Inhibition of Lung Tumor Development in ApoE Knockout Mice via Enhancement of TREM-1 Dependent NK Cell Cytotoxicity

Abstract

Apolipoprotein E (ApoE) is known to regulate lipid homeostasis and associated with atherosclerogenesis. Eventhough atherosclerogenesis is associated with tumor development, the role of ApoE in lung tumorigenesis and metastasis is not clear. Thus, the tumor growth and metastasis were compared in WT and ApoE knockout (KO) mice. Urethane-induced lung tumor incidence and B16F10 lung metastasis in ApoE knockout (KO) mice were significantly reduced in comparison to that in WT mice. Knockdown of ApoE expression in lung cancer cells and B16F10 cells also decreased cancer cell growth and metastasis. The inhibitory effect of ApoE KO on tumor development and metastasis was associated with increase of infiltration of NK cells. NK cells derived from ApoE KO mice showed much greater cytotoxicity than those from WT mice. These cytotoxic effect of NK cells derived from ApoE KO mice was associated with higher expression of Granzyme B, Fas Ligand, IFN-γ, TNF-α, NKG2D, NKp46, and DNAM-1 expression. Triggering receptor expressed on myeloid cell (TREM)-1 is a proinflammatory mediator expressed on NK cells, and is known to be associated with NK cell cytotoxicity. Thus, we investigated the role of TREM-1 on ApoE KO mice originated NK cell mediated cytotoxicity for cancer cells. Blockade of TREM-1 expression with a TREM-1 antagonist prevented NK cell-mediated cytotoxicity. TREM-1 antibody recovered cytotoxic effect of NK cells derived from KO mice of T-bet, which upregulating gene for TREM-1. These data indicate that ApoE KO suppressed lung tumor development and metastasis via increase of TREM-1-dependent anti-tumor activity of NK cells.

Keywords: NK cells; T-bet; TREM-1; apolipoprotein E; lung tumor development.

Figure 1

Effect of ApoE knockout on the lung tumor development. (A,B) Tumors were induced by a single intraperitoneal injection of 1 mg/g urethane once a week for 10 weeks (n = 6). Mice were sacrificed at 6 months after injection of the carcinogen. At the time of sacrifice, numbers of urethane-induced lung tumor were counted. (C) Lung tissues were processed and stained with H&E or analyzed by immunohistochemistry for detection of positive cells for ApoE, PCNA, and TREM-1. Scale bar, 100 μm. ^**p < 0.01.

Figure 2

Effect of ApoE knockout on B16F10 lung metastasis. (A,B) B16F10 cells were intravenously injected at mouse tail vein (4 × 10⁴ cells/mouse) (n = 6). After 21 days, mice were sacrificed, and lung metastatic nodules were visualized and counted. (C) Lung metastasis tissues were stained with haematoxylin and eosin or analyzed by immunohistochemistry for detection of positive cells for PCNA and TREM-1. Scale bar, 100 μm. ^*p < 0.05.

Figure 3

Effect of ApoE knockdown on cell growth and migration in cancer cells. (A) Lung cancer cells (A549 and NCI-H460) and B16F10 cells were plated on 96-well plates (1 × 10³ cells per well) and transfected with the negative control (NC) siRNA or ApoE siRNA (25, 50 or 100 nM) for indicated time points. Cell growth was measured by MTT assay (n = 5). (B) B16F10 cells were seeded on μ-Slide and transfected with NC siRNA or ApoE siRNA (50 or 100 nM). Cells were cultured to confluent on μ-Slide (n = 3). After silicon-wall removal, cells were allowed to migrate into cell-free zone. Cell migration was detected at various times post-silicon-wall removal by a microscopy at 100X. (C) B16F10 cells transfected with NC siRNA or ApoE siRNA (50 or 100 nM) were seeded onto transwell inserts pre-coated with collagen on the bottom side and loaded into culture well filled with growth medium containing 10% FBS as a chemoattractant (n = 3). After 18 h incubation, transwell inserts were fixed and stained by crystal violet solution. Bar graphs represent cell-migration distance or number of migrated cells. ^*p < 0.05 and ^***p < 0.01. (D) Cells were transfected with NC siRNA or ApoE siRNA (100 nM) for 24 h. Cell extracts were analyzed by Western blotting. Samples (30 μg) were resolved on SDS–PAGE and detected with specific antibodies against CDK4, CDK6, Cyclin D1, MMP-2, MMP-9 and ApoE. β-actin was used as a loading control.

Figure 4

Effect of ApoE KO on the infiltration of immune cells into urethane-induced lung tumor tissues and B16F10 lung metastasis tissues. (A,B) Representative immunohistochemical images showing MOMA2 (for monocytes/macrophages), CD8α (for cytotoxic CD8 T cells) and CD57 (for NK cells) positive cells in the urethane-induced lung tumor tissues (A) and B16F10 lung metastasis tissues (B) from WT and ApoE KO mice. Scale bar, 50 μm. (C,D) Bar graphs represent infiltrated cells in tumor area. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 5

Effect on ApoE KO on NK cell-mediated cytotoxicity. (A) NK cell cytotoxicity was determined using LDH assay co-cultured with WT or ApoE KO NK cells and B16F10 target cells for 4 h (1 × 10⁴ cells/well). (B) Perforin, Granzyme B, Fas L, TNF-α, IFN-γ, NKG2D, NKp46, and DNAM-1 mRNA expressions in WT or ApoE KO NK cells were determined by qPCR (n = 3). ^**p < 0.01 and ^***p < 0.001.

Figure 6

TREM-1 is involved in NK-cell mediated cytotoxicity. WT and ApoE KO NK (A) or T-bet KO NK (C) cell lysates were analyzed by Western blotting. NK cell cytotoxicity was determined using LDH assay co-cultured with control WT, LP-17 treated WT NK (B) or with control WT, non-treated or TREM-1 mAb treated T-bet KO NK cells (D) and B16F10 target cells for 4 h (1 × 10⁴ cells/well) (n = 3). E:T ratio is 4:1. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 7

ApoE, TREM-1, Cyclin D1, MMP-2, CD57, and T-bet expressions in the progression of lung tumors. (A) Tissue microarray analysis showing the expression of ApoE, TREM-1, Cyclin D1, MMP-2, CD57, and T-bet during lung tumor progression in normal and tumor tissue samples. Representative immunohistochemical images of each groups. (B) Bar graphs showing the ratio of ApoE, TREM-1, Cyclin D1, MMP-2, CD57, and T-bet expressions. Tissue microarray samples are contained of 10 samples from normal lung tissues and 140 samples from lung tumor tissues. ^***p < 0.001.