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Are over‑expressed alternative survivin transcripts in human bladder cancer Are over‑expressed alternative survivin transcripts in human bladder cancer suitable targets therapeutic targets?suitable targets therapeutic targets?

Daniela Wuttig *, Doreen Kunze, Susanne Fuessel, Joerg Stade, Oliver W. Hakenberg, Axel Meye, Manfred P. WirthDaniela Wuttig *, Doreen Kunze, Susanne Fuessel, Joerg Stade, Oliver W. Hakenberg, Axel Meye, Manfred P. Wirth

Department of Urology, Technical University Dresden, GermanyDepartment of Urology, Technical University Dresden, Germany

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

References:1 Zaffaroni N. et al. (2005): Survivin as a target for new anticancer interventions, J Cell Mol

Med 9 360-372.2 Fuessel S. et al. (2006): Chemosensi-tization of bladder cancer cells by survivin-directed

antisense oligodeoxynucleotides and siRNA, Cancer Lett 232 243-254.3 Ning S. et al. (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. et al. (2004): Systematic in vitro evaluation of survivin directed antisense

oligodeoxynucleotides in bladder cancer cells, J Urol 171 2471-2476.6 Belyanskaya L.L. et al. (2005): Cisplatin activates Akt in small cell lung cancer cells and

attenuates apoptosis by survivin upregulation, Int J Cancer 117 755-763.

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.

siRNA target-mRNA siRNA sequence (sense, 5´ → 3´) siRNA sequence (antisense, 5´→ 3´)

siSVV‑Bel survivin–wt, –2B, –Ex3 GGA GCU GGA AGG CUG GGA Gdtdt CUC CCA GCC UUC CAG CUC Cdtdt

siSVV284 survivin–wt, –2B GCA UUC GUC CGG UUG CGC Udtdt AGC GCA ACC GGA CGA AUG Cdtdt

siSVV‑D3 survivin–Ex3 GAC GAC CCC AUG CAA AGG Adtdt UCC UUU GCA UGG GGU CGU Cdtdt

siSVV‑2B survivin–2B UCA CGA GAG AGG AAC AUA Adtdt UUA UGU UCC UCU CUC GUG Adtdt

ns‑siRNA none UUC UUC GAA CGU GUC ACG Udtdt ACG UGA CAC GUU CGG AGA Adtdt

exon 1 exon 3exon 2 exon 4

siSVV-Bel siSVV-D3 siSVV284 siSVV-2B

exon 4exon 3exon 2Bexon 2exon 1

exon 4exon 2exon 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)

CD

DP

MM

C

un

trea

ted

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% CO2) 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 500-201, 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<0.001.

siS

VV

-Bel

siS

VV

284

siS

VV

-D3

siS

VV

-2B

ns-

siR

NA

siS

VV

-Bel

siS

VV

284

siS

VV

-D3

siS

VV

-2B

ns-

siR

NA

siS

VV

-Bel

siS

VV

284

siS

VV

-D3

siS

VV

-2B

ns-

siR

NA

siRNAconstruct

mRNA expression # mRNA expression protein expression apop-

tosis G2/Marrest

colonyformationwt Ex3 2B wt Ex3 2B wt Ex3 2B

siSVV-Bel -62 -53 -59 -58 -52 -60 -55 ±0 +17 + 46 + 28 - 30

siSVV284 -79 +38 -70 -78 -5 -81 -86 ±0 +77 + 33 + 92 - 32

siSVV‑D3 -16 -26 +43 +10 -10 +44 +74 ±0 +118 + 11 - 7 + 1

siSVV‑2B +23 +73 (-100) +68 +96 -72 +50 -8 +11 + 11 - 3 - 14

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)