Renal Cell Carcinoma: Evaluation of the mitochondrial long non-coding RNA as potential targets for therapy Borgna V.1,2,3, Peña E5, Rivas A1,2,4, Araya M1,2, Landerer E3, Fermandoise P5, Oliveira L1,2, Burzio V1,2,4,Villegas J1,2,4, Burzio L.O1,2,4 Andes Biotechnologies SA1, Fundación Ciencia para la Vida2 Facultad Medicina UNAB3, Facultad Ciencias Biológicas UNAB4, Hospital Barros Luco5 Introduction Cell Viability Anchorage-independent Growth of treated cells Fig 5- Dramatic decrease in anchorage-independent population after Andes-ASO treatment of RenCa cells. After treatment, 200 live cells were subcultured in soft agar and colonies were counted 2 weeks later. Renal cell carcinoma (RCC) is the seventh most common cancer in men. Patients with small organ-confined tumors have a 5 year survival rate of 90% in contrast to <10% in patients with distant metastasis. Unfortunately, metastatic RCC is uniquely resistant to chemotherapy and radiotherapy, so the treatment has focused on targeted therapies, but clinical results are less than optimal. We previously described a novel family of long non coding mitochondrial transcripts (ncmtRNA) that contain an inverted repeat (IR) linked to the 5´ region of the antisense 16SmtRNA transcribed from the L-Strand of the mtDNA (ASncmtRNA). These transcripts are express in normal proliferating cells, but are down regulated in tumor cells. We found that knocking down these transcripts in vitro with antisense oligonucleotide (ASO) induces selective death of several human and murine cell lines without affecting viability of normal cells. Fig 2.-Decreased cell viability 48hrs post treatment with Andes-ASO 150nM. Evaluated by Crystal Violet assay Apoptosis: Early Events Human Primary Culture Treatment Untreated ASO-control A B A 11,5% 27,2% A Andes-ASO 60,4% C B Knock-Down AS-ncmtRNA B Normal Cell Andes-ASO Cancer Cell Apoptosis TUNEL Fig 3.-Mitochondrial Membrane Potential (ΔΨm) 24hrs post ASO treatment. A.-Evaluation was performed with TMRE stain using flow cytometry. B.-Quantification of high and low mitochondrial membrane potential on different populations. Fig 6- Selective neoplastic cell death in human primary cultures. A.-Establishment of primary cultures derived from patients with Clear Cell RCC. Epithelial (Epcam+) cell selection was performed using magnetic beads. B.-Normal Renal mixed epithelial cells were treated for 48 hrs. with Andes-ASO 200nM. C.-RCC primary cultures were treated for 48 hrs. with Andes-ASO 200nM. Cell viability was evaluated with MTT assay A.-Structure and expression pattern of S-ncmtRNA and AS-ncmtRNA in normal and cancer cells B.-Knock-Down of the ASncmtRNA induces selective death of cancer cells. DAPI TUNEL The aim of this study was to evaluate in vitro the therapeutic value of silencing the ASncmtRNA with ASO in a murine renal adenocarcinoma cell line and in cells obtained from primary cultures of human RCC tumors. Main Goal Apoptosis: Late events Fig 4.- Massive DNA Fragmentation induced 48hrs post Andes-ASO treatment in RenCa Cells. A.-TUNEL Assay was performed and analyzed with fluorescence microscopy. B.-Quantification of apoptotic cells In Andes-ASO, ASO-control and untreated cells. A Conclusions DAPI TUNEL Knockdown of the ASncmtRNA induces massive cell death in the RenCa Cell line. Early and Late apoptosis hallmarks are detected post Andes-ASO treatment in the RenCa cell line. Anchorage-independent cell growth is diminished by the ASO therapy against ASncmtRNA. Andes-ASO induces cell death in human primary RCC cultures but not in normal renal cells. These results support the potential use of the ASncmtRNA as a novel target for RCC therapy. Andes-ASO Results 40X DAPI TUNEL AS-ncmtRNA Knock-Down in RenCa cells ASO-control A B 20X 40X B 20X Villegas, and Burzio, L.O. (2002) Localization of the 16S mitochondrial rRNA in the nucleus of mammalian spermatogenic cells. Molecular human reproduction, 8, 977-983. Villegas, J. et al. (2007) Expression of a novel non-coding mitochondrial RNA in human proliferating cells. Nucleic acids research, 35, 7336-7347. Burzio, V.A. et al. (2009) Expression of a family of noncoding mitochondrial RNAs distinguishes normal from cancer cells. Proceedings of the National Academy of Sciences of the United States of America, 106, 9430-9434. Fig 1.-Knock-down of the ASncmtRNA transcript. A.-Andes-ASO transfection of RenCa cells with lipofectamine 2000 (1ug/uL). B.-qPCR of ASncmtRNA 24 hrs post Andes-ASO treatment. RPS29 was used as House-keeping control gene.