We have previously shown that mTOR inhibitor (CCI-779) can restore cisplatin sensitivity in cisplatin resistant small cell lung cancer cell (SCLC) line (Mol. Cancer 4:25, 2005). However, not all cisplatin resistant cell lines are sensitive to mTOR inhibitor. In this report, we have investigated the molecular differences in these cisplatin resistant lung cancer cell lines and determined whether reversal of cisplatin resistance is by inhibition of mTOR. Two pairs of lung cancer cell lines and their resistant variants were used: SCLC1/SR-2 and MSCLCS/MSCLCSC. SR-2 exhibits 20 fold resistance to cisplatin and can be completely reversed with 30 ng/ml of CCI-779 whereas MSCLCSC exhibits 7 fold resistance to cisplatin and cannot be reversed with the same dosage. To confirm that the reversal effect is due to inhibition of mTOR, we have used SiRNA to knock down mTOR. Two SiRNA were used with similar result. The maximal effect occurs at 48 hrs. with 80% inhibition and lasts for 72hr. The ID50 of cisplatin was also decreased from 2.5 to 0.2ug/ml in the transfected cell lines which is similar to using CCI-779. The amount of phosphor-4E-BP was also decreased with maximal effect seen at 72 hr. Thus, our results further confirm that inhibition of mTOR can restore cisplatin sensitivity. Since SR-2 expressed higher levels of several genes including elongation factor alpha, hTERT, cyclin D3, Cyclin B replication A protein, RAD 52, RAD51, and Ku80, we determined whether these proteins decrease after addition of CCI-779. Our results show that Elongation factor alpha, hTERT, CyclinD3, Cyclin B, Replication A protein were decreased whereas RAD 52, RAD51 and Ku80 showed no changes. SR-2 cell lines also possess higher levels of pAKT and PDK1 which indicates activation of PI3, a growth stimulatory pathway upstream of mTOR. Thus, by inhibiting mTOR, the survival pathway of cisplatin resistant cells is inhibited. In contrast, MSCLCSC has minute amounts of pAKT as well as phosphor mTOR (cannot be detected by westernblot).In addition, by gene specific array analysis, MSCLCSC only showed increase in elongation factor alpha which can be inhibited by mTOR, whereas RAD51, 52, Ku80 are not affected by mTOR inhibitor. The remaining genes are being investigated. Thus, it is likely that mTOR inhibitor will restore cisplatin resistance in cell lines which have activated PI3 survival pathway and which possess high levels of replication proteins whose translation is controlled by mTOR. Supported by VA Research fund.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]