Abstract 1236: Combined transfer of p19Arf and interferon-beta genes to mouse melanoma cells causes LC3B- and caspase-3-independent cell death and alters the expression of critical genes

Tumors cells are able to resist cell death and evade the immune system. This resistance can be caused by alterations in Trp53 itself or p19Arf loss and the reduced anti-tumor immune response may be caused by loss of interferon-β (IFN), an important immune-stimulatory cytokine. Here, we investigate the impact of p19Arf and IFN gene transfer in melanoma cells with regards to the expression profile of genes responsible for the induction of cell death. Recombinant adenoviral vectors with a p53-responsive promoter (PG) driving expression of p19Arf or IFN were applied alone or in combination in B16F10 cells in vitro (mouse melanoma, p53 wt). Cell death was evaluated by annexin/PI staining and MTT assays. LC3B expression was verified by western blot analysis of cells extracts. Caspase-3 activity was evaluated using a lentiviral vector which constitutively expressing luciferase protein linked to an ubiquitin site that can be cleaved by caspase-3. RNA expression profile was evaluated by microarray analysis of treated samples and validation of gene expression was performed by real-time PCR. We verified that combined gene transfer of p19Arf and IFN resulted in 74% hypodiploid cells whereas single gene transfer yielded only half of this number, as shown by cell cycle and MTT analysis. Although there was a high rate of cell death, only 25% of cells were stained for annexin/PI and activation of caspase-3 was reduced in cells treated with p19Arf and IFN combination compared to treatment of p19Arf alone. Also, there was no increase in LC3B expression when cells were treated with the p19Arf and IFN combination. These observations suggest that B16F10 cells are dying by other mechanisms rather than apoptosis or autophagy. Transduced cells were analyzed for gene expression profile and critical genes were selected for validation. Combination of p19Arf and IFN transduction in B16F10 cells revealed a synergistic effect modifying exclusively the expression of 1054 genes involved in cell death, immune system activation and cell cycle arrest. There was an increase of 4-20 fold in expression of MDM2, p53, p21Waf1, PUMA, MDM4, FOXO1, NR3C1, PHLDA3, RANBP9 and WDR46, and also an increase of 400-fold in p73 expression in cells treated with p19Arf and IFN combination. Conclusions: The use of p53-responsive vectors to express p19Arf and IFN promotes complementation of the p53/Arf and IFN pathways, resulting in a synergistic gene regulation of many essential functions in cancer cells, destabilizing cell cycle and cell organization in general, while promoting expression of genes related to cell death. As p73 is a transcription factor of caspase-1 expression, we hypothesize that this could be the major participant involved in death of these cells. Financial support: 2011/10656-5, 2011/50911-4 and 2013/25167-5 (FAPESP). Citation Format: Aline H. Ribeiro, Paulo R. Del Valle, Ruan F.V. Medrano, Daniel G. Ferrari, Daniela B. Zanatta, Bryan E. Strauss. Combined transfer of p19Arf and interferon-beta genes to mouse melanoma cells causes LC3B- and caspase-3-independent cell death and alters the expression of critical genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1236. doi:10.1158/1538-7445.AM2015-1236