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 4

KSHV miR-K1-5p and -K4-5p inhibit CASTOR1 expression and activate mTORC1.

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KSHV miR-K1-5p and -K4-5p inhibit CASTOR1 expression and activate mTORC1...
miRNA suppressors reduced the level of miR-K1-5p (A) and -K4-5p (B) in KSHV-transformed cells. RT-qPCR examination of miR-K1-5p (A) and miR-K4-5p (B) in untransfected cells or in KMM cells transfected with a LNA-based scrambled control (NC), miRNA suppressor LNA-K1-5p (A), or LNA-K4-5p (B). 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) Knockdown of either miR-K1-5p or -K4-5p increased CASTOR1 expression in KMM but not MM cells. Untransfected cells or cells transfected with different concentrations of LNA-based NC, LNA-K1-5p, or LNA-K4-5p were examined for CASTOR1 protein (C) and mRNA (D) levels. Three independent experiments were repeated with similar results, and results from 1 representative experiment are shown. mRNA results from 3 biological replicates in D are shown as the mean ± SEM. (E) Knockdown of miR-K1-5p and -K4-5p additively increased CASTOR1 mRNA levels. Untransfected cells or cells transfected with different concentrations of LNA-based NC, LNA-K1-5p, LNA-K4-5p, or LNA-K1-5p plus LNA-K4-5p were examined for CASTOR1 mRNA expression. Three independent experiments were repeated with similar results, and results from 1 representative experiment with 3 biological replicates are shown as the mean ± SEM. Knockdown of miR-K1-5p (F) or -K4-5p (G) inhibited mTORC1 activation in KMM cells but not MM cells. Cells transfected with LNA-based NC or LNA-K1-5p (F) or LNA-K4-5p (G) were examined for p-S6K and p-4EBP1 by Western blotting. Three independent experiments were repeated with similar results, and results from 1 representative experiment are shown. For the Western blot analysis of KMM cells in G, the same set of samples were run in different gels but with the same loading calibration. 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.