Abstract
Alternative splicing has critical roles in normal development and can promote growth and survival in cancer. Aberrant splicing, the production of noncanonical and cancer-specific mRNA transcripts, can lead to loss-of-function in tumor suppressors or activation of oncogenes and cancer pathways. Emerging data suggest that aberrant splicing products and loss of canonically spliced variants correlate with stage and progression in malignancy. Here, we review the splicing landscape of TP53, BARD1 and AR to illuminate roles for alternative splicing in cancer. We also examine the intersection between alternative splicing pathways and novel therapeutic approaches.
Introduction
Alternative splicing generates multiple different mRNAs and downstream proteins from a single gene through the inclusion or exclusion of specific exons. This process occurs in 95% of all multi-exonic genes1 and is catalyzed by the spliceosome, a complex comprised from a core of five small nuclear ribonucleoproteins (U1, U2, U4, U5 and U6; reviewed in Burge et al.2). The spliceosome is aided by over 200 trans-acting factors that recognize cis-regulatory sequences within the pre-mRNA and direct the spliceosome to include or exclude specific exons (reviewed in Chen and Manley3 and Wahl et al.4). Splice variants can thus arise from mechanisms including alternative promoters, preferential usage of exons or splice sites, scrambling of exon order and alternative polyadenylation (Figure 1).
Common mechanisms of alternative splicing. Alternative splicing can occur although a number of different processes and give rise to different mature transcripts (right). Exons and final transcripts are illustrated as boxes while lines represent introns. Constitutively expressed exons are depicted in green, and alternatively spliced exons are depicted in blue or brown. Retained introns occur with the absence of splicing, with the intervening intron (black) included in the final transcript.
Alternative splicing affords a significant evolutionary advantage by providing a large source of proteomic diversity.5 Alternative splicing is often regulated at the tissue level, and tissue-specific variants cooperate to modulate protein–protein interaction networks.6 Stem cells express specific splice variants at each stage of differentiation, with undifferentiated stem cells maintaining the highest levels of diversity for splice isoforms.7 Alternative splicing is also critical in development and can be responsive to normal external stimuli (reviewed in Kalsotra and Cooper8 and Heyd and Lynch9). As with other developmentally related pathways, alternative splicing can be aberrantly regulated by cancer cells to their advantage. Genome-wide studies have long revealed the existence of cancer-specific splicing patterns.10, 11, 12 The ability to commandeer alternative splicing could be beneficial to cancer cells if early developmental stage isoforms critical for proliferation are also ectopically expressed, driving uncontrolled growth. This switch in splicing preference can be critical as numerous genes possess splice variants that are mutually antagonistic.
Escape from cell death is critical for tumorigenesis and some alternatively spliced genes in cancer modulate apoptosis. For example, BCL2L1 possesses an alternative 5' splice site after exon 2 that produces long and short isoforms that are translated into the BCL-XL and BCL-XS proteins, respectively (Figure 2a). BCL-XS promotes apoptosis whereas BCL-XL has anti-apoptotic effects.13 Cancers show predominant expression of the BCL-XL isoform.14, 15 Similarly, the FAS receptor (TNR6) is a cell surface receptor that can initiate cell death when bound to TNFS6, the FAS ligand.16 TNR6 is subject to alternative splicing (Figure 2b); in particular, a splice variant lacking the transmembrane domain results from exon skipping at exon 6.17 This shorter product is soluble and inhibits FAS-mediated cell death, presumably by binding competition for FAS ligand.18 Soluble FAS is detectable in serum, with higher concentrations found in cancer patients compared with healthy individuals.19, 20, 21
Alternative splicing leads to antagonistic variants. (a) BCL2L1 is spliced into two variants based on an alternative 5′ splice site (blue). Expression of the two canonical exons (green) results in the pro-apoptotic BCL-XS short isoform, whereas the long isoform, BCL-XL, arises from the splice site downstream of exon 1 and is anti-apoptotic. Exon sizes are relative to actual length, but the intron is not drawn to scale. (b) Exon 6 (blue) of TNR6 is a cassette exon. Inclusion in the mature mRNA leads to expression of the FAS receptor on the cell surface where it mediates cell death. Exclusion of exon 6 results in a soluble FAS isoform, which has anti-apoptotic effects by competing for ligand. Exons and introns are not drawn to scale. (c) PKM exons 9 (blue) and 10 (brown) are mutually exclusive exons. The embryonic variant PKM2 arises with incorporation of exon 10. Upon maturation to adulthood, this variant is typically switched to PKM1 by the alternative incorporation of exon 9. PKM2 promotes aerobic glycolysis whereas PKM1 promotes oxidative phosphorylation. Exons and introns are not drawn to scale.
Metabolic pathways are also frequently altered in cancer. The Warburg effect, a shift from oxidative phosphorylation to aerobic glycolysis, enables synthetic pathways at the expense of ATP production in cancer cells22 and is partly driven by alternative splicing of the pyruvate kinase M gene (PKM). Exons 9 and 10 are mutually exclusive (Figure 2c), giving rise to PKM1 (exon 9), the adult isoform, or PKM2 (exon 10), the embryonic or tumor isoform.23 PKM2 is expressed widely in cancer (reviewed in Mazurek et al.24), and replacement of PKM2 with PKM1 reverses the Warburg effect and increases oxidative phosphorylation.25
The regulation between antagonistic splice variants of the same gene can also be disrupted to affect proliferative pathways, interactions with proto-oncogenes and tumor suppressors, and the epithelial to mesenchymal transition, a pathway that promotes invasion and metastasis. Although the switch between antagonistic gene isoforms in cancer illustrates critical roles for alternative splicing, this subject has been covered recently26 and will not be reviewed here.
These splice variants are considered canonical products of alternative splicing because they are expressed in somatic tissue as part of normal development or to facilitate cell-specific functions. Although the definition of canonical splicing patterns will continue to evolve as transcript isoforms are dissected in more detail, alternative splicing does not need to follow these patterns to provide a tumorigenic advantage. This process may be harnessed to produce novel exon combinations and transcripts that are rarely, if ever seen in normal somatic cells. We focus here on examples of this aberrant splicing and its association with outcome as well as the therapeutic impact of the alternative splicing pathway. Aberrant splicing can arise through a couple of mechanistic models: alterations in core spliceosomal components can lead to global splicing deregulation and result in a large number of aberrant products. Similarly, alterations in an accessory splicing factor can lead to deregulation of splicing for the limited set of transcripts where the factor is required for accurate splicing. Alternatively, genomic mutations in a critical splicing motif of a single gene will change the splicing pattern of just that transcript.
Regardless of the mechanism, aberrant splicing of proto-oncogenes can produce constitutively active or even gain-of-function variants that confer new survival or proliferative abilities. In contrast, a tumor suppressor with aberrant splicing could result in an altered reading frame and a premature stop codon; subsequent destruction by the nonsense-mediated decay process would ultimately lower the total protein level and its tumor-suppressive capacity. Still, some transcripts may manage to avoid nonsense-mediated decay, and if the truncated protein gains dominant-negative regulatory functions, tumor suppression would be lowered even further. Until recently, technical reasons have led researchers to focus primarily on nonsynonymous coding mutations with easily predictable consequences. By doing so, the single largest source of protein diversity has been woefully neglected. This is best exemplified by the fact that there is perhaps no tumor suppressor as prominent as p53, yet its splicing has only recently gained significant appreciation.
p53
P53 is encoded by the gene TP53 and is one of the most well-known and well-studied tumor suppressors since its discovery over 30 years ago.27, 28 Although it was originally considered an oncogene for nearly a decade,29, 30, 31 p53 is a transcription factor32, 33 that enacts tumor suppression by mediating cellular functions including, but not limited to, apoptosis,34, 35 cellular senescence36, 37 and DNA repair38 (reviewed in Levine and Oren31 and Vogelstein et al.39). P53 activity is largely governed through a negative feedback loop with MDM2,40, 41, 42 an E3 ubiquitin-protein ligase that mediates proteasomal degradation of p53. Mutations in p53 that disrupt either of the tandem transactivating domains often contribute to tumorigeneisis (reviewed in Brosh and Rotter43).
Alternative splicing of TP53 was originally identified over 20 years ago in both mouse and humans,44, 45 however, investigation into the functional roles of these splice variants is still ongoing. TP53 contains 11 exons, which encode major functional domains including: the two tandem transactivation domains (TAD), the first of which also serves as the binding domain for MDM2, the DNA-binding domain and the oligomerization domain (Figure 3a). Owing to intron inclusion, normal human lymphocytes express an alternatively spliced variant of TP53 with a truncated C-terminal domain. This isoform is found in significant amounts only in quiescent cells and provided the first observation that TP53 splicing could have functional consequences; presumably due to the truncation of the oligomerization domain,46 it fails to bind DNA and possesses a transcriptional defect. Subsequently, an N-terminal truncated isoform, Δ40-p53, was discovered in the breast cancer cell line 21PT, arising from an internal transcriptional start site in the first exon.47 Additional splice variants carrying N-terminal deletions arise from an internal promoter before exon 5 to produce Δ133-p53 and Δ160-p53 isoforms that utilize start methionines at amino-acid positions 133 and 160, respectively48 (Figure 3b). Interestingly, local internal ribosomal entry sequences have been shown to produce N-terminal truncated isoforms, indicating the need for further study into how these isoforms are generated.49, 50 All three of these N-terminal variants can theoretically combine with three different C-terminal variants that arise from alternative splicing downstream of exon 9 (α, β and γ).48, 51 Canonical splicing of the transcript after exon 9 leads to the α-isoform with a complete oligomerization domain and the inclusion of exons 10 and 11. The β- and γ-isoforms are formed because of intron retention and stop codons, which follow after translation of 10 or 15 new amino acids, respectively (Figure 3c). Coexpression of full-length p53 and p53β enhances transcriptional activity on the p21 promoter but not the BAX promoter, and the two isoforms cooperate to promote senescence.52
Splicing schematic of TP53. (a) Exon structure of full-length TP53 with relative locations encoding the transactivation domains (TAD), DNA-binding domain (DBD) and oligomerization domain (OD) indicated (as described in UniProt). Exon sizes are relative to actual length, but introns are not drawn to scale. Start and stop codons are located at the indicated positions. Coding sequences are indicated as white-filled boxes, and untranslated regions are indicated as gray-filled boxes. Splice variants containing combinations between b and c have also been identified. (b) N-terminal TP53 splice variants. An alternative promoter between exons 4 and 5 leads to variants that utilize different start codons. Intron retention is indicated by the black-filled box. (c) C-terminal TP53 splice variants. Alternative 5′ or 3′ splice sites are indicated by brown-filled boxes, and alternative reading frames are indicated by blue-filled boxes.
Additional C-terminal variants were described recently. P53δ and p53ζ arise because of splice site point mutations and result in intron retention. P53δ, similar to the β- and γ-isoforms, truncates the oligomerization domain after exons 9 and 27 new amino acids. Intron retention in p53ζ results in a frame-shift and a premature termination codon in the middle of exon 7.53
In omitting the first 39–159 amino acids and truncating the transactivating domain, N-terminal variants of p53 likely act as dominant-negative regulators. Δ133-p53 inhibits full-length p53 activity as shown by in vitro reporter assays.48, 54 Additional in vitro studies indicate that Δ133-p53α does not bind p53 response elements and inhibits full-length p53 from binding p53 response elements.55 Δ133-p53 is expressed in a p53-dependent manner,56 raising the possibility that this isoform participates in a negative feedback loop to modulate full-length p53 activity. However, by encoding for an earlier translational start site, Δ40-p53 still retains the second TAD, and in a study analyzing p53 mutations within the TADs, activity of just the second TAD allowed for minimal transactivation of most p53 target genes.57 Indeed, the Δ40-p53 has impaired transcriptional activation and impairs growth suppression by oligomerizing with full-length p53.47, 58 In p53-null cell lines, transfection of Δ40-p53 alone was insufficient to initiate transcriptional activity of a p53 response element reporter, whereas co-transfection with full-length p53 decreased total p53 transcriptional activity in a dose-dependent manner. Interestingly, in Saos-2 cells, a lower ratio of Δ40-p53/full-length p53 increased transcriptional activity over full-length p53 alone, suggesting that this interaction varies upon cellular context. In these studies, full-length p53 was also protected from degradation by MDM2 when co-transfected with Δ40-p53.59 Although splice variants in p53 that alter the C-terminal domain require more investigation, p53β has been shown to display promoter-specific binding capabilities.48
Given functional differences among p53 splice variants, deregulation of these variants in cancer has come under scrutiny. Mutations affecting TP53 splicing are found in many different cancers, with some of these splice variants previously considered to be ‘neutral’ as they do not change amino-acid composition.60 In a reverse transcriptase–PCR analysis of pooled RNA from normal breast tissue, all three C-terminal splice variants could be detected, but Δ133-p53 variants were absent. In the same analysis on RNA extracted from a panel of 30 breast tumors, only 5 of which were identified to harbor p53 mutations, p53γ was not observed, whereas Δ133-p53α was found in 24 samples.48 In a separate cohort of 127 breast tumors, only 19% expressed both p53β and p53γ. By comparing tumors with p53 mutations, patients with concomitant loss of p53γ showed poor survival, whereas those who co-expressed p53γ had lower rates of recurrence, with survival similar to patients with functional wild-type p53.61 Both Δ40-p53 and p53β are expressed in a majority of melanoma cell lines and primary isolates of metastatic melanoma, but are absent or expressed at low levels in fibroblasts and melanocytes. Δ133-p53β is the only other splice variant expressed in melanoma cell lines, but at very low levels.62
In addition to the previously described N-terminal and C-terminal splice variants, a panel of 34 ovarian cancer cell lines revealed that internal exon skipping of TP53 can also occur. This was validated in a cohort of 245 primary ovarian cancers, with expression of the splice variants lower than observed in the cell lines. P53δ associated with decreased recurrence free and overall survival, supporting the idea that splice-site variants and nonsynonymous mutations can be similarly detrimental. Concomitant expression of p53β and functional wild-type p53 also correlated with decreased survival.53 In a separate study, Δ133-p53 and Δ40-p53 isoforms were identified in both stages I and stage III (n=83 and 86, respectively) ovarian cancers. Neither was associated with malignant progression, although the authors could not rule out a role in tumor initiation.63
Overall, it appears that the N-terminal and C-terminal splice variants of TP53 have distinct effects in cancer. The absent TAD in N-terminal variants provides an apparent mechanism to inactivate p53, and perhaps unsurprisingly, has been identified repeatedly in cancer. C-terminal variants require further investigation, however, some of these isoforms appear to be functional, given that expression of p53γ alongside mutant p53 phenocopies the survival of patients with wild-type p53.61
BRCA1-associated ring domain 1 (BARD1)
A majority of women who inherit mutations in the BRCA1 breast cancer susceptibility gene will develop breast cancer64 and 39% will develop ovarian cancer.65 The search for potential binding partners of this tumor suppressor led to identification of BARD1,66 a gene that spans 11 exons and encompasses nearly 85 kb on chromosome 2. Both BRCA1 and BARD1 have similar protein structures and form a heterodimer by binding through their RING finger domains (Figure 4a). This domain contains critical residues that are mutated in BRCA1 in association with breast and ovarian tumors. These data suggest that interactions between BRCA1 and BARD1 are required for tumor suppression.
Splicing schematic of BARD1. (a) Exon structure of full-length BARD1 with relative locations encoding the RING domain, ANK repeats and BRCT domains (as described in UniProt). BRCA1 and BARD1 interact through their respective ring domains. Exon sizes are relative to actual length, but introns are not drawn to scale. Start and stop codons are located at the indicated positions. Coding sequences are indicated as white-filled boxes, and untranslated regions are indicated as gray-filled boxes. (b) BARD1 splice variants exhibit various combinations of alternative polyadenylation and exon skipping. Alternative reading frames are depicted in blue. (c) Alternative promoters were found in exon 4 (brown) and encode for varying translational start methionines.
Given this critical interaction, it is surprising that BRCA1 shows frequent mutations (1653 as surveyed by the HGMD67), whereas BARD1 mutations in both familial and sporadic breast cancer are relatively rare.67, 68, 69, 70 BARD1 is more than just an accessory protein for BRCA1; the two genes are not co-expressed universally in all tissues,71 and in response to genotoxic stress, BARD1 functions in a BRCA1-independent manner to induce p53-dependent apoptosis.72 In addition, homozygous loss of BARD1 in mice results in an embryonic lethal phenotype, similar to BRCA1-null mice.73 Indeed, BARD1 itself has come to be recognized as a potent tumor suppressor.74
The relative paucity of BARD1 mutations is especially puzzling in light of several genome-wide association studies that identified BARD1 as a susceptibility gene for neuroblastoma.75, 76, 77 Among the first clues to this puzzle was the observation of aberrant BARD1 splicing in NuTu-19 cells, a rat ovarian cancer cell line78 that does not express full-length BARD1. Reintroduction of the full-length protein induced apoptosis, suggesting that this splice variant is defective in tumor suppression. Lacking the RING domain, this variant (BARD1δ, Figure 4b) is also expressed in several breast cancer lines, and co-immunoprecipitation experiments have shown that it is unable to interact with BRCA1.79
Could aberrant BARD1 splicing also confer dominant-negative regulation or other oncogenic properties? This scenario is suggested by the finding that BARD1 overexpression is associated with poor survival in breast and ovarian tumors.80 That BARD1 expression is inversely associated with outcome contrasts its purported role as a tumor suppressor. In addition, although the protein had been described as nuclear,66, 81 non-apoptotic cells exhibit cytoplasmic localization. Analysis at the RNA level revealed that many of these transcripts have N-terminal truncations and lead to decreased protein stability.82
An additional survey of cervical, breast, ovarian and endometrial cancer cell lines, along with ovarian tumor tissue arrays revealed that BARD1 spliced isoforms are often more abundant than the full-length product.83 This survey also identified and enumerated splice isoforms BARD1α-η (Figure 4b), as well as alternative transcriptional start sites in exon 4 (Ω1, Ω2, Ψ) (Figure 4c). RNA interference experiments directed only toward the full-length transcript did not affect cell growth, whereas small interfering RNAs targeting both full-length and BARD1 splice isoforms reduced proliferation, suggesting that BARD1 splice variants have functional roles.83, 84 Indeed, while full-length BARD1 normally promotes Aurora B degradation, BARD1β opposes this and promotes proliferation by scaffolding Aurora B and BRCA2 in late-stage mitosis in a BRCA1-independent manner.84 BARD1δ also has antagonistic functions relative to full-length BARD1. In MCF7 cells, the BRCA1-BARD1 complex leads to estrogen receptor alpha ubiquitination and degradation, but overexpression of BARD1δ instead enhances estrogen receptor alpha stability and transcriptional activity.85
Up to 19 different splice isoforms of BARD1 have been identified in colon cancer86 with a concomitant lack of full-length BARD1 expression.87 Furthermore, in analyses of human tumor biopsies with BARD1 epitope-specific immunohistochemistry, patients with expression of BARD1κ and BARD1π, both presumed to be internal deletion mutants, as well as expression of BARD1β showed poor outcome in non-small cell lung cancers.88 As aberrant splicing of BARD1 is not limited to hormonally regulated tumors, could the susceptibility locus in neuroblastoma demonstrate aberrant splicing as well? Analysis of BARD1 isoforms cloned from neuroblastoma cell lines and fetal sympathetic ganglia characterized a total of 15 unique splice variants in addition to the full-length transcript.89 In particular, expression of BARD1β was associated with the homozygous GG neuroblastoma risk allele at rs6435862. Similar to observations in ovarian cancer, knockdown and overexpression experiments in neuroblastoma cell lines reiterated the oncogenic characteristics of BARD1β.89
It remains unclear why BARD1 loss-of-function in cancer proceeds predominantly through alternative splicing as opposed to mutation. BARD1 has functions outside of the BRCA1 signaling axis, and mutations within BARD1 may be too deleterious for cancerous cells to overcome. In this case, alternative splicing could provide a mechanism in which activity is finely modulated, rather than abolished. The distinction between the loss of a tumor suppressor and the activation of oncogenic properties can be difficult. It is also possible that these splice variants gain oncogenic BRCA1-independent functions. The effects of BARD1 alternative splicing in neuroblastoma also demonstrate the importance of evaluating alternative splicing in conjunction with mutational surveys when considering loci and genes identified through genome-wide association studies or quantitative-trait loci mapping experiments.
Androgen receptor
Signaling through the androgen receptor (AR), a steroid hormone receptor transcription factor,90 is critical for prostate cancer development.91, 92 Ligand binding triggers receptor homodimerization and nuclear translocation, which in turn executes a transcriptional growth program through binding specific AR element sequences.93 AR spans approximately 200 kb on Xq11–12 and has eight exons that encode the full-length 110 kDa protein.94 Exons 2 and 3 encode most of the DNA-binding domain95 and exons 4–8 encode the ligand-binding domain96 (Figure 5a). A naturally occurring splice variant of AR exists and consists of an alternative first exon. This transcript is found in various tissues, with robust expression in the heart.97 Although the canonical first exon constitutes over 50% of the full-length transcript, this alternative N-terminal domain contains only seven amino acids and was shown to inhibit proliferation when overexpressed in LNCaP cells (Figure 5b). Dubbed AR45 due to its calculated 45 kDa molecular weight, this isoform forms heterodimers with full-length AR in a ligand-dependent manner and inhibits AR activity, presumably due to an inability of the alternative N-terminal domain to recruit a full set of transcriptional coactivators.97, 98, 99
Splicing schematic of AR. (a) Exon structures of full-length AR and the canonically spliced AR45 variant. Relative locations encoding the DNA-binding domain (DBD) and ligand-binding domain (LBD) are indicated (as described in UniProt). Exon sizes are relative to actual length, but introns are not drawn to scale. Start and stop codons are located at the indicated positions. Coding sequences are indicated as white-filled boxes, and untranslated regions are indicated as gray-filled boxes. Exons are numbered as they appear in full-length AR. Exon A has been previously described as Cryptic Exon 4 (CE-4), and exon C has been previously described as exon 9. Region B (brown) includes the previously described CE-2, CE-3, CE-4, CE-5 and various cases of intron retention. (b) Aberrant splicing of AR can include exon scrambling (AR-V2 and AR-V4) as well as exon skipping (AR-V12–AR-V14). The translated amino-acid sequence for the truncated C-terminus in region B is given for AR-V2 starting with the end of exon 3. (c) AR splice variants often include intron retention or the splicing of cryptic exons and subsequent truncation after exon 3. The translated amino-acid sequence for the truncated C-terminus in region B is given for the splice variants starting with the end of exon 3.
Androgen deprivation therapy has formed the backbone of prostate cancer therapy for over 70 years,100 achieved through surgical castration or drugs that block androgen biosynthesis.101, 102 Recurrent disease arises when the tumor loses androgen sensitivity, termed castration-resistant prostate cancer (CRPC). Comparative genomic hybridization revealed that amplification of AR may be utilized by CRPC to promote growth in low androgen conditions.103 The fact that normal AR isoforms show functional differences based on alternative splicing suggests another mechanism that could be exploited by CRPC. In fact, androgen insensitivity syndrome, a developmental disorder resulting in the impaired development of internal and external male phenotypes, is due to compromised androgen signaling.104 Interestingly, aberrant splicing of AR has been found in both partial and complete androgen insensitivity syndrome, resulting in receptors unable to bind ligand, as well as receptors that have no transcriptional activity even after ligand binding.105, 106, 107, 108, 109
All of these previously described alternatively spliced variants result in loss-of-function in AR. Might gain-of-function in AR isoforms be advantageous in CRPC? An AR isoform lacking the ligand-binding domain was first identified in the prostate cancer cell line 22Rv1110 and subsequently detected in prostate cancer tumor tissue.111 This isoform could act independently of ligand and was more efficient than full-length AR in activating transcription. Although this isoform was originally thought to result from proteolytic cleavage, knockdown experiments indicated that the truncated isoform arises because of alternative splicing. RNA interference targeting an exon partly encoding the ligand-binding domain only eliminated full-length AR, whereas small interfering RNAs targeting exon 1 knocked down all AR isoforms. In fact, the truncated isoform was found to use an alternative exon 2,112 which excluded post-translational modifications as a mechanism to generate this isoform. Further analysis of the 22Rv1 line has since revealed several other AR splice variants.113
Computational methods have identified three cryptic exons located between exons 3 and 4 and led to discovery of one of the best-characterized splice variants, AR-V7 (Figure 5b). Also cloned from 22Rv1 cells, this transcript contains a cryptic exon adjoined to exon 3 that harbors a stop codon after 17 amino acids.114, 115 Expression of this transcript was increased by ∼20-fold when comparing CRPC with hormone naive prostate cancer. A subset of the hormone naive samples with high expression levels indicated that high AR-V7 expression was a strongly associated with recurrence following surgical treatment.114 Expression of AR-V7 was examined by immunohistochemical analysis of 429 human prostate tissue samples, where it was also found to be upregulated in CRPC as compared with hormone naive samples. A higher cytoplasmic staining score also correlated with increased risk of recurrence after surgery.115 AR-V7 was constitutively active, with isoform-specific knockdown resulting in reduced proliferation in vitro and in vivo.114, 115
An additional novel AR splice variant, identified from a human bone metastasis, contains a partially retained intron between exons 2 and 3 that disrupts the DNA-binding domain.116 The LuCaP xenografts, 25 prostate cancers mostly derived from CRPC metastases, identified another splice variant lacking exons 5–7.117 Other cryptic exons, including one downstream of exon 8, were discovered in two CRPC samples using a custom tiling microarray that spanned 200 kb and encompassed the AR locus.118 Furthermore, a separate immunohistochemical tissue microarray analysis of 50 primary prostate cancers and 162 metastatic CRPC samples, using AR antibodies specific either for the N- or C-terminus, noted an increase of AR variants in CRPC but not primary prostate cancer.119
Additional AR splice variants have also been profiled in the VCaP (human) and Myc-CaP (murine) cell lines using deep sequencing methods.120 Interestingly, these variants were expressed at very low levels relative to full-length AR (∼0.1–2.5%) and gain-of-function was dependent on expression of full-length AR. Although these data suggest that AR splicing is inconsequential in these models, subsequent RNA interference experiments targeting full-length AR while measuring the activity of AR-V7 in the presence or absence of the AR agonist R1881 showed no difference.118 As constitutively active splice variants can be expressed in the absence of full-length AR,117 it appears that cellular context is critical to modulate the activity of conditionally active splice variants. Further functional analysis of truncated AR variants showed that C-terminal variation leads to differences in nuclear localization. However, regardless of the specific C-terminus, AR isoforms possess a basal level of nuclear localization and ligand independent, constitutive transcriptional activity. Indeed, the transcriptional ability of each individual variant is promoter dependent and is not affected by mutations that alter nuclear localization.121
Ultimately, the complex interplay between AR and its splice variants requires further investigation, but the abundance of aberrant splicing after exon 3 also adds to the intrigue of AR splicing. Transcripts involving out of order sequences (AR-V3 and AR-V4, Figure 5b) have been identified, but are these splice variants examples of exon scrambling,122, 123 or are there underlying genomic structural variations? This region remains a prime candidate to examine genetic control of splicing given the abundance of cryptic exons and diverse examples of intron retention (Figure 5c).
The splicing pathway
The serine/arginine rich (SR) family of proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) were among the first to be identified to regulate alternative splicing124, 125 and remain the best-studied trans-acting splicing factors. SR proteins typically facilitate splice site recognition and promote inclusion of alternatively spliced exons by binding to regulatory sequences present in the pre-mRNA, intronic and exonic splicing enhancers (Figures 6a and b). Upon binding, they can interact with other splicing co-activators or directly recruit spliceosomal small nuclear ribonucleoproteins to the splice site and activate splicing.126, 127 HnRNPs can oppose this action by binding to intronic and exonic splicing silencers on the pre-mRNA and inhibit recognition of the splice site (Figures 6a and c).3, 128 These splicing inhibitors are generally thought to sterically hinder spliceosomal small nuclear ribonucleoproteins or other splicing enhancers from binding, although additional mechanisms have been proposed to contribute to exclusion of alternatively spliced exons (reviewed in Chen and Manley3). Ultimately, whether an exon is included or excluded from the final mRNA transcript is determined by the relative concentrations of the activators and repressors at the splice site.129, 130
Genomic regulation of splicing. The decision to splice an alternative exon (blue) with constitutively expressed exons (green) is largely governed by the concentration of accessory splicing factors that recognize and bind splicing motifs. (a) SR proteins promote exon inclusion by binding intronic splicing enhancers (ISEs) or exonic splicing enhancers (ESEs) (gray) on the pre-mRNA and recruit spliceosomal small nuclear ribonucleoproteins (snRNPs—yellow) to the splice site. Other spliceosomal snRNPs are also recruited to splice the alternative exon, with successive rounds of splicing at downstream exons as in b. This can be opposed by heterogeneous nuclear ribonucleoproteins (hnRNPs) that recognize intronic splicing silencers (ISSs) or exonic splicing silencers (ESSs; red) on the pre-mRNA and inhibit SR protein binding or spliceosomal snRNPs from recognizing the splice site. The downstream splice site is recognized instead, and the alternative exon is excised with the rest of the intron as in c.
Although the cause of most aberrant splicing events remains unknown, an understanding of known splicing factors serves as a starting point for understanding mechanism. SF2/ASF, encoded by the gene SFRS1, is an SR protein that is also essential for genomic stability.131 Overexpression of this gene in a large panel of lung, colon and breast tumors identified SFRS1 as a proto-oncogene,132 and its transforming abilities have been shown to act through the mTOR Complex 1 signaling axis.133 Downregulation of another SR family member, SRSF3, promotes alternative splicing of p53, resulting in an increase of p53β and downstream cellular senescence.134
The transcription of several hnRNP proteins, including the polypyrimidine tract binding protein, is activated by the MYC oncogene. In cancer, these splicing factors bind to and inhibit inclusion of exon 9 for PKM, promoting the embryonic PKM2 isoform and aerobic glycolysis.135 Similarly, transcriptional upregulation of hnRNPA1 is downstream of EGFRvIII in glioma, and it affects alternative splicing of MAX to produce ΔMax and promote glycolytic gene expression and proliferation in glioma cells.136 HnRNPA1 deregulation is also prominent in lung, colon and renal cancers.137, 138, 139 Another hnRNP family member, hnRNPH, is overexpressed in glioblastoma where it may promote malignant progression by switching the splicing preferences of RON and MADD. RON encodes a receptor tyrosine kinase and is spliced into a ligand-independent, constitutively active form that promotes invasion and metastasis. MADD encodes a death domain containing adaptor protein that mediates apoptotic tumor necrosis factor-α signaling, and it is spliced into an antagonistic anti-apoptotic variant.140
Although these two protein families are perhaps the most ubiquitously expressed and well-studied splicing factors, others have also been found to contribute to tumorigenesis. For example, CD44 is known to undergo complex alternative splicing involving 10 variant exons,141 and in particular, exon v5 is critical for promoting metastasis.142 The transcriptional co-activator SND1 is also an effector of alternative splicing, as it interacts with the splicing factor SAM68 to promote CD44 exon v5 inclusion. Both SND1 and SAM68 are upregulated in prostate cancer, and knockdown of SND1 or SAM68 reduces proliferation and migration of prostate cancer cells.143
The splicing landscape as a whole has also come under recent scrutiny in cancer. Exon array analysis of a cohort of 47 neuroblastoma patients revealed numerous splicing differences separating stage 1 and high-risk stage 4 disease, including PKM, KIF1B and MAP2.144 Similarly, exon arrays have been used to examine alternative splicing in 102 normal and cancer tissue samples from colon, urinary bladder and prostate cancer. Over 2000 candidate alternative splicing events distinguished tumor and normal tissues, with a few genes (ACTN1, CALD1 and VCL) found in all three tumor tissues.145 In addition, whole-exome sequencing of 29 myelodysplasia specimens, a frequent precursor of acute myeloid leukemia, identified frequent mutations in the splicing pathway.146
Alternative splicing and cancer therapy
The tumorigenic advantages provided by alternative splicing are not limited to proliferation or apoptotic blockade. Aberrant splicing can also provide a means for therapeutic evasion by gain-of-function. As we improve our understanding of the molecular basis of different cancers, the development of highly specific small molecule inhibitors has yielded tremendous success.147 Once cancer is detected in the clinic and treatment commences, selective pressures within a tumor typically promote therapy resistance. Almost 80% of patients enrolled in a phase I clinical trial for metastatic melanoma observed partial or complete response to vemurafenib, an inhibitor of BRAF(V600E).148 Invariably, patients relapse, developing resistance through a number of mechanisms.149, 150, 151 Interestingly, in an in vitro model of inhibitor resistance, a novel splice variant lacking exons 4–8 has promoted BRAF dimerization and vemurafenib-resistant extracellular signal-regulated kinase signaling. Six of 19 patients with acquired resistance to vemurafenib also displayed BRAF(V600E) splice variants, indicating that therapy resistance could also be achieved through aberrant splicing.152
With emerging data associating splice variants with survival, can this information be used to improve therapy? If aberrant splicing leads to functional consequences, would correcting the splicing, perhaps in conjunction with conventional therapy, improve survival in cancer? A mouse model of spinal muscular atrophy provides clues in this regard. Spinal muscular atrophy is a genetic disease where loss of the SMN protein causes death of motor neurons in the anterior horn of the spinal cord, resulting in progressive and debilitating weakness. Although caused by loss-of-function mutations in SMN1,153 disease severity is modulated by the paralog SMN2, which is able to produce varying levels of functional protein. The variability in expression of functional SMN2 stems from the fact that exon 7 is predominantly skipped, resulting in a nonfunctional SMN.154 Anti-sense oligonucleotides targeting an intronic splicing silencer have been used to correct SMN2 splicing and restore SMN expression, providing effective long-term rescue of spinal muscular atrophy in mice.155 If pharmacologic anti-sense oligonucleotides can be developed to achieve specific knockdown in vivo, this approach could be directed to tumor-specific splice variants for cancer therapy.
As the pharmacologic development of anti-sense oligonucleotides continues, conventional therapy remains driven by small-molecule inhibitors and derivatives of naturally occurring compounds with antitumor effects. Herboxidiene,156 FR901464157 and the pladienolides158 are natural products that modulate spliceosomal components. These were of particular interest because of their low nanomolar IC50 and cytotoxic effects in cancer cell lines and animal models. They have led directly to the development of synthetic derivatives: E7107 from pladienolide B, spliceostatin A and meayamycin from FR901464, and the sudemycins, a set of molecules designed using a consensus pharmacophore gleaned from known structure-activity relationships (reviewed in Bonnal et al.159 and Webb et al.160). SF3B, a subunit of the U2 small nuclear ribonucleoprotein that facilitates splice site recognition (Figure 6), is the main target of these compounds,161, 162 and they modulate not only alternative splicing, but also expression of genes critical to cancer progression.163, 164, 165
By effecting intron retention or exon skipping, these drugs ultimately lead to activation of the nonsense-mediated decay pathway or the production of inactive cell cycle genes, resulting in cellular arrest.161, 162, 166 Interestingly, these drugs show tumor-specific effects with little toxicity, despite theoretical deleterious effects on normal cells. One plausible explanation is that drug dosage is critical to avoid complete inhibition of splicing, which would be universally toxic. At an appropriate dose, these drugs may still be effective at eliminating aberrant splicing or minimizing the splice isoforms that are critical for cancer progression. In this setting, cancers that have acquired deregulation of global splicing patterns may be best suited for spliceosome-inhibitory therapy. It is also possible that tumors driven in-part through functionally aberrant splicing may be exquisitely sensitive to these spliceosomal modulators, as the drugs reverse the splicing dependency. Future studies analyzing the molecular underpinnings of these drugs may reveal more about the functional roles of alternative splicing in cancer.
Conclusions and perspectives
The overexpression experiments in p53-null cells that revealed the dose-dependent dominant-negative regulatory effects of Δ40-p5359 also serve to highlight the importance of the ratio between splice variants. Is there an absolute threshold for expression of any particular splice isoform to produce a biological effect? The answer is certainly transcript-specific, as the normal function of the full-length product is just as critical as the distinction of the novel splice isoforms as gain-of-function or dominant-negative regulators. All three genes discussed here suggest that cellular context is also an important factor, and as evidenced by the inconsistent data between splice isoform ratios in AR, the answer is not always clear. Furthermore, minor amounts of aberrant splice isoforms may be indicative of global splicing deregulation with combinatorial effects in cancer. These issues may be better addressed as technology continues to improve in detecting splice isoforms and quantitating their expression.
Despite the uncertainty in this regard, the influence and impact that alternative splicing has in cancer is evident in the association of splice variants with outcome and the profound effects that splicing can have on therapy. Although interest in cancer-associated splicing continues to increase, the specific regulatory mechanisms harnessed to confer tumorigenic advantages are still poorly understood. This is true regardless of whether these events occur through aberrant splicing or modulation of existing alternative splicing.
Large-scale, unbiased proteomic approaches have been used to identify additional splicing factors.167 However, given the tissue- and cellular-specificity of splicing programs, can these methods be truly comprehensive? Along these same lines, splicing motifs have been so far defined by various methods. SELEX (systematic evolution of ligands by exponential enrichment) has identified exonic splicing enhancers in vitro168, 169 and in vivo,170 but this method necessitates a known splicing factor to begin. Computational methods have also identified exonic splicing enhancers171, 172 and intronic splicing enhancers173, 174 utilizing several approaches including motif comparisons between introns and exons, exons with weak splice sites and exons with strong splice sites, and evolutionary conservation. An unbiased approach using a splicing reporter assay to analyze all possible decamer motif combinations identified 109 intronic splicing enhancers.175 The fact that some of these motifs overlapped with previously identified exonic splicing silencers indicates the importance of positional effects on motif function. This assay utilized a reporter system in which random decamers were cloned into a specific position, but as human introns extend an average of 3.7 kb,176 a truly unbiased approach including positional effects remains to be examined.
The importance of defining and understanding these motifs should not be understated. The genetics of splicing clearly have crucial roles in cancer, as exemplified by the common variation associated with BARD1 splicing and the splice site mutations demonstrated in both TP53 and AR. Current technologies provide a wealth of sequencing data that has revolutionized the way we analyze mutations in cancer. However, mutational effects on splicing are typically ignored when distinguishing synonymous from nonsynonymous mutations. Similarly, a mutation that ultimately results in a premature stop codon often leads the resulting transcript and protein to be automatically classified as non-functional, without regard to additional splice variants. For example, some of the TP53 splicing experiments were performed in the K562 cell line, which has been categorized as ‘p53 null’ because of a premature stop codon at residue 148. These cells still expressed the Δ160-p53 isoform, indicating that functional splice variants are still being produced from transcripts carrying frameshift or nonsense mutations.177
In addition, high-throughput sequencing in cancer primarily focuses on the exome, mostly due to the benefits of the accompanying increase in coverage depth. Although these studies clearly yield insights into the genetics of disease, changes in splicing can easily be missed by failing to capture regulatory regions within the introns. It could be argued that as we do not have a definition for precise motifs at specific intronic positions to look for, there is no justification for whole-genome sequencing. Yet is there a better way to understand the genetic regulation of splicing without these data?
Next-generation precision medicine therapies are on the horizon and promise highly specific, perhaps even isoform specific, targeting abilities. In-depth genomic studies of splicing will be critical for defining splicing mutations that occur away from intron–exon boundaries. These data, coupled with biochemical and molecular analyses using splicing-modulating drugs, promise to uncover new targets in cancer and to enable novel approaches to block canonical cancer targets and pathways.
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Acknowledgements
We are grateful to Clay Gustafson, Miller Huang and Erin Simonds for reviewing the manuscript. The Weiss lab is supported by NIH grants CA176287, CA82104, CA133091, CA102321, CA148699, CA159859, CA163155 and CA081403; and the CureSearch, Katie Dougherty, Pediatric Brain Tumor, St Baldricks and Samuel G Waxman Foundations.
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Chen, J., Weiss, W. Alternative splicing in cancer: implications for biology and therapy. Oncogene 34, 1–14 (2015). https://doi.org/10.1038/onc.2013.570
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DOI: https://doi.org/10.1038/onc.2013.570
Keywords
- alternative splicing
- aberrant splicing
- p53
- BARD1
- AR
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