Abstract Prostate cancer is a major health problem for men in Western societies. Here we report a Prostate Cancer-Specific Targeting Gene-Viro-Therapy (CTGVT-PCa), in which PTEN was inserted into a DD3-controlled oncolytic viral vector (OV) to form Ad.DD3.E1A.E1B(Δ55)-(PTEN) or, briefly, Ad.DD3.D55-PTEN. The woodchuck post-transcriptional element (WPRE) was also introduced at the downstream of the E1A coding sequence, resulting in much higher expression of the E1A gene. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical bio-diagnostic marker. PTEN is frequently inactivated in primary prostate cancers, which is crucial for prostate cancer progression. Therefore, the Ad.DD3.D55-PTEN has prostate cancer specific and potent antitumor effect. The tumor growth rate was almost completely inhibited with the final tumor volume after Ad.DD3.D55-PTEN treatment less than the initial volume at the beginning of Ad.DD3.D55-PTEN treatment, which shows the powerful antitumor effect of Ad.DD3.D55-PTEN on prostate cancer tumor growth. The CTGVT-PCa construct reported here killed all of the prostate cancer cell lines tested, such as DU145, 22RV1 and CL1, but had a reduced or no killing effect on all the non-prostate cancer cell lines tested. The mechanism of action of Ad.DD3.D55-PTEN was due to the induction of apoptosis, as detected by TUNEL assays and flow cytometry. The apoptosis was mediated by mitochondria-dependent and -independent pathways, as determined by caspase assays and mitochondrial membrane potential.

Citation: Ding M, Cao X, Xu H-n, Fan J-k, Huang H-l, Yang D-q, et al. (2012) Prostate Cancer-Specific and Potent Antitumor Effect of a DD3-Controlled Oncolytic Virus Harboring the PTEN Gene. PLoS ONE 7(4): e35153. https://doi.org/10.1371/journal.pone.0035153 Editor: Ilya Ulasov, University of Chicago, United States of America Received: September 13, 2011; Accepted: March 9, 2012; Published: April 11, 2012 Copyright: © 2012 Ding et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Basic Research Program of China (973 Pro_ram) (No. 2010CB529901) (X. Liu), Important National Science & Technology Specific Project of Hepatitis and Hepatoma Related Program (2008ZX10002-023) (X. Liu), New Innovation Program (2009-ZX-09102-246) (X. Liu), the Zhejiang Sci-Tech University grant (1016834-Y), the National Basic Research Program of China (2009CB941704) (R. Li), the Shanghai Municipal Committee of Science and Technology (09140903200, 09DJ1400400, 08140901700 and 06ZR14072) (R. Li). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Prostate cancer is a major health problem for men in Western societies. Each year, approximately 230,000 American males are diagnosed with prostate cancer and nearly 30,000 die from this disease [1], [2]. The best available treatment for patients with the advanced disease is androgen ablation therapy, based on the observations of Huggins and Hodges [3] that clinical prostate cancer is under the trophic influence of male hormones. Tumor regression and improvement of clinical symptoms are temporary and the disease inevitably progresses to an androgen-independent state. Currently, no curative therapy is available for androgen-independent prostate cancers. Bio-therapy provides an attractive opportunity to target androgen-independent prostate cancers. Unlike traditional chemotherapy, it can be designed and customized to specifically target cancers according to our understanding of the disease at a molecular level. The adenoviral vector has been used as a transfer vehicle to introduce genes into cancer cells because it is more efficient than non-viral gene transfer methods [4], [5]. The adenoviral vector is stable in vivo, efficiently delivers genes to both dividing and non-dividing cells and rarely causes any significant disease itself [6], [7]. However, the traditional adenovirus used for gene therapy is a replication deficient one with low expression level of the therapeutic gene. Cancer-specific replication of the oncolytic vector, therefore, is absolutely required to prevent these problems. Two major strategies have been used to construct a replicative and cancer-specific oncolytic adenovirus. The first is to delete the viral element that is necessary for virus replication in normal cells, which is not required in tumor cells. For example, the E1B-55K gene, which was deleted in the oncolytic viruses ONYX-015 and ZD55 [8], [9], [10], is required for viral replication in normal cells but is dispensable in cancer cells due to compensatory mechanisms. The second strategy is to replace the promoter of a key gene in adenovirus replication, such as the E1A or E1B gene, with a tumor -specific promoter [11], [12]. Thus, the oncolytic virus is highly replicated when the promoter is activated. To overcome the limitations of traditional gene therapy with an replication deficient adenoviral vector, a Cancer-Targeting Gene-Viro-Therapy (CTGVT) was constructed by inserting an anti-tumor gene into a double-targeted oncolytic viral(OV) vector. It is actually an OV-gene, and has much better antitumor effect than that of either gene therapy alone or virotherapy alone. The oncolytic virus itself has anti-tumor power and selectively replicates several hundred-fold in tumor cells and thus, the therapeutic genes should also be selectively replicated several hundred-fold in tumor cells [11]. By innovatively integrating gene therapy and viro-therapy, the anti-tumor effect of the CTGVT is generally much higher than either method alone. On January 27, 2011, Amgen spent 1 billion USD to purchase OncoHSV-GM-CSF (an oncolytic virus from Herpes Simplex Virus-1), which is in phase III for advanced melanoma [13], [14], underlining the importance and potential of the CTGVT strategy. In this study, we first generated a double-regulated adenovirus, Ad.DD3.D55, in which the E1A gene is under the control of DD3 promoter and the E1B-55K gene was deleted. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical biomarker for prostate cancer [15]. Interestingly, a 214-bp fragment of the DD3 core promoter has a high promoter activity [16], and therefore it was used to control E1A expression in the oncolytic adenovirus. Because DD3 expression is highly restricted in prostate cancer, we previously used the minimal DD3 promoter to drive expression of E1A for generating an oncolytic virus [17]. The level of DD3 promoter-driven E1A expression was insufficient, therefore, WPRE was introduced to enhance the expression of the E1A gene in this study. WPRE promotes gene expression in a promoter- and cell-line-dependent manner [18]. WPRE has been exploited as an enhancer of transgene expression by enhancing nuclear export of an aberrantly retained messenger RNA from the nucleus to the cytoplasm [19]. In addition, PTEN is a well-known tumor suppressor gene that encodes a protein phosphatase [20]. Its deletion has been observed in high-grade prostate cancer and is very important for prostate cancer progression [21]. In this study, PTEN with a CMV promoter was inserted into Ad.DD3.D55 to form Ad.DD3.D55-PTEN, which has a prostate cancer replication specificity and antitumor gene specificity. Ad.DD3.D55-PTEN efficaciously induces apoptosis in prostate cancer cells, eliminating prostate cancer xenografts with higher antitumor efficiency.

Discussion In this study, Ad.DD3.D55-PTEN demonstrated an excellent antitumor effect in vitro and in vivo. Two major factors contribute to the cytotoxicity exerted by Ad.DD3.D55-PTEN. The first is that it replicates selectively in prostate cancer cells. We previously reported that the minimal DD3 promoter-controlled oncolytic virus replicated selectively in prostate cancer cells [17], although the promoter activity was not very satisfactory. PSA is a well-known biomarker of prostate cancer and its promoter has been widely used to study biotherapies for prostate cancer, including gene therapy with different vectors [22], [23], [24], [25]. Different recombinant promoters have been generated based on the Prostate Cancer-specific activity of a promoter or enhancer to increase the Prostate Cancer-specific activity of the promoter controlling the replication of the oncolytic virus [26], [27], [28], [29]. Cell killing efficacy could be improved by many methods, such as combining the oncolytic adenovirus with radiotherapy or chemotherapy. Clinical trials have shown that the combination of ONYX-15 virus and chemotherapy elicits a stronger anti-tumor response against head and neck cancer than ONYX-15 or chemotherapy alone [30]. Patients with prostate cancer have been treated most frequently with androgen ablation. Because the activity of the PSA promoter/enhancer is highly dependent on endogenous androgen [31], the in vivo cytotoxicity of a PSA promoter/enhancer-based oncolytic virus could be reduced significantly if it was used in patients who have been treated with androgen ablation. In this study, we introduced WPRE in the DD3-controlled E1A expression cassette. Interestingly, the presence of WPRE raised the levels of reporter expression considerably but did not significantly affect the restricted expression in prostate cancer cells. To our knowledge, this is the first reported use of WPRE in the generation of an oncolytic virus. In addition, our data show that Ad.DD3.D55-PTEN, in which E1A expression is under the control of the DD3 promoter and WPRE, exerted a strong inhibitory effect on the growth of androgen-dependent as well as androgen-independent cell lines, including CL1, DU145 and 22RV1 in cell culture and CL1 tumor growth in a xenograft nude mouse. Ad.DD3.D55-PTEN has a powerful cytotoxic effect in androgen-independent cell lines. Therefore, E1A expression under the control of the DD3 promoter and WPRE may be clinically significant for oncolytic viral therapy of patients with prostate cancer. Our results showed that the expression of PTEN played the second key role in the cytotoxicity of Ad.DD3.D55-PTEN. PTEN acts as a phosphatidylinositol phosphatase with a possible role in phosphatidylinositol 3′-kinase (PI3′K)-mediated signal transduction, suppressing the PI3K/AKT pathway by reducing the level of PI3′K in the cell [32], [33]. The oncolytic virus Ad.DD3.D55 and Ad.DD3.D55-PTEN led to an increase in phospho-Akt expression at 24 h after infection(Fig. 2.D); the activation of PI3K-Akt signaling is the pathway employed by viruses for viral endocytosis and replication [34], [35], [36], [37]. Ad.DD3.D55-PTEN inhibited phosphorylation of Akt at its activating residue (Ser473) as a result of PTEN expression, while the activation of Akt was maintained with the infection of Ad.DD3.D55(Fig. 2.D). The difference clearly indicated the powerful role of PTEN. In addition, PTEN has been identified as the inactivating alteration in multiple human cancer types, such as glioma, breast, lung, prostate, bladder, melanoma and kidney tumors, astrocytoma, and leukemia [38]. In prostate cancer, the loss of the PTEN protein is correlated with tumors of high grade and stage, suggesting that alterations in the PTEN gene may be associated with prostate cancer progression [21], [39]. Wu et al. established a mouse model with prostate-specific deletion of PTEN [40]. This mouse model recapitulates prostate cancer progression in humans: initiation of prostate cancer with prostatic intraepithelial neoplasia, followed by progression to invasive adenocarcinoma and subsequent metastasis. Therefore, PTEN is an optimal candidate for cancer gene therapy due to its unique properties. Introduction of PTEN by adenovirus or retrovirus has been studied for bio-therapy of various type of cancer, including ovarian, endometrial, bladder, gastric, colorectal, and esophageal cancers and glioblastoma [41], [42], [43], [44], [45], [46], [47]. For example, Gallick et al. reported that the adenoviral-mediated expression of PTEN inhibited proliferation and metastasis of human prostate cancer cells in vitro and in vivo [48]. Adenoviral-mediated PTEN transgene expression combined with radiotherapy, chemotherapy and other cancer-suppressing genes has been used to treat prostate cancer cells to boost the antitumor effect [49], [50], [51], [52]. In this study, high levels of PTEN expression driven by the CMV promoter were mediated by an oncolytic virus that replicated selectively in prostate cancer cells by using DD3 promoter to drive E1A expression. The antitumor efficacy of PTEN in the oncolytic virus was much enhanced compared with the adenovirus-mediated PTEN that was previously reported for use in prostate cancer [48], [52], it could be resulted from that Ad.DD3.D55-PTEN induced a massive apoptosis in prostate cancer cells by intrinsic and extrinsic pathways. We observed varying sensitivities of the different prostate cancer cell lines to the cytotoxicity of Ad.DD3.D55-PTEN. The level of reporter expression driven by the DD3 promoter and WPRE in LNCaP cells was only 21.79% of that in 22RV1 cells (Fig. 1), suggesting a lower replication of Ad.DD3.D55-PTEN in LNCaP cells, which would seem inconsistent with the higher killing efficacy of Ad.DD3.D55-PTEN in LNCaP cells than in 22RV1 cells (Fig. 3). However, 22RV1 cells express a functional PTEN, while LNCaP cells do not [53]. Given the above data, Ad.DD3.D55-PTEN probably exerts a stronger cytotoxic effect in PTEN-negative PCa cells than in PTEN-positive PCa cells. Volumes of tumor of CL1 cells was redued by 41.3% 20 days after infected with Ad.DD3.D55-PTEN in the nude mouse (Fig. 6A) strongly suggests that Ad.DD3.D55-PTEN has the therapeutic potential for the treatment of androgen-independent cancers. This study revealed that adenoviral replication slowed after approximately 12 days of active replication (Fig. 6B). Interestingly, even the rate of oncolytic virus kept very low in vivo after the period (Fig. 6B), the infected tumor still did not significantly grow(Fig. 6A). Its mechanism for this phenomenon remains unknown. One plausible explanation is that a vast majority of cancer cells was killed in the first days after injection of Ad.DD3.D55-PTEN, while growth of the remaining cancer cells is efficiently inhibited even in the presence of a minimal amount of oncolytic virus. The phenomenon is probably encouraging since most of virus will be cleared up in vivo when the oncolytic virus are used clinically. In conclusion, we developed a novel prostate-specific CTGVT(CTGVT-PCa), Ad.DD3.D55-PTEN, by controlling the expression of the E1A gene with a minimal DD3 promoter and WPRE. Ad.DD3.D55-PTEN had excellent anti-tumor efficacy in both androgen-dependent and -independent prostate cancer cell lines. This report also provides a new strategy for constructing a CTGVT-PCa with tumor types specificity that have few tumor-specific promoters available.

Materials and Methods Ethics Statement and Animal Experiment Male BALB/c nude mice (4-week-old) were maintained and used in a light and temperature controlled room in an AAALAC-accredited facility, and given water and lab chow ad libitum [17]. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Institute of Biochemistry and Cell Biology under protocol IBCB-SPF0029. To establish xenograft tumors, 5×106 22RV1 or CL1 cells in 150 µl DMEM were subcutaneously injected into the right flank of each mouse. When tumors reached 70–150 mm3 in size, mice were randomly assigned to one of four groups (six mice per group). Adenovirus (2×109 PFUs per mouse) or PBS was injected into the tumors every other day, with a total of three injections. The tumor volume (mm3) was measured with a vernier caliper every four days and calculated as (length×width2)/2. To study viral kinetics in vivo, tumors injected with Ad-PTEN or Ad.DD3.D55-PTEN were collected at 3, 6, 9, 12 and 15 days after the last injection and quickly frozen in liquid nitrogen. The total tumor DNA was extracted using the Genomic DNA MiniPreps Kit (Generay Biotech, Shanghai, China). Viral DNA was measured by real-time PCR using primers that anneal to the adenovirus E3 region. The oligonucleotide primers used for amplification are listed in Table 1. Genomic DNA amplified by β-actin primers was used as an internal control. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Nucleotide sequence of oligonucleotide primers used for virus identification, RT-PCR and real-time PCR. https://doi.org/10.1371/journal.pone.0035153.t001 Cells and Cell Culture The human cell lines used in this study were LNCaP, DU145, 22RV1,PC3 (prostate cancer), T-24 (bladder cancer), BEL-7404 (liver cancer), HeLa (cervical cancer), BEAS-2B (bronchial epithelial cell), HFL-I (embryonic lung fibroblast), L-02 (liver cell) and WISH (amnion cell). All cell lines were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). CL1 cells (prostate cancer) were grown as described by Tso et al. [54]. HEK293 was obtained from Microbix Biosystems, Inc. (Toronto, Ontario, Canada). The cell lines were incubated in DMEM or RPMI 1640 supplemented with 5–10% heat-inactivated fetal bovine serum (FBS) at 37°C in a humidified air atmosphere with 5% CO 2 . Luciferase Assay and Construction of Different Adenoviruses The minimal DD3 promoter (AF279290 nt309–522) was cloned as described previously [17]. The luciferase reporter plasmids pGL3-basic and pGL3-control were purchased from Promega Corp. (Madison, WI, USA). The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was a kind gift from Dr. Xiaoming Xie and was inserted downstream of the luciferase reporter gene between the XbaI and NheI sites in the plasmid pGL3-DD3 to construct the plasmid pGL3-DD3-WPRE. The luciferase reporter gene assay was performed as previously described [17]. All results were the average of three independent experiments. Different adenoviruses were generated by homologous recombination using Effectene Transfection Reagent (Qiagen, Germany) with the plasmid pBHGE3 and the plasmids pAd.DD3.D55 or pAd.DD3.D55-PTEN. Wild-type adenovirus (Ad.WT) was previously preserved in our laboratory [55]. The viral structure was confirmed by PCR. The sequences of the primers used for PCR are listed in Table 1. Western Blot Analysis The different cells were harvested at various times after infection with the indicated viruses at an MOI of 10. The total protein was extracted in cell lysis buffer (Beyotime, Shanghai, China). Protein concentrations were measured with the Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). The protein samples were electrophoretically separated on an 8–12% SDS-polyacrylamide gel and transferred to a PVDF membrane. The membranes were blocked for 30 min with 5% nonfat milk, sequentially incubated with primary and secondary antibodies and finally developed with ECL Western blot detection reagents (Pierce Biotechnology, Rockford, Illinois, USA) according to the manufacturer's instructions. Primary antibodies for E1A, E1B-55K, procaspase-3, poly(ADP-ribose) polymerase (PARP), Akt(Pan)(C67E7) and phospho-Akt(Ser473)(D9E) and all of the secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PTEN, cleaved-PARP and procaspase-8 antibodies were purchased from Cell Signaling Inc. (Danvers, MA, USA). The procaspase-9 antibody was purchased from Thermo Fisher Scientific Inc. (Fremont, CA, USA). The XIAP antibody was purchased from R&D Systems Inc. (Minneapolis, MN, USA) and the tubulin antibody was manufactured by Sigma-Aldrich (St. Louis, MO). Crystal Violet Cytopathic Assay Cells were seeded in 24-well plates and infected with Ad-PTEN, Ad.DD3.D55, Ad.DD3.D55-PTEN or wild type adenovirus at various MOIs. Five days after infection, cells were exposed to 2% crystal violet dissolved in 20% methanol for 5 min. The plates were then washed with distilled water (dH 2 O) and documented by photography. Colorimetric MTT (Tetrazolium) Cell Viability Assay Cells cultured in 96-well plates were incubated with viruses at an MOI of 10. MTT solution (20 µl, 5 mg/ml) was added to each well after various times and the absorbance was measured in dual wavelength mode (595 nm and 655 nm). The percentage of cell viability was calculated as follows: (mean A595/A655 of infected cells)/(mean A595/A655 of uninfected cells)×100. Apoptotic Cell Staining Cells grown on glass coverslips were treated with viruses at an MOI of 10 for 48 h and stained with 1 µg/ml Hoechst 33258 (Molecular Probes, Eugene, OR, USA). Immunohistochemical Analysis and TdT–mediated dUTP Nick End Labeling (TUNEL) Assay Frozen tumor sections were incubated in 1% H 2 O 2 and 0.4% Triton X-100 to eliminate endogenous peroxidase activity. The sections were then blocked with the blocking serum and incubated with the corresponding antibody. The subsequent procedures were performed using the ABC Staining System (Westang, Shanghai, China) according to the manufacturer's instructions. The apoptotic cells in tumor tissue sections were observed by using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining with a TACSTM 2 TdT-DAB In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer's instructions. The sections were counterstained with hematoxylin. All sections were visualized with an Olympus BX51 microscope (Olympus, Tokyo, Japan). Statistical Analysis The differences between the groups were assessed using the Student's t-test.

Acknowledgments We thank Lanying Sun for cell culture assistance and the Cell Analysis Center (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) for professional technical assistance.

Author Contributions Conceived and designed the experiments: MD XL XC JF DY. Performed the experiments: MD HX. Analyzed the data: MD HH. Contributed reagents/materials/analysis tools: YL JW. Wrote the paper: MD RL.