel functions (gain-of-function, GOF) that promote tumor malignancy. Here we report that mutant (mut) p53 promotes tumor malignancy by suppressing the expression of a natural occurring anti-inflammatory cytokine, the secreted interleukin-1 receptor antagonist (sIL-1Ra, IL1RN). We show that mutp53 but not wild-type (wt) p53 suppresses the sIL-1Ra production in conditioned media of cancer cells. Moreover, mutp53, but not wtp53, binds physically the sIL-1Ra promoter and the protein-protein interaction with the transcriptional co-repressor MAFF (v-MAF musculoaponeurotic fibrosarcoma oncogene family, protein F) is required for mutp53-induced sIL-1Ra suppression. Remarkably, when exposed to IL-1 beta (IL-1ß) inflammatory stimuli, mutp53 sustains a ready-to-be-activated in vitro and in vivo cancer cells' response through the sIL-1Ra repression. Taken together, these results identify sIL-1Ra as a novel mutp53 target gene, whose suppression might be required to generate a chronic pro-inflammatory tumor microenvironment through which mutp53 promotes tumor malignancy.

Che-1 modulates the decision between cell cycle arrest and apoptosis by its binding to p53.

The tumor suppressor p53 is mainly involved in the transcriptional regulation of a large number of growth-arrest- and apoptosis-related genes. However, a clear understanding of which factor/s influences the choice between these two opposing p53-dependent outcomes remains largely elusive. We have previously described that in response to DNA damage, the RNA polymerase II-binding protein Che-1/AATF transcriptionally activates p53. Here, we show that Che-1 binds directly to p53. This interaction essentially occurs in the first hours of DNA damage, whereas it is lost when cells undergo apoptosis in response to posttranscriptional modifications. Moreover, Che-1 sits in a ternary complex with p53 and the oncosuppressor Brca1. Accordingly, our analysis of genome-wide chromatin occupancy by p53 revealed that p53/Che1 interaction results in preferential transactivation of growth arrest p53 target genes over its pro-apoptotic target genes. Notably, exposure of Che-1(+/-) mice to ionizing radiations resulted in enhanced apoptosis of thymocytes, compared with WT mice. These results confirm Che-1 as an important regulator of p53 activity and suggest Che-1 to be a promising yet attractive drug target for cancer therapy.

Development of a murine orthotopic model of leukemia: evaluation of TP53 gene therapy efficacy.

The onco-suppressor gene TP53 has potential use in the gene therapy of many human cancers including leukemias. The latter indication derived from numerous experimental reports of p53-mediated suppressing effects on human and murine leukemia cells in vitro. However, few in vivo experiments have been performed, and those that have used a subcutaneous injection of p53-transduced leukemia cells. Thus, we developed an orthotopic leukemia model in adult, syngenic mice to evaluate the feasibility of TP53-mediated therapeutic approaches. We found that among other cells, v-src-transformed 32D myeloid progenitors induce leukemia when injected intravenously in syngenic mice. The resulting malignancy resembles the clinical manifestations of human acute myeloid leukemia because it is characterized by a massive invasion of bone marrow compartments, splenomegaly, generalized lymphadenopathy, and a macroscopic or microscopic infiltration of the kidneys, liver, and lungs. When these 32Dv-src cells were infected with a TP53-recombinant retrovirus before intravenous injection, we found a decreased mortality and, in those animals that develop leukemia, a drastic reduction of the generalized organ infiltration, suggesting that exogenous TP53 expression might be used for ex vivo bone marrow purging from leukemia cells.

Metastatic phenotype: growth factor dependence and integrin expression.

Homogeneous subpopulations, which are endowed with low or high metastatic potential, were selected from Lewis lung carcinoma (3LL) in an attempt to correlate metastatic phenotype with specific properties of tumor cells. Since the growth of malignant cells at secondary sites could depend on their ability to respond to microenvironments, the growth factor dependence of 3LL variants has been studied. The ability of variant lines to grow in monolayer and in soft agar cultures, either in the presence or absence of different growth factors or serum, was analyzed and correlated with their metastatic potential. The reported results demonstrate that tumor cells expressing higher metastatic potential also exhibit higher capacity to grow and proliferate in all the culture conditions tested, independently of the addition of exogenous growth factors or serum. Moreover, since highly metastasizing cells express a significant amount of TGF-beta 1 mRNA, a pattern of autocrine growth is postulated for 3LL metastatic cells. One relevant aspect of the phenotype of transformed cells is their reduced adhesion to solid substrates; this phenomenon is thought to reflect the invasive and metastatic potential of tumor cells. Since the adhesion of the cells to substrata is mediated by molecules of the extracellular matrix, the expression of extracellular matrix receptors (integrins) was studied on 3LL metastatic variants. In particular, through immunochemical and biochemical studies we investigated the expression of the laminin receptor(alpha 6/beta 1) and of a novel receptor (integrin: alpha 6/beta 4), of unknown function. The receptors were quantitated on the cell surface of 3LL variants by the use of specific monoclonal antibodies which recognize, respectively, different epitopes of alpha 6, beta 4 or beta 1 subunits. Results demonstrate that the novel integrin alpha 6/beta 4, is specifically expressed in highly metastasizing 3LL cells, whereas the laminin receptor alpha 6/beta 1 is expressed in all 3LL variants. In conclusion, data presented demonstrate that 3LL cells endowed with higher metastatic potential are more independent of the microenvironmental conditions in that they possess a higher autocrine capacity than the lower metastasizing ones, and could acquire higher capacity to invade through the expression on their cell surface of specific receptors for cell adhesion (the novel integrin, defined as alpha 6/beta 4).

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human lung carcinoma cells of different histotypes.

Structural and functional analyses of several integrin heterodimers were performed in non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines. The expression of beta 1, beta 3, beta 4, and beta 5 heterodimers was evaluated at protein and mRNA levels. By flow cytometry and immunoprecipitation experiments we demonstrate that NSCLC cells (A549 adenocarcinoma and DG 3 large cell carcinoma) coexpress integrin heterodimers composed of beta 1, beta 3, beta 4, and beta 5 subunits, whereas SCLC cells (AE2 and H69) express only beta 1 integrin heterodimers. Northern blot experiments confirmed immunochemical analysis: SCLC cells in contrast to NSCLC cells express only the mRNA coding for the beta 1 subunit. These data indicate that in lung carcinoma cells the diversity in the integrin repertoire depends upon differential gene expression. The functionality of integrin receptors has been studied using antibody blocking experiments. Data reported demonstrate that the alpha 6 beta 1 integrin is a laminin receptor in either SCLC or NSCLC cells. An antibody to the beta 4 subunit partially inhibits the adhesion of adenocarcinoma cells to lamin but does not block lamin adhesion of large cell carcinoma cells, even though alpha 6 beta 4 complexes are expressed on both cell types. Two antisera to vitronectin receptors inhibit the adhesion of NSCLC cels to both vitronectin and fibronectin. The same antisera inhibit the adhesion of SCLC cells only to laminin, indicating that the alpha v beta 1 integrin might function in these cells as laminin receptor.