TOR signaling in S-phase protects cells from DNA topoisomerase I poisons
Authors: Changxian Shen, Alice Gibson, Christopher Morton, Peter Houghton, Mary-Ann Bjornsti
Journal: Cancer Research (2008)

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA

2562 

The TOR (target of rapamycin) kinase regulates cellular responses to wide ranging environmental stresses, including conditions of restricted nutrients, growth factors or oxygen. In wild-type p53 cells, rapamcin inhibition of TOR-raptor complex (TORC1) signaling is associated with the accumulation of cells in G1-phase of the cell cycle, obviating a function for TORC1 in S-phase. Yet, the induction of DNA damage in S-phase induces a p53-dependent suppression of TORC1. These findings led us to consider the consequences of continued TORC1 signaling in the presence of DNA damage. Since the TOR pathway is conserved from yeast to human, we first used budding yeast as a genetically tractable model of a eukaryotic cell that lacks functional p53, yet retains wild-type cell cycle regulatory and S-phase checkpoint pathways. In synchronized cell populations exposed to the methylating agent MMS in S-phase, rapamycin inhibition of TORC1 both enhanced MMS-induced cytotoxicity and suppressed MMS-induced mutagenesis. An analysis of replication intermediates demonstrated that TORC1 signaling was required to maintain replication fork stability in cells exposed to DNA damage. Indeed, rapamycin also enhanced yeast cell sensitivity to the DNA toposiomerase I poison camptothecin. TORC1 signaling was required to sustain Rad53 checkpoint-mediated induction of the large RNR1 and RNR3 subunits of ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in the production of DNA precursor dNTPs. Thus, TORC1 signaling also maintained sufficient dNTPs necessary for translesion DNA synthesis, effectively promoting the acquisition of MMS-induced mutations. To determine if TORC1 signaling served a similar function in protecting human cells from DNA damaging agents in S-phase, we examined the activity of rapamycin in combination with the camptothecin class of chemotherapeutics, in several cell lines defective for p53 in vitro and a panel of pediatric tumor xenografts in vivo. Since the cytotoxicity of camptothecins requires on-going DNA replication, this combination obviated the need to use synchronized cells. As in yeast, we found that rapamycin enhanced the cytotoxicity of topotecan in HEK293 and rhabdomyosarcoma Rh30 cells as demonstrated by the loss of clonogenic survival and enhanced phosphorylation of histone H2AX. Even more surprising, we observed additive or greater than additive activity of rapamycin in combination with irinotecan in a panel of rhabdomyosarcoma and neuroblastoma xenograft mouse models. Contrary to predictions that rapamycin-induced G1 arrest would antagonize the S-phase-dependent toxicity of camptothecins, our findings suggest that TORC1 signaling protects cells from genotoxic stress in S-phase and, in the absence of a functional p53 pathway, may actually promote the acquisition of drug resistance as a consequence of DNA-damage induced mutagenesis.    Supported by ALSAC and NIH CA23009