Studies of the PTEN tumour suppressor in endometrial cancer.

Somatic mutations in the PTEN gene (Phosphatase and Tensin Homologue Deleted on Chromosome Ten) have been found in many types of cancer, but most frequently in cancer of the endometrium.The PTEN gene encodes a D3 lipid phosphatase of the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), which activates protein kinase B/Akt. Constitutive activation of Akt has been found in cells that lack functional PTEN, thus by dephosphorylating PIP3, this enzyme modulates several cellular functions e.g. proliferation, differentiation and migration.The PTEN protein comprises a 403 amino acid, 55KDa protein which is present in the cytoplasm but has also been detected in the nucleus of some cells. The function of nuclear versus cytoplasmic PTEN has not yet been determined. Little is known about modulation of PTEN expression by molecules such as hormones and cytokines. It has been reported, however, that NGF, BDNF and vitamin D3 analogues can up-regulate PTEN. The steroid hormones oestrogen and progesterone have been proposed as mediators of PTEN transcription, since their expression in endometrium reflects the menstrual cycle. The role of TGF-beta1 in PTEN expression of PTEN in human cells has been also investigated, but the results are contradictory. Compounds which stimulate up-regulation of PTEN represent potential anti-tumour therapies and therefore merit investigation. To further investigate the role of PTEN in endometrial cancer the following approaches were taken. The effect of TGF-beta1 on two endometrial carcinoma cell lines, HEC-1B and Ishikawa were investigated. The cell lines were stimulated with TGF-beta1 in the presence or absence of serum, and changes in mRNA and protein levels of PTEN and other genes analysed by RT-PCR and Western blotting. The morphology, cell number and cell viability were also assessed. Modest up-regulation of PTEN mRNA was detected in both cell lines, but little change in protein levels was observed. In accordance with published data, TGF-beta1 suppressed the growth of, and changed the morphology of both cell lines.To study PTEN sub-cellular localisation, full-length human PTEN cDNA was used in RT-PCR to generate a 1.2Kb fragment which was cloned into a green fluorescent protein expression vector pEGFP-N1 to create pRC-2. Sequencing of pRC-2 confirmed the in-frame cloning of wild-type PTEN. Lipid-basedtransfection was used to transiently transfect HEC-1B, Ishikawa and Cos-7 cells. Strong perinuclear and cytoplasmic localisation was detected in these cell lines, and localisation to the endoplasmic reticulum was observed. Stimulation with TGF-beta and 17-beta-estradiol had no discernable effect on sub-cellular localisation of PTEN in either HEC-1B or Ishikawa cell lines.A mutational study was performed using a large repository of archival endometrial carcinomas and normal cervical controls. PCR was used to amplify PTEN exons 5 and 8 from extracted DNA and the fragments separated by single-strand conformation polymorphism (SSCP) analysis. A number of samples exhibiting bandshifts were detected in both exons.