Circadian regulation of mTOR by the ubiquitin pathway in renal cell carcinoma Running title: Fbxw7 regulates the 24-h rhythm of mTOR

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT Circadian clock systems regulate many biological functions, including cell division and hormone secretion in mammals. In this study, we explored the effects of circadian control on the pivot cell growth regulatory mTOR, the activity of which is deregualted in tumor cells compared to normal cells. Specifically, we investigated whether the anti-tumor effect of an mTOR inhibitor could be improved by changing its dosing schedule in RenCa tumor-bearing mice. Active, phosphorylated mTOR displayed a 24h rhythm and levels of total mTOR protein (but not mRNA) also showed a circadian rhythm in RenCa tumor masses. Through investigations of the oscillation mechanism for mTOR expression, we identified the ubiquitination factor Fbxw7 as a mTOR regulator that oscillated in its expression in a manner opposite from mTOR. Fbxw7 transcription was regulated by the circadian regulator D-site binding protein (DBP). Notably, administration of the mTOR inhibitor everolimus during periods of elevated mTOR improved survival in tumor-bearing mice. Our findings demonstrate that the circadian oscillation of mTOR activity is regulated by circadian clock systems which influence the anti-tumor effect of mTOR inhibitors. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.