Abstract 3316: Oncolytic adenovirus-armed p53 induces apoptosis significantly through upregulating miR-93 and 106b in human osteosarcoma cells..
Authors: Hasei Joe , Sasaki Tsuyoshi , Tazawa Hiroshi , Hashimoto Yuuri , Kunisada Toshiyuki , Urata Yasuo , Ozaki Toshifumi , Fujiwara Toshiyoshi 
Published: August 2014

Bone and soft tissue sarcomasare the third most common cancer in children and account for 15.4% of all childhood malignancies. About one fourth of the patients show a poor response to conventional chemo-radiotherapy, resulting in subsequent recurrence and leading to a poor prognosis. Therefore, the development of a novel therapeutic strategy is required. We previously developed an oncolytic adenovirus, OBP-301 (Telomelysin), in which the hTERT gene promoter drives the expression of the E1A and E1B genes linked to an internal ribosome entry site (IRES). Recently, we revealed that OBP-301 shows the cytopathic activity in human bone and soft tissue sarcoma cells. However, some human osteosarcoma cells (MNNG/HOS, SaOS-2) were less sensitive to cytopathic activity of OBP-301. Therefore, we developed a telomerase-specific p53-armed oncolytic adenovirus (OBP-702). In this study, we examined the antitumor effect of OBP-702 in OBP-301-resistant osteosarcoma cells, and revealed the mechanism. We compared the antitumor effects of OBP-702 and OBP-301 using XTT assay. The 50% inhibiting dose (ID50) value of OBP-702 and OBP-301 for each cell was calculated using cell viability data obtained on day 5 after virus infection. We further evaluated the expression of p53, p21 and cleaved-PARP proteins using western blot analysis in OBP-301-resistant osteosarcoma cells after infection with OBP-702 or Ad-p53, which is a p53-expressing replication-defective adenovirus. OBP-702 showed more cytopathic activity compared to OBP-301. The ID50 value of OBP-702 was much lower than that of OBP-301 in all cell lines. Western blot analysis showed OBP-702 infection induced the cleavage of PARP in a dose-dependent manner and cell cycle analysis also showed that OBP-702 induced the highest percentages of sub-G1 population in MNNG/HOS cells compared to Ad-p53 or OBP-301. These results suggest that OBP-702 induces increased apoptosis compared to Ad-p53 or OBP-301. OBP-702 infection induced more profound p53 expression than Ad-p53, but p53-downstream target p21 proteins were not activated by OBP-702. The expression levels of miR-93 and miR-106b increased after OBP-702 infection. When SaOS-2 cells were infected with Ad-p53 at an MOI of 10 for 48 hours, pre-transfection with miR-93, miR-106b or both efficiently suppressed Ad-p53-induced p21 expression. These results suggest that OBP-702 suppresses p21 expression through E1A-dependent upregulation of miR-93 and miR-106b. In conclusion, we have clearly demonstrated that the p53-expressing oncolytic adenovirus OBP-702 has a much stronger antitumor effect than OBP-301 and Ad-p53. Oncolytic adenovirus-mediated p53 gene transduction would induce profound p53 expression without p21 activation regulated by miR-93 and 106b, resulting in the enhancement of antitumor effect.

Citation Format: Joe Hasei, Tsuyoshi Sasaki, Hiroshi Tazawa, Yuuri Hashimoto, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Oncolytic adenovirus-armed p53 induces apoptosis significantly through upregulating miR-93 and 106b in human osteosarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3316. doi:10.1158/1538-7445.AM2013-3316