Kaposi sarcoma–associated herpesvirus miRNAs suppress CASTOR1-mediated mTORC1 inhibition to promote tumorigenesis
Tingting Li, … , Enguo Ju, Shou-Jiang Gao
Tingting Li, … , Enguo Ju, Shou-Jiang Gao
Published July 15, 2019
Citation Information: J Clin Invest. 2019;129(8):3310-3323. https://doi.org/10.1172/JCI127166.
View: Text | PDF
Research Article AIDS/HIV Virology

Kaposi sarcoma–associated herpesvirus miRNAs suppress CASTOR1-mediated mTORC1 inhibition to promote tumorigenesis

Abstract

Cytosolic arginine sensor for mTORC1 subunits 1 and 2 (CASTOR1 and CASTOR2) inhibit the mammalian target of rapamycin complex 1 (mTORC1) upon arginine deprivation. mTORC1 regulates cell proliferation, survival, and metabolism and is often dysregulated in cancers, indicating that cancer cells may regulate CASTOR1 and CASTOR2 to control mTORC1 signaling and promote tumorigenesis. mTORC1 is the most effective therapeutic target of Kaposi sarcoma, which is caused by infection with the Kaposi sarcoma–associated herpesvirus (KSHV). Hence, KSHV-induced cellular transformation is a suitable model for investigating mTORC1 regulation in cancer cells. Currently, the mechanism of KSHV activation of mTORC1 in KSHV-induced cancers remains unclear. We showed that KSHV suppressed CASTOR1 and CASTOR2 expression to activate the mTORC1 pathway. CASTOR1 or CASTOR2 overexpression and mTOR inhibitors abolished cell proliferation and colony formation in soft agar of KSHV-transformed cells by attenuating mTORC1 activation. Furthermore, the KSHV-encoded miRNA miR-K4-5p, and probably miR-K1-5p, directly targeted CASTOR1 to inhibit its expression. Knockdown of miR-K1-5p and -K4-5p restored CASTOR1 expression and thereby attenuated mTORC1 activation. Overexpression of CASTOR1 or CASTOR2 and mTOR inhibitors abolished the activation of mTORC1 and growth transformation induced by pre–miR-K1 and -K4. Our results define the mechanism of KSHV activation of the mTORC1 pathway and establish the scientific basis for targeting this pathway to treat KSHV-associated cancers.

Authors

Tingting Li, Enguo Ju, Shou-Jiang Gao

×

Figure 8

mTOR inhibitors suppress pre–miR-K1 and -K4–induced cell proliferation.

Options: View larger image (or click on image) Download as PowerPoint
mTOR inhibitors suppress pre–miR-K1 and -K4–induced cell proliferation. ...
(A) The mTOR inhibitors rapamycin and Torin1 inhibited mTORC1 activation in ΔmiR-V, ΔmiR–pre–miR-K1, and ΔmiR–pre–miR-K4 cells. Cells were treated with DMSO, 200 nM rapamycin, or 50 nM Torin1 for 4 hours and analyzed for mTORC1 activation by examining expression of the downstream effectors p-S6K and p-4EBP1 by Western blotting. Results from 1 experiment are shown. (B) Rapamycin and Torin1 significantly inhibited pre–miR-K4 and -K4–induced cell proliferation. ΔmiR-mutant cells stably expressing vector control (ΔmiR-V), pre–miR-K4 (ΔmiR–pre–miR-K4), or pre–miR-K1 (ΔmiR–pre–miR-K1) were treated with DMSO, 100 nM rapamycin, or 50 nM Torin1, and cell numbers were counted daily. Three independent experiments were repeated with similar results, and results from 1 representative experiment with 3 biological replicates are shown as the mean ± SEM. (C and D) Rapamycin and Torin1 inhibit pre–miR-K4 and -K4-induced cell-cycle progression and induce apoptosis. ΔmiR-mutant cells stably expressing vector control (ΔmiR-V), pre–miR-K4 (ΔmiR-pre-K4) or pre–miR-K1 (ΔmiR-pre-K1) were treated with DMSO, 100 nM rapamycin or 50 nM Torin1 for 24 hours, and analyzed for cell-cycle progression (C) and apoptosis (D). Three independent experiments were repeated with similar results, and results from 1 representative experiment with 3 biological replicates are shown as the mean ± SEM. (E) Schematic illustration of KSHV miR-K4-5p and possibly miR-K1-5p direct suppression of CASTOR1, leading to activation of the mTORC1 pathway, enhanced cell proliferation, and cellular transformation. Data in B–D were analyzed by 1-way ANOVA followed by Tukey’s post hoc test for P values below 0.05. **P < 0.01 and ***P < 0.001.