Differential response of mTORC1 and mTORC2 to rapamycin in rapamycin-sensitive and -resistant tumor xenograft models

1425 Mammalian target of rapamycin (mTOR) is a large protein kinase that exists in two different complexes, mTORC1 and mTORC2. Rapamycin exhibits antitumor activity by inhibiting several processes including cell proliferation, cell survival and angiogenesis. The antitumor activity of rapamycin is variable and tumor type specific. The factors contributing to this differential activity are not known. Several in vitro studies have suggested that rapamycin universally inhibits mTORC1 (raptor complex) preventing the phosphorylation of down stream targets such as S6K. The response of mTORC2 (rictor complex) is tumor type specific. Furthermore, inhibition of mTORC1 alone leads to the activation of AKT via a feedback loop, which may protect cells from apoptosis. To understand the kinetic response of mTORC1 and mTORC2 to rapamycin in vivo, we analyzed phospho-S6 (pS6) and phospho-AKT (pAKT) expression in rapamycin-sensitive and -resistant tumor xenograft models including glioblastoma (U-87 MG), prostate (PC-3), non-small cell lung (NCI-H460) and colorectal (HCT 116) cancers. In rapamycin-sensitive PC-3 and U-87 MG xenograft models, rapamycin showed profound antitumor activity with a T/C ratio of 27% and 1.2%, respectively. In a refractory HCT 116 xenograft model, a moderate inhibition of tumor growth was observed with a T/C ratio of 51%. Response of tumor biomarkers was measured using western blots, Meso Scale Discovery (MSD) platform and immunohistochemistry. Robust endogenous expression of pS6 and pAKT was observed in all sensitive tumor types. In rapamycin-resistant HCT 116 model, tumor pAKT level was relatively low. Significant inhibition of pS6 was observed within 1 h after a single dose of rapamycin in all models and continued up to 48 h. In sensitive tumors, inhibition of pAKT was observed only after 24 h of rapamycin administration. In the HCT 116 model however, inhibition of pAKT was not observed at any time point studied even with multiple doses of rapamycin. Results from western blots correlated well with MSD analysis. Ease of quantification and the dynamic range of MSD makes it the preferable method of analysis. Immunohistochemical localization of total and phospho proteins of S6 and AKT revealed that the expression of phospho proteins was not uniform throughout the tumor. The expression was patchy and localized more in the peripheral zones of the tumor lobules. The expression of AKT was more abundant than S6 in terms of number of cells expressing the protein. Rapamycin significantly inhibited pS6 and pAKT in tumor cells but did not effect the expression of total S6 and AKT. Together these data suggest that a single dose of rapamycin treatment exerts a profound effect on mTORC1 in sensitive and resistant tumor types in vivo. Sustained activation of mTORC2/AKT even after chronic treatment may partially explain the modest growth inhibition of HCT 116 tumors.