Transforming growth factor-β gene silencing using adenovirus expressing TGF-β1 or TGF-β2 shRNA

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Abstract

Tumor cells secrete a variety of cytokines to outgrow and evade host immune surveillance. In this context, transforming growth factor-β1 (TGF-β1) is an extremely interesting cytokine because it has biphasic effects in cancer cells and normal cells. TGF-β1 acts as a growth inhibitor in normal cells, whereas it promotes tumor growth and progression in tumor cells. Overexpression of TGF-β1 in tumor cells also provides additional oncogenic activities by circumventing the host immune surveillance. Therefore, this study ultimately aimed to test the hypothesis that suppression of TGF-β1 in tumor cells by RNA interference can have antitumorigenic effects. However, we demonstrated here that the interrelation between TGF-β isotypes should be carefully considered for the antitumor effect in addition to the selection of target sequences with highest efficacy. The target sequences were proven to be highly specific and effective for suppressing both TGF-β1 mRNA and protein expression in cells after infection with an adenovirus expressing TGF-β1 short hairpin RNA (shRNA). A single base pair change in the shRNA sequence completely abrogated the suppressive effect on TGF-β1. Surprisingly, the suppression of TGF-β1 induced TGF-β3 upregulation, and the suppression of TGF-β2 induced another unexpected downregulation of both TGF-β1 and TGF-β3. Taken together, this information may prove useful when considering the design for a novel cancer immunogene therapy.

Introduction

Transforming growth factor (TGF)-β is a homodimeric protein that has three different isoforms (TGF-β1, TGF-β2 and TGF-β3), which have both overlapping and distinct functions.1, 2 These isoforms are secreted as latent 25 kDa active homodimer complexes that bind TGF-β receptors to initiate intracellular signaling.3 Because various oncogenic pathways directly inactivate the TGF-β receptor-Smad pathway, which inhibits proliferation and induces apoptosis, tumor cells can use TGF-β for their advantage to initiate immune evasion, growth factor production and metastasis.4, 5 All human tumors overexpress TGF-β and accordingly induce tumor cell invasion and metastasis.5 The TGF-β isoforms contribute to a number of cellular activities like inhibiting normal epithelial cell proliferation while inducing cancer cell proliferation, and promoting an invasive phenotype that is characterized by epithelial-to-mesenchymal transition.6 Another major role of TGF-β that is produced by tumors is to block the immune response.7 The local immunosuppressive environment of the tumor induced by TGF-β has proved to be the major obstacle to immunogene therapy with cytokines.8 Among the three isoforms of TGF-β, TGF-β1 is the most abundant, universally expressed,7, 9 and when silenced, increases antitumor immunity.10, 11 In addition to the effect on immunosuppression, TGF-β1 silencing also efficiently inhibits proliferation and migration.2 Recently, TGF-β1 has also been reported to suppress apoptosis.12, 13, 14

RNA interference is a regulatory mechanism of most eukaryotic cells that uses small double-stranded RNA to direct homology-dependent degradation of the target mRNA.15, 16 In mammalian cells, small interfering RNAs (siRNAs) are produced through cleavage of longer double-stranded RNA precursors by the RNaseIII endonuclease dicer.17, 18 After dicer processing, the 21–23 nucleotide siRNA is incorporated into a protein complex called RNA-induced silencing complex.19 Argonaute 2, a multifunctional protein contained within RNA-induced silencing complex, cleaves and releases the sense strand (passenger strand), thereby activating RNA-induced silencing complex with a single-stranded antisense strand (guide strand)16, 17 that selectively degrades complementary mRNA.

Because of the efficacy and specificity of siRNA, it is emerging as a potential new basis for the treatment of human diseases.20 However, one of the main drawbacks in the development of siRNA therapy is the instability of siRNA in vivo. To overcome this instability, DNA-based RNA interference drugs that utilize a vector delivery system are being developed. For the expression of siRNA in cells, short hairpin RNAs (shRNAs) are annealed to the vector system and processed into siRNAs by dicer after introduction into the cell.16 Here, we used adenovirus as a delivery vector for TGF-β shRNA. Adenoviral vectors are still frequently used as a method of gene transfer because of efficient transgene delivery, expression in both dividing and non-dividing cells and ease of propagation to high titers in spite of shortcomings like increased immunogenicity, the prevalence of pre-existing anti-adenovirus immunity in the human population and the inability to specific targeting.21, 22

Materials and methods

Cell culture

The human cancer cell lines, DU145 (human prostate adenocarcinoma) and A375 (human skin melanoma), were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (HyClone, Logan, UT, USA). B16BL6 and B16F10 (mouse melanoma) cells were also cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (HyClone). HeLa (human cervix adenocarcinoma) cells were cultured in minimal essential medium with 10% fetal bovine serum. Cells were maintained in a 37 °C humidified atmosphere containing 5% CO2.

Reagents

Trizol was purchased from Life Technologies (Carlsbad, CA, USA). All other chemicals were purchased from Sigma-Aldrich (St, Louis, MO, USA).

Construction of TGF-β shRNAs

To construct human TGF-β1 (hTGF-β1) shRNA, we screened three candidate sequences. The selected target sequence was 5′-IndexTermACCAGAAATACAGCAACAATTCCTG-3′, and the loop sequence was 5′-IndexTermTCTCTC-3′. Target selection was performed using an algorithm developed by Genolution Pharmaceuticals Inc. (Seoul, South Korea). For expression of hTGF-β1 shRNA in adenovirus, the top strand sequence was 5′-IndexTermGATCCGCCAGAAATACAGCAACAATTCCTGTCTCTCCAGGAATTGTTGCTGTATTTCTGGTTTTTTTA-3′ and the bottom strand sequence was 5′-IndexTermAGCTTAAAAAAACCAGAAATACAGCAACAATTCCTGGAGAGACAGGAATTGTTGCTGTATTTCTGGTG-3′ was selected and annealed for subcloning into pSP72ΔE3-U6. To construct mouse TGF-β1 (mTGF-β1) shRNA, we screened 11 candidate sequences. The selected target sequence was 5′-IndexTermCCCTCTACAACCAACACAACCCGGG-3′ and the loop sequence was 5′-IndexTermTCTCTC-3′. For expression of mTGF-β1 shRNA in adenovirus, the top strand sequence was 5′-IndexTermGATCGCCTCTACAACCAACACAACCCGGGTCTCCCCGGGTTGTGTTGGTTGTAGAGGGTTTT-3′ and the bottom strand sequence was 5′-IndexTermAGTCAAAACCCTCTACAACCAACACAACCCGGGGAGACCCGGGTTGTGTTGGTTGTAGAGGGG-3′ was selected and annealed for subcloning into pSP72ΔE3-U6. In the case of hTGF-β2 shRNA, we screened five candidate sequences. The selected target sequence was 5′-IndexTermGGATTGAGCTATATCAGATTCTCAA-3′ and the loop sequence was 5′-IndexTermTCTC-3′. For expression of hTGF-β2 shRNA in adenovirus, the top strand sequence 5′-IndexTermGATCCGGATTGAGCTATATCAGATTCTCAATCTCTTGAGAATCTGATATAGCTCAATCCTTTTA-3′ and the bottom strand sequence 5′-IndexTermAGCTTAAAAGGATTGAGCTATATCAGATTCTCAAGAGATTGAGAATCTGATATAGCTCAATCCG-3′ was selected and annealed for subcloning into pSP72ΔE3-U6.

Recombinant adenovirus

First, an adenovirus containing the green fluorescence protein (GFP) or LacZ gene was constructed to examine the infection efficiency. In the case of GFP, two different adenoviruses with the GFP gene in either the E1 or E3 region were produced. For the insertion of the GFP gene at the E1 region, pCA14 was used as a shuttle vector. After digestion of pEGFPN1 E1 (Clontech, Mountainview, CA, USA) with XhoI/XbaI, the insert was subcloned into XhoI/XbaI-digested pCA14. For the insertion of the GFP gene at the E3 region, pSP72ΔE3 was used as a shuttle vector. After pSP72ΔE3 was digested with KpnI, the ends were blunted with a Quick blunting kit (New England Biolabs, Ipswich, MA, USA) and further digested with BamHI, generating a linearized vector with one cohesive end. For the insertion of GFP, pEGFN1 was predigested with AflII, blunted, and then digested with BamHI to insert the linearized pSP72ΔE3. For the expression of LacZ, the LacZ gene was removed from pcDNA3.1/hygro/LacZ (Invitrogen, Carlsbad, CA, USA) by digestion with BamH/XbaI. Thereafter, it was inserted into linearized pCA14 with BamH/XbaI. Finally, pCA14-LacZ and pCA14-GFP were recombined into the dl324 adenoviral vector and pSP72-GFP was recombined into the dl324-IX adenoviral vector by homologous recombination in BJ5183 bacterial-competent cells, generating three different replication-defective adenoviruses to be used as control viruses.

The adenoviral shuttle vector, pCA14 (Microbix, Mississauga, ON, Canada), containing the IX gene of adenovirus type 5, was linearized by XmnI digestion. The adenovirus vector dl324-BstBI containing the Ad5 genome lacking the E1 region (340–4641 in nucleotide of Ad5) and E3 region (28 592–30 470 in nucleotide of Ad5) was linearized by BstBI digestion. The linearized vectors were cotransformed into Escherichia coli BJ5183 cells for homologous recombination, generating the recombinant adenoviral plasmid dl324-IX.

The shRNA construct and U6 promoter were subcloned into the pSP72ΔE3, E3 shuttle vector. The adenoviral shuttle vector, pSP72ΔE3-U6-shRNA, was linearized by XmnI digestion. The adenoviral vector dl324-IX was linearized by SpeI digestion and the linearized vectors were cotransformed into E. coli BJ5183 cells for homologous recombination. The homologously recombined adenoviral plasmids, dl324-IX-ΔE3-U6-NC and dl324-IX-ΔE3-U6-shTGF-β1, were then digested with PacI and transfected into 293 cells to generate the replication-incompetent adenovirus. The infectious titer of the adenovirus was determined by a limiting dilution assay using the Adeno easy vector system (Qbiogene, Carlsbad, CA, USA).

Quantitative real-time PCR

DU-145 or A375 cells were infected with a defective adenovirus expressing hTGF-β1, hTGF-β2, mTGF-β1 or scrambled shRNA. After 2 days of infection, cells were lysed with Trizol reagent (Life Technologies) and the total RNA was isolated via chloroform extraction. The RNA concentration was determined by using the Nanodrop 2000 (Thermo Scientific, Fremont, CA, USA). The real-time polymerase chain reaction (PCR) was assayed with the Power SYBR Green RNA-to-CT 1-Step Kit (Life Technologies). The reaction mixture was prepared with RT enzyme mix, reverse transcription (RT)-PCR mix, forward primer, reverse primer, RNA template and nuclease-free water. hTGF-β1 was amplified with the forward primer, 5′-IndexTermCAAGGGCTACCATGCCAACT-3′ and reverse primer, 5′-IndexTermAGGGCCAGGACCTTGCTG-3′. Human β-actin was amplified with the forward primer, 5′-IndexTermACTCTTCCAGCCTTCCTTC-3′ and reverse primer, 5′-IndexTermATCTCCTTCTGCATCCTGTC-3′. mTGF-β1 was amplified with the forward primer, 5′-IndexTermTTGCTTCAGCTCCACAGAGA-3′ and reverse primer, 5′-IndexTermTGGTTGTAGAGGGCAAGGAC-3′. Mouse β-actin was amplified with the forward primer, 5′-IndexTermGGCTGTATTCCCCTCCATCG-3′ and reverse primer, 5′-IndexTermCCAGTTGGTAACAATGCCATGT-3′.

ELISA

Enzyme-linked immunosorbent assays (ELISAs) were performed to estimate the level of secreted TGF-β1 after infection with an adenovirus expressing TGF-β1 shRNA. Adenovirus infection was carried out one day after seeding 2 × 105 A375 or DU-145 cells into a six-well plate. Because TGF-β1 protein is present in fetal bovine serum, infected cells were incubated for one more day after changing to serum-free medium and the ELISA was performed after harvesting the medium. After the harvested medium was activated, the activated sample or TGF-β1 standard protein was incubated for 2 h at room temperature. Then, the samples were completely washed and horseradish peroxidase-conjugated TGF-β1 antibody was added for 2 h at room temperature. Finally, after incubating with the substrate solution for 30 min and adding stop solution, the optical density of each well was determined at 450 nm by using a microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA). For the measurement of mTGF-β1, hTGF-β1 and hTGF-β2, ELISA was performed according to the manufacturer’s instructions for Quantikine mTGF-β1 (R&D Systems, Minneapolis, MN, USA) or Quantikine hTGF-β1 or Quantikine hTGF-β2 (R&D Systems), respectively. In the case of TGF-β3, ELISA was purchased from Koma Biotech Inc. (Seoul, South Korea) for the measurement of hTGF-β3, hTGF-β3 ELISA kit.

Statistical analysis

Data were presented as mean±standard error of mean (s.e.m.), and the significant differences between groups were determined by unpaired two-tailed t-test. P-values were calculated using GraphPad Prism version 6.0. A value of P<0.05 or P<0.01 was considered as statistically significant. All experiments were performed three times independently.

Results

Suppression of TGF-β1 with an adenoviral vector expressing TGF-β1 shRNA

To identify the best target sequence for suppression of hTGF-β1, three oligomers of TGF-β1 shRNA plus control shRNA (luciferase shRNA) were tested by Genolution Pharmaceuticals Inc. using real-time RT-PCR. Of the three selected TGF-β1 shRNAs (Figure 1a), TGF-β1 sh2 showed the greatest reduction, 89.7%, in TGF-β1 mRNA levels when expressed in HeLa cells (Figure 1b). In the case of mouse, 11 TGF-β1 shRNA oligomers and control shRNA (luciferase shRNA) were tested (Figure 1c). The analysis indicated that the greatest reduction of TGF-β1 mRNA levels (76.5%) was obtained with TGF-β1 sh10 (Figure 1d).

Figure 1
figure1

Screening of human and mouse transforming growth factor (hTGF and mTGF)-β1 short hairpin RNAs (shRNAs). (a) Sequences of three shRNA oligomers targeting hTGF-β1. The selected target sequence was indicated in bold. (b) Relative expression level of hTGF-β1 mRNA. Three oligomers of the target and control shRNA (luciferase shRNA) were transfected into HeLa cells. The knockdown efficiency of these oligomers was measured by quantitative real-time polymerase chain reaction (PCR) amplifying hTGF-β1. The relative expression level of hTGF-β1 was plotted after normalization to the luciferase shRNA control. (c) Sequences of 11 shRNA oligomers targeting mTGF-β1. The selected target sequence was indicated in bold. (d) Relative expression level of mTGF-β1 mRNA. All candidate shRNAs and control shRNA (transformation-related protein 53 (Trp53) shRNA) were transfected into NIH3T3 cells. The knockdown efficiency of these oligomers was measured by quantitative real-time PCR amplifying mTGF-β1 mRNAs. The relative expression level of mTGF-β1 was plotted after normalization to the transfection control.

TGF-β1 expression in cells after infection with an adenovirus expressing shTGF-β1

The infection efficiency of adenovirus type 5 was examined both in human and mouse cells before confirming the repression of TGF-β1 mRNA and protein. For this purpose, an adenovirus with either the GFP gene inserted at the E3 region or the LacZ gene inserted at the E1 region was constructed. We used A375 and DU-145 cells to determine infection efficiency in human cells and B16F10 and B16BL6 cells to determine the infection efficiency in mouse cells. We observed that the adenovirus could infect most of the human cells with a multiplicity of infection (MOI) of 50, whereas it could infect only a few mouse cells with an MOI of more than 500 (Supplementary Figure 1). Then, we examined the viral efficacy of adenoviruses expressing shTGF-β1 with various MOIs (1, 5, 10, 50 and 100) in human or mouse cells (100 and 500 MOI). To determine whether these viruses decreased TGF-β1 expression at the mRNA and protein level, we used real-time PCR and ELISA, respectively. TGF-β1 mRNA was decreased by 42% at only 1 MOI and TGF-β1 mRNA was suppressed by 98% at 200 MOI in DU-145 cells (Figure 2a), suggesting that the viral efficacy is dependent on the viral MOI. TGF-β1 protein was decreased similarly to TGF-β1 mRNA (Figure 2b). Similar results were observed in A375 cells (Figures 2c and d). Moreover, as expected, the viral efficacy of adenoviruses expressing shRNA no. 10 of mTGF-β1 in mouse cells could be confirmed only at 10-fold higher MOIs compared with human cells by determining the TGF-β1 protein level (Figure 2e).

Figure 2
figure2

Downregulation of human transforming growth factor (hTGF)-β1 mRNA by TGF-β1 short hairpin RNA (shRNA). Cancer cells (human A375 cells (a, b) or DU-145 cells (c, d)) were infected with adenovirus-expressing shRNA targeting human TGF-β1 (dl324-shTGF-β1) or scrambled DNA (dl324-sh-NC). TGF-β1 mRNA (a, c) and protein levels (b, d) were assayed by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. (e) After infection of adenoviruses (100 and 500 MOI) expressing shRNA targeting mouse TGF-β1 (dl324-sh-mTGF-β1) or scrambled DNA (dl324-sh-NC) to B16F10, which were samples for ELISA. See Materials and methods for more details. MOI, multiplicity of infection; NC, negative control.

Level of TGF-β isotypes correlates with the decrease in TGF-β1 or TGF-β2 mRNA

To examine the possibility that a compensatory mechanism was triggered during TGF-β1 suppression, the expression level of TGF-β isotypes was examined by real-time PCR. Strikingly, when the expression of TGF-β1 was decreased by adenovirus-expressing shTGF-β1 (at the highest MOI), the expression of TGF-β3 mRNA significantly increased, while the expression of TGF-β2 mRNA was not changed too much (Figure 3a). Then, we examined shRNA corresponding to the target sequence of hTGF-β2, which has a similar immunosuppressive function in tumor cells as TGF-β1. Unexpectedly, when the expression of TGF-β2 was suppressed by adenovirus-expressing shTGF-β2, the expression level of both TGF-β3 and TGF-β1 was decreased (Figure 3b). We further validated the interplay among TGF-β variants in response to treatment with shTGF-b1 RNA or shTGF-b2 RNA in DU-145 cells. We found that shTGF-β2-expressing adenovirus also suppressed the expression of TGF-β1 and TGF-β3, as well as TGF-β2, with the exception that the expression of TGF-β2 was suppressed by adenovirus-expressing shTGF-β1 (P<0.01) (Figure 3c). Then, we confirmed these patterns by measuring the secreted level of TGF-β variants (Figure 3d).

Figure 3
figure3

Correlation of the level of transforming growth factor (TGF)-β isotypes with the decrease in TGF-β1 or TGF-β2. A375 cells were infected with various multiplicity of infections (MOIs) of defective adenovirus carrying short hairpin RNA (shRNA) targeting human TGF-β1 (hTGF-β1) (dl324-shTGF-β1) or scrambled DNA (dl324-sh-NC) (a) or TGF-β2 (dl324-shTGF-β2) or scrambled DNA (dl324-sh-NC) (b). Infected cells were cultivated for 2 days and harvested for RNA preparation. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression level of hTGF-β1, hTGF-β2 and hTGF-β3 by using subtype-specific primer sets, with normalization to actin. (c) DU-145 cells were infected with 100 MOIs of defective adenovirus carrying shRNA targeting hTGF-β1 (dl324-shTGF-β1) or TGF-β2 (dl324-shTGF-β2), or scrambled DNA (dl324-sh-NC). Infected cells were cultivated for 2 days and harvested for RNA preparation. qRT-PCR was used to determine the expression level of hTGF-β1, hTGF-β2 and hTGF-β3 by using subtype-specific primer sets, with normalization to actin (t-test, P<0.001 or P<0.01). (d) A375 (left) or DU-145 cells (right) were infected with 100 MOIs of defective adenovirus carrying shRNA targeting hTGF-β1 (dl324-shTGF-β1) or TGF-β2 (dl324-shTGF-β2), or scrambled DNA (dl324-sh-NC). Infected cells were cultivated for 2 days and harvested for TGF-β1 or TGF-β2, or TGF-β3 by performing enzyme-linked immunosorbent assay (ELISA). NC, negative control.

Target specificity of TGF-β1 shRNA

Next, we investigated the requirement for sequence specificity in shRNA experiments by transfection of shuttle vector expressing human shTGF-β1 or shTGF-β2 and their corresponding one- or two-mismatched shRNA (Figure 4a). This analysis showed that even a single base pair difference in the shRNA completely abrogated the ability of the shuttle vector expressing shTGF-β1 or shTGF-β2 to suppress TGF-β1 or TGF-β2 at the mRNA level (Figure 4b).

Figure 4
figure4

Specificity of short hairpin RNAs (shRNAs) targeting human transforming growth factor (hTGF)-β1 or hTGF-β2. (a) Sequence alignment of human shTGF-β1 with the selected target sequence. In the case of TGF-β1 coding sequences (1–1173 base), the target sequences are 512–536 bases. The mismatched (mis) bases are indicated in bold (top). Sequence alignment of human shTGF-β2 with the selected target sequence. In the case of TGF-β2 coding sequences (1–1329 base), the target sequences are 578–602 bases. The mismatched bases are indicated in bold (bottom). (b) Human cell line (DU-145 (upper) or A375 (bottom)) were transfected with shuttle vectors expressing shRNAs targeting hTGF-β1 (pSP72ΔE3-shTGF-β1, pSP72ΔE3-shTGF-β1 with one mismatch, and pSP72ΔE3-shTGF-β1 with two mismatch) (left) or hTGF-β2 (pSP72ΔE3-shTGF-β2, pSP72ΔE3-shTGF-β2 with one mismatch, and pSP72ΔE3-shTGF-β2 with two mismatch) (right). After 2 days of transfection, total RNA was prepared from each sample, and quantitative real-time polymerase chain rection (PCR) was performed to investigate sequence specificity. Each shuttle vector expressing mismatched shRNA was constructed based on the ‘Construction of TGF-β shRNAs’ as in the Materials and methods section. NC indicates the shuttle vector expressing a scrambled shRNA. NC, negative control. Error bars represent the s.e. from three independent experiments. *Significant difference compared with NC (t-test, P<0.01 or P<0.05); **significant difference compared with NC (t-test, P>0.1).

Discussion

In this study, we showed the overriding of TGF-β1 downregulation by other TGF-β isoform (TGF-β3) due to the partly overlapping functions of three isoforms of TGF-β,23 whereas TGF-β2 downregulation was accompanied by downregulation of both TGF-β1 and TFG-β3, which can further attenuate the immunosuppressive effects of cancer cells induced by TGF-β.24 Despite the high similarity between the active domains of the TGF-β isoforms, TGF-β2 differs from the other isoforms in that it binds TβRII through different residues and is dependent on the coreceptor β-glycan for function.25, 26 Although TGF-β1 and TGF-β3 are both capable of binding directly to the type II receptor, the α3 helical region of TGF-β1 is structurally ordered, while that of TGF-β3 is structurally disordered, resulting in a more flexible TGF-β3 structure than TGF-β1.27, 28 These results suggest that the structure of the ligand/receptor complexes for TGF-β1 and TGF-β3 may be very different, leading to different downstream signaling pathways. Furthermore, Akhurst et al.29 found that the different temporal–spatial expression of the TGF-β isoforms in embryogenesis is a sign of uncompensated non-overlapping functions during development. However, our results (Figure 3) suggest that, although they may have some unique biological roles, TGF-β1 and TGF-β3 likely also have some similarities as they are able to compensate for one another in some situations. In cancer, TGF-β1 is generally more abundant than TGF-β2 or TGF-β3.30 Interestingly, when TGF-β1 was selectively downregulated by the shTGF-β1 adenovirus, endogenous TGF-β3 dramatically increased (Figures 3a (right) and c (right)). These results raise the possibility that at least one of the function of TGF-β1, suppression of the antitumor immune response, could still be intact when TGF-β1 is downregulated owing to the compensatory overexpression of TGF-β3.31 However, this compensatory mechanism did not include TGF-β2 (Figures 3a (middle) and c (middle)). Moreover, TGF-β2 downregulation by TGF-β2 shRNA strikingly correlated with downregulation of the other TGF-β isotypes. This was a surprising result and it is not yet fully understood how TGF-β2 could have an effect on the endogenous levels of the other TGF-β isotypes. One possibility is that a common transcription factor exists that regulates both TGF-β1 and TGF-β3, which is regulated by TGF-β2. This possibility is currently under investigation. For one reason or another, this study provides a novel therapeutic strategy to overcome TGF-β-related immunosuppressive activities by only TGF-β2 downregulation.

Abbreviations

TGF-β:

transforming growth factor-β

shRNA:

short hairpin RNA

GFP:

green fluorescence protein

ELISA:

enzyme-linked immunosorbent assay

References

  1. 1

    Massague J . The TGF-beta family of growth and differentiation factors. Cell 1987; 49: 437–438.

  2. 2

    Moore LD, Isayeva T, Siegal GP, Ponnazhagan S . Silencing of transforming growth factor-beta1 in situ by RNA interference for breast cancer: implications for proliferation and migration in vitro and metastasis in vivo. Clinical Cancer Res 2008; 14: 4961–4970.

  3. 3

    Annes JP, Munger JS, Rifkin DB . Making sense of latent TGFbeta activation. J Cell Sci 2003; 116 (Part 2): 217–224.

  4. 4

    Massague J . TGFbeta in Cancer. Cell 2008; 134: 215–230.

  5. 5

    Pardali K, Moustakas A . Actions of TGF-beta as tumor suppressor and pro-metastatic factor in human cancer. Biochim Biophys Acta 2007; 1775: 21–62.

  6. 6

    Massague J, Blain SW, Lo RS . TGFbeta signaling in growth control, cancer, and heritable disorders. Cell 2000; 103: 295–309.

  7. 7

    Elliott RL, Blobe GC . Role of transforming growth factor beta in human cancer. J Clin Oncol 2005; 23: 2078–2093.

  8. 8

    Kim S, Buchlis G, Fridlender ZG, Sun J, Kapoor V, Cheng G et al. Systemic blockade of transforming growth factor-beta signaling augments the efficacy of immunogene therapy. Cancer Res 2008; 68: 10247–10256.

  9. 9

    Ivanovic V, Demajo M, Krtolica K, Krajnovic M, Konstantinovic M, Baltic V et al. Elevated plasma TGF-beta1 levels correlate with decreased survival of metastatic breast cancer patients. Clin Chim Acta 2006; 371: 191–193.

  10. 10

    Conroy H, Galvin KC, Higgins SC, Mills KH . Gene silencing of TGF-beta1 enhances antitumor immunity induced with a dendritic cell vaccine by reducing tumor-associated regulatory T cells. Cancer Immunol Immunother 2012; 61: 425–431.

  11. 11

    Wei H, Liu P, Swisher E, Yip YY, Tse JH, Agnew K et al. Silencing of the TGF-beta1 gene increases the immunogenicity of cells from human ovarian carcinoma. J Immunother 2012; 35: 267–275.

  12. 12

    Ding Y, Kim JK, Kim SI, Na HJ, Jun SY, Lee SJ et al. TGF-{beta}1 protects against mesangial cell apoptosis via induction of autophagy. J Biol Chem 2010; 285: 37909–37919.

  13. 13

    Dogar AM, Towbin H, Hall J . Suppression of latent transforming growth factor (TGF)-beta1 restores growth inhibitory TGF-beta signaling through microRNAs. J Biol Chem 2011; 286: 16447–16458.

  14. 14

    Schafer H, Struck B, Feldmann EM, Bergmann F, Grage-Griebenow E, Geismann C et al. TGF-beta1-dependent L1CAM expression has an essential role in macrophage-induced apoptosis resistance and cell migration of human intestinal epithelial cells. Oncogene 2013 doi:10.1038/onc.2012.44; epub ahead of print 20 February 2012.

  15. 15

    Almeida R, Allshire RC . RNA silencing and genome regulation. Trends Cell Biol 2005; 15: 251–258.

  16. 16

    Aagaard L, Rossi JJ . RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev 2007; 59: 75–86.

  17. 17

    Whitehead KA, Langer R, Anderson DG . Knocking down barriers: advances in siRNA delivery. Nature reviews. Drug Discov 2009; 8: 129–138.

  18. 18

    Zhang H, Kolb FA, Jaskiewicz L, Westhof E, Filipowicz W . Single processing center models for human dicer and bacterial RNase III. Cell 2004; 118: 57–68.

  19. 19

    Rand TA, Ginalski K, Grishin NV, Wang X . Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity. Proc Natl Acad Sci USA 2004; 101: 14385–14389.

  20. 20

    Kumar LD, Clarke AR . Gene manipulation through the use of small interfering RNA (siRNA): from in vitro to in vivo applications. Adv Drug Deliv Rev 2007; 59: 87–100.

  21. 21

    Sharma A, Tandon M, Bangari DS, Mittal SK . Adenoviral vector-based strategies for cancer therapy. Curr Drug Ther 2009; 4: 117–138.

  22. 22

    Douglas JT . Adenoviral vectors for gene therapy. Mol Biotechnol 2007; 36: 71–80.

  23. 23

    Schlingensiepen KH, Jaschinski F, Lang SA, Moser C, Geissler EK, Schlitt HJ et al. Transforming growth factor-beta 2 gene silencing with trabedersen (AP 12009) in pancreatic cancer. Cancer Sci 2011; 102: 1193–1200.

  24. 24

    Olivares J, Kumar P, Yu Y, Maples PB, Senzer N, Bedell C et al. Phase I trial of TGF-beta 2 antisense GM-CSF gene-modified autologous tumor cell (TAG) vaccine. Clin Cancer Res 2011; 17: 183–192.

  25. 25

    De Crescenzo G, Hinck CS, Shu Z, Zuniga J, Yang J, Tang Y et al. Three key residues underlie the differential affinity of the TGFbeta isoforms for the TGFbeta type II receptor. J Mol Biol 2006; 355: 47–62.

  26. 26

    Laverty HG, Wakefield LM, Occleston NL, O’Kane S, Ferguson MW . TGF-beta3 and cancer: a review. Cytokine Growth Factor Rev 2009; 20: 305–317.

  27. 27

    Hinck AP, Archer SJ, Qian SW, Roberts AB, Sporn MB, Weatherbee JA et al. Transforming growth factor beta 1: three-dimensional structure in solution and comparison with the X-ray structure of transforming growth factor beta 2. Biochemistry 1996; 35: 8517–8534.

  28. 28

    Bocharov EV, Blommers MJ, Kuhla J, Arvinte T, Burgi R, Arseniev AS . Sequence-specific 1H and 15N assignment and secondary structure of transforming growth factor beta3. J Biomol NMR 2000; 16: 179–180.

  29. 29

    Akhurst RJ, Lehnert SA, Faissner A, Duffie E . TGF beta in murine morphogenetic processes: the early embryo and cardiogenesis. Development 1990; 108: 645–656.

  30. 30

    Bierie B, Moses HL . Transforming growth factor beta (TGF-beta) and inflammation in cancer. Cytokine Growth Factor Rev 2010; 21: 49–59.

  31. 31

    Li C, Wang J, Wilson PB, Kumar P, Levine E, Hunter RD et al. Role of transforming growth factor beta3 in lymphatic metastasis in breast cancer. Int J Cancer 1998; 79: 455–459.

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Acknowledgements

This work was supported by the Industrial Strategic Technology Development program (10035562: Development of nucleic acid-based anticancer drugs overcoming the immunotherapy resistance) funded by the Ministry of Knowledge Economy (MKE, Korea). This work was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education. Science and Technology (2012-0002108). S Oh and E Kim are funded by the Brain Korea 21 project for Medical Science, Yonsei University, College of Medicine, Seoul, South Korea.

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Correspondence to J-H Kim or J J Song.

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Supplementary Information accompanies the paper on Cancer Gene Therapy website

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Keywords

  • TGF-β isotype
  • shRNA
  • adenovirus
  • antitumor
  • immunosuppression

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