The Target of Rapamycin (TOR) kinase is a conserved member of the PI-3 kinase family, which has emerged as a central regulator of cellular responses to wide ranging environmental stress, including amino acid starvation, growth factor deprivation and hypoxia. TOR signaling regulates translation initiation, transcription, cell cycle progression and survival. Moreover, recent studies indicate that dysregulation of cap-dependent translation via activation of the TOR pathway or increased expression of eukaryotic initiation factor 4E (eIF4E) plays a role in genesis of cancer. Rapamycin bound to FKBP12 inhibits the function of TOR, with several rapamycin analogs in phase I-III oncology clinical trials. In combination with other chemotherapeutic agents, rapamycin generally show at least additive activity, although the underlying mechanisms are often unclear. Here we report that TOR signaling is also a determinant of cell survival in response to aberrations in DNA replication. Using the genetically tractable yeast Saccharomyces cerevisiae as a model system, we found that TOR signaling is required for S-phase progression and to maintain cell viability following intra-S phase checkpoint activation. This effect on survival in response to replication stress is conserved from yeast to humans, as similar effects were observed when synchronized HEK293 cells were released into S-phase in the presence of rapamycin and the DNA topoisomerase I poison, topotecan. When yeast cells transit S-phase in the presence of DNA damage, rapamycin inhibition of TOR signaling induced replication fork collapse, hyperphosphorylation of Rad53 and the downregulation of ribonucleotide reductase (RNR) regulatory subunits Rnr1 and Rnr3. DNA damage induced expression of RNR subunits provides the elevated dNTP pools necessary for translesion DNA synthesis. Although this transcriptional response to DNA damage is initiated in rapamycin-treated cells, TOR signaling is required to sustain this survival program. Moreover, the protective function of TOR signaling was dependent on an intact intra-S phase checkpoint. The mechanism by which cells maintain sufficient basal level of dNTPs to sustain cell viability and the slow progression of replication forks in response to RNR inhibitor HU is also unclear. Our results support a role for the TOR pathway in sustaining threshold levels necessary for S-phase transit and cell viability in response to long-term HU treatment. Thus, the TOR pathway cooperates with the MEC1/RAD53 checkpoint to regulate RNR levels and fork progression in response to DNA damage and replication stress. Supported by ALSAC and NIH grant CA23009.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]