Abstract 1496: Epithelial to mesenchymal transition and rapamycin sensitivity in cancer cell lines.
Authors: Holder Ashley M. , Adkins Farrell , Akcakanat Argun , Chen Huiqin , Do Kim-Anh , Hung Mien-Chie , Meric-Bernstam Funda 
Published: August 2014

Activation of the mTOR signaling pathway is common in multiple cancers. Rapamycin has inhibitory effects on solid tumors through inhibition of the mTOR kinase and alteration of downstream signaling. Though rapamycin and its analogs have yielded promising results from in vitro studies in sensitive cell lines, they have generated only a modest effect in clinical application. Epithelial to mesenchymal transition (EMT) in cancer cells involves not only the loss of cell-cell adhesion and acquisition of migratory and invasive properties but also resistance to cell death, senescence, and chemotherapy. We hypothesized that the EMT status of cancer cells regulates their sensitivity to rapamycin. We classified a panel of immortalized cancer cell lines as resistant or sensitive to rapamycin based on their IC50 as determined by Sulforhodamine B (SRB) assay. Using reverse phase proteomic array, we found that EMT markers were differentially expressed in sensitive compared to resistant immortalized cell lines. Rapamycin sensitive (RS) cell lines had increased expression of E-cadherin and N-cadherin while rapamycin resistant (RR) cell lines had increased expression of Smad3 (p&lt;0.02). To validate these findings, we performed western blotting for EMT markers in selected RR and RS cell lines. E-cadherin expression was higher in RS cell lines than RR cell lines; in turn, RR cell lines expressed increased levels of the mesenchymal marker Vimentin. To determine if inducing EMT decreases rapamycin sensitivity, we assessed E-cadherin and Vimentin expression by western blotting in the epithelial, immortalized breast cancer cell line, MCF7, which was transfected with a constitutively-active mutant of Snail, a known EMT driver. Constitutively-active Snail mutant MCF7 demonstrated loss of E-cadherin and gain of Vimentin expression compared to cells transfected with nonfunctional mutant Snail or Wild-type Snail. Furthermore, we found decreased growth inhibition with rapamycin by SRB assay in constitutively-active Snail mutant MCF7 compared to controls (p&lt;0.05). To determine if modulating EMT increases rapamycin sensitivity, we induced MET using ZEB1 siRNA knockdown and miR200b/c transfection in two mesenchymal cancer cell lines_ACHN and MDA-MB-231. Although miR200b/c transfection induced E-cadherin expression by immunoblotting in both cell lines, there was no significant increase in growth inhibition by SRB assay. In contrast, ZEB1 siRNA knockdown partially reversed EMT in these cells, which exhibited increased sensitivity to rapamycin by SRB assay. Most but not all mechanisms of EMT modulation altered rapamycin sensitivity in immortalized cancer cell lines. Future studies to determine the mechanism of EMT modulation that results in increased sensitivity to rapamycin could have important clinical applications for combination therapies utilizing rapamycin and its analogs.

Citation Format: Ashley M. Holder, Farrell Adkins, Argun Akcakanat, Huiqin Chen, Kim-Anh Do, Mien-Chie Hung, Funda Meric-Bernstam. Epithelial to mesenchymal transition and rapamycin sensitivity in cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1496. doi:10.1158/1538-7445.AM2013-1496