Tumorigenesis is an immortalization process in which the growth of normal cells is uncontrolled and programmed cell death is suppressed. Molecular biologic and immunologic studies have revealed that the aberrant expression of some proto-oncogenes boosts proliferation and inhibits apoptosis, which is vital for 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. Natural killer (NK), CD4+ T, and 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. Mol Cancer Ther; 13(6); 1503–13. ©2014 AACR.

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 hematologic 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 (CTL), 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 (TLR) 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 stimulators that could stimulate both innate and adaptive immune cells (12). Interferons (IFN) secretion following TLR-mediated activation of IFN-regulatory factors (IRF) 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, for example 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 (APC) and exhibit antitumor 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.

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 (BamHI at the 5′ and EcoRI at the 3′), and was cloned into vector pTZU6+1 vector that contains a U6 polymerase-III (pol-III) promoter. The shRNA template sequences are shown in Supplementary 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 Supplementary Table S1. To create the dual-functional vector, the U6+shRNA in pTZU6+1-shRNA was digested by HindIII and EcoRI, and inserted to pSIREN-ssRNA.

pSIREN-control, ssRNA, shRNA, or dual vectors were cloned into a lentiviral pGCSIL-GFP plasmid, and transfected into 293 T cells. Forty-eight hours 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 hours. 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) supplemented with 10% heat-inactivated FBS. 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 intratumorally once a week for 2 weeks. After another 2 weeks, the mice were sacrificed and the tumor volume was calculated by length × width2/2.

Human samples

HCC and nontumor 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 frozen and stored at −80°C for further use in real-time PCR and Western blotting assays.

Semiquantitative reverse transcription-PCR and real-time PCR analysis

Total RNA was extracted by the TRizol regent (Invitrogen) and cDNAs were synthesized using Superscript III Reverse Transcriptase (Invitrogen), followed by real-time PCR and semiquantitative reverse transcription (RT)-PCR analysis. For semiquantitative RT-PCR, cDNA was amplified using pairs of primers (RiboBio) 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). Relative gene expression was determined in comparison with that of GAPDH or β-actin. PCR primers are provided in Supplementary Table S2.

Western blotting

Cells were collected and lysed on ice using a total protein extraction reagent (Beyotime). The protein samples (30 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore). The membrane was blocked in Tris-buffered saline with 5% (w/v) nonfat dry milk, and then incubated with primary antibodies over night at 4°C, followed by incubation with horseradish peroxidase–conjugated secondary antibody for 50 minutes 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.). 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.

NK cytotoxicity assays

The ability of spleen lymphocytes to kill Hepa1-6 cells was evaluated by CFSE/7-AAD flow cytometry assay, as previously described (18). Briefly, Hepa1-6 cells were incubated with 1 mL of CFSE (2 mmol/L; Molecular Probes) for 10 minutes 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 hours. Following a further wash, cells were labeled for 15 minutes with 7-AAD (optimized at 0.25 μg/mL; Sigma-Aldrich) to identify dead cells. The cells were then analyzed via flow cytometer (FACScalibur). Cytotoxicity was calculated as follows: % lysis = (CFSE/7-AAD double positive cells/CFSE positive cells) × 100%.

Measurement of apoptosis

Staining for Annexin V-FITC/PI (BestBio) 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). Nuclear staining was evaluated under a light microscope via DAPI staining (Beyotime). A commercial Enzyme-Linked Immunosorbent Assay (ELISA) Kit (KeyingMei) 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).

Flow cytometry analysis

Splenic lymphocytes were isolated to analyze the percentages and activation of NK 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 minutes. For intracellular IFN-γ staining, splenic cells were cultured in RPMI 1640 containing 10% FCS, and treated with monensin (Sigma) for 4 hours to inhibit extracellular secretion of cytokines. The antibodies used were: FITC-conjugated NK1.1, PerCP-Cy5.5-conjugated CD3e (BD Biosciences); FITC-conjugated CD4, PerCP-Cy5.5-conjugated CD8, PE-conjugated CD69, APC-conjugated NKG2D, and PE-conjugated NKG2A (eBiosciences); and carboxyfluorescein-conjugated RAE-1, FITC-conjugated MULT-1, PE-conjugated H-60, and PE-conjugated IFN-γ mAb (R&D Systems). 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).

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 intraperitoneally into C57BL/6 48 hours before treatment (20). The LV-dual vector (MOI = 50) was then administered intratumorally once a week for 2 weeks. To ablate the function of TLR7, IRS661 (5′-TGCTTGCAAGCTTGCAAGCA-3′; TAKARA), a decoy analog that interferes with the combination of TLR7 and ssRNA (21, 22), was administered intravenously before LV-dual vector treatment.

Histochemical analysis

Tumor tissues were excised and fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned and stained with hematoxylin and eosin to assess morphologic changes and lymphocyte infiltration.

Statistical analyses

Statistical analysis was performed using a paired Student t test and Mann–Whitney U test. P values < 0.05 were considered significant.

Pim-3 is aberrantly expressed in mouse hepatoma cell lines and HCC tissues

First, 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 (Supplementary 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 (Supplementary Fig. S1B). Pim-3 expression in human primary hepatocellular carcinoma cells was significantly higher in tumor tissues than in nontumor tissues at both the mRNA and protein levels (Supplementary 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 4 siRNA duplexes to target the open reading frame encoding of Pim-3 mRNA by using BLOCK-iT RNAi Designer. The annealed siRNA oligonucleotides were knocked into the expressing vector pTZU6+1 to construct 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).

Figure 1.

The construction of a bi-functional vector bearing ssRNA and Pim-3–specific 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 hours 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 hours. Data are means ± SD of 3 independent experiments. **, P < 0.01, compared with the pSIREN transcription group.

Figure 1.

The construction of a bi-functional vector bearing ssRNA and Pim-3–specific 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 hours 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 hours. Data are means ± SD of 3 independent experiments. **, P < 0.01, compared with the pSIREN transcription group.

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We then confirmed the ability of the bi-functional vector to downregulate Pim-3 expression. Figure 2B shows that Pim-3 mRNA and protein levels were downregulated after transfection with both 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 D). Collectively, these results indicated a successfully constructed dual-function ssRNA-shRNA vector with both Pim-3 silencing and innate immune stimulation effects.

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 hours with indicated vectors using Annexin V/PI double staining. B, TUNEL staining to evaluate apoptosis of hepatoma cells after transfection for 24 hours via observation of red fluorescence. An arrowhead indicates shrinking nuclei. Data are shown as representatives (left) or means ± SD (right) from 3 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 hours with indicated vectors using Annexin V/PI double staining. B, TUNEL staining to evaluate apoptosis of hepatoma cells after transfection for 24 hours via observation of red fluorescence. An arrowhead indicates shrinking nuclei. Data are shown as representatives (left) or means ± SD (right) from 3 independent experiments. **, P < 0.01, compared with the pSIREN transcription group.

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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 proapoptotic 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 DAPI staining also showed obvious shrinking of nuclei and nucleosomes after transfection with the shRNA or bi-functional vector (Fig. 2B, bottom). Importantly, the dual-function vector showed a more significant proapoptotic effect than the shRNA vector.

We also observed that the shRNA and bi-functional vector significantly downregulated the mRNA and protein levels of antiapoptotic genes Bcl-XL and Bcl-2 (Supplementary 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 (Supplementary 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 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 and 36 hours, suggesting that the apoptotic-related pathway was activated (Supplementary 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 antitumor 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 intratumoral injection once a week for 2 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.

Figure 3.

Treatment with bi-functional vector delays tumor growth in vivo. A–C, C57BL/6 mice were subcutaneously 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 days 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 3 independent experiments with 3 mice per group. D, hepatoma-bearing mice were injected intraperitoneally 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 intraperitoneally 48 hours before treatment. The LV-dual vector (MOI = 50) was administered intratumorally once a week for two weeks. Two weeks later, the growth of hepatoma was observed and tumor volumes were calculated. Data are representative of 3 independent experiments with 5 mice per group. *, P < 0.05, **, P < 0.01, ***, P < 0.001, compared with the LV-ctrl group or isotype+LV-ctrl group.

Figure 3.

Treatment with bi-functional vector delays tumor growth in vivo. A–C, C57BL/6 mice were subcutaneously 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 days 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 3 independent experiments with 3 mice per group. D, hepatoma-bearing mice were injected intraperitoneally 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 intraperitoneally 48 hours before treatment. The LV-dual vector (MOI = 50) was administered intratumorally once a week for two weeks. Two weeks later, the growth of hepatoma was observed and tumor volumes were calculated. Data are representative of 3 independent experiments with 5 mice per group. *, P < 0.05, **, P < 0.01, ***, P < 0.001, compared with the LV-ctrl group or isotype+LV-ctrl group.

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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 (Supplementary Fig. S3A). Tumor apoptosis was also significantly higher in LV-dual–treated mice (Supplementary Fig. S3B), and the serum levels of IFN-α and IFN-β were higher in LV-dual therapy group, than in the shRNA and control groups (Supplementary Fig. S3C).

To further investigate which immune cells are involved in the antitumor responses, we depleted of NK, CD4+ T, and CD8+ T cells using depletion mAb, separately, by intraperitoneally 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 intraperitoneally 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, whereas 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 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.

Figure 4.

NK cells are involved in the suppression of hepatomas mediated by the bi-functional 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 hours. 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 3 independent experiments. *, P < 0.05, **, P < 0.01, compared with the LV-ctrl or LV-ctrl + isotype group.

Figure 4.

NK cells are involved in the suppression of hepatomas mediated by the bi-functional 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 hours. 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 3 independent experiments. *, P < 0.05, **, P < 0.01, compared with the LV-ctrl or LV-ctrl + isotype group.

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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 and 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 toward Th1, which contributed to the antitumor immune response.

Figure 5.

CD4+ T cells are important for NK-cell activation after stimulation with the bi-functional 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-bearing 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 3 independent experiments. *, P < 0.05, **, P < 0.01, compared with the LV-ctrl group or isotype control group.

Figure 5.

CD4+ T cells are important for NK-cell activation after stimulation with the bi-functional 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-bearing 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 3 independent experiments. *, P < 0.05, **, P < 0.01, compared with the LV-ctrl group or isotype control group.

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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 bi-functional 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 antitumor effect exerted by the bi-functional vector.

Activation of the TLR7 signal pathway is important for the antitumor effect mediated by the bi-functional vector

TLR7 and TLR8, also known as “nucleic acid-sensing TLRs,” were originally identified as recognizing imidazoquinoline derivatives such as imiquimod, resiquimod (R-848), and guanine analogs such as loxoribine (which have antiviral and antitumor properties; ref. 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 intravenously 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 (Supplementary Fig. S4). These results revealed that activation of the TLR7 signal pathway is essential for the antitumor effect of the bi-functional vector.

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 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+ splenic NK cells in mice treated with bi-functional vector were determined via flow cytometry. E, the percentages of IFN-γ+, CD25+, or CD69+ CD4+ T cells were detected via flow cytometry. Data are representative of 3 independent experiments with 4 mice per group. *, P < 0.05, **, P < 0.01, compared with the pSIREN transcription or solvent 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 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+ splenic NK cells in mice treated with bi-functional vector were determined via flow cytometry. E, the percentages of IFN-γ+, CD25+, or CD69+ CD4+ T cells were detected via flow cytometry. Data are representative of 3 independent experiments with 4 mice per group. *, P < 0.05, **, P < 0.01, compared with the pSIREN transcription or solvent group.

Close modal

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 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 antitumor 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 3 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, 4). 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 Supplementary 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 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 (MDSC; refs. 34 and 35). However, TLR7 expression and TLR7 signaling are often suppressed in tumor patients, suggesting impaired priming of host antitumor immune responses. For example, downregulation of TLR7 expression and function was found in hepatocytes from HCC, particularly HBV- or HCV-related HCC (36, 37). Therefore, it is necessary to stimulate an antitumor immune response through priming TLR7 signaling. Data have shown the potential therapeutic benefit in TLR7-based cancer immunotherapy (38, 39). 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 antitumor 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 proapoptotic effect than the shRNA vector. We hypothesize that ssRNA stimulation may contribute to tumor apoptosis. First, type I IFNs induce apoptosis in several tumor cells. Although we did not observe the direct induction of tumor apoptosis by ssRNA stimulation in Hepa1-6 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. Second, the ssRNA in the bi-functional vector may enhance the silencing effect of Pim-3-shRNA, and subsequently promote the proapoptotic effect. The exact mechanism of these effects needs to be further investigated.

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 and T cells are required for effective tumor suppression (Fig. 3D), whereas 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 antitumor responses has been reported (34, 40), 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 (34, 40, 41). 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 (42, 43). 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. First, type I IFNs induced by TLR7 signaling may directly contribute to the activation of immune cells and tumor suppression; second, type I IFNs-activated NK, CD4+ T, and CD8+ T cells exert enhanced cyotoxicity to tumor cells; third, 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 (44, 45). 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 promising therapeutic approach in future therapy for HCC or other solid tumors, in which Pim-3 is aberrantly expressed.

No potential conflicts of interest were disclosed.

Conception and design: Q. Guo, P. Lan, J. Zhang, Z. Tian, C. Zhang

Development of methodology: Q. Guo

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Q. Guo, P. Lan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Q. Guo, Q. Han, J. Zhang, C. Zhang

Writing, review, and or revision of the manuscript: Q. Guo, X. Yu, Z. Tian, C. Zhang

Study supervision: Z. Tian

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 costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data