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Bioeng Bugs. 2011 May-Jun; 2(3): 125–128.
Published online May 1, 2011. doi:  10.4161/bbug.2.3.15165
PMCID: PMC3225653
Modulation of RNA splicing as a potential treatment for cancer
John A Baumancorresponding author1 and Ryszard Kole1,2
1Department of Pharmacology; University of North Carolina; Chapel Hill, NC USA
2AVI Biopharma Inc.; Bothell, WA USA
corresponding authorCorresponding author.
Correspondence to: John A. Bauman; Email: bau/at/email.unc.edu
Received January 26, 2011; Revised February 12, 2011; Accepted February 15, 2011.
Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that upregulated the expression of an anti-cancer splice variant while simultaneously eliminating an overexpressed cancer-causing splice variant. This was accomplished by targeting pre-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs. SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., Δ7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy.
Key words: cancer, antisense, oligonucleotide, splicing, apoptosis
Pre-mRNA splicing, the co-transcriptional process of intron removal and exon joining seen in higher eukaryotes, was first thought to be an exception to the rule that mRNAs are carbon copies of their respective genes. Sequencing of the human genome revealed that not only splicing but also alternative splicing, which enables a single pre-mRNA transcript to give rise to multiple distinct splicing isoforms and therefore multiple variant proteins, are the norm. Alternative splicing explains how the relatively limited human genome (~30,000 genes) gives rise to more than 100,000 proteins. Recent analyses indicate that nearly 95% of all multi-exon gene transcripts undergo alternative splicing, 86% with a minor isoform frequency of 15% or more.1 In addition to regulating gene expression, pre-mRNA splicing and alternative splicing are being recognized for their roles in human disease. Indeed, up to half of disease-causing genetic mutations affect pre-mRNA splicing.2 The near-ubiquity of alternative splicing and the prevalence of splicing aberrations in human disease open up a new field that can result in the harvest of new gene- and sequence-specific medicines that achieve clinical benefits by modulating splicing. The SSO described in the Bauman et al. paper has the potential to be one such medication.
Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense drug candidates that modify pre-mRNA splicing. They are designed to block sequences in pre-mRNA essential for splicing, redirecting the splicing machinery to a new pathway. That this mechanism is feasible in vitro, in vivo in animals, and in patients has been demonstrated in numerous publications from this laboratory and by others.37
The SSO mechanism of action is distinct from conventional antisense oligonucleotides (ASOs) and siRNAs that inhibit gene expression by degrading the target mRNA through RNase H and the RISC complex, respectively. Instead, SSOs sterically block sequences in pre-mRNA without leading to their degradation, thereby shifting the pattern of splicing. This action requires that the SSO form very stable duplexes with its pre-mRNA target to enable successful redirection of the splicing factors to alternative, desired sequences. These requirements are met by incorporating various chemical modifications to the oligonucleotide backbone that improve binding affinity and do not support RNase H cleavage. Synthetic SSO chemistries include 2′-O-methyl, 2′-O-methoxyethyl (2′MOE), phosphorodiamidate morpholino, peptide nucleic acid and locked nucleic acid.8,9
Defective human cells can be pushed onto a suicide pathway through a programmed cell death mechanism called apoptosis. Apoptosis is essential for normal development and maintenance of tissue homeostasis; however, dysregulation of apoptotic signaling contributes to numerous pathological conditions, including cancer. Indeed, one of the hallmarks of cancer cells is their ability to evade and corrupt apoptosis, prompting the search for drugs that can restore or potentiate apoptotic signaling in tumor cells.10
Apoptosis is regulated by interactions among members of the Bcl-2 family of proteins, some of which (e.g., Bax and Bak) are pro-apoptotic and induce programmed cell death. Some are anti-apoptotic (e.g., Bcl-2 and Bcl-xL), and prevent the cell from committing suicide unnecessarily. A fine balance is maintained until an apoptotic event, such as DNA damage or growth factor deprivation, prompt Bax and Bak to trigger apoptosis.11,12 However, if anti-apoptotic Bcl-2 and/or Bcl-xL proteins are overexpressed they can overcome Bax and Bak, enabling the cell to survive with damaged or rearranged DNA and in the absence of growth factors. The cell is converted into a cancer cell that is free to proliferate, escaping normal controls. Because many chemotherapeutics induce apoptosis through Bax and Bak-like proteins, cancer cells with excess of Bcl-2 and Bcl-xL become resistant to chemotherapy.13 It follows that a drug that can reactivate the apoptotic pathway could be a good anti-cancer agent in its own right and/or could re-sensitize the cancer cell to chemotherapy.
Bcl-x undergoes splicing at two alternative 5′ splice sites of exon 2 (Fig. 1), yielding two protein with opposing functions, Bcl-xL and -xS.15 Use of the downstream splice site produces Bcl-xL, which exerts an anti-apoptotic effect by antagonizing and inhibiting the pro-apoptotic Bax and Bak proteins. It also induces growth of blood vessels that vascularize the tumor.16 Not surprisingly, Bcl-xL overexpression has been detected in a number of malignancies. A recent study of 3,131 cancer samples across more than two dozen cancer types found that the bcl-x gene was amplified in almost all of them, suggesting that upregulation of this gene may be a common mechanism by which cancers increase survival.17 Previously, this laboratory targeted Bcl-x in prostate and breast cancer cells,18,19 whereas the work discussed here focused on Bcl-x in metastatic melanoma.14
Figure 1
Figure 1
Splicing at the downstream or upstream 5′ alternative splice site of Bcl-x exon two produces Bcl-xL or Bcl-xS, respectively. Bcl-xL is highly expressed in many human cancers and confers resistance to a broad range of chemotherapeutic agents. It (more ...)
Use of the upstream 5′ alternative splice site of Bcl-x exon 2 produces an internally deleted protein, Bcl-xS (Fig. 1).15 Bcl-xS is anti-apoptotic because it directly binds and inhibits the pro-apoptotic Bcl-xL and Bcl-2 proteins.20 The authors show that targeting the downstream (Bcl-xL) splice site with a 2′MOE SSO redirects the splicing machinery to the upstream (Bcl-xS) splice site, effectively converting an anti-apoptotic molecule into a pro-apoptotic molecule. As a result, the tumor load in treated mice is reduced. Previously it was shown that newly generated Bcl-xS induced by SSO also sensitizes cancer cells to treatment with UV- and γ-irradiation and chemotherapeutic drugs, including etoposide, 5-fluorouracil, cisplatin, 5-fluorodeoxyuridine and doxorubicin.18,21
Two significant characteristics of splicing modulation that distinguish this approach from downregulation of anti-apoptotic genes by ASOs and siRNA are worth highlighting.22 First, in every instance of SSO-redirected splicing of Bcl-x pre-mRNA from Bcl-xL to Bcl-xS an anti-apoptotic Bcl-xL molecule is eliminated and the pro-apoptotic Bcl-xS molecule appears. The latter, as mentioned above, will inhibit existing Bcl-xL and Bcl-2 proteins, further reducing resistance of cancer cells to chemotherapy and apoptosis. The SSO thus produces a double or triple bang for the buck.
Second, the higher the level of expression of Bcl-x pre-mRNA in a cell, the more SSO-induced, anti-apoptotic Bcl-xS molecules that will be produced. This suggests that cells from aggressive cancers with higher levels of Bcl-x expression will be more susceptible to SSO-induced apoptosis than healthy, untransformed cells. This counterintuitive mechanism should thus reduce the undesirable side effects that are the bane of classic chemotherapeutics. Cancers with high Bcl-xL expression and those that depend on Bcl-xL expression for survival therefore represent good candidates for treatment with SSOs targeting Bcl-x.19 In contrast, RNase H-sensitive ASOs and siRNA become less effective as their target gene expression increases because as more target mRNA is expressed incomplete inhibition of target gene expression becomes more consequential.
For the above mechanisms to be fully exploited, obstacles associated with the delivery of macromolecules to tumors must be overcome. To this end, Bauman et al. delivered the Bcl-x SSO using lipid nanoparticles previously shown to be effective and well-tolerated in siRNA delivery to subcutaneous and lung tumor xenografts.2325 This treatment resulted in significant redirection of Bcl-x splicing in lung metastases and significant reduction in tumor burden.14 Such effects were not seen in animals treated with vehicle only, SSO only or nanoparticles formulated with a control SSO. The paper presents the first demonstration of sequence-specific SSO efficacy in tumors in vivo. Table 1 shows other potential targets for SSOs as anti-cancer drugs, indicating that this approach is not limited to the Bcl-x gene alone.
Table 1
Table 1
Examples of genes involved in the proliferation, survival and chemoresistance of cancer cells that express splice variants with different functions (reviewed in refs. 2628). This non-comprehensive list suggests a richness of potential SSO targets (more ...)
1. Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, et al. Alternative isoform regulation in human tissue transcriptomes. Nature. 2008;456:470–476. [PMC free article] [PubMed]
2. Lopez-Bigas N, Audit B, Ouzounis C, Parra G, Guigo R. Are splicing mutations the most frequent cause of hereditary disease? FEBS Lett. 2005;579:1900–1903. [PubMed]
3. Svasti S, Suwanmanee T, Fucharoen S, Moulton HM, Nelson MH, Maeda N, et al. RNA repair restores hemoglobin expression in IVS2-654 thalassemic mice. Proc Natl Acad Sci USA. 2009;106:1205–1210. [PubMed]
4. Hua Y, Sahashi K, Hung G, Rigo F, Passini MA, Bennett CF, et al. Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model. Genes Dev. 2010;24:1634–1644. [PubMed]
5. Graziewicz MA, Tarrant TK, Buckley B, Roberts J, Fulton L, Hansen H, et al. An endogenous TNFalpha antagonist induced by splice-switching oligonucleotides reduces inflammation in hepatitis and arthritis mouse models. Mol Ther. 2008;16:1316–1322. [PMC free article] [PubMed]
6. Kinali M, Arechavala-Gomeza V, Feng L, Cirak S, Hunt D, Adkin C, et al. Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: A single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol. 2009;8:918–928. [PMC free article] [PubMed]
7. van Deutekom JC, Janson AA, Ginjaar IB, Frankhuizen WS, Aartsma-Rus A, Bremmer-Bout M, et al. Local dystrophin restoration with antisense oligonucleotide PRO051. N Engl J Med. 2007;357:2677–2686. [PubMed]
8. Kurreck J. Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem. 2003;270:1628–1644. [PubMed]
9. Sazani P, Gemignani F, Kang SH, Maier MA, Manoharan M, Persmark M, et al. Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nat Biotechnol. 2002;20:1228–1233. [PubMed]
10. Fesik SW. Promoting apoptosis as a strategy for cancer drug discovery. Nat Rev Cancer. 2005;5:876–885. [PubMed]
11. Willis SN, Fletcher JI, Kaufmann T, van Delft MF, Chen L, Czabotar PE, et al. Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science. 2007;315:856–859. [PubMed]
12. Uren RT, Dewson G, Chen L, Coyne SC, Huang DC, Adams JM, et al. Mitochondrial permeabilization relies on BH3 ligands engaging multiple prosurvival Bcl-2 relatives, not Bak. J Cell Biol. 2007;177:277–287. [PMC free article] [PubMed]
13. Amundson SA, Myers TG, Scudiero D, Kitada S, Reed JC, Fornace AJ., Jr An informatics approach identifying markers of chemosensitivity in human cancer cell lines. Cancer Res. 2000;60:6101–6110. [PubMed]
14. Bauman JA, Li SD, Yang A, Huang L, Kole R. Anti-tumor activity of splice-switching oligonucleotides. Nucleic Acids Res. 2010;38:8348–8356. [PMC free article] [PubMed]
15. Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, et al. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 1993;74:597–608. [PubMed]
16. Karl E, Zhang Z, Dong Z, Neiva KG, Soengas MS, Koch AE, et al. Unidirectional crosstalk between Bcl-xL and Bcl-2 enhances the angiogenic phenotype of endothelial cells. Cell Death Differ. 2007;14:1657–1666. [PubMed]
17. Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J, et al. The landscape of somatic copy-number alteration across human cancers. Nature. 2010;463:899–905. [PMC free article] [PubMed]
18. Mercatante DR, Bortner CD, Cidlowski JA, Kole R. Modification of alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. analysis of apoptosis and cell death. J Biol Chem. 2001;276:16411–16417. [PubMed]
19. Mercatante DR, Mohler JL, Kole R. Cellular response to an antisense-mediated shift of Bcl-x pre-mRNA splicing and antineoplastic agents. J Biol Chem. 2002;277:49374–49382. [PubMed]
20. Minn AJ, Boise LH, Thompson CB. Bcl-x(S) anatagonizes the protective effects of Bcl-x(L) J Biol Chem. 1996;271:6306–6312. [PubMed]
21. Taylor JK, Zhang QQ, Wyatt JR, Dean NM. Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides. Nat Biotechnol. 1999;17:1097–1100. [PubMed]
22. Bauman J, Jearawiriyapaisarn N, Kole R. Therapeutic potential of splice-switching oligonucleotides. Oligonucleotides. 2009;19:1–13. [PubMed]
23. Li SD, Chen YC, Hackett MJ, Huang L. Tumor-targeted delivery of siRNA by self-assembled nanoparticles. Mol Ther. 2008;16:163–169. [PMC free article] [PubMed]
24. Li SD, Chono S, Huang L. Efficient gene silencing in metastatic tumor by siRNA formulated in surface-modified nanoparticles. J Control Release. 2008;126:77–84. [PMC free article] [PubMed]
25. Li SD, Chono S, Huang L. Efficient oncogene silencing and metastasis inhibition via systemic delivery of siRNA. Mol Ther. 2008;16:942–946. [PMC free article] [PubMed]
26. Mercatante DR, Kole R. Control of alternative splicing by antisense oligonucleotides as a potential chemotherapy: Effects on gene expression. Biochim Biophys Acta. 2002;1587:126–132. [PubMed]
27. Schwerk C, Schulze-Osthoff K. Regulation of apoptosis by alternative pre-mRNA splicing. Mol Cell. 2005;19:1–13. [PubMed]
28. David CJ, Manley JL. Alternative pre-mRNA splicing regulation in cancer: Pathways and programs unhinged. Genes Dev. 2010;24:2343–2364. [PubMed]
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