Page 31 - H. Cell signaling

Role of mTORC2 in NK cell‐mediated cytotoxicity

against tumor cells

Jaeho Cho, Mirae Kim,Gyu Tae Park, and Sung Su Yea

Dept. of Biochemistry, College of Medicine, Inje University, Korea

BACKGROUND AIM

Phosphoinositide 3-kinase (PI3K) • To assess how different classes of PI3K inhibitors and mTORC
• Lipid kinase family: 3'-hydroxy group of PtdIns and phosphoinositides inhibitors affect the function of NK cells, a key cell type for
• Mammalian PI3K: Class I, II, III; class I PI3Ks: generation of immunosurveillance and tumor immunotherapy
PtdIns(3,4,5)P 3 (PIP 3 ) • To assess how a promising new class of targeted agents impacts
• PIP 3 production: recruitment of protein effectors containing PH domain NK cell function.
• PI3K signaling is implicated in a variety of human diseases including
cancer.
Mechanistic target of rapamycin (mTOR) METHODS
• Complex and two type: mTORC1, mTORC2
• mTORC1 consists of Raptor and other factors(mTOR, mLST8, deptor, • Cells: Primary NK cells from C57BL/6 mice and NKL cell line were
PRAS40), mTORC2 is complex of Rictor and others(mTOR, mLST8, cultured in IL-2 and used.
mSIN1). • Cell-mediated cytotoxicity (CMC): CMC was determined by
Natural Killer (NK) cells calcein release assay with the NK cells and calcein AM-labeled
• Lymphocytes of the innate immune system that can kill tumor cells. target cells at the indicated effector:target (E:T) ratios.
• Critical role in eliminating tumors and virally infected cells by induction • Gene knock-down: NKL cells were transfected with Rictor siRNA,
of cytotoxicity and production of cytokines AKT siRNA, or control siRNA.
• Effector function is controlled by opposing signals from activating and • Western blot analysis: The whole-cell lysates were prepared and
inhibitory receptors analyzed for phosphorylation of indicated kinases.
RESULTS

Inhibitors of all class I PI3K isoforms significantly impaired cell-mediated cytotoxicity (CMC) of NK cells against tumor cells, whereas isoform-
selective inhibitors had little effect. CMC of NK cells against tumor cells was down-regulated by PP242, an inhibitor for both complex of mTOR,
but not by rapamycin inhibiting mTORC1 only. To study the role of mTORC2 on NK CMC, we evaluated the effect of knocking down RICTOR, a
critical factor for mTORC2. RICTOR knocked down found to inhibit NK CMC against tumor cells. In addition, regulation of AKT, a downstream
kinase of mTORC2, using siRNA as well as selective inhibitor also resulted in down-regulation of NK CMC against tumor cells.

Figure 1. Effect of PI3K and mTORC isoform-selective Figure 2. Effect of AKT and MAPKs on Figure 3. Effect of PI3K and mTORC isoform-selective
inhibitors on cell-mediated cytotoxicity in mouse NK cells. NK cell-mediated cytotoxicity in mouse NK inhibitors on the phosphorylation of AKT and ERK1/2 in
cells from C57BL/6 mice were purified from spleen and expanded cells. YAC-1 tumor cells were labeled with anti-NKG2D-stimulated mouse NK cells. NK cells from
for 8-10 days in IL-2. Cytotoxicity was determined by calcein calcein AM and co-cultured with NK cells at C57BL/6 mice were purified from spleen and expanded for 8-
release assay with the NK cells and calcein AM-labeled YAC1 the indicated E:T ratios in the presence of 10 days in IL-2. The NK cells were stimulated with plate-
target tumor cells at the indicated effector:target (E:T) ratios in the PD98059, SB203580, SP600125, and AKTi, bound anti-NKG2D mAb in the presence of indicated
presence of indicated inhibitors for 2h. The values are presented a specific inhibitor of ERK1/2, p38, JNK, and inhibitors for 5min. The whole-cell lysates were prepared and
as the mean±S.D. from three independent experiments. *p<0.05, AKT, respectively, for 2h. The values are analyzed for phosphorylation of AKT and ERK1/2 by
**p<0.01,***p<0.001, as determined by unpaired Student’s t test presented as the mean±S.E. from three Western blot analysis. Data presented are from three
and one-way ANOVA compared to the vehicle control group. independent experiments. independent experiments.
A. B. A. B.

Figure 5. Effect of mTORC2 on cell-
mediated cytotoxicity in human NK cells. Figure 6. Effect of AKT on cell-mediated cytotoxicity in
NKL human NK cells were transfected with human NK cells. NKL human NK cells were transfected with
Figure 4. Effect of PI3K and mTORC inhibitors on cell- Rictor siRNA or control siRNA. (A) The whole AKT siRNA or control siRNA. (A) The whole cell lysates from
mediated cytotoxicity in human NK cells. NKL human NK cells cell lysates were prepared and analyzed for AKT knocked down NKL cells were prepared and analyzed
were cultured in IL-2. Cytotoxicity was determined by calcein Rictor by Western blot analysis. (B) The Rictor for AKT and ERK1/2 by Western blot analysis. (B)
release assay with the NKL cells and calcein AM-labeled K562 knocked down NKL cells were cocultured with Cytotoxicity was determined by calcein release assay with
target tumor cells at the indicated effector:target (E:T) ratios in the calcein AM-labeled K562 cells ate 10:1 E:T the AKT knocked down NKL cells and calcein AM-labeled
presence of indicated inhibitors for 2h. The values are presented ratio. Data presented are from three K562 target tumor cells at 10:1 E:T ratio. Data presented are
as the mean±S.D. from three independent experiments. independent experiments. from three independent experiments.
CONCLUSION ACKNOWLEDGEMENTS Contact information

These results reveal the mTORC2 is important for This work was supported by the National
NK cell functions and indicate that isoform- Research Foundation of Korea (NRF) grant Sung Su Yea, Ph.D.
selective regulation would provide a better funded by the Korea government (MSIT) (grant
therapeutic window for specific disease states than number 2019R1F1A1062343). Tel: 051-890-6892
achieved by targeting all isoforms. E-mail: ssyea@inje.ac.kr