Volume 12 Issue 4 - January 15, 2010 PDF
Tumor-Selective Replication of an Oncolytic Adenovirus Carrying Oct-3/4 Response Elements in Murine Metastatic Bladder Cancer Models
Chao-Liang Wu1, Gia-Shing Shieh1, Ai-Li Shiau2,*
1Department of Biochemistry and Molecular Biology and 2 Departments of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan
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Oct-3/4 (also known as Oct-4, Oct-3 and POU5f1), a member of the POU family, is an octamer-binding transcription factor that is a key regulator in undifferentiated, pluripotent cells including human embryonic stem cells and germ cells. Oct-3/4 has been demonstrated to be a sensitive and specific immunohistochemical marker of gonadal germ cell tumors, specifically seminoma and embryonal carcinoma. Recent investigations have shown that Oct-3/4 gene was expressed in several human cancer cells but not in normal somatic tissues. It was believed to play a critical role in tumorigenesis. Although only a few targets of Oct-3/4 transcriptional regulation are known, it was revealed that Oct-3/4 can function as a homo or heterodimer on palindromic octamer DNA sequences (classic consensus ATTTGCAT) to repress or activate transcription according to flanking sequence or chromatin structure.

Oncolytic viruses have been used as a cancer treatment option since they selectively replicate in and destroy cancer cells. They utilize multiple mechanisms of action to kill cancer cells including cell lysis, cell apoptosis, antiangiogenesis and cell necrosis. Clinical trials of oncolytic viruses for head and neck, ovarian, brain and prostate cancers have been conducted and have been encouraging in terms of efficacy with minimal, if any, toxicity. However, current therapies for metastatic tumors have been a lack of efficacy. Successful treatment of metastases requires systemic effects and it still remains a major challenge for tumor therapy. Consequently, targeting of oncolytic adenoviruses to tumors to increase the efficacy and safety profile after systemic application has become an important issue for virotherapy.

A novel tumor-selective oncolytic virus, designated Ad.9OC (Ad5-9xORE-mini-CMV) was constructed and examined for the feasibility of this oncolytic virus with 9 copies of Oct-3/4 response elements to enhance tumor-selective viral replication, oncolysis, antitumor efficacy and survival advantage in murine tumor models.
Fig. 1. Ad.9OC replicates in bladder cancer cell lines and in bladder tumor tissues and exerts antitumor effects in vivo. A. Detection of adenoviral E1A, fiber, and hexon proteins in murine bladder cancer cells as well as human Hep3B hepatocellular carcinoma cells, but not in normal murine NMuMG epithelial cells after Ad.9OC infection. Various cells were infected with Ad.9OC at a multiplicity of infection (MOI) of 10 for 48 or 72 h, and their cell lysates were examined by immunoblot analysis. Expression of β-actin served as the quantitative control. Note that MBT-2/LM7 cells produced more adenoviral proteins than MBT-2 cells did. B. Detection of adenoviral fiber and hexon proteins in murine bladder tumors treated with Ad.9OC, but not in those treated with either Ad.YH or saline. Groups of three C3H/HeN mice bearing MBT-2 or MBT-2/LM7 tumors were inoculated intratumorally (i.t.) with Ad.9OC, Ad.YH (an adenovirus driven by the CMV minimal promoter), or saline at day 15. Tumors were excised from the treated mice at day 17, and the cryosections were detected for adenoviral fiber and hexon expression by immunohistochemical staining. The scale bar shown on 400× images corresponds to 100 μm. C, D. Antitumor effects of Ad.9OC in the subcutaneous tumor models. Groups of MBT-2 tumor- or MBT-2/LM7 tumor-bearing C3H/HeN mice were treated i.t. with 5 × 108 PFU of Ad.9OC or Ad.YH, or with saline at days 9, 11, and 13. Mean tumor volumes and Kaplan-Meier survival curves in the MBT-2/LM7 (C) and MBT-2 (D) models. *, p<0.05; **, p<0.01.

MBT-2 and its lung-metastasis subline, MBT-2/LM7, cells were inoculated subcutaneously into C3H/HeN mice respectively to evaluate the antitumor effects of Ad.9OC. Tumor growth and mouse survival were monitored after intratumoral Ad.9OC injection (Figure 1C, D). Ad.9OC treatment significantly retarded tumor growth on both MBT-2- or MBT-2/LM7-bearing mice compared with control virus, Ad.YH, or saline treatment. In addition, Ad.9OC exerted significantly greater antitumor effects on the metastatic, high Oct-3/4 expressing MBT-2/LM7 cells (p=0.0043) than on the non-metastatic MBT-2 cells (p=0.029) compared to the saline treatment. Taken together, these results indicated that Ad.9OC treatment resulted in significantly greater tumor growth inhibition and prolongation of survival in the metastatic MBT-2/LM7-bearing mouse model than in the non-metastatic MBT-2-bearing mouse model. To determine the therapeutic efficiency of Ad.9OC for lung metastasis, an experimental lung metastasis model was generated by intravenous injection of MBT-2/LM7 cells into the tail vein of C3H/HeN mice followed by intravenous injection of Ad.9OC, Ad.YH or saline. In lung colony formation assay, Ad.9OC reduced the lung colonization number of MBT-2/LM7 cells, resulting in fewer and smaller formation of tumor colony than the Ad.YH or saline groups. Histological sections also revealed that Ad.9OC-treated animals showed minor tumor lesions compared with the Ad.YH or saline group (Figure 2).

Oct-3/4 expression selectively modulated the replication of Ad.9OC, and therefore increased specificity and efficacy of Ad.9OC for Oct-3/4-expressing cancer cells compared to the control virus. Furthermore, Ad.9OC revealed higher antitumor efficacy in the Oct-3/4-high- expressing, metastatic bladder cancer tumor model than in the non-metastatic bladder cancer tumor model. Taken together, our data suggest that Ad.9OC may have therapeutic potential for the treatment of Oct-3/4 overexpressing metastatic cancers.
Fig. 2. Ad.9OC is effective in reducing pulmonary metastatic bladder tumors. C3H/HeN mice that had been injected with MBT-2/LM7-Luc cells (5 × 105) via the tail vein were treated i.v. with 5 × 107 PFU of Ad.9OC or Ad.YH, or with saline at days 3 and 5, and sacrificed at day 20. A. Gross appearances of formalin-fixed lungs from the treated mice. Note that the numbers and sizes of tumor nodules indicated by arrows were reduced in mice treated with Ad.9OC compared with those treated with either Ad.YH or saline. B. Measurement of the Luciferase activity in the lungs. Distribution of RLU normalized to protein levels for each mouse from the three treatment groups. The mean values are indicated by horizontal bar. C. Histological analysis of tumor nodules in the lungs. Note that H&E staining of 4-μm paraffin-embedded lung tissue sections reveals the presence of larger tumor nodules in the lungs from saline- or Ad.YH-treated mice compared with those from Ad.9OC-treated or normal mice. The scale bars shown on 40× and 100× images correspond to 2.0 mm and 500 μm, respectively. D. Quantitative analysis at the microscopic level of tumor nodules in the lungs. Growth index (mean area of metastasis/total area) of tumor nodules was calculated from at least three fields with highest density of tumor nodules in each section.
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