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David A. Foster

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Over the past few years it has become apparent that cancer cells require the activation of a set of intra-cellular signals that promote cell cycle progression and survival. One of the most common survival signals activated in human cancers is mediated by mTOR -the mammalian target of rapamycin. mTOR is a critical nutrient and energy sensor in cells that lets the cell know that there is sufficient material available for a cell to double its mass and divide. mTOR causes the phosphorylation of downstream targets ribosomal subunit S6 kinase and eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1), which promotes cell cycle progression. mTOR suppresses the activity of the tumor suppressor Transforming Growth Factor-β (TGF-β). TGF-β plays a central role in causing G1 cell cycle arrest. Rapamycin is a highly specific allosteric inhibitor of mTOR. In the presence of serum, rapamycin activates TGF- β signaling and causes G1 cell cycle arrest. This is one reason why rapamycin is frequently called a 'cytostatic drug'. While conventional low (nM) doses of rapamycin can retard G1 cell cycle progression, our lab has recently found that high (μ) doses of rapamycin are needed to induce complete G1 cell cycle arrest. However, it is unclear as to what causes the cells to be sensitive to high dose rapamycin treatment with regard to G1 cell cycle progression.

Prior studies in lab has shown that rapamycin in absence of serum induces apoptosis. High dose rapamycin inhibits eIF4E. In this study we revealed that knockdown of eIF4E causes apoptosis both in the presence and absence of serum. This was unexpected because rapamycin induces G1 cell cycle arrest in the presence of serum. Upon investigation, we have found that inactivated S6 kinase prevents the apoptotic effect observed by singular knockdown of eIF4E and results in G1 cell cycle arrest. This effect is dependent on TGF-β signaling which contributes to G1 cell cycle arrest. Suppression of S6 kinase phosphorylation alone is insufficient to cause complete cell cycle arrest, indicating that complete G1 cell cycle arrest is due to suppression of both S6 kinase and eIF4E. This proves that the cytostatic effect of rapamycin is suppression of both S6 kinase and eIF4E, while the cytotoxic effects are due suppression of eIF4E in the absence of S6 kinase-dependent activation of TGF-β signals.

This study also shows that nano-molar doses that inhibit S6 kinase were sufficient to activate TGF-β signaling. The high doses of rapamycin used to inhibit eIF4E correlated with inhibition of Rb phosphorylation. Consistent with these observations, knockdown of both Smad4 (an important player of TGF-β signaling) and Rb reversed the cytostatic effects of rapamycin. These data indicate that the G1 cell cycle arrest induced by rapamycin is due to the up regulation of TGF-β signaling and down-regulation of Rb phosphorylation via phosphorylation of the mTORC1 substrates S6 kinase and 4E-BP1 respectively.

Altogether, our findings not only place an importance to the evaluation of the activity/expression level of S6 kinase and eIF4E as readouts for rapamycin efficacy but also enhance the current understanding of the cytostatic effects of mTORC1 suppression with therapeutic implications.

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