Are over ‑ expressed alternative survivin transcripts in human bladder cancer suitable targets therapeutic targets? Daniela Wuttig *, Doreen Kunze, Susanne Fuessel, Joerg Stade, Oliver W. Hakenberg, Axel Meye, Manfred P. Wirth Department of Urology, Technical University Dresden, Germany Department of Urology, Technical University Dresden, Germany * References: 1 Zaffaroni N. et al. (2005): Survivin as a target for new anticancer interventions, J Cell Mol Med Fuessel S. et al. (2006): Chemosensi-tization of bladder cancer cells by survivin-directed antisense oligodeoxynucleotides and siRNA, Cancer Lett Ning S. et al. (2004): siRNA-mediated down-regulation of survivin inhibits bladder cancer cell growth, Int J Oncol Li F. (2005): Role of survivin and its splice variants in tumorigenesis, Br J Cancer Fuessel S. et al. (2004): Systematic in vitro evaluation of survivin directed antisense oligodeoxynucleotides in bladder cancer cells, J Urol Belyanskaya L.L. et al. (2005): Cisplatin activates Akt in small cell lung cancer cells and attenuates apoptosis by survivin upregulation, Int J Cancer Introduction wildtype (wt)-survivin [1-3]: - potent inhibitor of apoptosis, important for cell cycle progression - fourth most common transcript over-expressed in cancer -expression associated with bladder cancer (BCa) progression, poor patients´ outcome of disease, development of resistance to apoptosis-inducing agents alternative survivin splice variants [1, 4]: -four alternatively spliced gene products (survivin-  Ex3 and -2B in Fig. 1) -survivin ‑  Ex3 (  Ex3, presumably antiapoptotic) and survivin ‑ 2B (2B, presumably proapoptotic) expressed in numerous tumour entities -widely unknown role in tumor progression survivin inhibition in BCa cells by small interfering RNAs (siRNAs) or antisense oligodeoxynucleotides [3, 5]: - remarkable antiproliferative effects after simultaneous knock-down of different survivin transcripts (including survivin-wt) -survivin expression during G2/M cell cycle phase in normal cells  rather tumor cell selective anti-survivin treatment by specific inhibition of one specific alternative survivin transcript  aim:comparison of therapeutic efficiency of inhibition of one specific alternative survivin transcript vs. simultaneous knock-down of several survivin transcripts Fig. 1:Exon structure of survivin mRNAs and siRNA target sites within these transcripts. siRNAtarget-mRNAsiRNA sequence (sense, 5´ → 3´)siRNA sequence (antisense, 5´→ 3´) siSVV ‑ Bel survivin–wt, –2B, –  Ex3 GGA GCU GGA AGG CUG GGA GdtdtCUC CCA GCC UUC CAG CUC Cdtdt siSVV284survivin–wt, –2BGCA UUC GUC CGG UUG CGC UdtdtAGC GCA ACC GGA CGA AUG Cdtdt siSVV ‑ D3 survivin–  Ex3 GAC GAC CCC AUG CAA AGG AdtdtUCC UUU GCA UGG GGU CGU Cdtdt siSVV ‑ 2B survivin–2BUCA CGA GAG AGG AAC AUA AdtdtUUA UGU UCC UCU CUC GUG Adtdt ns ‑ siRNA noneUUC UUC GAA CGU GUC ACG UdtdtACG UGA CAC GUU CGG AGA Adtdt exon 1exon 3 exon 2 exon 4 siSVV-Bel siSVV-D3 siSVV284 siSVV-2B exon 4exon 3exon 2B exon 2 exon 1 exon 4 exon 2 exon 1 survivin-wt survivin-2B survivin-  Ex3 Table 1: siRNA sequences and targeted survivin transcripts. Results tissue sample analyses -survivin expression in BCa and non-malignant tissues: survivin-wt >  Ex3 > 2B -mean expression of survivin-wt,  Ex3 and 2B transcripts was significantly higher in malignant than in non-malignant tissues (p < 0.05) (Fig. 2) cell culture: siRNA-mediated survivin inhibition mRNA and protein expression: -survivin-wt,  Ex3 and 2B are expressed in EJ28 cells (data not shown) -effective target knock-down by all siRNAs 24h and 48h after trf start in EJ28 cells (Tab.2) -selective traget knock-down by siSVV-Bel, siSVV284 and siSVV-2B 24h and 48h after trf start (Tab. 2); siSVV-D3 knocked down non-targeted survivin-wt (by 16%) 24h after trf start (Tab. 2) -survivin-wt was also reduced on protein level by siSVV-Bel and siSVV284 (Tab. 2) cell growth: -all siRNAs increased apoptosis (Tab.2); strongest effects after siSVV-Bel and siSVV284 treatment -siSVV-Bel and siSVV284 increased G2/M arrest and reduced cell colony formation ability (Tab. 2) chemosensitization experiments: -significant chemosensitization effects to different concentrations of CDDP and MMC mainly by using siSVV-Bel and siSVV284 (examples in Fig. 3)  strongest antiproliferative effects after simultaneous inhibition of several survivin transcripts, including survivin-wt (siSVV-Bel, siSVV284) Discussion -over-expression of alternative survivin transcripts in BCa tissues indicates their accessibility for in vitro inhibition -antiproliferative effects following siSVV-Bel und siSVV284 treatment were described previously [3, 6]  we confirmed these data -inhibition of one specific alternative survivin transcript offers poorer antiproliferative effects than simultaneous inhibition of survivin-wt and its alternatively spliced transcripts  supposable cause: despite of an effective and a long-lasting mRNA reduction no reduction of  Ex3 or 2B proteins  specific inhibition of one alternative survivin transcript is not reasonable for anticancer treatment, at least for BCa Tab.2: Survivin mRNA expression and cellbiological effects following anti-survivin-siRNA treatment in EJ28 cells. + CDDP (2.1µg/ml) + MMC (0.9µg/ml) CDDP MMC untreated 63% ** 86% 107% 67% * 53% *** 92% 52% ** 94% 52% ** (Because of its 100% complementarity to EPR1-mRNA design of a survivin-wt specific siRNA was not possible.) Materials & methods tissue sample analyses -cryo ‑ preserved bladder tissue samples obtained from radical cystectomy specimens -18 BCa (1 pT1, 2 pT2, 10 pT3, 5 pT4; all high-grade) & 22 non ‑ malignant samples of 23 BCa patients (including 17 autologous pairs) -survivin transcript quantification by real time PCR (Roboscreen), normalized to glyceraldehyd-3-phosphate dehydrogenase (GAPDH) cell culture -cells cultivated under standard conditions (37°C, 5% CO 2 ) in DMEM (Invitrogen) supplemented with 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, normalized to GAPDH) -Western blot analyses (with BSA): antibodies: NB , Novus Biologicals (survivin-wt); ab3731, Abcam (  Ex3); ab3729, Abcam (2B); normalized to  -actin (A 5316, Sigma) -rate of apoptosis (Annexin V-FITC Apoptosis Detection Kit; BD Biosciences) -cell cycle distribution (Cycle Test Plus DNA Reagent Kit; BD Biosciences) -colony formation chemosensitization experiments: - chemotherapeutic treatment 24h after siRNA trf -incubation times: mitomycin c (MMC) 2h, cisplatin (CDDP) 24h -cell viability (WST ‑ 1 test; Roche) Fig. 3: Viability of EJ28 cells following siRNA mono-treatment or siRNA / chemotherapeutic combination-treatment 72 or 96h after siRNA trf. Viabilities of siRNA and siRNA/chemothera- peutic 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< siSVV-Bel siSVV284 siSVV-D3 siSVV-2B ns-siRNA siSVV-Bel siSVV284 siSVV-D3 siSVV-2B ns-siRNA siSVV-Bel siSVV284 siSVV-D3 siSVV-2B ns-siRNA siRNA construct mRNA expression # mRNA expressionprotein expression  apop- tosis  G2/M arrest  colony formation wt  Ex3 2Bwt  Ex3 2Bwt  Ex3 2B siSVV-Bel ±0± siSVV ±0± siSVV ‑ D ±0± siSVV ‑ 2B (-100) Table 2: siRNA-mediated knock-down of survivin-wt,  Ex3 and 2B and their effects on cell proliferation. All parameters were measured 48h (# 24h) after siRNA treatment. Data are normalized to those of ns-siRNA treated cells and declared in % as differences to ns-siRNA treated control. Fig. 2: mRNA expression of survivin-wt,  Ex3 and 2B in BCa (tu) and non-malignant (tf) tissue samples. Boxplots represent values within the 25. and 75. percentiles, error bars values to 10. and 90. percentile, respectively. Dashed lines show the mean values and solid ones median values. Points out of the error bars display outliers. Because of excluding expression values with a mean deviation >35% within a repeated determination, different numbers of samples (n) were used for creation of different plots. (n=18) (n=21) (n=15) (n=16) (n=16) (n=22)