| SIRNA
AND ANTISENSE OLIGONUCLEOTIDES AS THERAPEUTIC UTILITIES
FOR PROSTATE CANCER.
Dr Badar A Usmani, Dr Ian R Graham University of Leeds |
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Prostate cancer is the most common cancer in the U.K, and remains a major cause of early death in males throughout the developed world. Prostate cancer cells often undergo changes that stop them responding to some treatments, and cause them to break away and cause secondary tumours, especially in the bones. We now know that one of these changes leads to an increase in the amount of an enzyme called Endothelin-converting enzyme-1 (ECE-1), which can exist in several related forms. One of these forms appears to be instrumental in the migration of cancer cells away from the prostate. The grant we have been awarded by the Prostate Cancer Campaign is designed to look at ways of abolishing this harmful form of ECE-1 by affecting the way it is produced from its gene. One way is to actually block its production, and another is to trick the cells into switching from producing the harmful form to producing its less harmful relatives by affecting a process known as RNA splicing.
In the latter approach, we investigated the use of a different class of RNA reagents to trick the prostate cancer cells into switching from one type of ECE-1 to another. As mentioned in our interim report, we have concluded that ECE-1c is responsible for the ability of prostate cancer cells to invade, migrate and form secondary tumours. We have therefore tried to block production of ECE-1c using antisense oligonucleotides (AO) to interfere with the process of RNA splicing. The structure of the gene for ECE-1 is shown in Figure 2, and is quite complex, with many different splicing events giving rise to the four different forms of ECE-1.
Initially, we undertook a comprehensive analysis of the known sequence of the ECE-1 gene in this region, which included the sequences at the 5' (donor) splice site (indicated by arrows above), and exonic splicing enhancers (ESE), which promote RNA splicing. We then designed a panel of ten AOs against these potential targets, five of which would block the production of ECE-1c alone, and the other five blocking the production of ECE-1b, c and d together. This was expected to result in an up-regulation of ECE-1a, which counteracts prostate cancer cell migration (see figures 2 and 3).
Figure 2 is an analysis of exon 2 (i.e that which is common to the three isoforms other than 1a), and Figure 3 an analysis of exon 1c. The sequences have been analysed using a program called ESE-finder (1, 2), which predicts binding sites for splicing-promoting proteins. Masking these binding sites results in silencing of the exon. The oligos were designed to bind to clusters of ESEs within each exon. There are also two for each exon (1C4 & 5, 1D4 & 5) which are designed to interfere with the exon/intron boundary (5' or donor splice site), which we have found to be a good target in the dystrophin gene (3, 4).
The oligonucleotides are all 20mer 2'-O-methyl RNA on a phosphorothioate backbone. These AOs were tested in DU145 cells and revealed the following:
Figure 4: Probing of a Western blot using a polyclonal antibody specific for Ece-1c (panel A), indicating a prominent doublet at the previously reported apparent molecular weight of ~120-130kDa. A similar membrane has also been probed with a monoclonal antibody capable of detecting all four Ece-1 isoforms (Panel B, 40 minute exposure; Panel C, 5 minute exposure). The major band is most likely to be Ece-1c, but there is also a band at about 75kDa, consistent with this being a low level of Ece-1a There is an apparent reduction in the level of Ece-1c and a concomitant increase in Ece-1a after treatment with oligonucleotide 1c5, compared with the untransfected negative control (LF). Identical amounts of DU145 protein extract were loaded in each lane.
Figure 5: Probing of a Western blot using a polyclonal antibody specific for Ece-1a, indicating a prominent band at the previously reported apparent molecular weight of ~75kDa. The upper panel is a 5 minute exposure; the lower panel is a 1 minute exposure. The intensity of the band in lane 1c1 appears to be higher than the adjacent control (LF), despite identical amounts of DU145 protein extract being loaded.
Conclusion:
Essentially, the experiment involved transfecting prostate DU145 cells with each of the ten novel oligonucleotides, complexed with Lipofectamine 2000. Protein extracts were made 48 hours later, and run on a Western gel. Gels were Ponceau S stained to check for equal loading.
Ece-1a was detected in all the transfected cells, but the level of 1a appears to be slightly increased in the presence of oligo 1C1 (compare to the untransfected control), and maybe with 1D2 (1C5 is clearly underloaded). The level of Ece-1c seems to be reduced in those two transfections, when compared with the monoclonal which detects all iosoforms (short exposure shows it best).
Ece-1c is identified as a doublet band. Lane 1D1 shows a substantially enhanced upper band of this doublet compared to the untransfected cells in the right-hand lane, which is echoed in the experiment with the monoclonal, which shows a higher level of a band running at about 125kD (presumably 1c).
Given the above results we shall continue to evaluate the efficacy of our siRNA reagents and PMOs, in tissue that has been removed from patients suffering from either prostate cancer or benign prostatic disease, and to incorporate these objectives into a subsequent full research funding proposal.
Exon splicing may be the new therapeutic utility of the future.
References:
1. Smith, P. J., Zhang, C., Wang, J. Chew, S. L., Zhang, M. Q. and Krainer,
A. R. 2006. An increased specificity score matrix for the prediction
of SF2/ASF-specific exonic splicing enhancers. Hum. Mol. Genet. 15(16):
2490-2508.
2. Cartegni L., Wang J., Zhu Z., Zhang M. Q., Krainer A. R.; 2003. ESEfinder:
a web resource to identify exonic splicing enhancers. Nucleic Acid Research,
2003, 31(13): 3568-3571
3. Graham IR, Hill VJ, Manoharan M, Inamati GB, Dickson G. Towards a
therapeutic inhibition of dystrophin exon 23 splicing in mdx mouse muscle
induced by antisense oligoribonucleotides (splicomers): target sequence
optimisation using oligonucleotide arrays.J Gene Med. 2004 Oct;6(10):1149-58
4. Arechavala-Gomeza V, Graham IR, Popplewell LJ, Adams AM, Aartsma-Rus
A, Kinali M, Morgan JE, van Deutekom JC, Wilton SD, Dickson G, Muntoni
F. Comparative analysis of antisense oligonucleotide sequences for targeted
skipping of exon 51 during dystrophin pre-mRNA splicing in human muscle.
Hum Gene Ther. 2007 Sep;18(9):798-810.
Final research report dated 31 October 2007
Project 2005/09