Insights Into the Role of ASPP2 (Apoptosis Stimulating Protein of p53-2) in Lymphomagenesis.

Abstract 1967

Poster Board I-990

ASPP2 is a member of a family of p53 binding proteins that enhance apoptosis, in part through selective stimulation of p53 transactivation of pro-apoptotic target genes. Low ASPP2 expression is found in many human cancers and has been associated with poor clinical outcome in patients with aggressive lymphoma. Using an ASPP2+/- mouse model, we have previously demonstrated that ASPP2 is a haploinsufficient tumor suppressor and that reduced ASPP2 expression results in attenuated damage-response thresholds (Kampa et al., PNAS 2009). While ASPP2-/- mice are not viable, ASPP2+/- mice have an increased incidence of -irradiation-induced tumors compared to ASPP2+/+ mice.γspontaneous and ASPP2+/- mice develop high-grade thymic T-cell lymphomas after -irradiation. Moreover, primary ASPP2+/- thymocytes have an attenuatedγ -irradiation compared to ASPP2+/+ thymocytes.γapoptotic response after To explore the mechanisms of how attenuated ASPP2 expression could increase thymic lymphomagenesis and attenuate apoptosis, we performed global gene expression profiling on unirradiated, and 5 Gy irradiated ASPP2+/+ and ASPP2+/- thymocytes using an Affymetrix Mouse GeneChip® Array. We found significant differences in gene expression between ASPP2+/+ and ASPP2+/- thymocytes, in both unirradiated and irradiated sets. Using Ingenuity Pathway Analysis software, we found that amongst the highest scoring pathways displaying differences were those associated with cell growth, tumor formation, hematologic malignancies, immune response, cell death and cell cycle regulation. We additionally studied global phosphorylation patterns using 2-dimensional gel electrophoresis, fluorescent phosphoprotein dye Pro-Q Diamond staining, and liquid chromatography tandem mass spectrometry to determine the posttranscriptional mechanisms mediated by attenuated ASPP2 expression. Analysis of the phosphoproteome of ASPP2+/+ and ASPP2+/- mouse embryonic fibroblasts (with and without irradiation) revealed differences in the phosphorylation status of 108 peptides/proteins including those involved in regulating cell cycle checkpoints, T-cell receptor signaling, cell stress response, DNA repair mechanisms, cell growth, translation and transcription. Differential expression of the identified genes and proteins was verified by PCR and Western Blot. Thus, reduced ASPP2 expression affects global transcriptional as well as post-transcriptional networks intimately involved in the development of hematologic disorders–suggesting that ASPP2 function is by far more complex than solely enhancing the expression of pro-apoptotic p53 target genes. Given that ASPP2 is a bona fide tumor suppressor, reduced ASPP2 levels result in global dysregulation of pathways engaged in tumor suppression networks and the cellular damage response, which may ultimately promote genomic instability and tumor formation. Our findings provide insights into the role of ASPP2 in lymphomagenesis and reveal possible new targets for cancer therapy.