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Potential molecular targeting of splice variants for cancer

treatment
Christopher A Blair
1,2
and Xiaolin Zi
1,2,3
1
Departments of Urology, University of California, Irvine, Orange, CA 92868, USA
2
Pharmaceutical Sciences, University of California, Irvine, Orange, CA 92868, USA
3
Chao Family Comprehensive Cancer Center, University of California, Irvine, Orange, CA 92868,
USA
Abstract
Array of new targets for investigation as cancer therapeutics has great potential to grow as new
splice-variants are identified and characterized in cancer cell-lines and tumor samples. Tumor-
specific splice variants are being discovered at an increasing rate and their functions are also
investigated in cancer progression. The tumor-specific splice variants whose expression patterns
and activities are successfully characterized may become attractive targets for ablation or splicing
modification. The extreme specificity of their expression suggests that a variant-specific treatment
may allow for targeting of cancerous cells with minimal impact to healthy tissues. Clinical
investigation of applying antisense oligonucleotides to down-regulate mRNAs that contribute to
cancer cell survival and to modify splicing patterns in muscular dystrophy has shown promising
results. These results show that antisense therapy may be applied effectively and safely in humans.
As these treatment strategies continue to improve and novel tumor-specific splice-variants are
identified, modification of splicing patterns will become an important field of investigation to
develop more effective and safe cancer therapies.
Keywords
Cancer therapeutics; Expressed sequence tags; Osteopontin-c; SiRNA; Tumour specific splice
variants
Alternative splicing
Alternative splicing is the process by which a single gene may produce many different
transcripts that can show a wide range of activities, and is responsible for much of the
diversity of the human proteome
1
. There also exists a high degree of tissue-specific and
condition-specific alternative splicing
2
that creates a virtual guarantee that uncharacterized
splice-variants with significant functional differences from the normally expressed version
will continue to be discovered, and many of the potential differences such as ligand binding/
specificity or effect on cell cycle regulation
3
have strong implications for cancer
development and progression. Identification of tumor-specific splice variants is a key first
step in the selection of potential novel treatment targets. While direct analysis of tumor
samples and cancer cell lines can discover small numbers of new splice variants at a time,
large scale sequencing efforts such as the Cancer Genome Project have collected vast
amounts of data on cancer genes that, when analyzed, presents potential therapeutic targets
*
Correspondent author Telehone: (714) 456-8316 Fax: (714)456-1786 xzi@uci.edu .
NIH Public Access
Author Manuscript
Indian J Exp Biol. Author manuscript; available in PMC 2012 J anuary 9.
Published in final edited form as:
Indian J Exp Biol. 2011 November ; 49(11): 836839.
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in the form of genes with characteristic splicing mutations
4
. The usage of expressed
sequence tags (ESTs) has allowed the prediction of large numbers of tumor-specific splice
variants, however this can only be used as a preliminary means of identification, requiring
confirmation of true tumor-specificity by independent methods
5-7
. As more information
continues to emerge on the expression patterns of individual splice variants and their
implications in cancer development and progression, the promise of new and effective
therapeutic targets grows ever stronger.
Role of splice variants in cancer progression
True cancer-specific splice variants present particularly attractive potential targets for
therapy when they have been associated with cancer progression, and provide a broadly
applicable treatment approach due to the ubiquitous nature of alternative splicing events.
Recent report in ovarian cancer has identified tumor-specific splice variants and tested the
viability of treatments targeting them, with encouraging results. For example osteopontin-c
(OPNc) was found to be expressed in ovarian malignant and borderline tumor samples and
cell lines, and was completely absent in healthy tissues. When overexpressed, OPNc, which
lacks exon 4 of the full length osteopontin, was observed to dramatically increase cell
proliferation, invasion, migration, colony formation, and tumor growth, while other non
tumor-specific osteopontin variants did not. This activity suggests that targeted down-
regulation of OPNc, which leaves the full-length and non tumor-specific variant expression
levels unaltered, may provide a means of preventing tumor progression while leaving
healthy tissues unaffected
8
. It was also reported that in taxane-resistant ovarian cancer,
siRNA silencing of survivin (an anti-apoptotic protein) variant 2B inhibited both cell growth
in vitro and tumor growth in vivo. Additionally, survivin 2B silencing caused increased
sensitivity to docetaxel in taxane-resistant cells, demonstrating that treatment resistance
associated with splice variants can potentially be overcome by targeting the variant mRNAs
themselves rather than the full length mRNA
9
. Another recently identified splice variant
that may develop into a therapeutic target is the coxsackie adenovirus receptor (CAR)
variant CAR4/6, which is expressed in cervical cancer samples, but not in normal cervical
epithelial tissue. Ectopic expression of CAR4/6 has been found to significantly enhance both
proliferation and invasion of multiple cell lines, suggesting that investigation into its
targeted down-regulation is warranted and may mitigate the variants harmful effects
10
.
In prostate cancer (PCa), inhibition of androgen receptor (AR) signaling by androgen
ablation is a common therapy applied when cancer metastasizes beyond the prostate or
recurs after radical prostatectomy
11
. In response to androgen depletion, however, PCa will
eventually recur with a castration resistant phenotype (CRPC) that retains its dependence on
AR signaling, which the cells are able to maintain despite castrate levels of circulating
androgens
12
. One proposed mechanism for progression to CRPC is the significant
upregulation of constitutively active AR isoforms lacking the ligand-binding domain present
in normal AR
13-17
. The role of splice-variants in CRPC is particularly complex, since
evidence seems to indicate that the gain of function granted by the isoforms associated with
castration resistance is dependent on the presence of full length androgen receptor
molecules, though the exact mechanism of this dependence remains to be determined
18
.
Approaches for targeting splice variants
The use of custom designed oligonucleotides to bind to specific target sequences is currently
the leading means of correcting conditions associated with pre-mRNA mis-splicing
19
. The
potential of applying antisense oligonucleotides (AONs) as a means of splicing modification
by inducing the exclusion of aberrant exons has been well established as a viable treatment
method, notably in the treatment of Duchenne Muscular Dystrophy (DMD), in which AON-
Blair and Zi Page 2
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mediated exon skipping can greatly reduce the severity of the disorders impact
20-22
. Two
phase I/IIa clinical trials for AON-based DMD therapy have shown encouraging results that
demonstrate the safety and effectiveness of antisense therapy application in human
subjects
23, 24
.
Investigation of the concept of using antisense therapy to restore normal cell-cycle
regulation in cancer cell lines is becoming a promising approach in mitigating many of the
factors that allow cancer development, progression, and survival. A phase II clinical trial is
currently underway using antisense inhibition of survivin as a treatment for prostate cancer
in combination with docetaxel, after the antisense oligonucleotide (LY2181308) was found
to induce apoptosis and also sensitize cells to apoptosis induced by chemotherapy
25
.
Inhibiting the expression of full length, fully functional transcripts may, however, be a
dangerous approach when the gene also has an important function in healthy cell
populations, as may be the case for survivin
26
. However, when a tumor-specific splice
variant with activity vital to the cancer cells survival is identified, it provides an elegant
solution to the problem of ablating problematic variant proteins in tumor cells while leaving
healthy cells with normal transcripts alone
9
.
Another interesting application of antisense therapeutics is the usage of splice switching
oligonucleotides (SSOs) to take advantage of the antagonistic effects of the anti-apoptotic
Bcl-xL and pro-apoptotic Bcl-xS splice variants of Bcl-x, an apoptotic regulator. By
targeting the 5 splice site of exon 2 of Bcl-x pre-mRNA, the investigators were able to
decrease the expression of Bcl-xL and increase expression of Bcl-xS, inducing apoptosis and
reducing in vivo tumor load
27
. A similar approach may potentially be applied in CRPC if
splicing of the AR splice-variant AR-V7, which provides a gain-of-function allowing
androgen-independent growth, can be switched to increase the expression of AR-V1, which
acts as a dominant-negative inhibitor of AR-V7 (Ref. 18). Use of this approach is likely to
be more difficult in the case of AR splicing, however, due to the diversity of AR splice-
variants and the recently described detection of intragenic rearrangement and duplication in
AR splicing deregulation, which may limit the possibility of manipulating the expression of
specific variants
28
.
An alternative approach to the inhibition of AR signaling was recently described in which
the antibiotic nigericin inhibited both androgen-dependent and androgen-independent
growth of AR and truncated AR positive cell lines via destabilization of AR mRNAs and an
additional but unidentified post-translational mechanism. The overall effect of nigericin
treatment in this study proved to be very similar to the result of siRNA mediated knockdown
of full-length and variant transcripts
29
. While this may develop into a promising treatment
for CRPC, drugs with similar activities that target both full-length and truncated mRNAs
may not be as applicable in cases where the full-length transcript is necessary for vital
processes in healthy cells, as in the case of survivin
26
. Additionally, discovering drugs with
this sort of activity may not be achieved as reliably as designing an AON or siRNA
mediated approach to targeted expression modification simply by nature of the need to
screen compounds for activity.
Conclusion
The continuing identification of novel splice-variants in cancerous tissues and cell lines
provides a large and rapidly-expanding array of potential therapeutic targets. The steps for
identifying novel splice variants as therapeutic targets and approaches for targeting splice
variants for cancer are summarized in Figs 1, 2. The expression patterns of many of these
variants, once determined, will provide investigators with information about which variants
are largely harmless or even beneficial, and which are strongly up-regulated or only
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expressed in cancerous cells. While this information alone is valuable as an indicator of a
patients prognosis, more investigation into the in vivo and in vitro effects of these splice
variants may become possible in order to determine which variants result in cell-cycle
deregulation or treatment resistance. Such splice variants may be targeted for ablation either
as a treatment in itself or to improve the outcomes of existing treatments. The understanding
of the role of specific splice-variants in cancer progression improves, an increase in the use
of patient-specific expression patterns for treatment decisions and in development of more
viable molecularly targeted treatments are expected. While current policy precludes the
ability to run clinical trials for all but the most widespread potential targets for alternative-
splicing modification, it seems inconceivable that this will remain the case as the body of
work demonstrating the efficacy and specificity of splice-variant targeting continues to
grow.
Acknowledgments
This work was supported by NIH award 5R01CA122558-04 and 1R21CA152804-01A1, and DOD idea
development award PC100869 (X Z).
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Fig. 1.
Steps to validate splice-variants as treatment targets
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Fig. 2.
Application of treatment strategies
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