Cyclooxygenase-2 (COX2) gene silencing with siRNA could enhance DNA vaccination to inhibit established ErbB-2 carcinomas

Background: Cyclooxygenase-2 (COX2) is the key enzyme in the synthesis of prostaglandin E2 from its precursor, arachidonic acid. The level of COX2 is normally very low in cells but increased amounts of COX2 are commonly detected in both premalignant and malignant tissues. In order to investigate COX2 as a potential target for the prevention and treatment of cancer, we propose a novel immunotherapeutic approach for the prevention of ErbB-2 positive breast carcinomas, based on DNA vaccination against a specific antigen (ErbB-2) in combination with the silencing of COX2. Material and methods: The short hairpin RNA interference cassette targeting COX2 mRNA, containing the mouse RNA promoter U6, was amplified and cloned into the Eco72I site of pVAX-ratECTM (RRT), a vector carrying the extracellular and transmembrane domains of rat ErbB-2, and into pVAXI, to obtain respectively RRT-COX2 and pVAXCOX2. The gene silencing efficacy of both plasmids was checked by transfecting COX2 over-expressing A17 cells. The plasmids were used for electroporation-based vaccination of female BALB-neuT mice of different ages, corresponding to various stages of cancer progression, from atypical hyperplasia to invasive lobular carcinoma. We are evaluating mammary cancer progression in vivo, the titer of anti-ErbB-2 antibodies in the immune sera, and the ability of spleen cells to release IFNg in response to the H2d immune dominant peptide of ErbB-2. Results: Vaccination of 10 week-old female BALB-neuT mice with RRT plasmid significantly triggers a protective immune response toward the development of autochthonous mammary cancer in BALB-neuT mice (Quaglino et al. 2004, Cans Res). 30% of treated mice were still fully free from palpable tumors one year after vaccination, when all control animals had already died because of mammary cancer. Till now (week 46 of age) we see a similar protection using RRT-COX2. The level of anti-ErbB-2 antibodies in the sera from RRT-COX2 vaccinated mice is slightly higher than that of RRT vaccinated mice, but not significantly different. We are now collecting spleen cells from vaccinated mice to evaluate the specific CD8 response against ErbB-2. Experiments with mice vaccinated when they already have lobular carcinomas are ongoing. Conclusions: COX2 suppression induced by shRNA might help to overcome tumormediated immunosuppression and generate an effective anti-tumor immunity not only in prophylactic but also in therapeutic vaccination.