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 August 1, 2019; First published July 15, 2019
Citation Information: J Clin Invest. 2019;129(8):3310-3323. https://doi.org/10.1172/JCI127166.
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Categories: 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

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Figure 1

KSHV-transformed cells activate the mTORC1 pathway and are sensitive to mTOR inhibitors.

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KSHV-transformed cells activate the mTORC1 pathway and are sensitive to ...
(A) mTORC1 was activated in KSHV-transformed cells. Cells were analyzed for p-4EBP1 at Ser65 and p-S6K at Thr389. Three independent experiments were repeated with similar results, and results of 1 representative experiment are shown. (B) Rapamycin and Torin1 inhibited mTORC1 activation in KSHV-transformed cells. Cells were treated with DMSO, 100 nm rapamycin, or 50 nm Torin1 for 16 hours and analyzed for mTORC1 activation. Three independent experiments were repeated with similar results, and results from 1 representative experiment are shown. The same set of samples were run in different gels but with the same loading calibration. (C) Rapamycin and Torin1 significantly inhibited proliferation of KSHV-transformed cells. Cells 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 4 biological replicates are shown as the mean ± SEM. (D) Rapamycin and Torin1 significantly inhibited colony formation of KMM cells in soft agar. KMM cells treated with DMSO, 200 nM rapamycin, or 100 nM Torin1 were examined for colony formation in soft agar. Representative images acquired with a ×4 objective are shown. Graph shows the quantification of colonies with a diameter of greater than 50 μm. Three independent experiments were repeated, and results are shown as the mean ± SEM. (E and F) Rapamycin and Torin1 induced cell-cycle arrest but no significant apoptosis in MM and KMM cells. Cells were treated with DMSO, 100 nM rapamycin, or 50 nM Torin1 for 24 hours and analyzed for (E) cell-cycle progression or (F) apoptosis. Three independent experiments were repeated, and results are shown as the mean ± SEM. Data were analyzed by 1-way ANOVA followed by Tukey’s post hoc test for P values below 0.05. *P < 0.05, **P < 0.01, and ***P < 0.001.