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.
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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 3

Pre–miR-K1 and -K4 mediate KSHV downregulation of CASTOR1 and activation of mTORC1.

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Pre–miR-K1 and -K4 mediate KSHV downregulation of CASTOR1 and activation...
(A and B) The miRNA cluster and vFLIP mediated KSHV downregulation of CASTOR1. Analysis of CASTOR1 mRNA levels by RT-qPCR (A) and protein levels by Western blotting (B) in mock (MM), WT (KMM), ΔmiR, ΔvFLIP, and ΔvCyclin, cells. Three independent experiments were repeated with similar results, and results from 1 representative experiment are shown. mRNA results from 3 biological replicates are shown as the mean ± SEM. (C) The miRNA cluster mediated KSHV activation of the mTORC1 pathway. Analysis of the mTORC1 downstream effectors p-S6K and p-4EBP1 in mock, WT, ΔmiR, ΔvFLIP, and ΔvCyclin cells by Western blotting. 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. (D and E) Pre–miR-K1 and -K4 mediated KSHV downregulation of CASTOR1. Analysis of CASTOR1 mRNA (D) and protein (E) levels in WT cells and ΔmiR cells complemented with vector control or individual KSHV pre-miRNAs. mRNA results in D were from 3 biological replicates and are shown as the mean ± SEM. Three independent experiments were repeated with similar results, and results from 1 representative experiment are shown. Mut, mutant. (F) Pre–miR-K1 and -K4 mediate mTORC1 activation. Western blot analysis of the mTORC1 downstream effectors p-S6K and p-4EBP1 in mock and WT cells and ΔmiR cells complemented with vector control (V) or pre–miR-K1 or -K4. Three independent experiments were repeated with similar results, and results from 1 representative experiment are shown. Data were analyzed by 1-way ANOVA followed by Tukey’s post hoc test for P values below 0.05. *P < 0.05 and ***P < 0.001.