The use of conditionally replicative adenoviruses (CRAd) is an attractive tool for cancer therapy. Many types of CRAd have been developed to date, some of which are currently undergoing clinical trials [1
]. Two types of genetically engineered CRAd have been examined in detail [2
]. The first group of viruses have mutations in the genes required for viral replication. For example, the E1A or E1B55k gene deleted-virus [3
] has been effective at killing pRB- or p53-deficient cancer cells. The other group consists of viruses that possess a cancer-specific transcription system in the virus genes required for replication, such as E1A. For example, promoters of the telomerase gene [5
] or prostate-specific antigen gene [6
] are inserted into the 5′- untranslated region (UTR) of the E1A gene to produce CRAds that are specifically activated in cancer cells.
The control of mRNA decay is one of the important mechanisms of the gene expression system. AU-rich elements (AREs) are RNA elements commonly present in the 3′-UTR of certain mRNAs that encode many early response genes or growth-related genes such as proto-oncogenes, growth factors, and cytokines [7
]. Multiple copies of the typical sequence AUUUA exist in ARE and target ARE-mRNA for rapid degradation [7
HuR, a member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, binds to ARE in order to protect ARE-mRNA from rapid degradation [9
]. Although HuR is predominantly localized in the nucleus, it has the ability to shuttle between the nucleus and cytoplasm, and the stabilization of ARE-mRNA by HuR has been linked to its localization in the cytoplasm [10
In cells transformed by the adenovirus oncogene product E4orf6, ARE-mRNA and its associated proteins, such as pp32 and HuR, are exported to the cytoplasm in a chromosome region maintenance 1 (CRM1)-independent manner [12
]. E4orf6 may also stabilize ARE-mRNA, which endows it with the potential to transform cells [13
]. The export of HuR and concurrent stabilization of ARE-mRNA do not solely depend on virus gene products and have been reported in many cancer cells. The cytoplasmic expression of HuR has been implicated in the malignancy of several types of carcinomas, such as colon cancer, and has also been suggested to contribute to the cancerous malignant phenotype [11
]. Increased cytoplasmic HuR level was recently identified as an important prognostic marker in several cancers [15
In the present study, we developed the oncolytic adenoviruses AdARET and AdAREF, possessing the ARE of the TNF-α
genes in the 3′-UTR of the E1A
gene, respectively. The ability of these viruses to replicate was markedly higher in cancer cells than in normal cells and occurred in an E1A expression-dependent manner. These viruses exhibit cytolytic activity for cancer cells in vitro and in vivo. These findings indicate that the viruses have potential as oncolytic viruses. In the previous study, a virus with a COX-2 ARE in the 3′-UTR of E1A was developed [17
]. This virus was developed primarily for cancer cells with ras mutations. AdARET and AdAREF were also effective in cancer cells that do not have the ras mutation. In addition, our virus also has reduced E1A expression, which means less damage to normal cells.
In this study, we described the construction and features of AdARET and AdAREF, two new oncolytic adenoviruses. These viruses contain the ARE of the TNF-α
genes in the 3′-UTR of the E1A gene, which allows them to replicate specifically in ARE-mRNA-stabilized cancer cells. Indeed, E1A expression was high specifically in cancer cells and the productive rate of these viruses were very high in several cancer cells, but were very low or negligible in normal cells. These viruses exhibited selective cytolytic activity for cultured cancer cells in vitro. In the nude mouse xenograft model, infection with these viruses significantly reduced tumor volume. In order to produce a virus that would exert less damage to normal cells, the viruses were created with the E1 region inserted in the reverse direction. Furthermore, these viruses could not express the E1B55k gene due to its interruption. The expression of E1A protein from these viruses was lower and slower than in wild-type adenovirus infected-cells and the E1B55k protein was not visible by western blot analysis. These results indicate that AdARET and AdAREF have potential as oncolytic viruses. To the best of our knowledge, there are few reports describing oncolytic viruses with tumor selectivity based on the level of mRNA stability. As mentioned, AdARET and AdAREF failed to express E1B55k due to the truncation in this gene. This feature is the same with the E1B55k gene deleted-adenoviruses such as Onyx-015 or H101, which have already been applied clinically [3
Since these viruses were expected to replicate in cancer cells through E1A-ARE mRNA stabilization, we determined whether the ARE-mRNA stabilization system was required for virus replication. In order to estimate this, we used three kinds of experiments. Two assays were performed to examine the down-regulation HuR expression and one experiment was used to observe the effect of the up-regulation of cytoplasmic HuR. We confirmed the virus titer in heat shock (HS)-treated cancer cells, as the amount of HuR was down-regulated, and HuR-targeted mRNA was also decreased in HS-treated cells [18
]. This HS treatment would down-regulate the virus titer if AdAREF actually replicated using the ARE-mRNA stabilization system. The titer of AdAREF in HS-treated cells was lower than that of non-treated cells (Figure 2
B). The reduction of AdAREF virus replication has also been seen with HuR-KD cells. HuR-KD, with two kinds of siRNA specific for HuR, reduced HuR expression and virus production (Figure 2
C). Conversely, when HuR export was activated by LPS treatment, replication of AdARET increased (Figure 2
D). These results indicate that the developed viruses grow in an ARE-mRNA stabilization system dependent manner. Since ARE-mRNA is usually known to be stabilized in many types of cancer cells [11
], AdARET and AdAREF can be expected to be effective in numerous cancers.
In a previous study, an adenovirus including the ARE of COX-2 in the 3′-UTR of the E1A gene was shown to be a conditionally replicating virus [17
]. This virus replicated selectively in RAS/P-MAPK-activated cancer cells because ARE-mRNA was stabilized under the activated RAS pathway. In the case of AdARET and AdAREF, cytolytic activity was not dependent on the RAS/P-MAPK pathway, because the virus exhibited oncolytic activity for HeLa cells, which carry a normal ras gene. These findings indicate that these viruses are effective for cancer cells carrying unmutated ras.
Since the stability of ARE-mRNA has been reported in many biological or pathological events, such as inflammation, viral infection, hypoxia, and UV irradiation, AdARET and AdAREF have potential in the treatment of diseases with these biological features.
4. Materials and Methods
4.1. Cell Lines and Antibodies
The human lung cancer cell lines A549 and H1299, cervical carcinoma cell lines HeLa, HeLa S3, and C33A, breast cancer cell lines MCF-7 and MDA-MB-231, osteosarcoma cell lines U2OS and Saos-2, prostate cancer cell line PC3, oral cancer cell line HSC-3, human embryonal kidney cell line (transformed by the adenovirus E1 gene) 293, human foreskin fibroblast cell line BJ, gingival fibroblast cell line HGF1, lung fibroblast MRC5, mammary epithelial cell line HMEC, and small airway epithelial cell line SAEC were all used in the present study. All cells were obtained from the American Type Culture Collection or Lonza and cultured in DMEM containing 10% FBS with antibiotics at 37 °C in a 5 -% CO2 atmosphere under humidified conditions.
For western blot analysis, the following antibodies were used: antibodies specific to E1A (M58, sc-58658, Santa Cruz Biotechnology, Dallas, TX, USA), E1B55k (2A6, generous gift from T. Shenk), HuR (3A2, sc-5261, Santa Cruz Biotechnology, Dallas, TX, USA), Hexon (Abcam, Cambridge, United Kingdom), β-actin (Actin(C4) hrp, sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), β-tubulin (05–661, EMD Millipore Corp., Darmstadt, Germany), Lamin B (C-20, sc-6216, Santa Cruz Biotechnology, Dallas, TX, USA), and Anti-Adenovirus Type 5 antibody (ab6982, Abcam, Cambridge, United Kingdom) as primary antibodies. Anti-mouse (m-IgGκ BP-HRP: sc-516102, Santa Cruz Biotechnology, Dallas, TX, USA) and Anti rabbit (mouse anti-rabbit IgG-HRP: sc-2357, Santa Cruz Biotechnology, Dallas, TX, USA) were used as secondary antibodies.
4.2. Construction of AdARET and AdAREF
AdARET and AdAREF were constructed using a pXhoIC plasmid [25
] and pAxcwit2 cosmid (Takara Bio., Kusatsu, Shiga, Japan). pXhoIC contained the E1 region of the type 5 human adenovirus genome. A 40-base pair of synthesized ARE fragment (5′-gtgattattt attatttatt tattatttat ttatttacag-3′) from the TNF-α
and a 69-base pair of synthesized ARE fragment (5′- ttttattgtg tttttaattt atttattaag atggattctc agatatttat atttttattt tattttttt -3′) from the c-fos
genes were inserted into the HpaI site in the 3′-UTR of the E1A gene of pXhoIC. A fragment of the E1 region was then amplified by PCR, and inserted into the deleted E1 region of the adenovirus genome in the pAxcwit2 cosmid using SmiI-restricted endonuclease. The entire genome of AdARET and AdAREF were isolated by cutting using PacI and each fragment was then transfected into 293 cells. Virus particles were concentrated by several rounds of viral infection and cells were collected in order to prepare a virus lysate by subjecting them to three cycles of freezing and thawing. The titers of the infectious unit (ifu) of these viruses were determined using the Adeno-XTM
Rapid Titer Kit (Cat no-632250, Clontech Laboratories, Mountain View, CA, USA) and 293 cells according to the manufacturer’s instructions. In order for a virus to be used in in vivo experiments, its extract was purified using a Fast Trap adenovirus purification and concentration kit (MILLIPORE) according to the manufacturer’s protocols. A Quick titer adenovirus quantitation kit (VPK-106, Cell Biolabs, San Diego, CA, USA) was used to count virus particles.
4.3. Western Blot Analysis
Western blot analysis was performed as previously described [26
]. Cells were lysed with RIPA buffer (150 mM NaCl; 25 mM Tris-HCl, pH 7.6; 1% Nonidet P-40; 1% sodium deoxycholate; 0.1% SDS) containing protease inhibitors. Equal amounts (20 µg) of total protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The binding of antibodies was visualized using Supersignal West Femto Maximum Sensitivity Substrate (Cat-34095, Thermo Fisher Scientific, Waltham, MA, USA).
4.4. In Vitro Virus Proliferation Assay
Human tumor and normal cells were seeded at 5.0 × 104 cells/well 24 h before infection. Cells were infected with AdARET and AdAREF at an MOI of 100 vp/cell. These infected cells were incubated at 37 °C for 72 h, then collected, and a virus lysate was prepared as described above. Titers of the viruses were determined using the Adeno-XTM Rapid Titer Kit (Clontech, Laboratories, Mountain View, CA, USA) and 293 cells.
4.5. HuR Manipulation
For HuR knockdown, Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) was used to transfect HeLa cells with 20 nM each of siRNAs targeting HuR 1 (5′-AAG UGC AAA GGG UUU GGC UUU UU-3′) or HuR 2 (5′-AAU CUU AAG UUU CGU AAG UUA UU-3′) or a negative control siRNA (5′-TCT TAA TCG CGT ATA AGG CTT-3′; Qiagen, Hilden, Germany). Forty-eight hours after transfection, A549 cells were infected with AdARET and AdAREF. After 48 h of infection, all cells were scrapped to purify total protein, and the virus lysate was prepared with three freeze-thaw cycles. Viral titers were detected by using the Adeno-X Rapid Titer kit (Clontech Laboratories, Laboratories, Mountain View, CA, USA) and 293 cells.
For heat shock treatment, A549 cells were kept at 43 °C for 2 h immediately after being infected with AdARET and AdAREF. Cells were heat shocked (for 2 h) again 24 h after infection. Cells were harvested at 24 h after infection and the viral titers were determined as described above.
In order to promote cytoplasmic localization of HuR, cells were treated with 25 ng/mL LPS (E. Coli 0111: B4, Sigma-Aldrich, St Louis, MO, USA).
A549 and HeLa cells were grown over coverslips in 24 well plates at 60–80% confluence and treated with LPS (25 ng/mL). For HuR staining, cells were rinsed twice with phosphate buffered saline (PBS), fixed for 10 min at RT with 4% paraformaldehyde in PBS at room temperature, blocking and permeabilization was completed using 1% BSA plus 0.25% Triton X-100 in PBS at room temperature. The fixed cells were subsequently incubated with HuR primary antibody for overnight at 4 °C followed by Alexa Fluor 488 secondary antibodies for 1 h at room temperature, respectively. DAPI was used to counterstain cell nuclei before cells were mounted on slides using Mountant permafluor (Thermo scientific, FM 111212A, Waltham, MA, USA). Cells were observed with an IX71 inverted microscope (Olympus, Tokyo, Japan). Image acquisition was performed with the Olympus Fluoview Software (FV10-ASW 4.2 viewer).
4.7. Cytopathic Effect Assay and Cell Viability Assay
Human cancer and normal cells were plated on 24-well plates (5 × 104 cells/well). Twenty-four hours later, cells were infected with AdARET and AdAREF at a multiplicity of infection (MOI) of 1, 10, 20, 50, or 100 virus particles (vp)/cell and maintained for an additional seven days. Cells were then fixed and stained with Coomassie brilliant blue. A 2–3-bis [2-methoxy-4-nitro- 5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay was used to examine cell viability. Cells were seeded on 96-well plates at a density of 3.0 × 103 cells/well 24 h before viral infection. Twenty-four hours later, AdARET and AdAREF were infected at a MOI of 100 vp/cell. Cell viability was determined using an XTT assay on days 3 and 7 with Cell proliferation kit II (Cat no-11465015001, Roche Diagnostics, Roche, Germany) according to the manufacturer’s protocol.
4.8. In Vivo Human Tumor Model
Human Cervical Cancer HeLa S3 cells (1.0 × 106 cells/mouse) were injected subcutaneously into the flanks of female BALB/cAJc1-nu/nu nude mice (5-week-old and 20–22 g) and permitted to grow to approximately 9–10 mm in diameter. Mice were randomly divided into three groups (five per group), and 109 vp (100 µL) of dl312, AdAREF and AdARET were injected twice (at days 1 and 4) directly into the tumor. The perpendicular diameters of the HeLa S3 tumors were measured every four or five days. Tumor volumes were calculated using the following equation: volume (mm3) = A × B2 × 0.5 (where A is the longest diameter and B is the shortest diameter). The body weight and motor activity of each animal was monitored as indicators of general health and toxicity. The mice were sacrificed by cervical dislocation 25 days following injection of the virus. All techniques involving animals in this study were performed according to the ethical standards of the Animal Care and Use Committee of the Hokkaido University. Sapporo, Japan (Permission number for the animal experiment: 19–0099).
Immunohistochemical staining was performed with serial tissue sections (4.5 µm thick) from formalin-fixed, paraffin-embedded tissue blocks. In brief, all sections were deparaffinized in xylene, rehydrated in graded alcohol, and subjected to antigen retrieval by heat treatment in Tris-ethylenediaminetetraacetic acid (TE) buffer. In order to inhibit endogenous peroxidase activity, the slides were then immersed in 3% hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA) for 5 min, and then blocking solution [1% bovine serum albumin (Sigma-Aldrich) in phosphate-buffered saline (PBS)] for 30 min. The immunohistochemical detection of Adenovirus 5 (Abcam, ab6982) was performed using anti-Adenovirus Type 5 (Rabbit Adenovirus antibody, dilution, 1:1,000; catalog no., ab6982; Abcam) in PBS in a humidified chamber at 4 °C overnight. The sections were then subjected to anti-rabbit secondary antibody (H1901, Nichirei Bioscience, Tokyo, Japan) at 37 °C for 30 min, followed by antibody detection using a peroxidase-conjugated streptavidin-diaminobenzidine (DAB) readout system (DAKO), and counterstaining with DAPI. Rinses were carefully performed with several changes of PBS between each stage of the procedure (5 min washes repeated three times). Images were randomly captured using a nanozoomer slide scanner and NDPViewer (NanoZoomer 2.0 HT, version 2.3.27, Hamamatsu, Shizuoka, Japan).