Abstract A35: Identification of pro-survival rapamycin-dependent microRNA (Rapa-miRs) in tuberous sclerosis complex
Authors: Lam Hilaire C. , Trindade Anil J. , Medvetz Douglas A. , Yu Jane , Priolo Carmen , Chan Stephen Y. , Henske Elizabeth P. 
Published: August 2014

Abstract
Background: Tuberous sclerosis complex (TSC) is a multisystem, hamartomatous disease associated with tumors of the brain, skin and kidney, as well as progressive cystic lung destruction characteristic of lymphangioleiomyomatosis (LAM). TSC is caused by mutations in TSC1 (hamartin) or TSC2 (tuberin), resulting in hyperactive mechanistic Target of Rapamycin (mTOR) signaling. mTOR is part of two multimeric complexes, mTORC1 and mTORC2. Rapamycin and related rapalogs inhibit mTORC1 activity and stabilize tumors in TSC patients, but tumor progression proceeds at pretreatment rates following cessation of therapy. We sought to determine whether miRNA contribute to the response of TSC2-deficient cells to mTOR inhibition.
Methods: Using TSC2-deficient patient angiomyolipoma-derived 621-101 cells, we identified rapamycin-dependent miRNA (“Rapa-miRs”) with two separate platforms (Signosis and Exiqon) and validated them by qRT-PCR. Published expression profiling data were used in conjunction with network analysis overlayed with KEGG and Reactome data to identify Rapa-miR targeted pathways.
Results: Rapamycin-dependent regulation of miR-29b, -21, -24, -221, -106a and -199a were common to both platforms and were classified as candidate “Rapa-miRs.” Interestingly, several Rapa-miRs, including miR-21 and -221, are known pro-survival oncogenic miRNA (oncomiRs). Rapamycin induced miR-21 in diverse cell lines, including C3H-10T1/2 with and without knockdown of TSC2, suggesting that rapamycin regulates Rapa-miRs in a TSC2-independent manner. Induction of miR-21 by rapamycin was associated with enhanced processing of pri-miRNA into pre- and mature miRNA, indicating mTORC1 may function in miRNA production. The catalytic mTOR inhibitor Torin 1 induced miR-221 and -29b, but did not induce miR-21. These data suggest that miR-21 induction by rapamycin requires active mTORC2 complex; however, this effect was Akt-independent as inhibition of Akt did not block the induction of miR-21 by rapamycin. Finally, network analysis using the Consolidated Interactome (CI) revealed that Rapa-miRs inhibit multiple key pro-apoptotic nodes, including cytochrome c and BAK1. These data suggest that Rapa-miRs regulate proteins critical for determination of cellular fate and survival in response to rapamycin treatment.
Conclusions: Rapamycin induces pro-survival oncomiRs in TSC2-deficient cells; therefore, targeting Rapa-miRs may potentiate rapamycin therapy in TSC patients.
Citation Format: Hilaire C. Lam, Anil J. Trindade, Douglas A. Medvetz, Jane Yu, Carmen Priolo, Stephen Y. Chan, Elizabeth P. Henske. Identification of pro-survival rapamycin-dependent microRNA (Rapa-miRs) in tuberous sclerosis complex. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A35.