Download Cancer syndromes and therapy by stop-codon readthrough

Review
Cancer syndromes and therapy by
stop-codon readthrough
Renata Bordeira-Carriço1, Ana Paula Pêgo2, Manuel Santos3, and Carla Oliveira1,4
1
Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-465 Porto, Portugal
INEB - Instituto de Engenharia Biomédica, Universidade do Porto, 4150-180 Porto, Portugal
3
RNA Biology Laboratory, Department of Biology and CESAM, University of Aveiro, 3810-193 Aveiro, Portugal
4
Faculty of Medicine of the University of Porto, 4200-319 Porto, Portugal
2
Several hereditary cancer syndromes are associated
with nonsense mutations that create premature termination codons (PTC). Therapeutic strategies involving
readthrough induction partially restore expression of
proteins with normal function from nonsense-mutated
genes, and small molecules such as aminoglycosides
and PTC124 have exhibited promising results for treating
patients with cystic fibrosis and Duchenne muscular
dystrophy. Transgenic expression of suppressor-tRNAs
and depleting translation termination factors are,
among others, potential strategies for treating PTC-associated diseases. In this review, the potential of using
readthrough strategies as a therapy for cancer syndromes is discussed, and we consider the effect of
nonsense-mediated decay and other factors on readthrough efficiency.
Inherited diseases and premature truncation
One-third of all inherited disorders are caused by truncating mutations (nonsense, frameshift, or splice-site) in
genes encoding fundamental, disease-associated proteins
[1]. These mutations produce premature termination
codons (PTCs), which are potentially deleterious because
the truncated proteins generated are often non-functional
(Figure 1a and b) or exert dominant-negative effects [1,2].
Specifically, the incidence of nonsense mutations in individual cases of genetic disorders range from 5 to 70% [3],
depending on the population analyzed. A collection of
4631 genes deposited in the Human Genome Mutation
Database (HGMD Professional release 2012.1; http://
www.hgmd.org), documented nonsense mutations as
11.2% (13 802/123 656 mutations) of all mutations known
to cause, or be associated with, inherited human diseases
[3,4].
Clinical trials of therapies aimed at suppressing the
effect of premature truncation have been preferentially
used in patients with autosomal recessive or X-linked
disorders carrying nonsense mutations in at least one
allele [3]. The most relevant examples include cystic fibrosis (CF) [5], Duchenne muscular dystrophy (DMD) [6],
hemophilia A (HA) and B (HB), Hailey–Hailey disease
Corresponding author: Oliveira, C. ([email protected]).
Keywords: aminoglycosides; hereditary cancer syndromes; nonsense-suppression;
PTC124; readthrough; suppressor-tRNA; nonsense-mediated decay (NMD); premature termination codon (PTC).
(HHD), McArdle disease (MD), and methylmalonic acidemia (MMA) [3].
Cancer syndromes caused by premature protein
truncation
At its most basic, a hereditary cancer syndrome is a genetic
predisposition to develop cancer. These syndromes increase a person’s lifetime chance of developing cancer,
usually at early stages in life. Although cancer syndromes
display an autosomal dominant pattern of inheritance,
they behave as recessive diseases at the somatic level,
given that cancer arises only when both alleles of a tumor-suppressor gene are inactivated within a cell [7].
Glossary
Aminoglycosides: molecules that inhibit prokaryotic protein synthesis when
used in high doses, which is the reason they are commonly used as antibiotics.
In low doses they have the ability to induce codon misreading, allowing the
introduction of either non-cognate or near-cognate amino acids at a sense
codon, or the introduction of an amino acid at a stop codon, leading to
translational readthrough.
Negamycin: dipeptide antibiotic with the ability to bind rRNA and alter
translational accuracy, inhibiting protein synthesis or inducing misincorporation of amino acids, similar to some aminoglycosides. Nonetheless, the exact
mechanism of negamycin miscoding activity remains to be elucidated.
Nonsense-mediated decay (NMD): mRNA quality-control process, occurring in
eukaryotic organisms, that is responsible for the degradation of transcripts
carrying disease-causing PTCs and a variety of physiologic transcripts. Among
the latter are transcripts with upstream open-reading frames, transcripts
containing introns in the 30 -untranslated region (UTR), and transcripts derived
from alternative splicing. NMD usually reduces the level of PTC-bearing
transcripts but does not eliminate them completely. Hence, low levels of the
encoded truncated proteins are often observed.
Nonsense-suppression: misreading of nonsense codons by introduction of an
amino acid at the PTC location, avoiding translation termination. The insertion
of the cognate or a non-cognate amino acid at the PTC depends on whether it is
recognized by the cognate or a near-cognate tRNA, respectively.
Premature termination codons (PTC): stop codons that may result from a
single nucleotide change as a consequence of a nonsense mutation or from an
out-of-frame insertion or deletion (frameshift mutations).
PTC124: a 284.24 Da chemical compound (1,2,4-oxadiazole linked to fluorobenzene and benzoic acid rings) that is orally bioavailable when prepared in
aqueous suspension. It has the ability to induce PTC readthrough, although it
has no structural similarity to aminoglycosides or other clinically developed
drugs. Commercially known as Ataluren.
Readthrough (of PTCs): misreading of PTCs, allowing translation to progress (it
has the same meaning as nonsense-suppression).
Release factors: proteins responsible for promoting translation termination by
recognizing stop codons. In eukaryotes, stop codons are recognized at the
ribosome by the eukaryotic release factors eRF1 and eRF3.
Suppressor-tRNA: mutant tRNAs with an anticodon sequence complementary
to a stop codon, allowing its reading as a sense codon. Introduction of a
cognate amino acid at the PTC site by a suppressor-tRNA can restore the
synthesis of a full-length protein.
1471-4914/$ – see front matter ! 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2012.09.004 Trends in Molecular Medicine, November 2012, Vol. 18, No. 11
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Treatments for patients with these syndromes do not
differ from those of sporadic cases, and the most frequent
therapeutic approach is surgery combined with conventional chemo- and radio-therapy, with limited success.
Most mutations underlying cancer syndromes, as for
many other inherited disorders, generate PTCs [8–10].
Approximately 10–30% of all patients suffering from inherited cancer carry nonsense mutations [8–10]. In this setting,
nonsense-suppression therapy embodies an attractive strategy to recover protein expression.
In this section, we briefly present the clinical and molecular aspects of several cancer syndromes that arise in
the context of PTC-causing mutations.
Hereditary diffuse gastric cancer (HDGC; MIM #137215)
HDGC is an autosomal dominant cancer predisposition
syndrome caused by alterations of CDH1 (the gene encoding E-cadherin) in approximately 45% of all families [11].
Heterozygous CDH1 mutation carriers generally develop
cancer before the age of 50 and have an estimated 80%
lifetime risk of developing diffuse gastric cancer by the age
of 80 [12]. In addition, females have an additional 52%
lifetime risk of developing lobular breast cancer by the age
of 75 [12]. Nearly 75% of all HDGC families described to
date carry truncating mutations [8,11]. Available data on
100 of these families show the following distribution of
mutations: 30% small frameshift, 24% splice-site, 19%
nonsense, 22% missense mutations, and 5% large deletions
[8,11] (Oliveira et al., unpublished).
In these patients, cancer arises when a somatic inactivation event hits the remaining normal CDH1 allele at the
target organ [8]. This generally occurs during the third
decade of life, and no treatment is currently available [12].
Prophylactic gastrectomy has so far been the only option
presented to asymptomatic mutation carriers [12]. In these
circumstances, at least those families carrying nonsense
mutations could benefit from a less invasive clinical approach such as a nonsense-suppression therapy.
Familial adenomatous polyposis (FAP) and attenuated
adenomatous polyposis coli (AAPC; MIM #175100)
FAP is an autosomal dominant disorder that accounts for
less than 1% of all cases of colorectal cancer (CRC) [13]. It is
characterized by the development of hundreds to thousands
of adenomatous polyps during the second and third decades
of life, with a lifetime risk of developing CRC of nearly 100%
[13]. The adenomatous polyposis coli (APC) gene is affected
in more than 90% of patients [13], mostly by nonsense (30%)
or frameshift mutations (68%) that generate truncated
proteins [14]. Missense mutations have also been described
as predisposing to development of colorectal tumors [15].
Attenuated adenomatous polyposis coli (AAPC) is characterized by the occurrence of fewer than 100 colonic adenomas, a milder colorectal phenotype with later onset of
colorectal cancer (after 40 years of age) [16], and characteristic mutations in the 50 and 30 ends of the APC gene [17].
Hereditary non-polyposis colorectal cancer (HNPCC;
MIM #120435)
HNPCC is an autosomal dominant disorder characterized
by a limited number of adenomas, early onset of CRC
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Trends in Molecular Medicine November 2012, Vol. 18, No. 11
(before 50 years), and the development of extra-colonic
cancers: gastric, endometrial, ovarian, renal, and hepatobiliary [13]. HNPCC is associated with DNA microsatellite
instability (MSI) due to mutations in the MMR genes [13];
50% of these mutations occur in MLH1, 40% in MSH2, and
10% in all the other genes described to be affected in this
syndrome: MSH6, PMS2, PMS1, and MLH3 [18]. According to the HGMD, nonsense, splice-site, and small frameshift mutations that potentially create PTCs constitute at
least 55% of all mutations occurring in MLH1, 51% in
MSH2, 64% in MSH6, and 43% in PMS2. Although deletions (small and large) are overall the most frequent
alterations, of all mutations described for each of these
genes missense mutations make up from 15–30% while
nonsense mutations constitute about 10% (http://
www.hgmd.org). Very few mutations have been described
for the PMS1 and MLH3 genes (http://www.hgmd.org).
Cowden Syndrome (CS; MIM #158350)
CS is a rare autosomal dominant inherited cancer syndrome, characterized by a high risk of developing carcinomas and hamartomas of the breast, thyroid, and
endometrium [10]. It is associated with PTEN mutations
in 85% of cases [13]. PTEN germline mutations have been
described in CS families with the frequencies of 20%
missense, 20% insertions, 13% deletions, 10% splice-site,
3% referred to as deletion/insertion mutation, and 33%
nonsense [19].
Peutz-Jeghers syndrome (PJS; MIM #175200)
PJS is an autosomal dominant disorder associated with a
30–50% increased risk of developing breast cancer [10], as
well as increased risk of other cancer types such as gastric,
colon, or pancreatic [20]. PJS is associated with mutations
in STK11/LKB1, a gene encoding serine/threonine kinase
11, which is a master regulator of AMPK and the AMPKrelated kinases [21]. STK11 mutations have been described in 69% of PJS probands, from which 27% were
missense, 27% insertions, 18% deletions, 5% affected a
splice-site, and 18% were nonsense [21].
Li-Fraumeni syndrome (LFS; MIM #151623)
LFS is an autosomal inherited cancer predisposition syndrome, clinically defined by the occurrence of familial
sarcoma and characterized by a cluster of early onset
cancers (before 45 years), including brain cancer, adrenal
cortical carcinoma, and breast cancer [22]. Germline TP53
mutations have been detected in approximately 80% of
families that comply with LFS criteria, and TP53 is the
only gene found to be associated with this syndrome [22].
Most mutations described are missense (>72%), approximately 14–16% are frameshift and splice-site, and about
7% are nonsense [22].
Familial breast-ovarian cancer (BROVCA; MIM #604370,
#612555)
Approximately 5% of all cases of breast cancer are associated with a hereditary cancer susceptibility syndrome with
early onset (before 50 years) and are caused by mutations
in high penetrance susceptibility genes, most involved in
DNA repair [10]. Nearly 16% of hereditary breast cancers
Review
are associated with germline mutations in either of the
BRCA (breast cancer 1 and 2) genes [10]. One defective
copy of BRCA1 or BRCA2 in the germline is sufficient for
cancer predisposition, although the loss of the second allele
is required for cancer development [23]. Germline BRCA
mutations are associated with a 50–80% risk of breast
cancer, a 60% risk of contralateral breast cancer, and a
15–25% risk of ovarian cancer [24]. Most BRCA1 (!70%)
and BRCA2 (!90%) mutations are truncating, namely
small insertions and deletions, nonsense substitutions,
and splice-site mutations [10]. Although rare, the contribution of missense mutations to breast cancer predisposition has also been demonstrated [10]. Variable mutation
frequencies have been described in BROVCA families,
depending on the population studies, but the most frequent
are frameshift mutations, 43–61%, whereas splice-site and
missense mutations are less frequent [25,26]. Nonsense
mutations account for 14–25% of the genetic changes
observed [25,26].
The examples provided above demonstrate that between
10% and 30% of families affected by most of these cancer
syndromes carry nonsense mutations, which could potentially be reverted by a readthrough strategy. However,
because missense mutations are frequent and often deleterious in most of these syndromes, readthrough therapy
specifically replacing the PTC by the cognate amino acid
might constitute a safer strategy. In some specific cases,
such as BROVCA families, in which most BRCA1 and
BRCA2 mutations that introduce PTCs are associated with
greater cancer susceptibility than most missense mutations
[27], a readthrough strategy replacing PTC by a nearcognate amino acid, could also be considered.
In addition, because mRNA quality control mechanisms
largely degrade the PTC-containing transcripts generated
by nonsense mutations, the amount of transcripts available for readthrough therapies may not be sufficient to
recover the function of mutant tumor suppressor genes
(TSG). Therefore, the nonsense-mediated decay (NMD)
mRNA quality control mechanism emerges as a crucial
factor affecting readthrough efficiency.
NMD in disease
NMD effect in hereditary diseases
Transcripts carrying PTCs can be detected and degraded
by the NMD system, a post-transcriptional mechanism of
mRNA quality control found in eukaryotes [1,2]. In mammalian cells, and generally after mRNA splicing, NMD is
elicited by stop codons located >50–55 nucleotides upstream of an exon–exon junction. After splicing, the exon-junction complex (EJC) is loaded onto mRNAs
approximately 24 nucleotides upstream of exon–exon junctions [1]. During the first round of translation, the ribosome stalls when it encounters a PTC and cannot progress
far enough to displace the EJC [2], as it normally would [1].
The continued presence of both the ribosome and the EJC
allows for the stable formation of additional proteins that
trigger NMD. The NMD-competent region comprises the
full mRNA sequence >50–55 nucleotides upstream of the
last exon–exon junction [28]. NMD is not triggered when
PTCs are located beyond this location, which constitutes
the NMD-incompetent region [28].
Trends in Molecular Medicine November 2012, Vol. 18, No. 11
NMD seems to have evolved as a protection against the
potentially damaging effects of PTCs [2], and the phenotype of many monogenic diseases caused by premature
protein truncation is affected by NMD. In some cases, the
degradation of PTC-bearing transcripts avoids the dominant-negative effect of truncated proteins, reducing the
severity of the disease. NMD has been described as protective, for instance in b-thalassemia [29] and osteogenesis
imperfecta [30,31]. In other cases, the downregulation of
PTC-bearing mRNAs encoding truncated proteins that
retain residual function may cause more severe phenotypes due to haploinsufficiency. NMD deleterious effect has
been described in diseases such as Duchenne muscular
dystrophy (DMD) [6] and Ullrich’s disease [32]. In fact, the
inhibition of NMD components attenuates the Ullrich
disease phenotype in human fibroblasts [32].
NMD effect in hereditary cancer syndromes
The effect of NMD over nonsense mutant TSGs is largely
dependent on the site of the mutation in the mRNA, the
effect of the truncated protein, and the context of the
germline or somatic cancer. In this section, the relationship between NMD, nonsense mutant TSGs, and cancer
syndromes will be discussed.
Despite the apparently protective role for NMD, currently available data on patients inheriting PTC-containing alleles in NMD-competent regions of TSG is quite
divergent. For instance, in BRCA1, most germline mutations causing PTCs occur in its NMD-competent region and
NMD is triggered by 80% of the PTC-positive alleles [33].
Supporting a protective role for NMD, truncating mutations in the central region of BRCA1 (NMD-competent) are
associated with a lower risk of breast cancer [33]. Moreover, the two most common BRCA1 mutations occur in the
NMD-incompetent region and generate high levels of truncated proteins [33], which presumably possess deleterious
effects.
In FAP, most mutations occur in the last exon of APC
(NMD-incompetent region) and generate proteins that are
functionally impaired [34]. Nevertheless, mutations very
close to the N terminus or C terminus of APC, which also
presumably constitute NMD-incompetent regions, lead to
an attenuated phenotype [13]. In the latter cases, it is
possible that N-terminal mutations allow some kind of
translation re-initiation, giving rise to functional proteins,
and that APC molecules lacking only the very last Cterminal amino acids retain residual function.
HDGC also provides a good example of the relationship
between the mutation site and clinical presentation. CDH1
mutation carriers with PTC-containing alleles in NMDcompetent regions have an earlier age-of-onset, whereas
those with PTC-containing alleles in NMD-incompetent
regions develop gastric cancer later in life [28]. These
observations point towards a residual function for C-terminal truncated proteins that are not degraded by NMD.
These contrasting effects of nonsense mutations, truncated proteins, and NMD on the clinical presentation of
patients with cancer syndromes ultimately reflects particular aspects of protein function and cellular and tissue
contexts. Each case should therefore be carefully analyzed
before moving forward with a readthrough-based therapy.
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(a) Normal transla!on
P
A
Readthrough
Full-length func!onal protein
Canonical
STOP
(b) Premature termina!on in the presence of a PTC
P
Readthrough
A
eRF3
eRF1
PTC
Canonical
STOP
Truncated protein
(c) Readthrough of PTC by aminoglycosides, negamycin or PTC124
P
Readthrough
A
Full-length func!onal protein
Full-length missense func!onal protein
PTC
Canonical
STOP
Aminoglycoside
Negamycin
PTC124
Full-length missense non-func!onal protein
(d) Readthrough of PTC by deple!on/modula!on o"ermina!on factors
P
Readthrough
eRF3
A
eRF1
Full-length func!onal protein
Full-length missense func!onal protein
PTC
Canonical
STOP
Full-length missense non-func!onal protein
(e) Readthrough of PTC by suppressor-tRNA
Readthrough
P
A
Suppressor-tRNA
PTC
Key:
rRNA
Canonical
STOP
tRNA
Full-length func!onal protein
mRNA
protein
TRENDS in Molecular Medicine
Figure 1. The effect of readthrough strategies on protein translation. Several ways exist for modifying the normal processes that occur during termination by a premature
termination codon (PTC). The readthrough efficiency is modest and readthrough agents do not act over all transcripts, so production of truncated and full-length proteins is
predicted to occur simultaneously. (a) During normal translation, a tRNA carrying the appropriate amino acid enters the A site of the rRNA, upon recognition of the mRNA
codon by the tRNA anticodon. The peptidyl tRNA, with the nascent polypeptide, is located in the P site. The amino acid in the A site binds to the nascent polypeptide, and
the ribosome moves along the mRNA three nucleotides, with transfer of the tRNA from the A site to the P site [1]. (b) In the presence of a PTC, there is no tRNA matching the
stop codon. Instead, the release factors eRF1 and eRF3 bind and terminate translation by releasing the polypeptide, which is a truncated protein [1]. (c) When
aminoglycosides, PTC124, or negamycin bind to the rRNA there is no premature termination of translation, despite the presence of a PTC. An alteration of rRNA
conformation is induced upon binding of the small molecule, reducing the accuracy of the codon–anticodon interaction [1]. This enables incorporation of an aminoacylated
tRNA, moving translation towards the canonical stop codon and originating a full-length protein [1]. These proteins often contain missense mutations at the PTC location
because near-cognate tRNAs may recognize the codon sequence by two nucleotides, allowing the insertion of near-cognate amino acids instead of the cognate amino acid
[73]. Therefore, the proteins produced may be functional or non-functional depending on whether or not the missense mutations affect conformation and binding to other
proteins. The interaction of aminoglycosides, negamycin, and PTC124 with the rRNA may be different. In this picture, and for the sake of simplicity, the interaction between
the readthrough agent and the rRNA is depicted at the same spot in the three strategies. (d) Depletion of release factors eRF1 and/or eRF3 leaves the A site available to the
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NMD effect and the potential use of readthrough
therapies
Patients carrying a PTC-containing allele in the NMDincompetent region will continuously produce truncated
proteins [28,34], which may have a dominant-negative
effect by interfering with wild-type protein function [7]
(Figure 2). Despite the disease severity in these cases,
large amounts of mRNA molecules are available for readthrough therapy because NMD is not a limiting factor.
However, the choice of therapeutic approach should not
disregard the effects and phenotypes described for missense mutations. In syndromes where missense mutations
produce a milder phenotype, readthrough therapy introducing a near-cognate amino acid could be considered. In
syndromes where missense mutations are highly deleterious, the specific replacement of the PTC by the cognate
amino acid would be more appropriate.
Another possible consequence of PTC-containing alleles
in the NMD-incompetent region is the production of truncated proteins retaining residual function and lacking
dominant-negative effects. These generally originate
milder clinical phenotypes and are not predicted to greatly
benefit from the therapies discussed here (Figure 2).
Patients carrying PTC-containing alleles in the NMDcompetent region have a single allele sustaining the production of wild-type protein, while the mutant is downregulated, preventing dominant-negative effects [7].
Availability of 50% wild-type protein, although generally
sufficient to maintain tissue function, may lead to haploinsufficiency [28,35]. The haploinsufficiency in TSG is a rare
and highly debatable issue [7]. Generally, tumour initiation requires inactivation of both alleles from a TSG, but it
has been claimed that haploinsufficiency may cause cancer
initiation, for instance in TP53 or in the BRCA1 and
BRCA2 mutant contexts [7]. Correction of nonsense mutant alleles to recover functional protein could be nevertheless advantageous independent of haploinsufficiency.
In a scenario where haploinsufficiency induces cancer, the
increase in protein expression levels, due to correction of
the mutant allele, could prevent cancer initiation. In a
situation where a single allele is enough to sustain normal
protein function, without promoting cancer, the recovery of
protein expression from the mutant allele could prevent
cancer initiation even after the second allele inactivation in
the target organ. In summary, for carriers of PTC-containing alleles in the NMD-competent region, NMD is limiting
and its function may hamper the efficacy of readthrough
therapies. Therefore, apart from choosing a strategy for
inducing readthrough, a NMD inhibition strategy should
also be considered.
Readthrough strategies to suppress protein premature
truncation
Many efforts have been made to develop therapeutic strategies to overcome PTC-causing mutation effects. Substitution
Trends in Molecular Medicine November 2012, Vol. 18, No. 11
of a PTC for a near-cognate amino acid can be induced by
administering aminoglycosides, negamycin [36,37], or
PTC124 [38], or by depleting termination factors [39,40].
Substituting a PTC with the cognate amino acid is also
possible using suppressor-tRNAs [41]. In addition, the expression of smaller, though functional, proteins by inducing
skipping of the PTC-carrying exon or mini-gene delivery may
also overcome the effects of PTC [42]. These strategies have
been extensively tested in DMD and were recently reviewed
elsewhere [42].
Readthrough efficiency inversely correlates with translation-termination efficiency and may be influenced by
several factors [1]. A determining factor is PTC identity;
readthrough has been shown to be more efficient for the
UGA stop codon, followed by UAG, and, to a lesser extent,
UAA [1]. The sequences surrounding a stop codon, both
upstream and downstream, also have an important role in
determining readthrough efficiency, especially the fourth
nucleotide immediately after the stop codon [1].
NMD is determinant in PTC-suppression efficiency, and
thus its reduction leaves more PTC-carrying transcripts
available for readthrough [1]. Depleting the NMD factors
UPF1 or UPF2 enhanced CFTR expression after gentamicin
treatment in CF patients [43]. A different response to gentamicin was described in different cells with variable NMD
efficiency, although carrying the same PTC [43,44]. Studies
in yeast and fibroblasts suggest that inhibition of NMD
occurs upon PTC readthrough [45,46]. Hence, the efficiency
of readthrough strategies will depend on the effect of NMD
on PTC-carrying transcripts and on the balance between
termination suppression and mRNA degradation.
The specific advantages and pitfalls of different readthrough strategies are discussed below.
Substitution of a premature stop codon by a nearcognate amino acid
Aminoglycosides, when used in low doses, induce translational misreading in both prokaryotes and eukaryotes by
inserting an amino acid at the site of the PTC, producing
full-length, proteins that often have function, even if only
residual [1,47] (Figure 1c).
The potential of aminoglycosides as therapeutic tools
has been demonstrated in several genetic disorders such as
hemophilia, b-thalassemia, or spinal muscular atrophy
(SMA), but most extensively in DMD and CF [3]. The
efficiency of full-length functional protein production in
these studies varied from 1% to 25%, depending on the
brand of aminoglycoside and chemical composition, the
stop codon, and the surrounding nucleotide sequence
[1,47,48]. Trials in humans have been conducted for some
diseases, as shown in Table 1. The majority of studies were
performed in CF and DMD/Becker muscular dystrophy
(BMD) patients, generally using gentamicin, with variable
degrees of clinical improvement (Table 1). The use of
gentamicin nevertheless revealed limited functional
entrance of any tRNA that can interact with the PTC, promoting missense readthrough [40]. This may lead to the production of a full-length protein carrying missense
mutations. (e) The suppressor-tRNA anticodon is mutated to be complementary to the PTC, so it is able to recognize the PTC and insert the cognate amino acid [48]. A
competition occurs between suppressor-tRNA and the release factors eRF1 and eRF3 for the A site of rRNA [58]. When the suppressor-tRNA enters the A site, with
successful interaction with the mRNA PTC, the cognate amino acid is bound to the nascent polypeptide, with readthrough of the PTC, and a normal full-length protein is
produced [58].
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PTC in the NMD-competent region
Trends in Molecular Medicine November 2012, Vol. 18, No. 11
PTC
NMD-competent
region
NMD-incompetent
region
50% of normal protein expression
PTC
PTC in the NMD-incompetent region
NMD-competent
region
Truncated protein
with dominant
nega!ve effect
NMD-incompetent
region
Truncated protein
without dominant
nega!ve effect
PTC
NMD-competent
region
NMD-incompetent
region
Truncated protein with
residual func!on
TRENDS in Molecular Medicine
Figure 2. Effect of nonsense-mediated decay (NMD) in protein expression from a premature termination codon (PTC)-causing germline mutation. This simplified scheme
shows how NMD can influence protein expression resulting from a PTC-causing germline mutation. When one allele is inactivated by a PTC-causing mutation in the NMDcompetent region, truncated transcripts are downregulated by NMD with a loss of 50% of protein expression (upper panel). When the allele is inactivated by a PTC-causing
mutation in the NMD-incompetent region, it can generate a truncated protein with or without a dominant-negative effect (middle panel). Another possibility is that the PTCcausing mutation generates a near full-length protein that, although truncated, maintains residual wild-type protein function (lower panel).
outcomes. Translational misreading induced by aminoglycosides often introduces errors [40] that may result in the
production of non-functional proteins. Moreover, high concentrations and/or long-term treatments, requiring intravenous administration, can cause severe side effects [1]
related to nephrotoxicity and ototoxicity [48,49], which
may be irreparable. Excessive use can also lead to the
emergence of drug-resistant bacterial infections [50]. Removing elements of aminoglycoside structure that induce
toxicity has generated several derivatives (NB30, NB54,
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NB74, and NB84) that induce readthrough with a 10-fold
decrease in cellular toxicity [32,51]. NB54 [52], NB74, and
NB84 demonstrate higher suppression efficiency than gentamicin [51]. Apart from these derivatives, unrelated compounds with significantly less toxic effects have been
developed.
Negamycin is a dipeptide antibiotic with the ability to
bind rRNA and alter translational accuracy, but the mechanism remains unknown [3,36] (Figure 1c). Negamycin
administration restored dystrophin expression in a DMD
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Trends in Molecular Medicine November 2012, Vol. 18, No. 11
Table 1. Clinical trials and tentative treatments with readthrough strategies in PTC-causing diseases patients
Disease
Gene
affected
OMIM
reference
Therapy
applied
Duchenne muscular
dystrophy/
Becker muscular
dystrophy (DMD/BMD)
DMD
300377
Gentamicin
Ataluren
(PTC124)
Clinical trials/
tentative
treatments in
patients [Refs]
[74]
[75]
[76]
NCT00264888
(phase 2)
NCT00759876
(phase 2a)
NCT00592553
(phase 2b)
Cystic fibrosis (CF)
CFTR
602421
Gentamicin
NCT00847379
(phase 2b)
NCT01009294
(phase 2a)
NCT01247207
(phase 3)
[77]
[78]
[79]
[80]
[81]
[43]
Tobramycin
[80]
Ataluren
(PTC124)
NCT00234663
(phase 2)
NCT00237380
(phase 2)
[82]
NCT00458341
(phase 2)
[83]
NCT00351078
(phase 2b)
[84]
NCT00803205
(phase 3)a
NCT01140451
(phase 3)
Clinical outcome
No clinical improvement reported.
No clinical improvement.
Increased dystrophin expression in muscle
tissue, to a potentially therapeutic range in 3
out of 16 patients.
Without noticeable side effects.
Limited functional outcomes that would
provide better quality of life.
Visible improvement in dystrophin
expression in muscle in vivo.
Improvement in muscle strength and
functions were not statistically significant.
Extension study from NCT00264888.
174 patients tested with increased walking
ability.
Slower disease progression.
No adverse events reported.
Extension study from NCT00592553.
Suspended.
In recruiting status.
Response to treatment in 7 out of 9 patients.
Response to treatment in 4 out of 5 patients.
Response to treatment in 17 out of 19
patients.
No response to treatment in 11 patients
tested.
Response to treatment in 6 out of 9 patients
tested with the same nonsense mutation.
Recovery of protein expression at the
membrane of the nasal epithelial cells, with
improvement of respiratory status.
No response to treatment in all the 4
patients tested with different nonsense
mutations.
Response to treatment in 17 out of 19
patients.
Enhanced CFTR chloride channel activity.
No response to treatment in 11 patients
tested.
No clinical improvement in 24 patients
tested.
Response to treatment in 17 out of 23
patients.
Reduced the epithelial electrophysiological
anomalies with clinical benefits.
Adverse effects were mild or moderate.
Response to treatment in 15 out of 30
patients.
Increased the proportion of nasal epithelial
cells expressing apical full-length CFTR
protein.
Adverse effects were mild or moderate.
Response to treatment in 11 out of 18
patients.
Increased pulmonary function.
Adverse effects were mild or moderate.
Significant improvements in lung function.
In recruiting status.
673
Review
Trends in Molecular Medicine November 2012, Vol. 18, No. 11
Table 1 (Continued )
Disease
Gene
affected
OMIM
reference
Therapy
applied
Hemophilia A/B
300841
300746
300841
300746
613878
Gentamicin
Ataluren
(PTC124)
NCT00947193
(phase 2)
Factor VII deficiency
FVIII
FIX
FVIII
FIX
FVII
Clinical trials/
tentative
treatments in
patients [Refs]
[85]
Gentamicin
[86]
Hailey–Hailey disease
ATP2C1
604384
Gentamicin
[36]
McArdle disease
PYGM
608455
Gentamicin
[87]
LAD1
CD18
600065
Gentamicin
[88]
Methylmalonic acidemia
MUT
MMAA/
MMAB
609058
607481/
607568
Ataluren
(PTC124)
NCT01141075
(phase 2)
Clinical outcome
Response to treatment in 1 out of 3 patients.
Response to treatment in 1 out of 2 patients.
Suspended.
Suspended.
Minimal sub-therapeutic effects in 2 out of 2
patients.
Response to treatment in only 1 patient
tested.
No response to treatment in 4 patients
tested.
Corrected full-length protein in 2 out of 2
patient leucocytes.
No therapeutic effect because the protein
was predicted to be either dysfunctional or
mislocalized.
Suspended.
Suspended.
a
http://www.drugs.com/clinical_trials/top-line-data-phase-3-trial-ataluren-patients-nonsense-mutation-cystic-fibrosis-show-promising-13812.html?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+Drugscom-ClinicalTrials+%28Drugs.com+-+Clinical+Trials%29.
mouse model [50] and promoted PTC readthrough stabilizing mRNA levels in congenital muscular dystrophy
patient’s myotubes, although it was not sufficient to recover functional full-length protein expression [37].
PTC124 is a minimally toxic compound that can be
orally administered, and is one which has no structural
similarity to aminoglycosides [38]. This drug is more efficient than gentamicin in stable cell lines and promotes
dystrophin expression in primary muscle cells from DMD
patients and mdx mice [38]. PTC124 also restored 24–29%
of the wild-type protein function in a CF mouse model [53].
It has been the most successful readthrough agent in
clinical trials for DMD/BMD and CF patients, with no
significant side effects observed (Table 1). PTC124 nonsense-suppression efficiency did not affect normal termination in humans, rats, and dogs [38], which was supported
by preclinical safety pharmacology and toxicology studies
in healthy volunteers [54]. Despite the promising examples
mentioned above, the successful use of PTC124 depends on
the function of the particular gene and protein, the disease
context, and whether a near-cognate amino acid substitution is adequate.
Another strategy to promote PTCs readthrough is by
reducing the efficiency of translation termination by downregulating levels of the eukaryotic release factors (RF)
eRF1 and eRF3 (Figure 1d), responsible for decoding stop
codons in eukaryotic ribosomes [40]. High eRF1 activity is
critical for translation termination, preventing readthrough of PTCs [40]. eRF1 competes with readthrough
activity, hence decreasing eRF1 levels may be an alternative or complementary approach to elevating readthrough
of a PTC in human cells [40]. Depleting eRF1 and eRF3
mRNA and protein levels, using small interfering RNAs
(siRNAs) and antisense oligonucleotides (AONs, also
known as ASOs), increased PTC readthrough in human
cell lines [39,40,55], although with lower efficiency than
other strategies [56].
674
Substitution of a premature stop codon by the cognate
amino acid
Suppressor-tRNAs are mutant tRNAs that have an anticodon complementary to a stop codon [48]; they can read a
stop codon as sense codon, restoring the synthesis of an
active gene product [57]. In this way, whenever a ribosome
contains a stop codon in the A site two paths are available:
translation can be terminated by the action of eRF1-eRF3,
or an amino acid can be incorporated into the nascent
peptide by the suppressor-tRNA and translation elongation will continue to the next in-frame stop codon [58]
(Figure 1e). These two reactions occur at a ratio determined by the balance between the activities of translation
termination factors and translation elongation machinery
[58].
Suppressor-tRNAs have effectively suppressed nonsense and frameshift mutations in mammalian cells [46]
and Xenopus oocytes [59], and they have been shown in
vitro as possible therapeutic agents for xeroderma pigmentosum [60], b-thalassemia, and DMD [48]. The introduction of multi-copy suppressor-tRNA plasmids restored
chloramphenicol acetyltransferase (CAT) activity in the
heart of transgenic mice [57], and frameshift-suppression
by suppressor-tRNAs restored COL6A protein expression
in Ullrich patient fibroblasts [46].
Compared with other readthrough strategies, suppressor-tRNAs have several advantages: the ability to suppress
not only nonsense but also frameshift mutations, high
specificity for one of the three stop codons [56], and the
ability to insert the correct (cognate) amino acid at the PTC
site [41]. This overcomes the risk of inserting missense
mutations, emerging as an interesting alternative
approach for treating PTC-related cancer syndromes.
However, this ability may be compromised for some suppressor-tRNAs because the ‘identity elements’, nucleotides
that determine the recognition of a tRNA by the cognate
aminoacyl-tRNA synthetase and consequent charging of
Review
the tRNA with the respective amino acid, are mainly
located in the tRNA anticodon loop [61].
Suppressor-tRNAs have been shown to induce higher
readthrough levels, compared with aminoglycosides and
the depletion of translation termination factors [56]. However, this was observed using a UAG stop codon in a single
cell line [56], thus suppression efficiency could vary in
other contexts. It is possible to improve suppressor-tRNAs
readthrough efficiency by concomitant eRF1/eRF3 inhibition, overcoming the hierarchy in stop codon selection that
limits UGA and UAA termination suppression in higher
eukaryotic translation systems [62]. Moreover, tRNAs
have features that facilitate the inclusion of tRNAs into
delivery vectors and their expression in different cell types.
tRNA promoters are strong and active in all cell types, are
located within the structural sequences encoding the tRNA
molecule, and the transcriptionally active tRNA sequence
is less than 500 bp [61]. Nevertheless the in vivo delivery of
these vectors may be difficult to achieve, as will be discussed below.
Overall drawbacks of readthrough strategies
Clear drawbacks of readthrough strategies are the lack of
gene specificity and the potential for readthrough of normal stop codons that may result in toxic aggregates or
molecules with dominant-negative activities [48]. However, it has been suggested that normal and premature
termination differ mechanistically [63] and that translational readthrough is somehow specific for PTC-carrying
transcripts, given that stop codon fidelity seems to be
higher at the original stop codons [5]. Even if translation
termination fails at the cognate stop codon, multiple inframe stop codons are frequently found at the 30 end of
many open-reading frames, terminating translation [32].
Accordingly, it has been demonstrated in vitro that no
protein elongation occurred beyond the native stop codons
[38], in accordance with safety profiles in clinical studies
[5].
A third important pitfall is the lack of specificity for
target cells, with potentially deleterious effects in normal
cells, and this will be discussed in the following section.
Recognizing the drawbacks of overall readthrough strategies and the results from clinical trials with aminoglycosides and PTC124, despite their limited success, provide
important background for improving readthrough
approaches. Although aminoglycosides, negamycin, and
PTC124 can be administered systemically, nucleic acidbased strategies depend on delivery systems, and are
therefore less advanced.
Can delivery be improved and enhance the therapeutic
outcome?
Progress has been made in identifying and understanding
PTC-caused diseases, and a number of readthrough and
gene-replacement therapies are being explored, but one of
the major challenges of this field is finding a method to
make these therapies effective and overcome many of their
disadvantages and side effects. For many years, improvements in drug delivery strategies have contributed to the
development of new vectors that direct a therapeutic agent
more precisely to the cells/tissues of interest, precluding
Trends in Molecular Medicine November 2012, Vol. 18, No. 11
toxicity and other unwanted side effects, as well as maintaining these molecules at therapeutic concentrations,
over long periods of time. To achieve the full realization
of gene therapy, an effective delivery system should do the
following: (i) assure the protection of the nucleic-acids from
degradation in the extracellular environment; (ii) provide
for a sufficient circulation time by avoiding premature
clearance if the vector is to be administered intravenously;
(iii) allow the targeting of specific cell populations; (iv)
efficiently deliver the nucleic acid to a sufficient number
of cells; and (v) assure sufficient persistence to produce a
therapeutic effect.
Although viruses have been the most popular vectors in
this context, fundamental drawbacks including pathogenicity, insertional mutagenesis, toxicity, limitations in
scaling up, cost, and ethical concerns [64] have drawn
interest towards non-viral vectors. Most often cationic in
nature, non-viral vectors based on liposomes, polymers, or
dendrimers, among others, allow greater flexibility in
terms of the size of the nucleotide cargo that can be
transported, they can bypass the immune system, and
there are fewer safety concerns [65]. Furthermore, these
can be functionalized with targeting moieties of interest,
allowing the exploration of receptor-mediated uptake into
the cell of interest, as was recently shown for the targeting
of neuronal cells [65]. At present, there are a number of
products in the market based on controlled delivery systems, although none in the gene therapy context. With
advances in the area of nanomedicine, faster progress is
expected in the near future [66].
Readthrough therapies and cancer
Very few studies describe TSG readthrough in cancer
models, and all use antibiotics. Aminoglycosides and the
macrolide antibiotic tylosin induced readthrough of mutant APC and restored its function in human CRC cell lines
and mouse models [67]. Nonsense mutations in TP53 were
suppressed by aminoglycosides [68], recovering functional
full-length protein in several cancer cell lines [69].
Substitution of a premature stop codon for a near-cognate
amino acid may not have severe functional consequences in
some genes, such as CFTR or DMD, but in cancer syndromes
missense mutations can cause disease [8,11,15], affecting
protein secondary structure, post-translational modifications, and interactions with other molecules [15,70–72].
Suppressor-tRNAs arise then as an interesting strategy
for these cases, due to their ability to suppress nonsense
mutations by replacing PTCs with the cognate amino acid in
the protein sequence. Nevertheless, there are no guarantees
concerning the efficiency of this strategy because demonstrations of suppressor-tRNAs efficiency in vivo are scarce.
However, given their potential to promote PTC readthrough
without introducing missense mutations, further studies
are warranted to develop suppressor-tRNAs as readthrough
agents.
Combined use of NMD inhibitors and readthrough
agents may constitute an option to enhance readthrough
efficiency. In patients carrying germline mutations in
the NMD-competent region, NMD inhibitors could increase nonsense-suppression efficiency by raising mRNA
levels available for readthrough. Patients with germline
675
Review
Box 1. Outstanding questions
" Can readthrough strategies achieve successful clinical outcomes
in cancer syndromes as has been observed for recessive
hereditary diseases?
" Could readthrough be used as a prophylactic strategy for carriers
of nonsense-mutation that underlie cancer syndromes?
" Would nonsense-suppression therapy improve the clinical outcome of cancer patients in the early stages of disease?
" Could suppressor-tRNAs (cognate amino acid substitution) be
considered a therapy for cancer syndromes?
" Can tRNAs be as efficient as chemical compounds and synthetic
molecules, such as aminoglycosides, PTC124, or AONs?
" Can they work as nonsense-suppressor agents with mild toxic
effects?
" What is the range of protein expression and functional recovery
after tRNA suppression?
mutations in the NMD-incompetent region, which can
generate deleterious truncated proteins, could also benefit
from nonsense-suppression therapy, without the need for
NMD inhibition.
The hypothetical applicability of nonsense-suppression
therapy in patients with hereditary cancer syndromes will
always be dependent on the type of mutation, the location
of the PTC, the nature of the gene, protein function, and
cellular and tissue contexts. Nonsense-suppression could
constitute a prophylactic strategy to delay cancer initiation
in disease-free germline mutation carriers, or could prevent cancer progression when applied to cancer patients at
an initial stage, by recovering tumor-suppressor protein
expression (Box 1).
Concluding remarks
The unveiling of the molecular and genetic features of
hereditary cancer syndromes, and its relationship with
clinical phenotypes, is extremely important for developing
new therapies that can be applied to each patient in a more
specific and personalized manner. Considering the substantial frequency of nonsense mutations in cancer syndromes, nonsense-suppressing strategies could constitute
a new therapeutic option, in combination with other anticancer agents, to improve clinical outcomes. There is much
to be done to achieve the levels of re-expressed protein that
are sufficient to obtain clinical benefits. Given the influence
of NMD in PTC-carrying transcripts levels, the use of NMD
inhibitors concomitantly with readthrough therapy could
generate better results. Furthermore, safe delivery systems must be developed to allow specific targeting of the
affected organs with mild side effects to the organism. An
‘ideal therapy’ should be efficient enough to ensure, in vivo,
both the recovery of protein expression from the affected
genes in specific tissues as well as a considerable effect in
disease regression or prevention.
Acknowledgment
This work was supported by the Calouste Gulbenkian Foundation
through the project ‘Mutated suppressor tRNAs as a therapeutic tool
for cancer associated syndromes: HDGC as a model’ and the Portuguese
Foundation for Science and Technology (FCT) [PhD grant: SFRH/BD/
46462/2008-RBC; salary support to C.O. from POPH – QREN/Type 4.2,
European Social Fund and Portuguese Ministry of Science and
Technology (MCTES)]. IPATIMUP and INEB are Associate
Laboratories of the Portuguese Ministry of Science, Technology and
Higher Education and are partially supported by FCT. The authors
676
Trends in Molecular Medicine November 2012, Vol. 18, No. 11
acknowledge Hugo Pinheiro, Denisa Mateus, and Cecı́lia Durães for
critical reading of the manuscript.
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