Recently, a candidate tumor suppressor gene, PTEN, has been identified on human chromosome 10q23 that is mutated in sporadic cancers of the brain, breast, and prostate. Germline mutations of the PTEN gene are observed in Cowden disease, an inherited predisposition in breast and thyroid cancer. Analysis of the codon sequence of PTEN has revealed a tyrosine phosphatase/dual specificity phosphatase domain. The phosphatase domain sits within a large domain homologous to tensin and auxilin. Mutations have been observed within the phosphatase domain and non-overlapping portions of the tensin domain. The carboxy-terminal half of the codon sequence has no significant homology to other genes; nevertheless, it is a target for mutations as well. Thus, it represents a potential functional domain of unknown significance. The first specific aim will attempt to identify the frequency of PTEN inactivation in brain and breast tumors. Tumor DNA paired with normal DNA samples will be analyzed for PTEN mutations in exonic sequences. Homozygous deletions of exons will be investigated using a variety of techniques, including Southern blotting, PCR, with polymorphic markers within and surrounding the gene, and fluorescent in situ hybridization (FISH). The potential inactivation of PTEN expression through methylation will be explored. Finally, tumors at different phases of development will be tested for alterations. The second specific aim is to characterize the PTEN protein. Both polyclonal and monoclonal antibodies will be developed. These antibodies will be utilized to identify the location of PTEN in cells, normal tissues, and tumors. PTEN will be expressed as a fusion protein in e. coli, purified, and tested for phosphatase activity. Mutants of PTEN found in people will be expressed to determine their effects on phosphatase activity. Finding proteins that specifically interact with the phosphatase or other domains of the protein will be a high priority. Approaches to find such proteins include co-immunoprecipitation, two-hybrid analysis, and affinity purification.
The third aim i s to determine the physiological function of PTEN and uncover its mechanisms of tumor suppressor. To identify the role of PTEN in normal and tumor development, the gene will be mutated in the germline of mice. As an alternative approach, tumor cell lines mutated for PTEN will be complemented with the wild type gene. Phenotypes to be tested include cell proliferation and tumorigenesis.
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