Suppression of lung cancer with siRNA targeting PTTG

Abstract

Abstract. Pituitary transforming gene (PTTG) is frequently expressed at high levels in malignant tumors. We report high levels of expression of PTTG in various lung tumors and tumor-derived cell lines. For a better understanding of its role in maintaining the cancer phenotype, we used RNA interference (RNAi) directed against PTTG. Transfection of H1299 cells with PTTG siRNA duplex (5'-UGG GAG AUC UCA AGU UUC A-3') to a final concentration of 100 nmol/l resulted in almost complete depletion of PTTG mRNA within 24 and 48 h when compared to expression in untransfected cells or cells transfected with control siRNA. Western blot analysis showed nearly a 60% reduction in PTTG protein within 48 h of transfection. In phenotype analysis, we investigated the effect of PTTG siRNA on colony formation on soft agar, and tumor development in nude mice. Transfection of H1299 cells with PTTG siRNA duplex significantly reduced colony formation compared to untransfected cells or cells transfected with control siRNA. Mice injected with H1299 cells transfected with PTTG siRNA followed by injection of siRNA developed no tumors within two weeks of injection, but developed small tumors (67.85±45.87 mg) within 4 weeks of injection, whereas untransfected cells, or cells transfected with control siRNA developed large tumors (232.12±102.78 and 231.57±83.76 mg respectively). Suppression of PTTG as well as Ki67, bFGF and CD34 was observed in H1299 tumors treated with PTTG siRNA compared to untreated and control-treated siRNA in nude mice. In conclusion, decreasing PTTG expression through PTTG siRNA inhibits tumor growth both in vitro and in vivo. Future studies are needed to test whether PTTG expression can be efficiently depleted by siRNA expressed from a DNAbased expression vector combined with a specific-promoter, such that RNAi can specifically target PTTG in cancer cells without affecting normal cells.

Small interfering RNA against PTTG: A novel therapy for ovarian cancer

“…Based on these results, we hypothesized that a correlation exists between the level of expression of PTTG and tumorigenesis, and that down-regulation of PTTG expression in tumors will lead to suppression of tumor growth and development. In our recent studies, we showed that down-regulation of PTTG in lung tumor cells (H1299) by PTTG siRNA resulted in the inhibition of colony formation in vitro and tumor development and growth in nude mice (6). Consistent with our results, Cho-Rok et al (24), using an adenovirus system expressing PTTG siRNA to downregulate PTTG expression, recently showed a significant reduction in tumor growth in vitro and in vivo for hepatoma cells.…”

“…To assess in vivo activity of PTTG silencing, A2780 cells transfected with control plasmid, control siRNA, or PTTG siRNA were trypsinized, washed twice with PBS, and resuspended in RPMI media without serum and antibiotic. Cells (2x10 6 /site) were injected subcutaneously into both flanks of 4 to 6-week-old nu/nu mice (NCI). Five mice were used in each group.…”

“…Initially cloned from the rat pituitary tumor (3), pituitary tumor transforming gene (PTTG), also known as securin, is an oncogene that is highly expressed in many tumors including pituitary (4,5), lung (6), colon, and ovarian tumors (7)(8)(9)(10). On the other hand, PTTG expression in normal tissue is very low except for the testis (8).…”

Development of pituitary tumor-transforming gene (PTTG) transgenic mice for the treatment of ovarian cancer.

“…In the lung cancer cell line H1299 [120], a 60% reduction in PTTG protein resulted in null males can be rescued from diabetes development by undergoing surgical gonadectomy followed by estradiol treatment through increased serum adiponectin levels resulting in increased insulin sensitivity [122]. As a follow-up study, the mechanism of reduced pancreatic ~-cells proliferation by PTTG deletion was determined [123].…”

“…Due to the lack of specific ovarian promoter, we selected a strong non-specific CMV promoter, to induce the necessary level of PTTG to achieve this biological function, as the level of PTTG is critical for tumorigenesis [116,120] . However, due to the nature of this promoter, it was necessary to analyze other tissues to determine if tumorigenesis has occurred.…”