The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome ten) is frequently mutated in a broad range of sporadic tumors, as well as in a group of overgrowth syndromes including Cowden and Proteus syndromes. PTEN encodes a dual-specificity phosphatase and has been identified as an antagonist for the PI3-kinase signaling pathway. The aim of this study was to determine the functionality of five missense and five truncating PTEN mutations that have been previously associated with cancer or overgrowth. Wild-type PTEN cDNA was first subcloned into the mammalian expression vector, pcDNA4/TO. The ten PTEN mutations were generated using site-directed mutagenesis and were transiently transfected into PC3, a PTEN-null prostate cancer cell line. The effects of reintroducing either wild-type or mutant PTEN into PC3 were assessed in regards to their ability to modulate PI3-kinase and ERK signaling pathway activity, as well as altering rates of cell death and proliferation. Immunoblotting of phosphorylated Akt was used as a measure of the effect of mutant PTEN on downregulation of PI3-kinase signaling. As predicted, reintroduction of wild-type PTEN into PC3 cells resulted in a decrease in Akt phosphorylation compared with vector alone transfectants. Preliminary data showed that some of the mutants appeared to have reduced activity, while some had similar activity to wild-type PTEN in inhibiting Akt activation. The expression level of protein and mRNA transcripts of wild-type and mutant PTEN were investigated using immunoblotting and quantitative real time PCR (qRT-PCR), respectively. For the qRT-PCR analysis, a Taqman probe analysis was designed to specifically prevent co-amplification of the PTEN pseudogene, which shares over 98% homology with the coding sequence of functional PTEN. Differential PTEN gene and protein expression was observed, with three mutants showing comparable mRNA expression to wild-type PTEN while the remaining seven mutants had greatly reduced expression. Post-transcriptionally, full length missense mutants, with the exception of the R173C mutant, generally showed a higher expression level of the tumor suppressor protein compared to truncation mutants. These results suggest that mutation may impact on the stability of PTEN, both at the transcriptional and translational level. In combination with functional studies currently underway, new information will be revealed on the role of PTEN as a major determinant of both tumor development and generalized overgrowth.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA