PTEN is a tumor suppressor highly mutated in a variety of human cancers. We found that Pten inactivation in the mouse results in early embryonic lethality and that Pten can act as a bona fide tumor suppressor in vivo. We have also shown that concomitant Pten and p27 losses are critical events in cooperative oncogenesis and demonstrated that Pten is haploinsufficient in some of its biological functions. We hypothesize that Pten is also haploinsufficient for tumor suppression and that its inactivation perturbs key developmental programs. We will test this hypothesis and study the molecular mechanisms by which PTEN suppresses tumorigenesis through a direct genetic approach with the following Specific Aims: 1. To define, in conditional Pten mice, the role of Pten in ontogenesis and tumorigenesis. We have generated Pten/floxed mice which we will utilize to conditionally inactivate Pten in: i) the developing limb given the fact germ line PTEN mutations in humans have been associated with bilateral hand malformations; ii) the hemopoietic mesoderm; and iii) in Pten null mosaic mutant embryos, an approach which has allowed us to delay and in some cases circumvent embryonic lethality. Ontogenesis and tumorigenesis will be studied in these conditional mutants, 2, To assess genetically the consequence and relevance of Pten haploinsufficieney in tumorigenesis in various tissues. We have generated compound mutants (Pten Hy/-) in which the expression of Pten is further reduced as compared to Pten +/- mutants (Pten hypomorph mutants). These mice are viable and preliminary results indicate that tumorigenesis is accelerated in these mutants. We will assess whether and in which tissue complete or partial Pten inactivation is required for tumorigenesis and metastasization. 3. To determine in conditional inducible mutants the consequence of Pten inactivation throughout aging. Aging and telomere shortening have a dramatic impact on tumor type, onset and incidence. We will utilize conditional inducible Pten mutants (Pten/in) in which the gene can be disrupted employing a tetracycline (Tet) regulated system, to inactivate Pten at various times during the mouse life span. Furthermore, Pten /in will be crossed with rnTer """"""""1""""""""(for telomere shortening), p66 she (mice whose life span is markedly prolonged) or TTD _m (mice with premature aging) mutants. Tumorigenesis and metastasization will be studied in these mutants. 4. To determine the basis of the functional cross talk between PTEN and p27 KiP in tumor suppression: the role of Skp2. We find that Pten inactivation results in increased expression of the F-box protein Skp2 and, in turn, in p27 Kwdegradation, but does not affect p27 KIPlocalization. We will dissect the molecular basis of the Pten/Akt/Skp2/p27 KxP cross talk and assess the genetic relevance of Skp2 in Pten inactivation-driven tumorigenesis. To this end, Pten +/- mutants will be intercrossed with Skp2 transgenic and KO mice to determine whether overexpression or inactivation of Skp2 cooperates or blocks tumorigenesis caused by Pten inactivation. We will also determine whether concomitant Skp2 inactivation rescues the early embryonic lethality observed in Pten /"""""""" mutants. 5. To determine the molecular mechanisms of growth suppression by PTEN through cell-cycle arrest. PTEN can suppress cell growth (Gl-arrest) independently of its phosphatase activity. Several cancer associated PTEN mutations do not affect PTEN catalytic activity. We propose to study the molecular basis of this phosphatase independent activity and the biological and molecular consequence of these cancer associated mutations.
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