FUNCTIONAL AND IMMUNOPHENOTYPIC ANALYSIS OF TUMOR SUPPRESSOR GENES IN STS

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Th main objective of this proposal focuses on the continued characterization of mutations and altered patterns of expression of cell cycle regulators as they relate to processes of tumorigenesis and tumor progression in adult soft tissue sarcomas. It is the investigators hypothesis that molecular abnormalities of TP53 and RB genes, as well as those that directly or indirectly affect their encoded products, produce a selective advantage for tumor growth and an aggressive behavior in soft tissue sarcoma patients. This is supported by the facts that TP53 mutations and altered expression of p;53 and pRB are frequent events in adult soft tissue sarcomas and are associated with poor clinical outcome and reduced patient survival. The goals of this project are to translate basic research findings into clinical studies, and to collaborate with other Program members in the evaluation of novel potential tumor markers. The Specific Aims are; Aim #1: To further define TP53 mutations and altered patterns of p53 and pRB expression in primary and metastatic soft tissue sarcomas. The investigators plan to prospectively validate previous reports from their laboratory and other groups that have shown the prognostic value of TP53. They will also determine if different TP53 mutations have an impact on the outcome of the disease, and if there is any correlation between altered p53 and pRB expression patterns. These studies will be performed in collaboration with Projects 1 and 3, as well as with pathology and Biostatistics Cores. Aim #2: To characterize the functional activities of the p53 mutant products and pRB altered proteins identified in soft tissue sarcomas and STS cell lines established as a component of the program. Functional studies of p53 and pRB will be conducted to distinguish silent mutations from those contributing to the malignant phenotype. In addition, the investigators will also study down- stream events of their pathways, including mdm2 and E2F proteins. These studies will be conducted in collaborative with Dr. Pavietich and with Dr. Berrtino's laboratory (see Project #3). Aim #3: To transfer wild-type TP53 into soft tissue sarcoma cells processing TP53 mutations, including STS cell lines established in the Program. The investigators propose to introduce the TP53 gene in soft tissue sarcoma cells by an adeno-associated viral vector and by cationic liposomes in an attempt to restore TP53 tumor suppression functions. They will also analyze specific molecules that may undergo differential expression upon TP53 wild-type gene transfer, including mdm2 and E2F1; cdk2 and cdk4; cyclins D1, A and E; and certain cyclin dependent kinase inhibitors (ie, p21/WAF1). The investigators will also assess apoptosis by the analysis of DNA breaks (TUNEL method).