Reprogramming of the microRNA transcriptome mediates resistance to rapamycin

Abstract

The mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation that is often deregulated in cancer. Inhibitors of mTOR, including rapamycin and its analogues, are being evaluated as antitumor agents. For their promise to be fulfilled, it is of paramount importance to identify the mechanisms of resistance and develop novel therapies to overcome it. Given the emerging role of microRNAs (miRNAs) in tumorigenesis, we hypothesized that miRNAs could play important roles in the response of tumors to mTOR inhibitors. Long-term rapamycin treatment showed extensive reprogramming of miRNA expression, characterized by up-regulation of miR-17-92 and related clusters and down-regulation of tumor suppressor miRNAs. Inhibition of members of the miR-17-92 clusters or delivery of tumor suppressor miRNAs restored sensitivity to rapamycin. This study identifies miRNAs as new downstream components of the mTOR-signaling pathway, which may determine the response of tumors to mTOR inhibitors. It also identifies potential markers to assess the efficacy of treatment and provides novel therapeutic targets to treat rapamycin-resistant tumors.

FIGURE 1.

Rapamycin inhibits cell growth in parental BC3H1 cells but not in RR1 cells. A, 10⁴ BC3H1 or RR1 cells were plated in 24-well plates and treated with 100 nm or 1 μm rapamycin, respectively, and cells were counted after 5 days. The results are presented as mean ± S.D. (n = 3); *, denotes p < 0.01 compared with DMSO-treated cells. B, representative immunoblots for the indicated proteins of whole cell extracts of BC3H1 cells treated with vehicle or 100 nm rapamycin (Rap) for 1 or 24 h or RR1 cells grown without rapamycin for 1 week and then treated with vehicle or 100 nm rapamycin for 1 or 24 h or constantly treated with 1 μm rapamycin (R).

FIGURE 2.

Differential miRNAs expression in rapamycin-sensitive and -resistant cells. A, heat map of the unsupervised hierarchical clustering performed on 526 filtered miRNA probes. Normalized and background-corrected data and gene values are Z-score transformed. B, principle component analysis of the data set after normalization, background correction, and filtration. C, clustering heat map of the miRNA that contribute to PC1. D, clustering heat map of the miRNA that contribute to PC2.

FIGURE 3.

increased Myc and miR-17–92 cluster mediate the resistance to rapamycin. A, total RNA was extracted from BC3H1 cells treated for 24 h with DMSO or from RR1 or RR3 cells grown continuously in the presence of 1 μm or 100 nm rapamycin, respectively. Total RNA was extracted, and qRT-PCR was performed for the indicated miRNAs. Data shown are mean ± S.D., significantly different from DMSO-treated BC3H1 cells; *, p < 0.01 by t test. B, cumulative distribution plots of -fold changes in mRNA expression in RR1 versus BC3H1 cells. Separate curves are displayed for all mRNAs (black curve) or subsets of mRNAs predicted to be targeted by selected dysregulated miRNA (colored curves). The median change in expression for each mRNA subset is depicted by a respective dot on the x axis. C, total RNA was extracted from BC3H1 and RR1 cells treated as described in A, and qRT-PCR was performed for Myc Tgif2 and Thbs1. Data shown are mean ± S.D., n = 3, significantly different from DMSO-treated BC3H1 cells; *, p < 0.01, by t test. D, representative immunoblots for the indicated proteins from whole cell extract of BC3H1 or RR1 cells treated with DMSO or with rapamycin, as indicated, for 24 h. E, GSEA plots of TGFβ positive response gene sets, which were repressed in RR1+R cells: panel 1, PLASARI_TGFB1_TARGETS_10HR_UP, genes up-regulated in MEF cells upon stimulation with TGFβ1 for 10 h; panel 2, LABBE_TGFB1_TARGETS_UP, up-regulated genes in NMuMG cells (mammary epithelium) after stimulation with TGFβ1; panel 3, PLASARI_TGFB1_SIGNALING_VIA_NFIC_10HR_UP, genes up-regulated after 10 h of TGFβ1 stimulation in MEF cells with NFIC knock-out versus wild type MEFs; panel 4, MCBRYAN_PUBERTAL_TGFB1_TARGETS_UP, pubertal genes up-regulated by TGFβ1; panel 5, TGFB_UP.V1 _UP, genes up-regulated in a panel of epithelial cell lines by TGFβ1. F and G, RR1 cells were transfected with 10 nm Myc-specific siRNA, the indicated miRNA inhibitors (100 nm), or control and treated for 3 days with 100 nm rapamycin followed by cell count. Data shown are mean ± S.D. *, significantly different compared with untreated cells (p < 0.001 by t test).

FIGURE 4.

Myc and let-7 antagonize the expression of each other. A, total RNA was extracted from BC3H1 cells, VSMC, and C2C12 cells treated for 24 h with 100 nm rapamycin or vehicle, and qRT-PCR was performed for the indicated miRNA. Data shown are mean ± S.D., significantly difference from untreated cells; *, p < 0.01 by t test. B, BC3H1 or RR1 cells were transfected with 100 nm let-7c mimics, let-7c inhibitors, or control. Total RNA was extracted after 48 h, and Myc qRT-PCR was performed. C–E, BC3H1 cells were transfected with control plasmid or Myc-expressing plasmid, and RR1 cells were transfected with Myc-specific siRNA or control siRNA. Total RNA was extracted after 48 h, and qRT-PCR was performed for the indicated miRNA. Data shown are mean ± S.D.; *, significantly different from untreated cells (p < 0.001 by t test).

FIGURE 5.

Let-7 mediates the response of cells to rapamycin. BC3H1 or RR1 cells were transfected with the indicated miRNA mimics (100 nm) (A and B), miRNA inhibitors (C and D), or control. BC3H1 cells and RR1 cells were treated with 100 nm rapamycin for 5 days followed by cell count. Data shown are mean ± S.D., n = 3; *, significantly different from untreated cells; **, significantly different from rapamycin-treated BC3H1 cells (p < 0.001 by t test).

FIGURE 6.

Model of the role of miRNAs in the response of cells to mTOR inhibitors. In RR1 cells, an increase in Myc expression induces oncogenic miR-17–92 and related clusters and inhibits tumor suppressor miRNAs. Shown are the up-regulated (red) and down-regulated (black) miRNAs or genes found in RR1 cells.