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Inhibition of histone deacetylase 1/2 enhanced NK cell mediated

anticancer immunity through induction of NKG2D ligands

Haeryung Cho 1 , 2 , Eun Jung Yun 1 , 2 , Jaeho Bae 1 , 2

Department of Biochemistry, PNU GRAND Convergence Medical Science Education Research Center, Pusan National
University School of Medicine, Yangsan 50612, Korea

Abstract Introduction
Histone acetylation is an epigenetic mechanism that Lung cancer is one of the most common cancers and
regulates the expression of various genes such as the due to a high relapse rate and distant metastasis has
natural killer group 2, member D (NKG2D) ligand, and the highest mortality cancer (1). The important factors
reduced by overpressure in cancer cells. NKG2D for survival of lung cancer patients is the host's
ligand is a molecule that activates NKG2D receptor immunity against cancer and the prognosis of lung
expression in immune cells and is overexpressed by cancer patients can be improved by enhancing the
histone deacetylases (HDACs) inhibitors. It is not activity of immune cells (2). NKG2D that expressed on Figure 3. Silencing HDAC1 or HDAC2 using specific siRNA. The
known which type of HDAC enzyme in humans human NK cells and some T cells is an active receptor (A) NCI-H23, (B) A549 were incubated for 48 hours after
transfection with 150 nM siH1 (siRNA targeting HDAC1), siH2
involved in NKG2D ligand expression. Observing the that binds to the NKG2D ligand of cancer cells and (siRNA targeting HDAC2), and scRNA. The levels of HDAC1 and
expression of NKG2D ligand and major transmits an activation signal to immune cells (3). The HDAC2 expression were analyzed by Western blotting using anti-
histocompatibility complex (MHC) class I molecules in expression of the NKG2D ligand is increased in HDAC1 and anti-HDAC2 monoclonal antibodies. β-actin was used
lung cancer cells treated with a few HDAC inhibitors infected and transformed cells, rarely in normal cells as the loading control.
and small interfering RNAs (siRNA), we found HDAC (4,5). (In order) To increase the level of NKG2D ligands
genes that affect NKG2D ligand expression. When adequate for NK cells activation for therapeutic
HDAC1/2 inhibitor was used, it was found that the strategies, it is necessary to understand the
induction of NKG2D ligand expression and regulatory mechanism of NKG2D ligands. Histone (A) (B)
susceptibility to NK cells were increased and deacetylation is one of the mechanisms regulating the
HDAC1/2 was silenced, the expression of NKG2D gene transcription of NKG2D ligands and an
ligand was increased. This study suggests that epigenetic process that affects gene transcription and
HDAC1/2 is involved in NKG2D ligand expression and has been associated with NKG2D ligand transcription,
NK cell-mediated anticancer immunity. but what type of histone deacetylases are involved is
unknown (6). This study will give better understanding
of HDAC inhibitors by identifying the HDAC types that
play a role in regulating the expression of NKG2D
ligands and by confirming whether the selective
inhibitors have NK cell cytotoxic effects on lung
cancer cells (7). (C) (D)
Results
(A) (B) (C)

Figure 4. Surface expression of NKG2D ligands after HDAC1 or
HDAC2 silencing. Surface protein expression of NKG2D ligands
was analyzed using flow cytometry 48 hours after transfecting
NCI-H23 (A,B) and A549 (C,D) with siH1 or siH2. The solid gray,
dotted, and black lines represent isotype control, scRNA, and
siRNA, respectively.
(D) (E) (F)

Figure 1. Altered transcription of NKG2D ligands after treatment with HDAC inhibitors. The transcription of NKG2D ligands was analyzed in
lung cancer cells using multiplex RT-PCR. The NCI-H23 cells were each treated with 1, 10, 100, and 1000 nM of the following selective
HDAC inhibitors: (A) FK228, (B) RGFP966, (C) MC1568, (D) Tubacin, and (E) PCI34051 for 18 hours. (F) The A549 were treated with FK228 Figure 5. Selective cell death by NK cell cytotoxicity after
for 18 hours. Changes in transcription were normalized to the β-actin (ACTB) and presented as the mean fold change in comparison to the treatment with FK228. NCI-H23 were treated with FK228 for 24
controls.
hours at 100 nM and then stained with CFSE. The target cells
were co-cultured with NK-92 for 4 hours at effector-to-target
ratios of 1:1 and 5:1. Flow cytometry was used to measure the
proportion of PI-positive cells, which indicated NK cell-mediated
lysis.
(A) (B) (C)
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Figure 2. Treatment with HDAC inhibitors alters the surface expression of NKG2D ligands. Histograms show the surface expression of Acknowledgements
NKG2D ligands in NCI-H23 that were treated for 24 hours with 100 nM of (A) FK228, (B) RGFP966, (C) MC1568, (D) Tubacin, (E) PCI34051. This study was supported by the National Research Foundation of Korea
(F) The A549 were treated with 100 nM FK228 for 24 hours. Solid gray, dotted, and black lines represent the isotype control, media-treated (NRF) grant (No. NRF-2018R1D1A1B07047437) and the National Research
control, and HDAC inhibitor-treated samples, respectively. Foundation of Korea (DIRAMS) grant by the Korean government (MSIP)
(50593-2021) of Korea.