Immunotherapy for hepatoma using a dual-function vector ... · 4/10/2014 · NK, CD4+T, CD8+T...

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Immunotherapy for hepatoma using a dual-function vector with both

immunostimulatory and Pim-3-silencing effects

Qie Guo1,3, Peixiang Lan1, Xin Yu1, Qiuju Han1, Jian Zhang1, Zhigang Tian1,2, Cai Zhang1

1Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong

University, Jinan 250012, Shandong, China

2Department of Microbiology and Immunology, School of Life Sciences, University of Science and

Technology of China, Hefei 230027, Anhui, China

3Department of Pharmacy, The Affiliated Hospital of Medical College, Qingdao University, Qingdao

266000, Shandong, China

Corresponding author: Cai Zhang, Institute of Immunopharmacology & Immunotherapy, School of

Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan 250012, China. Tel &

Fax: 86-531-8838-3782; E-mail: [email protected]

Running title: Immunotherapy for hepatoma using a dual-function vector

Key words: immunotherapy; hepatoma; Pim-3; TLR7; dual-function vector

Financial support: C. Zhang was supported by grants from the National 973 Basic Research Program

of China (#2013CB944901), the Natural Science Foundation of China (#81273220, # 31200651), and

the Young and Middle-aged Scientist Award of Shandong Province (# BS2010YY033).

The authors disclose no potential conflicts of interest

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on October 11, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 10, 2014; DOI: 10.1158/1535-7163.MCT-13-0722

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6 figures (Fig. 1-Fig. 6); 2 supplementary Tables (Table S1-S2) and 4 supplementary figures (Fig.

S1-S4)




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Abstract

Tumorigenesis is an immortalization process where the growth of normal cells uncontrolled

and programmed cell death is suppressed. Molecular biological and immunological studies have

revealed that the aberrant expression of some pro-oncogene boosts proliferation and inhibits apoptosis,

which is vital for the tumor development. The hypofunction of the host immune system also drives the

development and metastasis of malignant tumors. Pim-3, a member of the Pim family, is aberrantly

expressed in several cancers. Data suggest that Pim-3 inhibits apoptosis by phosphorylating the

proapoptotic BH3-only protein, Bad. Here, we constructed a dual-function small hairpin RNA

(shRNA) vector containing an shRNA targeting Pim-3 and a TLR7-stimulating ssRNA. Stimulation

with this bi-functional vector in vitro promoted significant apoptosis of Hepa1-6 cells by regulating

the expression of apoptosis-related proteins and induced secretion of type I IFNs. Most importantly,

this bi-functional vector more effectively inhibited subcutaneous Hepa1-6 cell growth than did single

shRNA and ssRNA treatment in vivo. NK, CD4+T, CD8+T cells and macrophages were required for

effective tumor suppression, and CD4+T cells were shown to play a helper role in the activation of NK

cells, possibly by regulating the secretion of Th1 or Th2 cytokines. This ssRNA-shRNA bi-functional

vector may represent a promising approach for tumor therapy.




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Introduction

Accumulated genetic and epigenetic changes that alter the proliferation and survival pathways

of normal cells have resulted in cellular transformation and progressive tumor growth (1). Evasion of

apoptosis and self-sufficiency in growth signals are essential for malignant growth. The

proto-oncogene family Provirus integrating site Moloney murine leukemia virus (Pim) is a highly

conserved serine/threonine kinase family that has been implicated in cancer progression (2,3). Three

Pim kinases (Pim-1, -2 and -3) have been identified. Pim-1 and Pim-2 induce cell cycle progression in

cooperation with c-Myc, acting as inhibitors of apoptosis in hematological malignancies and some

solid tumors (2,3). The newest member of the family, Pim-3, is aberrantly expressed in several

cancers, particularly those of endoderm-derived organs, including liver, pancreas, colon and stomach

(4-7). Data suggest that Pim-3 inhibits apoptosis by phosphorylating and inactivating the proapoptotic

BH3-only protein Bad (4-6). Pim-3 mRNA and protein were detected in human hepatocellular

carcinoma (HCC) tissues and cell lines, but not in normal hepatocytes and liver tissues. Silencing of

Pim-3 by RNA interference inhibited growth and enhanced apoptosis in hepatoma cells (7). Thus,

Pim-3 kinase may be a candidate molecular target for cancer therapy.

Tumor pathogenesis also involves a process called cancer immunoediting, a temporal transition

from immune-mediated tumor elimination in early phases of tumor development to immune escape of

established tumors (8). The ability to evade immune recognition and to suppress immune reactivity are

the main methods whereby cancers evade immune destruction (9,10). Host immunosuppression,

mediated by tumor cells, is characterized by incompetence of cytotoxic T lymphocytes (CTLs),

massive secretion of suppressing cytokines (such as IL-10 and TGF-β) and activation of Treg cells,

leading to functional deficiencies in CTLs, CD4+ Th1 cells or natural killer (NK) cells (9-11). Thus,

tumor therapy must restimulate the immune response, in addition to suppressing oncogene expression.

Toll-like receptors (TLRs) are pattern recognition receptors that trigger the innate immune

response and prime the antigen-specific adaptive immune response by recognizing conserved

structures in pathogens. TLRs are important in protective immunity against cancer and infection (12).

TLRs are expressed by immune and non-immune cells, and their ligands represent promising immune




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stimulators that could stimulate both innate and adaptive immune cells (12). Interferons (IFNs)

secretion following TLR-mediated activation of IFN-regulatory factors (IRFs) is regarded as the

central coordinator of immune revival (13). TLR7 or TLR8, expressed in endosomes, recognizes

natural nucleoside structures, such as viral single-stranded RNA (ssRNA) and synthetic compounds,

e.g. imidazoquinolines (14,15). U- or GU-rich ssRNAs, such as ssRNA40 derived from HIV-1, are

potent TLR7 activators (12,14,16). Binding of TLR7 with its agonists triggers a signaling cascade,

which comprises recruitment of MyD88, activation of the NF-κB and IRF7 pathway, and production

of type I IFN and inflammatory cytokines. TLR7 stimulation can prime activation of NK and T cells

directly or with the help of activated antigen presenting cells (APCs) and exhibit anti-tumor immune

responses (16,17).

Here, we constructed a dual-function small hairpin RNA (shRNA) vector containing an shRNA

targeting Pim-3 and a TLR7-stimulating ssRNA. Stimulation with this bi-functional vector in vitro

promoted significant apoptosis of Hepa1-6 cells and induced secretion of type I IFNs. Importantly, the

vector more effectively inhibited subcutaneous Hepa1-6 cell growth than did single shRNA and

ssRNA treatment in vivo. NK, CD4+, CD8+ T cells and macrophages were required for effective tumor

suppression. CD4+T cells were shown play a helper role in activating NK cells. The bi-functional

vector may represent a promising approach for tumor therapy.

Materials and Methods

Plasmid construction and lentiviral packaging

Transcription of each shRNA oligonucleotide targeting Pim-3 (sense-loop-antisense) was designed as

a synthetic duplex with overhanging ends identical to those created by restriction enzyme digestion

(BamH I at the 5' and EcoR I at the 3'), and was cloned into vector pTZU6+1 vector which contains a

U6 polymerase-III (pol-III) promoter. The shRNA template sequences are shown in Table S1.

Transcription of each ssRNA oligonucleotide synthetic duplex sequence (sense-terminator) was

designed using a similar overhanging ends procedure to the shRNA, and cloned into expression vector

pSIREN, which contains a U6 pol-III promoter. ssRNA template sequences are shown in Table S1. To




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create the dual functional vector, the U6+shRNA in pTZU6+1-shRNA was digested by Hind III and

EcoR I, and inserted to pSIREN-ssRNA.

pSIREN-control, ssRNA, shRNA, or dual vectors were cloned into a lentiviral pGCSIL-GFP

plasmid, and transfected into 293T cells. 48 h later, culture supernatant was collected and filtered

through a 0.45 μm filter. Viruses (LV-ctrl, LV-ssRNA, LV-shRNA, LV-dual) were harvested by

centrifugation at 70,000 × g at 4°C for 2 h. Harvested viruses were aliquoted and stored at -80°C.

Cell culture

Mouse hepatoma cell lines Hepa1-6 and H22 (Cell Bank of the Chinese Academy of Sciences,

Shanghai, China) and normal mouse hepatocyte cell line BNL.CL2 (American type culture collection,

ATCC) were maintained in DMEM medium (GIBCO/BRL, Grand Island, NY, USA) supplemented

with 10% heat-inactivated fetal bovine serum. These cell lines were used within 6 months of receipt.

Cells were never used above 10 passages and were cultured at 37°C in a humidified atmosphere with

5% CO2.

Animals, tumor challenge and treatment

C57BL/6 mice (6-8 weeks old; Experimental Animal Center of Beijing University, Beijing,

China) were maintained under specific pathogen-free (SPF) conditions. The Committee on the Ethics

of Animal Experiments of the Shandong University approved all the animal studies.

1 × 106 Hepa1-6 cells were injected subcutaneously into the right flank of C57BL/6 mice. After 2

weeks, LV-ctrl, LV-ssRNA, LV-shRNA, LV-dual (MOI = 50) were administered intra-tumorally once a

week for two weeks. After another two weeks, the mice were sacrificed and the tumor volume was

calculated by length × width2/2.

Human samples

HCC and non-tumor liver tissue samples were obtained from the Shandong Provincial Hospital (Jinan,

China) under the National Regulation of Clinical Sampling in China. Both were immediately fresh




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frozen and stored at -80° for further use in Real-Time PCR and western blotting assays.

Semi-quantitative RT-PCR and Real-Time PCR analysis

Total RNA was extracted by the TRIzol Regent (Invitrogen, Carlsbad, CA, USA) and cDNAs

were synthesized using Superscript III Reverse Transcriptase (Invitrogen), followed by Real-Time

PCR and semi-quantitative RT-PCR analysis. For semi-quantitative RT-PCR, cDNA was amplified

using pairs of primers (RiboBio, GuangZhou, China) that specifically amplify Pim-3 or other genes,

according to the manufacturer’s protocol. For Real-Time PCR analysis, cDNA was amplified with the

assistance of SYBR green (Bio-Rad, USA). Relative gene expression was determined in comparison

with that of GAPDH or β-actin. PCR primers are provided in Table S2.

Western blotting

Cells were collected and lysed on ice using a Total Protein Extraction Reagent (Beyotime,

Jiangsu, China). The protein samples (30 μg/lane) were separated by sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane

(Millipore, Bedford, MA, USA). The membrane was blocked in Tris-buffered saline with 5% (w/v)

non-fat dry milk, and then incubated with primary antibodies over night at 4°C, followed by

incubation with horseradish peroxidase-conjugated secondary antibody for 50 min at room

temperature. Immunoreactive proteins were visualized using Molecular Imager ChemiDoc XRS

System BioradChemiDoc XRS (Bio-Rad). Rabbit anti-human pim-3, anti-mouse pim-3, anti-mouse

Bad, anti-mouse p-Bad, anti-mouse NF-κB, and anti-mouse p-NFκB mAbs were purchased from Cell

Signaling Technology (New England BioLabs Inc., USA). Rabbit anti-mouse p-PKR, anti-mouse

IκB-α, anti-mouse bcl-2, anti-mouse bcl-XL, anti-mouse Bim, and anti-mouse TLR7 mAbs were

obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

NK cytotoxicity assays

The ability of spleen lymphocytes to kill Hepa1-6 cells was evaluated by CFSE/7-AAD flow




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cytometry assay, as previously described (18). Briefly, Hepa1-6 cells were incubated with 1 ml of

CFSE (2 mM) (Molecular Probes, Eugene, OR, USA) for 10 min at 37 °C and then washed. Spleen

lymphocytes were isolated and added to the target cells at effector/target ratios of 50:1, 25:1 and 12.5:1,

respectively, for 4 h. Following a further wash, cells were labeled for 15 min with 7-AAD (optimized at

0.25 μg/ml, Sigma-Aldrich) to identify dead cells. The cells were then analyzed via flow cytometer

(FACScalibur, USA). Cytotoxicity was calculated as follows: % lysis = (CFSE/7-AAD double positive

cells /CFSE positive cells) × 100%.

Measurement of apoptosis

Staining for AnnexinV-FITC/PI (BestBio, Shanghai, China) via flow cytometry was used to

detect apoptosis of tumor cells . The percentage of cells that were Annexin V-positive represented the

proportion of apoptotic cells. Alternatively, apoptosis was also measured by TUNEL staining using a

One Step TUNEL Apoptosis Assay Kit (Beyotime, China). Nuclear staining was evaluated under a

light microscope via DAPI staining (Beyotime). A commercial enzyme-linked immunosorbent assay

(ELISA) kit (KeyingMei, Beijing, China) detected the level of caspase-8 in cell lysates, according to

the manufacturer’s instruction.

ELISA for cytokine detection

The levels of cytokines (IFN-α, IFN-β, IFN-γ, TNF-α, IL-4, and IL-10) in culture supernatants

from Hepa1-6 cells and in the serum of tumor-bearing mice were detected by ELISA kits

(ExCellBiology, Shanghai, China).

Flow Cytometry analysis

Splenic lymphocytes were isolated to analyze the percentages and activation of NK cells and T

cells after the treatment with shRNA, ssRNA and dual vectors. The expression of NKG2D, NKG2A,

and PD-1 on lymphocytes and NKG2D ligands on Hepa1-6 cells was also detected. Cells were

harvested, blocked with anti-FcγR mAb, and stained with labeled mAbs at 4°C for 45 min. For




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intracellular IFN-γ staining, splenic cells were cultured in RPMI 1640 containing 10% FCS, and

treated with monensin (Sigma, St. Louis, MO, USA) for 4 h to inhibit extracellular secretion of

cytokines. The antibodies used were: FITC-conjugated NK1.1, PerCP-Cy5.5-conjugated CD3e (BD

Biosciences, San Diego, CA, USA); FITC-conjugated CD4, PerCP-Cy5.5-conjugated CD8,

PE-conjugated CD69, APC-conjugated NKG2D and PE-conjugated NKG2A (eBiosciences, San

Diego, CA); and Carboxyfluorescein-conjugated RAE-1, FITC-conjugated MULT-1, PE-conjugated

H-60 and PE-conjugated IFN-γ mAb (R&D Systems, Minneapolis, MN, USA). All stained cells were

analyzed using a flow cytometer, and the data were processed with WinMDI 2.9 software (Scripps

Research Institute, La Jolla, CA, USA).

Lymphocyte depletion and TLR7 inhibition

Cell depletion mAbs were purified from PK136 (α-NK1.1), GK1.5 (α-CD4) and 2.43 (α-CD8α)

hybridoma cell lines. To deplete cells, tumor-bearing mice were injected intraperitoneally with 1 mg

of mAb for 3 days (19). To deplete macrophages, 1 mg liposomes containing DMDP

(dichloromethylene diphosphonate, Sigma) was administered i.p. into C57BL/6 48 h before treatment

(20). The LV-dual vector (MOI = 50) was then administered intra-tumorally once a week for two

weeks. To ablate the function of TLR7, IRS661 (5'-TGCTTGCAAGCTTGCAAGCA-3') (TAKARA,

Otsu, Shiga, Japan), a decoy analog that interferes with the combination of TLR7 and ssRNA (21,22),

was administered i.v. before LV-dual vector treatment.

Histochemical analysis

Tumor tissues were excised and fixed in 10 % neutral buffered formalin; embedded in paraffin;

sectioned and stained with hematoxylin and eosin to assess morphological changes and lymphocyte

infiltration.

Statistical analyses

Statistical analysis was performed using a paired Student’s t-test and Mann–Whitney U-test. P




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values < 0.05 were considered significant.

Results

Pim-3 is aberrantly expressed in mouse hepatoma cell lines and human hepatocellular

carcinoma tissues. Firstly, we detected the expression of Pim-3 in mouse hepatoma cell lines Hepa1-6

and H22, and in the normal hepatocyte cell line BNL.CL2. Pim-3 was highly expressed in Hepa1-6

and H22 cell lines, but was weakly expressed in normal hepatocytes (Fig. S1A). Western blotting also

showed that Pim-3 expression was higher in hepatoma Hapa1-6 and H22 cell lines, and low in

BNL.CL2 cells (Fig. 1B). Pim-3 expression in human primary hepatocellular carcinoma cells was

significantly higher in tumor tissues than in non-tumor tissues at both the mRNA and protein levels

(Fig. S1C and S1D). These results suggested a critical role of Pim-3 in tumorigenesis and

pathogenesis of liver cancer.

Construction of a dual function vector with both immunostimulatory ssRNA and

Pim-3-silencing shRNA. To clarify the role of Pim-3 in cell growth and apoptosis of hepatomas and

to stimulate an immune response and silence Pim-3 expression simultaneously, we constructed a dual

function vector containing an immunostimulatory ssRNA and a Pim-3-gene–silencing shRNA (Fig.

1A). We designed four siRNA duplexes to target the open reading frame encoding of Pim-3 mRNA by

employing BLOCK-iTTM RNAi Designer. The annealed siRNA oligonucleotides were knocked into

the expressing vector pTZU6+1 to construct short-hairpin RNAs (shRNAs). Four different ssRNA

oligonucleotides were designed and inserted into pSIREN plasmid (ssRNAs). The shRNA and ssRNA

vectors were transfected into Hepa1-6 cells separately to test the Pim-3 silencing and

immunostimulatory effect. The shRNA oligonucleotide with most effective silencing effect was

selected and inserted into the pSIREN plasmid containing the most potent ssRNA oligonucleotide to

form a bi-functional vector (Fig. 1A).

We then confirmed the ability of the bi-functional vector to downregulate Pim-3 expression. Fig.

2B shows that Pim-3 mRNA and protein levels were downregulated after transfection with both




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shRNA and the bi-functional vector compared with the pSIREN or ssRNA transfection group (Fig.

1B). The ssRNA is regarded as a ligand of TLR7, and ssRNA stimulation can activate TLR7 signal

pathway, leading to the production of type I IFN and inflammatory factors (14-17); therefore, we

detected whether the bi-functional vector could stimulate the TLR7 pathway by measuring the

production of IFN-α and IFN-β. The mRNA of IFN-α and IFN-β in Hepa1-6 cells was induced and

the concentration of IFN-α and IFN-β in the supernatants increased after transfection with the ssRNA

and bi-functional vector (Fig. 1C and 1D). Collectively, these results indicted a successfully

constructed dual function ssRNA-shRNA vector with both Pim-3 silencing and innate immune

stimulation effects.

The dual function vector transfection induces the apoptosis of murine hepatoma Hepa1-6 cells.

Pim-3 is reported to inhibit the apoptosis of tumor cells by phosphorylating and inactivating

pro-apoptotic protein Bad (4,7). Silencing of Pim-3 significantly promoted apoptosis of Hepa1-6 cells,

as detected by Annexin V/PI double staining (Fig. 2A). As depicted in Fig. 2B, the apoptotic cells

were induced markedly after transfection with both shRNA and dually function vector as shown by in

vitro TUNEL staining. Nuclear DAP I-staining also showed obvious shrinking of nuclei and

nucleosomes after transfection with the shRNA or bi-functional vector (Fig. 2B, lower). Importantly,

the dual function vector showed a more significant pro-apoptotic effect than the shRNA vector.

We also observed that the shRNA and bi-functional vector significantly downregulated the

mRNA and protein levels of anti-apoptotic genes Bcl-XL and Bcl-2 ( Fig. S2A). The expression of

Bim, another member of Bcl-2 family, did not change. Bad plays a crucial role in promoting cell

apoptosis, probably by causing the release of cytochrome C from mitochondria and the disintegration

of membranes (4,23). Phosphorylated Bad (p-Bad) represents an inactive form of Bad, which is

induced by proto-oncogenes and exploited by tumor cells to avoid apoptosis (4,24). Silencing of

Pim-3 by the shRNA and bi-functional vector markedly suppressed the phosphorylation of Bad, but

did not alter the protein abundance of total Bad (Fig. S2B). These results suggested that ablation of

endogenous Pim-3 increased apoptosis by downregulating the expression of Bcl-XL and Bcl-2 and by




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blocking the phosphorylation of Bad. In Hepa1-6 cells, the activity of caspase-8 (an apoptosis

activator) was enhanced by both shRNA and dual function transfection at 24 h and 36 h, suggesting

that the apoptotic-related pathway was activated (Fig. S2C). These results suggest that transfection

with the Pim-3 shRNA and dual function vector promotes apoptosis of hepatoma cells by silencing

Pim-3, suppressing the phosphorylation of Bad and downregulating Bcl-XL and Bcl-2, resulting in the

activation of the apoptosis-related signal pathway.

Treatment with bi-functional vector inhibits subcutaneous tumor growth of hepa1-6 in vivo. To

explore the anti-tumor effect of the dual function vector in vivo, C57BL/6 mice were administered

subcutaneously with 1 × 106 Hepa1-6 cells. After 2 weeks of tumor challenge, LV-ctrl, LV-ssRNA,

LV-shRNA, and LV-dual (MOI = 50) were administered separately via intra-tumoral injection once a

week for two weeks. Tumor volume was calculated at 4 weeks. Treatment with ssRNA, shRNA and

dual function LV-vector significantly suppressed tumor growth (Fig. 3A), with the dual vector

treatment displaying the most significant inhibition (Fig. 3B). Tumor weight showed a similar trend

(data not shown). These results indicated that silencing of Pim-3 and stimulation of the immune

response contribute to the antitumor activity of the bi-functional vector in vivo.

Both NK and T cells are required for suppressing the growth of hepa1-6 by the bi-functional

vector. The more efficient tumor inhibition of the bi-functional vector suggested that the

immunostimulatory effect exerted by the ssRNA plays important role in suppressing tumor growth. To

explore the mechanism of suppression of tumor growth, particularly the mechanism of immune

stimulation, we observed the activation of immune responses induced by the bi-functional vector in

C57BL/6 mice. The proportion and activation (CD69+) of splenic NK and CD4+ T cells, but not

CD8+T cells, increased significantly in both LV-ssRNA- and LV-dual-treated mice (Fig. 3C).

Meanwhile, lymphocyte infiltration was observed in tumor tissue of both LV-ssRNA and LV-dual

treatment groups, and tumor necrosis was found in LV-ssRNA, LV-shRNA and LV-dual treatment

groups (Fig. S3A). Tumor apoptosis was also significantly higher in LV-dual-treated mice (Fig. S3B),




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and the serum levels of IFN-α and IFN-β were higher in LV-dual therapy group, than in the shRNA

and control groups (Fig. S3C).

To further investigate which immune cells are involved in the anti-tumor responses, we depleted

of NK, CD4+T, and CD8+T cells using depletion mAb, separately, by i.p. for 3 days after solid tumors

were established. LV-coated vectors were then administered to tumor-bearing mouse via intratumoral

injection. Depletion of NK, CD4+ and CD8+T cells significantly attenuated the dual function

vector-induced tumor inhibition (Fig. 3D). Depletion of NK and CD4+T cell exhibited a more obvious

effect. Thus, NK, CD4+T and CD8+T cells are all required for bi-functional vector-mediated growth

suppression of hepa1-6 cells. We also observed the role of APCs in tumor growth by depleting

macrophages using 1 mg of liposomes containing DMDP i.p. into tumor-bearing mouse before dual

functional vector treatment. Depletion of macrophages also significantly impaired dual function

vector-induced tumor suppression (Fig. 3D).

To explore the exact mechanism of NK cells in the inhibition of the growth of Hepa1-6 during

dual function vector administration, we isolated splenic NK cells from tumor-bearing mice treated

with indicated vectors and tested their ability to kill Hepa1-6 targets. The cytotoxicity of NK cells

from mice treated with both LV-ssRNA and LV-dual was higher than that from LV-ctrl-treated mice,

with highest cytolysis observed in the LV-dual group (Fig. 4A). We further examined the expression of

NK cell receptors NKG2D and NKG2A, the co-inhibitory receptor PD-1 and intracellular IFN-γ by

FACS. The expressions of activating receptor NKG2D and IFN-γ markedly increased, while the

inhibitory receptor NKG2A and PD-1 were suppressed in both ssRNA and dual-treated groups, with

larger changes in dually function vector treatment group (Fig. 4B). We detected the expression of

NKG2D ligands MULT-1, RAE-1 and H-60 on hepa1-6 cells via FACS. MULT-1 and H-60

expressions were upregulated after transfection with both ssRNA and bi-functional vectors in vitro

(Fig. 4C). However, RAE-1 was not detected on hepa1-6 cells (data not shown). To further determine

whether the enhanced cytolytic capacity of NK cells induced by ssRNA and dual vector was mediated

by NKG2D and its ligands, we blocked the interaction of NKG2D and its ligands with neutralizing

anti-NKG2D antibody before detecting NK cell lysis. NKG2D blockade significantly attenuated the




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cytotoxicity of NK cells against Hepa1-6 cells (Fig. 4D). These results showed that treatment with

ssRNA and bi-functional vector induced NKG2D expression and IFN-γ production, while reducing the

expression of NKG2A and PD-1, which promoted NK cell activation. ssRNA and bi-functional vector

treatment also augmented the expression of NKG2D ligands, and the interaction of NKG2D and its

ligands contributed to the enhanced NK lysis.

CD4+ T cells are involved in the activation of NK cells.

CD4+T cell depletion also impaired tumor inhibition, suggesting that CD4+T cells play an important

role in the suppression of tumor growth mediated by bi-functional vector (Fig. 3C-D). ssRNA and

bi-functional vector administration downregulated the expression of PD-1 on CD4+ T cells, suggesting

the reverse of CD4+ T cell inhibition in tumor-bearing mice (Fig. 5A). The secretion of Th1 and Th2

cytokine in serum, represented by IFN-γ, TNF-α and IL-4, IL-10 respectively, were examined. Both

ssRNA and dual vector administration promoted the production of Th1-type cytokine IFN-γ and

TNF-α, but reduced the secretion of Th2-type cytokine IL-4 and IL-10 (Fig. 5B). We hypothesized

that treatment with ssRNA and bi-functional vector promoted CD4+T cell proliferation and activation

(Fig. 3C), and shifted the balance of Th1 and Th2 cytokine secretion by CD4+ T cells towards Th1,

which contributed to the anti-tumor immune response.

CD4+T cells provide help for NK cell activation (25,26); therefore, we determined whether

depletion of CD4+T cells impaired the activation and function of NK cells. CD4+T cell elimination did

not influence the proportion of splenic NK cells; however, NK cell activation was inhibited

significantly in the bifunctional vector treatment group (Fig. 5C). Moreover, the cytotoxicity of

splenic NK cells was significantly reduced after CD4+T cells were depleted (Fig. 5D). Thus CD4+T

cells provide a necessary help for NK cell activation and play important role in the anti-tumor effect

exerted by the bi-functional vector.

Activation of the TLR7 signal pathway is important for the anti-tumor effect mediated by

the bi-functional vector. TLR7 and TLR8, also known as ‘nucleic acid-sensing TLRs’, were




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originally identified as recognizing imidazoquinoline derivatives such as imiquimod, resiquimod

(R-848), and guanine analogs such as loxoribine (which have antiviral and antitumor properties) (17).

TLR7 also recognizes ssRNA derived from RNA viruses, such as vesicular stomatitis virus, influenza

A virus and human immunodeficiency virus (27). To further determine the mechanism whereby

ssRNA recognition leads to increased NK and CD4+ T cell activation during dual vector treatment, we

evaluated TLR7 and TLR8 expression in Hepa1-6 cells after dual vector transfection. The gene

expression of TLR7 and TLR8, but not TLR3, in Hepa1-6 cells was significantly induced after

transfection with ssRNA and dual function vectors (Fig. 6A). The protein level of TLR7 also increased

(Fig. 6B). Both vectors also promoted phosphorylation of NF-κB and degradation of IκB-α, indicating

the activation of the TLR7 signal pathway. Interestingly, the phosphorylation of another

pattern-recognition receptor, PKR, was also upregulated after transfection with ssRNA and

bi-functional vectors (Fig. 6B). To further confirm the role of TLR7 in the dual vector-induced

immunostimulatory effect and tumor inhibition, we suppressed the TLR7 signal pathway by

administering a TLR7 inhibitor, IRS661, a decoy analog that interferes with the combination of TLR7

and ssRNA (21), to tumor-bearing mice i.v. before LV-dual vector treatment. We found that IRS661

treatment nearly completely eliminated dual vector-induced tumor suppression (Fig. 6C). In addition,

IRS661 treatment significantly attenuated the expression of IFN-γ, perforin and CD69 in/on NK cells

(Fig. 6D), as well as the expression of IFN-γ, CD25 and CD69 in/on CD4+T cells (Fig. 6E),

suggesting the functional impairment of NK and CD4+T cells. Similarly, IRS661 treatment markedly

decreased the percentages of NK and CD4+T cells, as well as the activation of NK, CD4+T and CD8+T

cells in tumor-infiltrating sites (Fig. S4). These results revealed that activation of the TLR7 signal

pathway is essential for the anti-tumor effect of the bi-functional vector.

Discussion

Evidence indicates that tumor cells have evolved mechanisms to evade single-targeted treatments such

as chemotherapy and radiotherapy. The proliferation and migration of tumors rely on their genetic and




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epigenetic plasticity and the suppression of host immune responses. Genetic plasticity includes

aberrant expression of some proto-oncogenes that are associated with malignant growth and evading

apoptosis (1,9,10). Meanwhile, the immunosuppressive microenvironment induced by tumors further

contributes to their escape from immunosurveillance (28). Therefore, combined therapies that silence

oncogene expression and stimulate anti-tumor immune responses represent a novel therapeutic

strategy.

Pim kinases are important downstream effector molecules of some oncogenes, such as ABL,

JAK2 and Flt-3, and are closely related to tumorigenesis (29). Although belonging to the Pim kinase

family, the expression and regulatory mechanisms may be different for the three Pim members. Pim-1

protein is highly expressed in the liver and spleen during hematopoiesis and is overexpressed in

lymphoma and leukemia. Pim-2 is largely expressed in both solid and hematological tumors (29).

Pim-3 expression seems to be restricted to solid tumors, in particularly adenocarcinomas from the

liver, pancreas, colon and stomach [4-7]; however, it is not found in the normal colon, thymus, liver

and small intestine. Pim-3 is highly expressed in HCC tissues and cell lines, and is reported to

accelerate HCC development when induced by the hepatocarcinogen diethylnitrosamine (DEN) in

Pim-3 transgenic mice in which Pim-3 is selectively expressed in the liver (30). Thus, Pim-3

represents an attractive target for cancer therapy, particularly for HCC.

Pim-1 and Pim-2 can be upregulated by cytokines such as IL-12 and IFN-α via STAT proteins

activation, and are involved in T cell differentiation (31,32). However, for Pim-3, ETS-1 and Sp1 are

major regulators of its gene expression. Although the human Pim-3 gene contains putative binding

sites for STAT3, STAT3 showed little contribution to Pim-3 gene expression in human pancreatic

cancer cells (33,34). Accordingly, we did not see any changes in Pim-3 expression after ssRNA

transfection, although increased levels of type I IFN were produced (Fig. 1 and Fig. S2). We proposed

that the STAT protein does not regulate Pim-3 gene expression in hepatomas. There may be different

regulatory mechanisms for different Pim genes and in hematopoietic or solid tumor cells.

TLR7 recognizes specific viral ssRNA sequences, such as GUGUU, U-rich sequences, and a

GU-rich 4-mer (14,16,17). Activation of TLR7 signaling leads to production of type I IFN and




17

inflammatory cytokines, which further prime innate and adaptive immune responses (14,16). Systemic

application of TLR7 ligands not only functionally activates both CD8+ T cells and NK cells, but also

blocks the suppressive function of regulatory T cells and myeloid-derived suppressor cells (MDSCs)

(35,36). However, TLR7 expression and TLR7 signaling are often suppressed in tumor patients,

suggesting impaired priming of host anti-tumor immune responses. For example, downregulation of

TLR7 expression and function was found in hepatocytes from HCC, particularly HBV- or

HCV-related HCC (37,38). Therefore, it is necessary to stimulate an anti-tumor immune response

through priming TLR7 signaling. Data has shown the potential therapeutic benefit in TLR7-based

cancer immunotherapy (39,40). Here, we constructed a dual function vector containing both a

Pim-3-targeting shRNA and a TLR7-based immunostimulatory ssRNA. This bi-functional vector not

only promoted apoptosis of hepatoma cells by silencing Pim-3, but also induced production of type I

IFN by activating TLR7 signaling. It further stimulated the activation of NK, CD4+T and CD8+T cells,

leading to enhanced anti-tumor immune responses and suppression of tumor growth. This is the first

bi-functional vector that inhibits the growth of hepatomas by promoting tumor apoptosis via

Pim-3-silencing and stimulating TLR7-dependent anti-immune responses. Similarly, we have also

constructed a dual-function TLR7-based immunostimulatory HBx-shRNA vector and used to treat

chronic HBV persistence in a mouse model. The vector showed not only potent HBV inhibition, but

also reversal of HBV-induced immunotolerance, by stimulating both intrinsic innate and systemic

adaptive immune responses (19).

Interestingly, this bi-functional ssRNA-shRNA vector showed a more significant pro-apoptotic

effect than the shRNA vector. We hypothesize that ssRNA stimulation may contribute to tumor

apoptosis. Firstly, type I IFNs induce apoptosis in several tumor cells. Although we did not observe

the direct induction of tumor apoptosis by ssRNA stimulation in Hepa 1-6 cells, we think it may

increase the susceptibility of tumor cells to apoptosis. Thus, ssRNA treatment renders tumor cell more

prone to undergo apoptosis when Pim-3 is silenced. Secondly, the ssRNA in the bi-functional vector

may enhance the silencing effect of Pim-3-shRNA, and subsequently promote the pro-apoptotic effect.

The exact mechanism of these effects needs to be further investigated.




18

To determine which immune cells are responsible for the tumor regression process, we depleted

of NK, CD4+T, CD8+T cells and macrophages using depletion mAb or liposomes containing DMDP,

respectively. We determined that both NK cells and T cells are required for effective tumor

suppression (Fig. 3D), while NK cells show enhanced cytotoxicity against hepatoma via augmented

NKG2D-NKG2D ligands interaction (Fig. 4). The critical role of NK cells in TLR7/8

activation-mediated anti-tumor responses has been reported (35,41), and most studies showed that the

activation of NK cells through TLR7/8 recognition requires the help of APCs; however, TLR7/8

signaling may exert a direct activating role on NK cells (35,41,42). We also demonstrated the critical

role of macrophages in the tumor suppression mediated by the dual function vector. We hypothesize

that macrophages and other APCs provide indispensable helper role for both NK and T cell activation,

possibly by activation of TLR7 on APCs. Therefore, macrophages, CD4+T, NK, and CD8+T cells all

contribute to the observed tumor regression. In addition, we found that CD4+T cells provide a helper

role in NK cell activation, predominantly by secreting Th1-type cytokines. This is in agreement with

observations in other cancer models (43,44). Surprisingly, the inhibition of TLR7 with IRS661

completely abrogated vector-induced tumor regression (Fig. 6). We assume that TLR7 activation is the

first issue for immune cells activation-induced tumor suppression. Firstly, type I IFNs induced by

TLR7 signaling may directly contribute to the activation of immune cells and tumor suppression;

secondly, type I IFNs-activated NK, CD4+T and CD8+T cells exert enhanced cyotoxicity to tumor

cells; thirdly, type I IFNs might contribute to tumor apoptosis, as described above. However, why

TLR7 inhibitor treatment can nearly completely eliminate the dual vector-induced tumor suppression

requires further investigation.

Despite accumulating evidence showing the strong immune activation induced by TLR7

stimulation and the successful immunotherapy of skin tumors by TLR7 agonists when applied

topically, their systemic use for the treatment of cancer has been delayed because of TLR7 tolerance

by repeated administration (45,46). Here, therapy with the dual function vector provided a sustained

and long-lasting stimulation rather than short-lived immune activation by TLR7 agonists, and thus will

avoid the TLR7 tolerance induced by repeated administration. This strategy might represent a




19

promising therapeutic approach in future therapy for HCC or other solid tumors, in which Pim-3 is

aberrantly expressed.

Acknowledgements

This work was supported by grants from the National 973 Basic Research Program of China

(#2013CB944901), the Natural Science Foundation of China (# 81273220, # 31200651), and the

Young and Middle-aged Scientist Award of Shandong Province (# BS2010YY033).




20

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Figure Legends

Figure 1. The construction of a bi-functional vector bearing ssRNA and Pim-3-specific short

hairpin RNA (shRNA). A, Schematic of the construction of the dual functional vector. B, The

expression of Pim-3 in Hepa1-6 cell line was measured by RT-PCR (left) and western blotting (right)

after transfection for 24 h with indicated vectors. C, Real-Time PCR analysis of IFN-α or IFN-β gene

expression in Hepa1-6 cells after transfection in vitro. D, The levels of IFN-α or IFN-β in the culture

supernatants of Hepa1-6 cells, measured by ELISA after transfection with the indicated vectors for 24

h. Data are means ± SD of three independent experiments. **, P < 0.01, compared with the pSIREN

transcription group.

Figure 2. Transfection with shRNA and bi-functional vector promotes the apoptosis of hepatoma

cells in vitro. A, Flow cytometric analysis of apoptosis in Hepa1-6 cells after transfection for 24 h

with indicated vectors using AnnexinV/PI double staining. B, TUNEL staining to evaluate apoptosis

of hepatoma cells after transfection for 24 h via observation of Red fluorescence. An arrowhead

indicates shrinking nuclei. Data are shown as representatives (left) or means ± SD (right) from three

independent experiments. **, P < 0.01, compared with the pSIREN transcription group.

Figure 3. Treatment with bi-functional vector delays tumor growth in vivo. A-C, C57BL/6 mice

were subcutaneous challenged with 1 × 106 Hepa1-6 cells, and 2 weeks later LV-ctrl, LV-ssRNA,

LV-shRNA, and LV-dual (MOI = 50) were administered intratumorally for 14 d once a week. Tumor

volumes were calculated (B). The percentages of NK, CD4+T , CD8+T cells and CD69+ NK or T cells

(C) in splenic lymphocytes from hepatoma-bearing mice were determined by flow cytometry. Data are

representative of three independent experiments with three mice per group. D, Hepatoma-bearing

mice were injected i.p. with 1 mg of depleting antibodies (α-CD8β, α-CD4, α-NK1.1) for 3 days to

deplete T and NK cells. To deplete macrophages, 1 mg liposomes containing DMDP was administered

i.p. 48 h prior to treatment. The LV-dual vector (MOI = 50) was administered intra-tumorally once a




26

week for twice. Two weeks later, the growth of hepatoma was observed and tumor volumes were

calculated. Data are representative of three independent experiments with 5 mice per group. *, P <

0.05, **, P < 0.01, compared with the LV-ctrl group.

Figure 4. NK cells are involved in the suppression of hepatomas mediated by the bifunctional

vector in a NKG2D-dependent manner. A, The cytotoxicity of NK cells was determined by

measuring (using CFSE/7-AAD assay) the ability of splenic lymphocytes in treated mice to kill

Hepa1-6 cells. B, The percentages of NKG2D+, NKG2A+, IFN-γ+ or PD-1+ NK cells were determined

via FACS. C, The expression of NKG2D ligands H-60 and MULT-1 on Hepa1-6 cells was confirmed

via FACS after transfection with indicated vectors for 24 h. D, The cytotoxicity of splenic NK cells

from hepatoma-bearing mice treated with bi-functional vectors against Hepa1-6 cells, determined by

the CFSE/7-AAD assay, after incubation with or without NKG2D blocking mAbs. Data are

representative or means ± SD of three independent experiments. *, P < 0.05, **, P < 0.01, compared

with the LV-ctrl or LV-ctrl + isotype group.

Figure 5. CD4+T cells are important for NK cell activation after stimulation with the

bifunctional vector. A, Flow cytometry of PD-1+ cells in splenic CD4+T cells. B, Serum levels of

Th1- and Th2- type cytokines detected by ELISA. C, The proportion of total NK or CD69+ NK cells

in spleens identified by FACS after CD4+T cell depletion. D, Splenic NK cells from tumor mice (with

or without CD4+T cell depletion) treated with bi-functional vector were isolated and their cytotoxicity

against Hepa1-6 cells was confirmed via the CFSE/7-AAD assay. Data are shown as means ± SD of

three independent experiments. *, P < 0.05, **, P < 0.01, compared with the LV-ctrl group or isotype

control group.

Figure 6. The TLR7 pathway is critical for NK cell activation and the inhibition of the growth of

hepatoma resulting from bi-functional vector therapy. A, TLR expression was analyzed in Hepa1-6




27

cells after transfection with indicated vectors via real-time PCR. B, western blotting of the expression

of TLR7 and related signaling molecules in Hepa1-6 cells after transfection in vitro. C, Tumor

performance in hepatoma-bearing mice treated with bi-functional vector with or without

co-administration of TLR7 inhibitor. Tumor volumes were calculated. D, The percentages of IFN-γ,

Perforin or CD69 positive splenic NK cells in mice treated with bi-functional vector were determined

via flow cytometry. E, The percentages of IFN-γ, CD25 or CD69 positive CD4+T cells were detected

via flow cytometry. Data are representative of three independent experiments with four mice per

group. *, P < 0.05, **, P < 0.01, compared with the pSIREN transcription or solvent group.




Published OnlineFirst April 10, 2014.Mol Cancer Ther Qie Guo, Peixiang Lan, Xin Yu, et al. both immunostimulatory and Pim-3-silencing effectsImmunotherapy for hepatoma using a dual-function vector with

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