Adenomatous polyposis coli (APC) is a multifunctional tumour suppressor protein that negatively regulates the Wnt signalling pathway. The APC gene is ubiquitously expressed in tissues and organs, including the large intestine and central nervous system. The majority of patients with sporadic and hereditary colorectal cancer have mutations in the gene encoding APC. Approximately 30% of these mutations are single nucleotide changes that result in premature stop codons (nonsense mutations). A potential therapeutic approach for treatment of this subset of patients is the use of aminoglycosides and macrolides that induce nonsense mutation read-through and restore levels of full-length protein. We have used reporter plasmids and colorectal cancer cell lines to demonstrate that several aminoglycosides and tylosin, a member of the macrolide family, induced read-through of nonsense mutations in the APC gene. In xenograft experiments and in the ApcMin/+ mouse model, these compounds ameliorated the tumorigenic clinical symptoms caused by nonsense mutations in the APC gene.
- Adenomatous polyposis coli (APC)
- colorectal cancer (CRC)
- nonsense mutations
- aminoglycoside and macrolide antibiotics
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- Adenomatous polyposis coli (APC)
- colorectal cancer (CRC)
- nonsense mutations
- aminoglycoside and macrolide antibiotics
Colorectal cancer (CRC), a cancer of the large bowel, is one of the most common cancer types.1 Nearly 85% of all CRC cases are sporadic in origin and the rest occur as a result of an inherited genetic mutation.2 Colorectal cancers arise from adenomas, which are dysplastic but non-malignant precursor lesions in the colon. Progression to carcinoma occurs through the accumulation of multiple somatic mutations, ultimately leading to malignant transformation and the formation of an invasive cancer. One of the most critical genes mutated in the progression to CRC is the adenomatous polyposis coli (APC) tumour suppressor.3 Since APC mutations are detected very early in the adenoma–carcinoma sequence, the APC protein is thought to act as a ‘gatekeeper’ of colorectal carcinogenesis and functional loss of APC appears to be a prerequisite to progression towards malignancy.2 Around 85% of all sporadic and hereditary colorectal tumours do not express functional APC.2
APC is a large (312 kDa) protein that has many well-characterised functional domains: (1) an oligomerisation and (2) an armadillo region in the N-terminus, (3) a number of 15- and 20-amino acid β-catenin binding repeats in its central portion, and (4) a C-terminus that contains binding sites for EB1 and the human disc large (DLG) protein. The multiple domains of the APC protein allow it to interact with numerous protein partners, including β-catenin, Axin, EB-1 and DLG, and it is involved in a wide variety of cellular processes.4 APC's critical role in tumorigenesis is based on its negative regulation of the Wnt signalling pathway through control of β-catenin levels.5 6 Mutations in APC result in the accumulation and nuclear translocation of β-catenin. In the nucleolus, β-catenin binds to the TCF/LEF transcription factors and initiates transcription of a wide variety of genes. The downstream transcriptional activation targets of β-catenin include a number of genes involved in the development and progression of colorectal carcinoma. In addition to its role in the Wnt signalling pathway, β-catenin is also involved in cell adhesion. In cell–cell adherence junctions, β-catenin binds to the cell adhesion molecule E-cadherin and links E-cadherin to the actin cytoskeleton. Cell–cell adhesiveness is generally reduced in human cancers.7 Indeed, recently it has also been shown that APC functions in cell adhesion8 and that restoration the expression of full-length APC in a colon cancer cell line enhances cell adhesion.9 In addition, APC has important roles in other basic cellular functions such as chromosomal stability, neuronal polarisation and directed migration.4 10–13 It is thus likely that the truncation of APC is involved in the initiation of colorectal cancer by leading to high levels of Wnt signalling and by disrupting important cellular functions, such as intercellular adhesion.
Almost all (95%) APC mutations in CRC are nonsense or frameshift mutations that result in a truncated protein product with abnormal function.14 Of these nonsense and frameshift mutations, a large number are mutations that are caused by a single nucleotide change that leads to the replacement of an amino acid with a stop codon.14 As in CRC, a large number of other human genetic diseases result from mutations that cause the premature termination of the synthesis of the protein encoded by the mutant gene15 and one way of treating these diseases would be to supplement with a wild-type copy of the gene. Interestingly, aminoglycoside antibiotics are known for their ability to suppress disease-associated premature stop mutations through their binding to the decoding site of the ribosome.16 17 In both prokaryotes and eukaryotes, aminoglycosides induce miscoding by mimicking the conformation change in the 16S rRNA that would be induced by a correct codon–anticodon pair, thereby compromising the integrity of codon–anticodon proofreading during translation.18 19 The efficiency of the stop codon read-through induced by aminoglycosides depends on the stop codon type (the ranking order is generally UGA → UAA → UAG)20 and is influenced by the sequence surrounding the stop codon, especially the +4 nucleotide.21 22 Importantly, this aminoglycoside-induced stop codon read-through may lead to expression of a gene containing a nonsense mutation. The levels of restoration induced by aminoglycosides are in the range of 2–10%, which may produce sufficient amounts of protein to restore physiological function.23 24
Recent studies show that aminoglycosides can induce read-through of nonsense mutations in a number of genes, including the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene and the dystrophin gene.25–28 However, aminoglycosides have limited use as therapeutic agents due to their nephro- and oto-toxicities.29 Like the aminoglycosides, members of the macrolide antibiotic family induce stop codon read-through in prokaryotes.30 In this study, we have investigated the ability of several aminoglycosides and macrolides to induce read-through of nonsense mutations in the APC gene. Our results demonstrate that both aminoglycosides and a member of the macrolide family of antibiotics, tylosin, can induce read-through and restore the function of mutant APC, resulting in reduced oncogenic phenotypes in both in vitro and in vivo CRC models.
Materials and methods
Cell culture, transfections and luciferase reporter assays
Human embryonic kidney cell lines 293T (HEK293T) and the human colon carcinoma cell lines SW1417, HT-29, COLO205 and HCT116 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 100 U/ml penicillin–streptomycin. Cells were kept in a humidified 5% CO2 atmosphere at 37°C. HCT116 cells were transfected using JetPEI (PolyPlus Transfection NY, USA) following the manufacturer's instructions. For HEK293T cells, the standard calcium phosphate precipitation method was used. For dual luciferase reporter assays, cells were seeded at 1×105 cells per well in 24-well plates 24 h prior to transfection. Cells were transfected with 0.75 μg of reporter plasmid. Cells were supplemented with fresh medium or fresh medium containing the appropriate antibiotic 24 h post-transfection. After 24 h, cells were lysed using passive lysis buffer (Promega, Madison, WI) and luciferase activity was determined using the dual luciferase reporter assay (Promega). Stop codon read-through levels were calculated by determining the firefly/Renilla ratio in each transfection reaction.
Plasmid construction and reagents
The human colorectal adenocarcinoma cell line SW1417 was used as a DNA template for APC promoter amplification. In brief, the SIGMA REDExtract-N-Amp Tissue PCR Kit was used to purify genomic DNA. The APC gene promoter was amplified from the genomic DNA using the following primers: forward, 5′-AGC CCG CCG ATT GGC TGG GTG-3′; reverse 5′-GAA AGG CCA TCG GTT TAA G-3′. To yield restriction ends for ligation, sequences 5′-GCG GTA CC-3′ and 5′-GCA GAT CT-3′ were added to the 5′ ends of the forward and reverse primers, respectively. The PCR products were digested with restriction enzymes KpnI and BglII and ligated to the KpnI- and BglII-digested plasmid pGL3-Basic (Promega), which carries the firefly luciferase gene. Next, the Renilla luciferase sequence was amplified from the PRL-TK vector (Promega) plasmid using the primers and subcloned downstream of the APC promoter using the BglII and HindIII restriction sites. Finally, wild-type or mutated (at codon 1450) APC sequence spanning amino acids 1447–1456 was amplified using the following primers: forward wild-type, 5′-AGC TTC ACC AAG CGA GAA GTA AAA CAC CTC CAC ATG GAA GAC GCC AAA AAC ATA AAG AAA GGC CCC TCG AGG G-3′, reverse 5′-CGC CCT CGA GGG GCC TTT CTT TAT GTT TTT GGC GTC TTC CAT GTG GAG GTG TTT TAC TTC TCG CTT GGT GA-3′, forward mutated, 5′-AGC TTC ACC AAG TGA GAA GTA AAA CAC CTC CAC ATG GAA GAC GCC AAA AAC ATA AAG AAA GGC CCC TCG AGGG-3′, reverse 5′-CGC CCT CGA GGG GCC TTT CTT TAT GTT TTT GGC GTC TTC CAT GTG GAG GTG TTT TAC TTC TCA CTT GGT GA-3′. The fragments were inserted in frame between the Renilla luciferase and the firefly luciferase genes using the HindIII and NotI sites. The upstream Renilla luciferase gene serves as an internal normalisation control for transfection efficiency, mRNA abundance and the levels of translation initiation. Translation of the firefly luciferase originates from the same translation initiation signal but is downstream of the APC sequence. Following normalisation, the difference between Renilla and firefly luciferase activities reflects the frequency of translation through the APC sequence. To generate the construct that encodes the Hemagglutinin (HA) epitope, the Renilla ORF, wild-type or mutated APC sequence and the firefly luciferase ORF were amplified by PCR and subcloned downstream of an HA epitope that was added to pcDNA3.1 (Invitrogen, Carlsbad, California using the BamHI and XhoI sites. All constructs were sequenced to confirm the identity and fidelity of subcloning steps.
Cells grown on glass coverslips were fixed for 20 min in PBS containing 3.7% paraformaldehyde. Fixed cells were washed three times with PBS, permeabilised with 0.1% Triton X-100 for 1 h and blocked in PBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100 for 1 h. Subsequently, cells were incubated at room temperature with primary and secondary antibodies for 60 and 30 min, respectively. For immunofluorescent analysis of intestinal polyps, the intestines of 12-week-old untreated or tylosin-treated ApcMin/+ mice were fixed in 10% formalin overnight at room temperature; paraffin embedded and 5-μm thick sections were cut using a microtome. Sections were dewaxed and rehydrated through a graded ethanol series, unmasked (Vector H-3300; Burlingame, CA) and blocked 2 h in PBS/0.2% Tween20/0.2% gelatin (Sigma G-1890; Sigma, Rehovot, Israel). Sections were then incubated overnight at 4°C with primary antibody and for an additional 2 h with secondary antibody at room temperature. Finally, sections were incubated 2 min with 4′-diamidine-2′-phenylindol dihydrochloride (DAPI; Sigma) and mounted with Gel/Mount aqueous mounting medium (Sigma–Aldrich). Immunofluorescence microscopy was performed using a confocal laser microscopy system (LSM510; Carl Zeiss, Thornwood, NY). Mouse anti-E-cadherin (1:500, Transduction Laboratories) and rabbit anti phospho-histone H3 (1:200; Santa Cruz Biotechnology, Santa Cruz, California, USA) primary antibodies were used. The secondary antibodies, used at 1:500 dilutions, were Alexa-Fluor-633 (Molecular Probes, Carlsbad, California) conjugated anti-rabbit and anti-mouse FITC conjugate (Sigma–Aldrich, Rehovot, Israel).
The entire gastrointestinal tract from stomach to anus of C57BL/6J-ApcMin/+ mice were removed, opened along the length and rinsed with ice cold PBS. Small and large adenomas were seen throughout the intestine with more lesions found in the ileum of untreated compared to treated mice. After scoring, specimens were fixed in 10% formalin overnight at room temperature and paraffin embedded. Sections of 5 μm were cut using a microtome and stained with H&E for pathological examination.
Immunoprecipitation and western blot analysis
Total cell lysates from HCT116 cells transfected with 4 μg plasmid were prepared by solubilisation in lysis buffer (150 mmol/l NaCl, 50 mmol/l Tris, pH 7.5, and 0.2% NP-40). Lysates from freshly dissected tumours were prepared by homogenisation in H buffer (0.5 mol/l β-glycerophosphate, 25 mmol/l EGTA, 10 mmol/l EDTA, 10 mmol/l benzamidine, 1 mmol/l orthovanadate, 10 mmol/l NaF and 1 mmol/l DTT). Protein concentrations were determined by a Bradford kit. Cell lysates (2 mg) were immunoprecipitated with 8 μg of HA-probe (Y-11) antibody (Santa Cruz Biotechnology) or rabbit-anti striatin (Millipore, Billerica, MA, USA) overnight at 4°C. Protein A/G plus agarose beads (Santa Cruz Biotechnology) were added and incubated on a rocker platform at 4°C for 1 h. Beads were collected by centrifugation and washed three times in lysis buffer. Protein samples were analysed by SDS–PAGE and transferred to nitrocellulose membranes, blocked with 5% low fat milk, and detected with the appropriate antibody. For western blots untransfected (SW1417, HCT116) cells in 10-cm dishes or transfected (HEK293T) cells in 24-well plates were washed with PBS and solubilised in lysis buffer (100 mmol/l NaCl, 50 mmol/l Tris, pH 7.5, 1% Triton X-100, 2 mmol/l EDTA) containing complete EDTA-free protease inhibitor mixture (Roche Applied Science, Palo Alto, CA). Protein extracts from xenografts were prepared using lysis buffer (50 mmol/l Hepes pH 7.5, 1% Triton X-100, 2 mmol/l EDTA, 2 mmol/l EGTA, 0.15 mol/l sucrose) containing complete EDTA-free protease inhibitor mixture. Following SDS–PAGE, proteins were transferred to nitrocellulose membranes, blocked with 5% low fat milk and incubated with the specific primary antibody. Membranes were washed in 0.001% Tween 20 in PBS and incubated for 45 min with a secondary antibody. After washing in Tween/phosphate-buffered saline, membranes were subjected to enhanced chemiluminescence (ECL) detection analysis. The following antibodies were used for western blot analysis: rat anti-HA (Roche; 1:1000), mouse anti-APC (Ab-2 1:300; Calbiochem, Darmstadt, Germany), mouse anti-actin (1:10000; MP Biomedicals, Solon, OH), anti E-cadherin (1:5000; BD Transduction Laboratories, Lexington, KY), mouse anti-striatin (1:5000; BD Transduction Laboratories), mouse anti-GSK-3β (1:5000; BD Transduction Laboratories), mouse anti-PP2A (1:200; Upstate, Billerica, MA), mouse anti-tubulin (1:10 000; Sigma), and rabbit anti-COX-2 (1:1000; Cell Signaling, Danvers, MA). Anti-rat (1:2500), anti-rabbit (1:10 000) and anti-mouse (1:10 000) IgG horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology.
Cell proliferation determination
SW1417 cells were seeded at 2000 cells/well in six-well plates. After 1 day, the medium was replaced with normal medium or medium containing 0.012 mg/ml tylosin. The medium was replaced every 3 days in all wells. The number of cells was evaluated at the intervals indicated in the figure legend.
Six-week-old male BALB/c nu/nu athymic mice, referred to as nude mice, were obtained from Harlan (Rehovot, Israel) and housed in our animal quarters in individual cages; the room was temperature-controlled with a 12-h light–dark period. Mice were given standard mice food pellets and water ad libitum. C57BL/6J-ApcMin/+ mouse progenitors were purchased from the Jackson Laboratory (Bar Harbor, Maine). Our breeding colony was established by crossing heterozygous male C57BL/6J-ApcMin/+ mice with wild-type female C57BL/6J mice. Genomic DNA was prepared from tail-snips, using Tail Lysis Buffer (10 mmol/l Tris pH 8, 100 mmol/l NaCl, 25 mmol/l EDTA, 0.5% SDS) and 10 mg/ml Proteinase K (Roche) as described previously.31 Offspring were characterised for the Min genotype by PCR; the general PCR conditions, including primer pairs and amplification conditions, have been described previously.32
Tumorigenicity assay in nude mice
On day 0, exponentially growing HT-29 or SW1417 cells were harvested and resuspended in PBS at a concentration of 5×106 or 5×107 cells/ml, respectively. Three (HT-29) or eight (SW1417) days later, mice were arbitrary divided into control and treatment groups. Antibiotics were provided in the drinking water. The drinking water of both control and treated mice was replaced every 3 days. Tumour development was followed daily. After the mice had been killed (day 14 or day 28 post-injection for HT-29 or SW1417 cells, respectively), the tumours were dissected from neighbouring connective tissues and weighed. One half of each tumour was frozen in liquid nitrogen and stored at −80°C and the other half was fixed in 4% paraformaldehyde and paraffin embedded.
Growth in soft agar
SW1417 or HCT116 cells (105 cells/well) were suspended in 1 ml of 0.3% Difco Noble agar (Difco, Sparks, MD) supplemented with complete culture medium. This suspension was layered over 1 ml of 0.6% agar-medium base layer in six-well cluster dishes and treated with antibiotics as indicated in the legend to figure 2a. After 14 days, colonies >0.05 mm were counted.
SW1417 or COLO205 cells (102cells) were seeded into a 10-cm culture dish. The growth medium was replaced every 3 days with medium or with medium containing the indicated antibiotic. Three weeks later the colonies were fixed with cold methanol and stained with 10% Giemsa solution (Sigma).
SW1417 cells (102 cells) were untreated or treated for 4 days with the indicated antibiotic. Cell viability was determent using The Alamar blue reagent (Biosource, Carlsbad, California)33 according to the protocol provided by the manufacturer.
Band intensity determination
The protein bands were quantified by a computer-assisted densitometer (TINA 2.0c; Fuji BAS, Tokyo, Japan) described at http://www.tina-vision.net/about.php.
Freshly dissected slices (0.5 cm in length) for the small intestines of untreated or treated Min mice were placed on a LB-agar plate and incubated at 37°C over night. Bacterial colonies (derived from the intestine flora) were scraped, diluted in a drop of water and smeared on a glass slide. The samples were heat-fixed and Gram stained using a Gram Stain Kit (BD, Transduction Laboratories). Lexington, KY
Results were presented as mean±SEM. Statistical analysis among groups was performed using the Student t test. A value of p<0.05 was regarded as statistically significant.
Aminoglycosides and macrolides induce read-through of the APC nonsense mutation
A dual luciferase reporter plasmid system was used to measure the effect of aminoglycosides and macrolide antibiotics on read-through of a mutation in the APC gene at codon 1450, which is a ‘hot-spot’ for nonsense mutations. This mutation is found at this site in the APC genes of a large number of patients with CRC.34 35 The basic features of this reporter system are similar to previously described reporter plasmids36: The Renilla luciferase and firefly luciferase reporter genes are located on either side of APC wild-type or mutated sequences and are under the transcriptional control of the APC promoter (figure 1A). This dual reporter system provides an internally controlled system permitting calibration of individual experiments as both reporter proteins are expressed from the same message consequently minimising the potential variation between different experiments. HCT116 cells were transfected with these constructs. Cells were lysed after 48 h and the luciferase levels were determined. As expected, replacement of the amino acid arginine with a stop codon (CGA to TGA) at codon 1450 of the APC gene resulted in decreased levels of firefly luciferase expression (figure 1B). Next, we evaluated the effects of aminoglycosides and macrolides on luciferase activity. Results shown in figure 1C indicate that these antibiotics caused read-through of the stop codon. Of those tested, the macrolide tylosin (at 0.012 mg/ml) resulted in the highest levels of stop-codon read-through.
In order to measure protein levels, we constructed a similar reporter plasmid (figure 1D). In this plasmid, the coding region of the HA epitope was inserted upstream of the Renilla luciferase sequence and the APC promoter was replaced with the stronger cytomegalovirus (CMV) promoter. HEK293T cells were transiently transfected with this plasmid containing wild-type or mutated APC sequences. Cells were treated with different aminoglycosides and macrolides 24 h after transfection. Cells were lysed 24 h later and lysates were subjected to SDS–PAGE and immunoblotted using an anti-HA antibody. As shown in figure 1E, the plasmid encoding the wild-type APC sequence expressed relatively high levels of an HA-fusion protein of the expected 110 kDa (figure 1E). Geneticin, gentamicin and tylosin (at different concentrations) treatment all led to expression of the same size HA-fusion protein. In parallel, similar cell lysates were subjected to immunoprecipitation experiments using an anti-HA antibody. Results show that a read-through product of 110 kDa was detected in the treated cells only (figure 1F). Densitometer quantification of this band revealed that the gentamicin treatment resulted in 12% read-through while geneticin, and tylosin led to approximately 8% read-through.
Aminoglycosides and macrolides reduce oncogenic phenotypes of SW1417 CRC cells
SW1417 cells are a CRC cell line that contains a nonsense mutation in codon 1450 (CGA to TGA) of the APC gene and are thus a good model for investigation of APC nonsense mutation read-through.37 HCT116 CRC cells contain a wild-type APC and a mutated β-catenin protein37 and therefore serve as a control. Anchorage-independent growth in soft agar is a good indicator of the in vivo tumorigenicity of several cell types, including CRCs.38 We evaluated the effects of different types of aminoglycosides or of tylosin on the growth of SW1417 and HCT116 cells in soft agar. Figure 2A shows that treatment of SW1417 but not of the control, HCT116 cells, with the different antibiotics reduced the size and number of the colonies growing in soft agar. Similar results were found in a plating efficiency assay. In this assay the ability of sparse cells to grow and form colonies is tested. The antibiotic treatment led to a decrease in the ability of the cells to form colonies and colonies formed were mostly smaller than those formed by untreated cells (figure 2B). As a control COLO205 cells were used. COLO205 are CRC cells that contain a frame-shift mutation (addition of one nucleotide) in the APC gene.37 Although COLO205 form larger colonies then WS1417 cells, the number and size of the colonies were not affected by the antibiotic treatment (figure 2B). As the side effects of read-through compounds on normal protein translation termination is a major concern we examined the size of several proteins by western blot analysis (figure 2C). The results indicate that the different treatment types did not lead to detectable read-through of the normal termination signals of the proteins examined since we did not observe any change in protein size.
Next, untreated and treated cells were stained for E-cadherin, which is a protein important for maintaining correct cell–cell adhesion, a property that is compromised in many types of cancer.39 40 As shown (figure 2D), the E-cadherin expression pattern in cells treated with the antibiotics was predominantly localised to sites of cell–cell contact at the periphery of the cell as compared to untreated cells where no such staining is observed. Interestingly, it has been shown that restoration of APC expression in CRC cells leads to membrane localisation of E-cadherin and enhanced cell adhesion.9 The expression of APC in untreated and tylosin-treated cells was determined using western blot analysis. Results shown in figure 2E demonstrate that treated cells expressed low levels of full-length APC protein (no APC was detected in untreated cells).
Next, we examined the proliferation rates of untreated and tylosin-treated SW1417 cells. Treatment of the cells for 2 weeks with tylosin led to a dramatic decrease in cell proliferation rates (figure 2F). Similar experiments were conducted in HCT116 cells that express a wild-type APC protein. No difference in proliferation was observed in untreated compared to tylosin-treated HCT116 cells (not shown). To assess whether the treatment affected cell viability an Alamar Blue assay41 was performed with the different antibiotics at the concentrations used to induce APC nonsense mutation read-through (figure 2G). The results demonstrate that the treatment did not affect cell viability. Thus, treatment of CRC cells that contain a nonsense mutation in the APC gene with agents that induce stop codon read-through resulted in reduced oncogenic phenotypes and enhanced expression of E-cadherin in the areas of cell–cell contact. Importantly, the treatment did not affect normal termination codons or overall cell viability.
Aminoglycosides and macrolides reduce tumour growth in vivo
The APC gene in HT-29 cells contains a nonsense mutation at codon 853;37 these cells are widely used to form xenografts in nude mice. HT-29 cells (5×106 cells/ml) were inoculated subcutaneously into athymic nude mice. Once the tumours reached a measurable size of 100 mm2 (after 4 days), the mice were assigned to control and treatment groups. The tumours were removed after 2 weeks. As shown in figure 3A, treatment with geneticin, gentamycin or tylosin led to a reduction of approximately 50% in the tumour size compared to the control, untreated mice. Similar experiments were performed with SW1417 cells using tylosin treatment (figure 3B). Athymic nude mice were inoculated subcutaneously with 5×107 cells/ml. Tumours reached measurable size after 14 days and were removed 4 weeks later. Tylosin treatment led to a reduction in tumour size compared to controls. Representative H&E-stained sections of the centres of the tumours demonstrated that tumours from treated mice showed dramatic cell death compared to tumours from controls (figure 3C). Western blot analysis of protein extracted from xenografts of control and tylosin-treated mice indicated that the treatment led to expression of full-length APC protein (figure 3D,E). Moreover, the treatment resulted in decreased expression of the Wnt target gene COX-2 (figure 3D). A number of experiments conducted in our laboratory indicate that COX-2 can form a cellular complex with APC (Personal communication A. Zilberberg 2010) and, interestingly, immunoprecipitation of xenograft proteins using striatin, an APC binding protein42 revealed that the treatment led to a decrease in COX-2 protein levels (figure 3E). HCT116 cells that do not express COX-243 served as a negative control. As a control to these experiments, athymic nude mice were injected with 1×107 HCT116 cells. HCT116 are CRC cells that have a wild-type APC protein. Treatment of these mice with tylosin did not result in any change in tumour size or other tumour properties (not shown). To assess the toxic effects of the treatment on the mice, 5-week-old nude mice were treated with the different antibiotics and individually weighed at 10-day intervals. Results indicate that the different treatment types did not affect mice weight (figure 3F) or general behaviour (not shown). To determine the levels of serum gentamicin the fluorescence polarisation immunoassay (FPIA) was performed. Mice (n=4) were untreated or treated with gentamicin in the drinking water for 1 week. Results show that while gentamicin was not detected in the serum of untreated mice (<0.6 mg/l), in the treated mice the gentamicin levels ranged between 1.8 and 2.2 mg/l indicating that the gentamicin was absorbed and reached the bloodstream.
Tylosin treatment reduces intestinal polyp size and extends the life span of the Min mouse
Min mice (ApcMin/+) carry a nonsense mutation in the APC gene at codon 850 that results in the expression of a truncated, non-functional APC product.44 These mice develop intestinal polyps as a result of this mutation.45 Thus, the Min mouse is a suitable in vivo model for examining the effects of APC nonsense mutation read-through. We chose to use tylosin treatment for these experiments since this treatment offered the best results in experiments described above and has low oral toxicity and few side effects.46
The intestines of age-matched (12 weeks) untreated and tylosin-treated Min mice were examined. The tylosin treatment led to a significant reduction in the total number of polyps, with the most profound decreases observed in polyps that were larger than 2.0 mm in size (figure 4A) as observed by gross analyses of the whole intestine stained with 0.5% methylene blue and polyp counting. As the (ApcMin/+) mice display splenomegaly and decreased haematocrit levels when compared with wild-type animals, the spleen size acts as a marker for chemoprevention in the Apcmin mouse.47 Therefore, we examined the effect of tylosin treatment on spleen size and haematocrit. Tylosin treatment reduced the spleen size, sometimes to near normal (figure 4B) and dramatically increased haematocrit levels (not shown). We then examined the effect of tylosin on cell proliferation in the intestinal polyps. We isolated intestines from 12-week-old age-matched animals and examined intestinal polyps for the presence of phospho-histone H3-positive cells. Immunofluorescent imaging showed a significant decrease in levels of cell proliferation in intestinal polyps obtained from the treated mice compared to control, untreated animals (figure 4C). As the antibiotics were administrated in the mice drinking water we next examined whether antibiotics reached the intestine where they can affect the tumours. Bacteria recovered from intestinal sections of equal size from untreated and treated mice were Gram stained. Both tylosin and geneticin treatment led to a decrease in total bacteria number. Moreover, most bacteria derived from tylosin-treated mice stained pink (indicative of Gram-negative bacteria) in the Gram staining whether the bacteria from the geneticin were mostly purple (indicative of Gram-positive bacteria). This is consistent with the fact that tylosin mostly inhibits Gram-positive bacteria48 and gentamicin mostly inhibits Gram-negative bacteria.49 Min mice usually die from anaemia, which is presumably secondary to the development of multiple adenomas that cause bleeding into the intestinal lumen. As the tylosin treatment led to a reduction in tumour size and number, we compared the life span of treated and untreated animals, with mice being humanely killed when moribund. Untreated mice had a median life span of 198 days, whereas treated animals lived an average of 282 days (figure 4E). Our data show that the tylosin treatment leads to a reduction in polyp size and number and to an increase in the life span of Min mice.
The majority of sporadic and hereditary colorectal cancer cases, one on the most common cancer types, are initiated by activating mutations in proteins of the Wnt pathway.3 Over 80% of these mutations are mutations in the APC gene.3 In agreement with the Knudson's two-hit hypothesis, inactivation of both APC alleles is detected in the majority of intestinal tumours at early stages of tumour development.50 The vast majority of these mutations result in frameshift or stop codons.2 Around 30% of the known mutations are nonsense mutations that result in the introduction of a pre-mature stop codon in the APC gene.14 Attenuation or prevention of CRC development could be achieved in these patients by supplementing the cells with a wild-type APC protein. This could be achieved through gene therapy, but this type of therapy has not yet been broadly successful.51 The subset of patients with CRC who develop tumours as a result of APC nonsense mutations could be helped by small-molecule antibiotics that induce APC nonsense mutation read-through.
Aminoglycoside antibiotics and PTC124, a new chemical entity, have been shown to suppress clinically relevant premature stop codons in vitro and in vivo with variable efficiency.52 In this study, we evaluated aminoglycoside antibiotics and a member of the macrolide antibiotic family, tylosin, for induction of read-through of nonsense mutations in the APC gene. We initially screened a number of aminoglycosides and tylosin in in vitro experiments using reporter plasmids. The nonsense mutation, at codon 1450, accounts for approximately 8–10% of all somatic APC mutations35 and this mutation was used in our reporter constructs. All aminoglycosides tested (gentamicin, geneticin and paromomycin) induced similar levels of read-through at concentrations ranging from 0.5 to 1 mg/ml, with gentamicin most effective at inducing protein expression. In the reporter assay the macrolide tylosin (at 0.0012 mg/ml) induced the highest read-through levels compared to all other compounds tested. The intensity of the read-through product revealed an antibiotic induced read-through level of 8–12% which is consistent with other findings showing read-through levels of 2–15% using similar constructs.53 Most APC point mutations are C → T transitions, with the vast majority (97%) at CGA, thus generating the TGA stop codon.35 The APC nucleotide sequences in our reporter plasmid were identical to the wild-type codon 1450 or to the disease causing CGA to TGA mutation and surrounding sequence.
Next, the effect of APC nonsense mutation read-through was examined in tissue culture. The growth characteristics of SW1417 CRC cells that harbour an APC nonsense mutation at codon 1450 reflect their tumorigenic phenotype as shown by the anchorage-independent growth and the capability of these cells to form colonies when grown in sparse cultures. Treatment of the cells with aminoglycosides or tylosin resulted in reduction of these oncogenic phenotypes and in reduced proliferation rates compared to untreated cells. Loss of cell-adhesive properties is involved in the development of human cancers. The E-cadherin-mediated cell adhesion in cancer cells is inactivated by multiple mechanisms.54 It has been shown that loss of APC in CRC cells reduces E-cadherin-dependent adhesion and restoration of APC induces cell adhesion.8 9 Moreover, APC binds to the tight junction complex in epithelial cells.42 In this study we found that treatment of CRC cells with aminoglycosides or with tylosin induced higher levels of expression of E-cadherin in areas of cell–cell contact than observed in untreated cells, suggesting that this treatment may lower the invasive properties of these cells. In the reporter assays the antibiotics induced between 8% and 12% read-through levels, while on the endogenous APC protein the effect was more significant (around 30%). This result indicates that antibiotic induced read-through of APC nonsense mutations may be an efficient way to restore relatively high levels of wild-type APC protein when used in vivo.
Antibiotic treatment also successfully induced full-length APC expression in animal models. Nude mice with SW1417 xenografts treated with aminoglycosides or tylosin expressed APC and had reduced tumour size compared to untreated animals. These tumours expressed lower levels of the oncogenic Wnt target gene cyclooxygenase-2 (COX-2) than tumours from untreated animals. The Min mouse (ApcMin/+) carries a nonsense mutation in the APC gene at codon 85045 that converts the codon for amino acid leucine into a stop codon; this mouse develops intestinal polyps. We used this mouse model to examine the effect of tylosin on APC nonsense mutation read-through. Our results show that treated mice had fewer intestinal polyps and a decreased level of cell proliferation within the intestinal polyps compared to untreated controls. Additionally, the treatment restored normal spleen size and haematocrit levels and, importantly, dramatically increased the life span of the treated mice.
Our experiments demonstrate that induced read-through of nonsense mutations in the APC gene results in a full-length APC protein product with functional properties. Aminoglycoside-induced read-through of human nonsense mutations has the potential to correct a large number of genetic disorders that result from premature nonsense mutations 15 and is being evaluated for treatment of cystic fibrosis and muscular dystrophy. Unfortunately, one of the major limitations in the use of aminoglycosides is their toxicity; thus, other types of compounds that induce read-through are currently being developed.55 Interestingly, tylosin, a member of the macrolide family of antibiotics with limited toxicity in animals, induced high levels of APC nonsense read-through and may prove to be suitable for treatment of patients with CRC and other genetic diseases that result from pre-mature stop codons.
One concern of using drugs to suppress pathogenic nonsense mutations is the read-through of normal termination codons that may result in toxic aggregates or dominant negative read-through products.55 However, available evidence suggests that normal and premature termination differ mechanistically56–58 and it has been shown that two different compounds negamycin59 and PTC12452 have reduced toxicity and selectively induce read-through of premature but not normal termination codons. While tylosin treatment did not lead to read-through of the normal stop codons in a number of proteins that were examined, the effects of tylosin on normal termination read-through are currently been examined.
Although both alleles are mutated in APC-defective CRC cells, these cells typically express high levels of the mutated mRNA and, as a result, a truncated N′-APC protein. It has been demonstrated that stability of the mutated mRNA is an important factor in the ability of an agent to induce nonsense read-through.60 In addition, most APC nonsense mutations produce the TGA stop codon that has been shown to be the most prone to read-through.35 The mutant APC mRNA stability and the type of stop codon most often observed make CRC patients good candidates for induced read-through. Restoration of even 1% of normal protein function may result in near-normal or a clinically less severe phenotype and it is reasonable to speculate that rescue of APC function by stop-codon read-through could significantly ameliorate the oncogenic phenotypes of CRC cells.
Aminoglycosides are known to promote nonsense mutation read-through by binding to the ribosomal A-site and induce conformational changes that stabilise near-cognate mRNA–tRNA complexes, instead of inserting the release factor.23 61 62 Tylosin that also binds the ribosomal large unit,63 binds the E-site48and thus may function in a similar manner. Another possibility is that tylosin, as it binds the E-site, may disrupt the mechanism of peptide release.30 Further studies of the ways in which tylosin affect the proper termination of translation, should provide detailed insights on the function of tylosin in promoting nonsense mutation read-through.
The authors thank Lea Shochat for help with the mouse work.
Funding Israeli Academy of Sciences, The Association for International Cancer Research (AICR) from the Public Committee for Allocation of Estate Funds, Ministry of Justice, Israel (grant no. 0113406), Israel Cancer Research Fund (ICRF), Israel Cancer Research fund, NIH grant R03CA113252 and the Recanati Foundation.
Competing interests None.
Ethics approval The study was performed according the guidelines of the Institutional Animal Care Committee of Tel Aviv University.
Provenance and peer review Not commissioned; externally peer reviewed.
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