urologie.uniklinikum-dresden.de, *e-mail: daniela.wuttig@uniklinikum-dresden.de

1. exon 1 exon 3 exon 4 exon 2 exon 4 exon 1 exon 2 exon 1 exon 2 exon 2B exon 3 exon 4 survivin-WT survivin-2B survivin-DEx3 siSVV-Bel307 siSVV-D3-332 siSVV284 siSVV-2B-405 53% *** 63% ** 52% ** 52% ** 94% 67% * 92% 107% 86% CDDP MMC untreated + CDDP (2.1µg/ml) + MMC (0.9µg/ml) • Therapeutic effects of siRNA‑mediated inhibition of different • survivin transcripts in bladder cancer cells Daniela Wuttig *, Doreen Kunze, Susanne Fuessel, Axel Meye, Manfred P. Wirth Department of Urology, Technical University Dresden, Germany http://urologie.uniklinikum-dresden.de, *e-mail: daniela.wuttig@uniklinikum-dresden.de • Introduction • wildtype (WT)-survivin [1-3]: • potent inhibitor of apoptosis, indispensable for cell cycle progression • - fourth most common transcript overexpressed in cancer • - expression associated with bladder cancer (BCa) progression, poor patient´s outcome of disease, development of resistance to apoptosis-inducing agents • survivin splice variants [1, 4]: • - four alternatively spliced gene products • survivin‑DEx3 (antiapoptotic) and survivin‑2B (proapoptotic) expressed in numerous tumour entities • - unknown role in tumor progression • survivin inhibition in BCa cells by small interfering • RNAs (siRNAs) or antisense oligodeoxynucleotids [3, 5]: • simultaneous knock-down of different survivin trans-cripts (including survivin-WT) •  remarkable antiproliferative effects • side effects of anti-survivin therapies on healthy cells could be reduced by inhibiting only one specific survivin transcript instead of multiple ones •  aim: comparison of therapeutic effectiveness of simultaneous siRNA-mediated knockdown of different survivin isoforms versus down-regulation of one specific splice variant • Materials & methods • cells cultivated under standard conditions (37°C, 5% CO2) in DMEM (Invitrogen; +1% non essential amino acids, +10% fetal calf serum, +1% HEPES) • lipid-mediated transfection (trf) of siRNAs (200nM; siRNA:DOTAP=1:3, w/w) against different survivin transcripts (Tab.1,Fig.1) in EJ28 cells (4h, 37°C) • examinations: survivin transcript quantification (real time PCR; Roboscreen; referenced to glycerinaldehyd-3-phosphatdehydrogenase {GAPDH}), cell viability (WST‑1 test; Roche), rate of apoptosis (Annexin V-FITC Apoptosis Detection Kit; BD Biosciences) • chemosensitization experiments: chemotherapeutic treatment (incubation: mitomycin c {MMC} 2h, cisplatin {CDDP} 24h) was carried out 24h after siRNA transfection Fig.1: Exon structure of survivin mRNAs and siRNA target sites within these transcripts. Table 1: siRNA sequences and targeted survivin transcripts. (Because of its 100% complementarity to EPR1-mRNA design of a survivin-WT specific siRNA was not feasible.) • Results • survivin transcripts were quantified in 7 BCa cell lines (Fig.1) •  EJ28: remarkable overexpression of survivin & splice variants  selected as model system of human BCa • - mRNA expression: • - siRNAs effectively inhibited their targets 24h and 48h after trf start in EJ28 cells (Tab.2) • - siSVV‑Bel307, siSVV284 & siSVV‑2B‑405 selectively reduced their targets 24h and 48h after trf • - siSVV-D3-332 knocked down non-targeted survivin-WT (to 84%) 24h after trf start • cell biology: • - siRNAs increased apoptosis (by 33%) & reduced cell colony formation ability (by ~30%) (Tab.2) • - chemosensitization: • - siSVV-Bel307 & siSVV284 statistical significantly sensitized EJ28 cells to CDDP and MMC; • - siSVV-D3-332 and siSVV-2B-405 did not (Fig.3) • Thestrongest antiproliferative effects were observed after simultaneous inhibition of different survivin transcripts, including survivin-WT (siSVV-Bel307, siSVV284). Fig.2: mRNA expression of survivin and its splice variants in human BCa cell lines. Tab.2: Survivin mRNA expression and cellbiological effects following anti-survivin-siRNA treatment in EJ28 cells. # denoted in % of non silencing (ns)-siRNA, * differences to ns-siRNA control • Discussion • transcripts survivin-WT, -2B and -DEx3 are expressed in all examined BCa cell lines  cells suitable for inhibition of these transcripts • antiproliferative effects following siSVV-Bel307 und siSVV284 treatment were described previously [3, 6]  we confirmed these data •  but: inhibition of one specific survivin splice variant  despite of a long-lasting mRNA reduction  offers poorer effects in apoptosis induction & reduction of long-term cell proliferation •  because of up-regulation of remaining survivin transcripts? •  either simultaneous inhibition of different survivin transcripts or reduction of survivin-WT is responsible for strong antiproliferative effects •  since survivin-WT expression is associated with course of disease, the latter is more supposable •  Therefore, we showed for the first time that the specific inhibition of a survivin splice variant is not reasonable for anticancer treatment, at least in BCa therapy. siSVV-Bel307 siSVV284 siSVV-D3-332 siSVV-2B-405 ns-siRNA siSVV-Bel307 siSVV284 siSVV-D3-332 siSVV-2B-405 ns-siRNA siSVV-Bel307 siSVV284 siSVV-D3-332 siSVV-2B-405 ns-siRNA References: 1 Zaffaroni N., Pennati M., Daidone M.G. (2005): Survivin as a target for new anticancer interventions, J Cell Mol Med 9 360-372. 2 Fuessel S., Herrmann J., Ning S., Kotzsch M., Kraemer K., Schmidt U., Hakenberg O.W., Wirth M.P., Meye A. (2006): Chemosensi-tization of bladder cancer cells by survivin-directed antisense oligodeoxynucleotides and siRNA, Cancer Lett 232 243-254. 3 Ning S., Fuessel S., Kotzsch M., Kraemer K., Kappler M., Schmidt U., Taubert H., Wirth M.P., Meye A. (2004): siRNA-mediated down-regulation of survivin inhibits bladder cancer cell growth, Int J Oncol 25 1065-1071. 4 Li F. (2005): Role of survivin and its splice variants in tumorigenesis, Br J Cancer 92 212-6. 5 Fuessel S., Kueppers B., Ning S., Kotzsch M., Kraemer K., Schmidt U., Meye A., Wirth M.P. (2004): Systematic in vitro evaluation of survivin directed antisense oligodeoxynucleotides in bladder cancer cells, J Urol 171 2471-2476. 6 Belyanskaya L.L., Hopkins-Donaldson S., Kurtz S., Simoes-Wust A.P., Yousefi S., Simon H.U., Stahel R., Zangemeister-Wittke U. (2005): Cisplatin activates Akt in small cell lung cancer cells and attenuates apoptosis by survivin upregulation, Int J Cancer 117 755-763. Fig.3: Viability of EJ28 cells following siRNA mono-treatment or siRNA/chemotherapeutic combination-treatment. Viabilities of siRNA and siRNA/chemotherapeutic treatments are denoted in % of ns-siRNA, chemotherapeutic mono-treatments in % of untreated control. Percentages represent residual viability compared to respective control. Student´s t-test: *p<0.05, **p<0.01, ***p<0.001.