Our data show that localization and transcriptional activity of FKHR is aberrant in PTEN-null cells. Reconstitution of wild-type PTEN, but not lipid phosphatase-inactive mutants, restores both localization and transcriptional activation of FKHR in these cells. While wild-type FKHR is relatively inactive in PTEN null cells, a phosphosite mutant of FKHR (FKHR;AAA) that is no longer phosphorylated by Akt can still localize to the nucleus and activate transcription in such cells. This mutant induces death in a cell line susceptible to PTEN-mediated cell death. Surprisingly, it does not induce apoptosis but, rather, induces a G1
arrest in cells that likewise arrest with wild-type PTEN. Together, the data derived from the cell death assays and the cell cycle arrest assays support the notion that an intact and active FKHR protein is capable of carrying out PTEN function in its absence. That is, activated FKHR complements the loss of PTEN in two different functional assays. These data support the idea that FKHR is sufficient for PTEN function in cells. Finally, previous data have shown that PTEN-null cells have low levels of p27 and that reintroduction of PTEN up regulates p27 levels (33
). We find that FKHR;AAA dramatically induces p27 levels in PTEN-null cells. These data suggest that the finding of aberrant p27 levels in the absence of PTEN might arise as a consequence of the lack of FKHR function in such cells. In keeping with these data, Medema et al. recently reported similar data which demonstrated a role for Forkhead factors as regulators of cell cycle progression and, using defined genetic cells, showed that such regulation does indeed depend on the induction of p27 (40
The PI3K/Akt pathway is a well-known oncogenic signaling pathway (75
). Cell survival and cell proliferation have been linked to this pathway in multiple systems. For example, interleukin-3-dependent cell lines require Akt for survival, as do cells in which anoikis is blocked by Ras activation (17
). On the other hand, expression of activated PI3K in the absence of serum can induce DNA synthesis (30
). Furthermore, PTEN is capable of inducing apoptosis or a cell cycle arrest, and loss of PTEN in primary cells leads to either excessive proliferation or defects in apoptosis. In mammalian cell-based assays, a diverse group of substrates have been linked to Akt activation. In C. elegans
, on the other hand, the insulin/PI3K/Akt signaling pathway that regulates aging, while conserved with mammalian cells, has thus far yielded only the Forkhead homologue daf-16
as a downstream target (46
). It is possible that the deregulated activity of multiple Akt substrates contributes to the neoplastic properties inherent to a PTEN-null tumor cell and that certain substrates might individually contribute to the regulation of apoptosis and cell proliferation. Our data, however, support the notion that the pathway linking PI3K and PTEN to transformation of mammalian cells is essentially identical to the pathway regulating aging in C. elegans
. This pathway is comprised of a receptor tyrosine kinase such as IGF-IR (daf-2
), PI3K (ageI
), Akt-1 (akt1
) and Akt-2 (akt2
), PDK-1 (pdk1
), PTEN (daf-18
), and the daf-16
homologues (FKHR, FKHRL1, and AFX) (23
It is interesting that elements of this pathway that are linked genetically in C. elegans
are the same elements of the pathway that have been associated with genetic alterations in human tumors. The PI3KCA gene is amplified in ovarian cancer and is also found as a retroviral oncogene (1
). Akt-1 and Akt-2 are amplified in a limited number of tumors, and Akt-1 is the cellular homologue of v-Akt (2
). Finally, PTEN
is widely mutated in cancer, and FKHR has been the target of translocation in rhabdomyosarcoma. Interestingly, in this tumor, two different translocations give rise to the fusion proteins PAX3-FKHR or PAX7-FKHR (16
). Our data support the notion that FKHR could act as a tumor suppressor; thus, one untested possibility is that these translocations might produce chimeric proteins that could act in a dominant negative manner to inactivate FKHR function.
The notion that a transcription factor might induce a G1 arrest or induce cell death is not new. Indeed, this is precisely the case for p53. The parallels between these pathways are striking. p53 receives signals that reflect the state of the genome (DNA damage) at least in part from a PI3K family member, ATM. This signal may be transmitted through phosphorylation of p53. p53 can then enact a G1 arrest through transcriptional regulation of p21. p53 induces apoptotic cell death through both transcription-dependent and -independent mechanisms. FKHR, on the other hand, receives signals primarily from the environment external to the cell. These signals are transmitted through a type I PI3K and result in the phosphorylation of FKHR and its subsequent inactivation. In its active state FKHR, can promote a G1 arrest through the induction of p27 and can induce apoptosis perhaps through regulation of Fas signaling or through regulation of FasL itself.
How does FKHR regulate p27? p27 is primarily regulated posttranscriptionally, both through ubiquitin-mediated proteolysis and through translation controls. There is limited information to suggest that transcriptional regulation of p27 is important. Furthermore, PTEN did not alter p27 mRNA levels (33
). On the other hand, Medema et al. (40
) have demonstrated activation of the p27 promoter by AFX, and we have shown that both wild-type PTEN and wild-type FKHR, but not mutant controls, were capable of inducing activation of the p27 promoter (data not shown). In addition, Medema et al. reported a modest induction in p27 mRNA levels (40
). We have also seen a modest (1.3- to 1.5-fold induction in mRNA upon adenovirus expression of FKHR;AAA (Fig. B) and upon adenovirus expression of PTEN. In addition, however, the half-life of p27 protein is significantly prolonged. Here, it is possible that a modest increase in p27 levels induced through transcription might lead to inhibition of cyclin-dependent kinase activity followed by a decrease in p27 phosphorylation and then a change in the half-life of p27 protein. Since this process involves a catalytic mechanism, a small change in p27 mRNA levels could lead to a large difference in protein half-life. For example, an increase in the transcription of p27 could alter the balance between the two proposed complexes of p27 and cyclin E-cdk2, one inhibitory and one in which p27 is degraded (60
). Alternatively, it is possible that FKHR;AAA directly alters or regulates components of the p27 degradation apparatus. Specifically, it will be of interest to know whether Forkhead factors can alter the levels of any of the components of the Skp-Cul-F box (SCF) complex.
The mechanism that underlies FKHR induction of apoptosis is likewise not yet clear. FKHRL1 can regulate the FasL promoter, suggesting that these transcription factors might directly regulate the levels of this death effector (5
). In keeping with this notion, PTEN+/−
mice develop an autoimmune lymphoid hyperplasia syndrome that phenocopies mutations in the murine Fas gene (18
). On the other hand, cells from the PTEN+/−
animals did not demonstrate defects in FasL or Fas but, rather, were defective in the apoptotic response to Fas (18
). In either case, it would appear that PTEN-mediated and, by extension, FKHR-mediated apoptosis probably involves the Fas pathway.
Finally, our data support the notion that, as is the case in C. elegans, signaling pathways might be more linear, at least with respect to transformation, than is commonly suspected. This would lead one to further suspect that PTEN-null cells might be particularly sensitive to inhibitors directed against members of this pathway; if true, such dependence would bode well for the future success of therapeutics aimed at intervening in PI3K signaling.