Oncogene (2008) 27, 4255–4260 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $30.00 www.nature.com/onc SHORT COMMUNICATION The NMD mRNA surveillance pathway downregulates aberrant E-cadherin transcripts in gastric cancer cells and in CDH1 mutation carriers

R Karam1,2,3, J Carvalho1, I Bruno2, C Graziadio4, J Senz5, D Huntsman5, FCarneiro 1,3, R Seruca1,3, MFWilkinson 2 and C Oliveira1,3

1Cancer Genetics Group, Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP), Porto, Portugal; 2Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, USA; 3Medical Faculty, University of Porto, Porto, Portugal; 4Department of Genetics, FFFCMPA, Porto Alegre, Brazil and 5Hereditary Cancer Program, British Columbia Cancer Agency, Vancouver, Canada

Germline mutations in the gene encoding the tumour invasion and metastasis, as a result of its ability to suppressor E-cadherin (CDH1) are the underlying genetic mediate cell-to-cell adhesion (Suriano et al., 2003). defect responsible for hereditary diffuse gastric cancer Germline mutations in the E-cadherin gene (CDH1) (HDGC). A remarkably high percentage (B80%) of are the underlying genetic defect responsible for CDH1 mutations in HDGC patients and carriers generate hereditary diffuse gastric cancer (HDGC; OMIM No. premature termination codons (PTCs). Here, we exam- 137215), an autosomal dominant syndrome character- ined whether CDH1 transcripts harbouring PTCs are ized by high susceptibility to early onset diffuse gastric downregulated by nonsense-mediated decay (NMD), an carcinomas (Guilford et al., 1998). Heterozygous RNA surveillance pathway that degrades PTC-bearing carriers of E-cadherin germline mutations frequently transcripts. Using an allele-specific expression (ASE) remain asymptomatic at least until the second decade of assay to differentiate between mutated and wild-type life, when inactivation of the remaining wild-type allele CDH1 alleles, we found that PTC-bearing CDH1 of the CDH1 gene occurs (Oliveira et al., 2004). This two mRNAs are strongly downregulated in normal gastric hit inactivation mechanism is believed to determine the tissue from several CDH1 mutation carriers. We show initiation of diffuse gastric cancer, consistent with the that NMD is responsible for this robust downregulation, two-hit inactivation model proposed by Knudson as CDH1 transcripts harbouring PTCs in the KATO-III (1971). Interestingly, the vast majority of such germline gastric tumour cell line were upregulated in response to CDH1 mutations (B80%) generate premature termina- protein synthesis inhibitors or depletion of the NMD tion codons (PTCs) (Oliveira et al., 2006; Kaurah et al., factors UPF1 and eIF4AIII. Analysis of HDGC patients 2007). In this report, we investigated whether PTCs in harbouring CDH1 alleles with PTCs at a wide variety of the CDH1 gene engage nonsense-mediated mRNA different positions indicates an association of their predicted decay (NMD), a conserved mRNA surveillance ability to induce NMD and an earlier age of onset of gastric mechanism that typically degrades mRNAs harbouring cancer. This suggests that NMD may be detrimental for PTCs. The NMD response in mammals discriminates HDGC patients and therefore NMD is a potentially useful normal stop codons from PTCs using the exon junction therapeutic target for CDH1 mutation carriers. complex (EJC), a set of proteins recruited to mRNAs Oncogene (2008) 27, 4255–4260; doi:10.1038/onc.2008.62; near exon–exon junctions after RNA splicing (Chang published online 21 April 2008 et al., 2007). Most normal transcripts escape NMD since the stop codon is in the final exon and hence EJCs are Keywords: NMD; E-cadherin; CDH1; gastric cancer; only deposited upstream the stop codon, permitting the HDGC; KATO-III ribosome to displace them. In contrast, an aberrant transcript harbouring a stop codon in an internal exon (where most PTCs reside) will have at least one EJC downstream, thereby triggering NMD (Chang et al., E-cadherin is an adhesion molecule that acts as a 2007). tumour suppressor protein by inhibiting tumour cell Evidence suggests that NMD modulates the pheno- type of numerous diseases (Holbrook et al., 2004; Wilkinson, 2005). In some diseases, NMD appears to Correspondence: Professor C Oliveira, Cancer Genetics Laboratory, IPATIMUP, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal. reduce the severity of the phenotype by downregulating E-mail: [email protected] or the expression of truncated proteins with dominant- Professor MFWilkinson, Department of Biochemistry and Molecular negative activity (for example, in neurological syn- Biology, Unit 1000, The University of Texas MD Anderson Cancer dromes caused by SOX10 deficiency, b-thalassemia, Center, 1515 Holcombe Boulevard, Houston, TX77030, USA. E-mail: [email protected] osteogenesis imperfecta and Marfan syndrome). In Received 27 September 2007; revised 3 January 2008; accepted 21 other diseases, NMD appears to increase disease February 2008; published online 21 April 2008 severity because of haploinsufficiency by degrading NMD downregulates aberrant E-cadherin mRNA R Karam et al 4256 truncated protein retaining some normal function (for there would not be an EJC downstream of this stop example, in cystic fibrosis, ataxia-telangiectasia-like codon. disorder and Duchenne muscular dystrophy). In our We first examined an individual with a nonsense study, we examined the role of NMD in the phenotype mutation at position 283 (C>T), which generates a PTC of HDGC patients caused by truncated mutations in the at codon 95 in exon 3 (Dussaulx-Garin et al., 2001). CDH1 gene. ASE analyses were performed using Q-SnapShot, To test whether NMD downregulates truncated a quantitative fluorescent single-nucleotide extension CDH1 transcripts, we performed allele-specific expres- assay that measures expression by the rate of incorpora- sion (ASE) analysis on normal gastric mucosa from six tion of fluorescent dideoxy-nucleotides at polymorphic/ asymptomatic individuals, belonging to five different mutated sites (Carrel and Willard, 2005). This analysis HDGC families carrying five different truncating CDH1 demonstrated that the carrier with this mutation had mutations. Four of these mutations generate a PTC that B14-fold less expression of the mutant allele compared has at least one exon–exon junction downstream (in the with the wild-type (PTCÀ) allele (Figure 1a). We next NMD competent region) and thus we predicted that examined a carrier of mutation 1137G>A, which is in transcripts harbouring these mutations would be down- the exon 8 splice donor site, and leads to the generation regulated by NMD. The fifth mutation generates a PTC of an alternatively spliced transcript harbouring a PTC in the last exon (in the NMD incompetent region) and in codon 386 (Frebourg et al., 2006). Three independent hence we predicted it would not trigger NMD, because families have been described to carry this 1137G>A

Figure 1 CDH1 ASE in normal gastric tissue of CDH1 mutation carriers. Patient’s biological material was obtained with informed consent. RNA was isolated from fresh tissue in B, C, D and E with TRIzol (Invitrogen, Carlsbad, CA, USA) and from paraffin embedded tissue in A and Fwith PureScript RNA Purification System (Gentra, Minneapolis, MN, USA), following the manufacture’s protocol. 1 mg of total RNA was reversed transcribed using SuperScript (Invitrogen). Flanking primers were designed for each mutation/polymorphism and Q-SnapShots were performed using PCR products with a 310ABI sequencer following manufacturer’s protocol. Specific primer sequences were used for ASE determination, through the rate of incorporation of fluorescent dideoxy- nucleotides at polymorphic/mutated sites: (a) 283C>T (mutation site); (b) 1137G>A; (c and d) 1135del8ins5 (CDH1 2076T>C SNP to differentiate mutated and wild-type allele expression); (e) 2061delTG (mutation site); (f) 2398delC (mutation site). Q-SnapShot measurement of relative expression of the PTC þ allele, for each individual, is indicated as the ratio with the wild-type allele (gray bars on the right side of the figure). Transparent peak area represents the expression of the WT (PTCÀ) allele and the full black peak represents PTC þ allele. Exon number and location of PTCs in the CDH1 gene are represented in the schematic figure on the left. Note: Every primer sequence will be available upon request. Experiments performed in triplicate. ASE, allele-specific expression; PTC, premature termination codon; WT, wild type.

Oncogene NMD downregulates aberrant E-cadherin mRNA R Karam et al 4257 mutation (Kaurah et al., 2007). ASE analysis revealed cycloheximide and puromycin on these cells. These two that the PTC-bearing (PTC þ ) mutant allele was protein synthesis inhibitors have previously been Bnine-fold less expressed than the PTCÀ allele in the shown to reverse NMD and upregulate PTC þ asymptomatic carrier we analyzed (Figure 1b). We transcripts (Carter et al., 1995) by virtue of the fact also examined the effect of another mutation (1135de- that NMD requires protein synthesis to recognize PTCs l8ins5) that generates a PTC at codon 386 (Oliveira (Chang et al., 2007). To evaluate whether NMD et al., 2004). We found that two individuals from a selectively reduces the level of PTC þ and not family harbouring this deletion had reduced expression PTCÀ CDH1 transcripts, we distinguished between of the PTC þ allele (4.8-fold and 7.7-fold) relative to the these two classes of transcripts using radioactive reverse PTCÀ allele (Figures 1c and d). The fourth mutation transcriptase PCR analysis on polyacrylamide gels. We we tested was 2061delTG, which generates a PTC chose this approach because it permits bands to be at codon 688 in exon 13 (Kaurah et al., 2007). ASE analysed after few PCR cycles (before components analysis of normal gastric tissue from an asymptomatic become limiting), allowing for quantitative analysis. carrier of this mutation demonstrated that the PTC þ This analysis demonstrated that cycloheximide and allele was reduced in expression by B6-fold relative to puromycin selectively upregulated the three PTC þ the PTCÀ allele (Figure 1e). Finally, we examined a transcripts (harbouring PTCs at codons 351, 357 patient carrying a 2398delC mutation, which generates a and 374) but not the PTCÀ (wild-type) transcript PTC in the final exon (codon 815) (Kaurah et al., 2007), (Figures 2c and d). and hence would not be predicted to elicit NMD. In To definitively verify the role of NMD in down- agreement with this prediction, ASE analysis demon- regulating PTC þ CDH1 transcripts, we used RNAi to strated that the mutant allele was not expressed at lower deplete the levels two NMD factors known to be levels than the wild-type allele (Figure 1f). essential for NMD: (i) the RNA helicase UPF1; and We found that the mRNAs harbouring PTCs located (ii) the RNA-binding anchor of the EJC, eIF4AIII at the predicted NMD competent region were in fact (Chang et al., 2007). We found that siRNAs targeting expressed at a lower abundance compared with the wild these two factors decreased the level of their corres- type, albeit at different levels. Differential decay of ponding mRNAs (Figure 2e) and protein (Figure 2f), frameshift mutation-derived mRNAs has been demon- and significantly increased the level of CDH1 mRNA in strated before (El-Bchiri et al., 2005). Interestingly, the KATO-III cells, indicating that NMD was reversed mRNA carrying the PTC 815 in the predicted NMD (Figure 2g). incompetent region was not degraded, but on the Our finding that CDH1 truncated transcripts are contrary, was 4–5-fold more abundant than the wild- downregulated by NMD is interesting in light of the type. It is possible that this result actually indicates not fact that an extremely high percentage (B80%) of only insensitivity of the mutated gene to NMD, but also CDH1 mutations in HDGC patients generate PTCs decreased expression of the wild-type allele as result of a (Oliveira et al., 2006). This suggested the hypothesis second hit event. that rather than having a protective role, NMD may To assess whether NMD is responsible for the promote disease in CDH1 mutation carriers. To assess downregulation of mutant PTC-bearing CDH1 tran- this possibility, we analyzed the medical data from a scripts, we used the KATO-III cell line. This diffuse large set of published HDGC families (a total of 264 gastric tumour cell line was previously reported to patients) carrying truncating germline mutations harbour a splice-site mutation in the CDH1 exon 7 (Oliveira et al., 2006; Kaurah et al., 2007) in both splice donor (1008G>A), leading to the generation of NMD competent and incompetent regions of the alternatively spliced CDH1 transcripts containing PTCs CDH1 gene (Figure 3a). This analysis revealed that (Oda et al., 1994). We first performed a complete there is a significant association (P ¼ 0.03) between the characterization of CDH1 genomic sequences and RNA ability to induce NMD and the age of onset of gastric transcripts in the KATO-III cell line. Short tandem cancer (Figure 3b). Patients carrying PTCs in the repeat analysis with amelogenin and 15 single nucleotide NMD competent region developed cancer earlier polymorphisms demonstrated that the KATO-III (Figure 3b, left panel) than patients carrying PTCs in cell line cultured for our studies exactly matched the the NMD incompetent region (Figure 3b, right panel). KATO-III DNA profile provided by the American Type Supporting this notion, a previous study reported four Culture Collection (www.atcc.org) (data not shown). families carrying the 2398delC mutation (located in the DNA sequencing of the CDH1 exon sequences NMD incompetent region) had a cumulative risk of and exon–intron boundaries detected the previously acquiring gastric cancer of only 40% for men and 63% reported 1008G>A mutation and also demonstrated for women, which was considerably lower than for that KATO-III cells harbour a wild-type allele of HDGC patients in general: 67% for men and 83% CDH1 (Figure 2a). Cloning and sequencing of CDH1 for women (Kaurah et al., 2007). cDNA identified three alternatively spliced PTC- The association of predicted NMD activity with earlier bearing species harbouring different lengths of IVS7 age of onset of gastric cancer, as well as the observation of (Figure 2b). lower cumulative risk in patients harbouring a CDH1 To determine whether the alternatively spliced PTC- mutation that escapes NMD, suggests that NMD may bearing CDH1 transcripts are downregulated by NMD, have a detrimental effect on the clinical progression of we examined the effect of the protein synthesis inhibitors HDGC. The simplest explanation for this is that after

Oncogene NMD downregulates aberrant E-cadherin mRNA R Karam et al 4258 PTC - PTC 351 PTC 357 120 PTC 374 100 80 60 40 20 % of Transcripts Transcripts % of compared to PTC- 0 NT PURO CHX

ATG 1008G Normal Stop Treatment 120 WT 1 2 3 45678 9101112131415 16 100 80 60 1008G>A PTC 351 40 PTC351 20 % of Expression ATG 6 7 8 0 siLuc-UPF1 siUPF1-UPF1 siLuc-eIF4AIII sieIF4AIII-eIF4AIII +7bp 1008G>A PTC 357 siRNA [50nM] - mRNA expression PTC357 ATG 6 7 8 siRNA siRNA

+25bp Luc UPF1 Luc eIF4AIII 1008G>A PTC 374 Anti-UPF1 Anti-eIF4AIII PTC374 ATG 6 7 8 Anti-Actin Anti-Actin

+intron 7

SENSE PRIMER END-LABELED ANTI-SENSE PRIMER END-LABELED 3 * NT PURO CHX MARKER PURO CHX * NT 2.5

PTC 374 2

1.5

1

0.5

PTC 357 CDH1 mRNA level Relative 0 PTC 351 PTC - (WT) siLUC siUPF1 sieIF4AIII Treatment Figure 2 Characterization of KATO-III cell line (purchased from American Type Culture Collection (ATCC; HTB-103)). Cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin (Gibco, Grand Island, NY, USA) in a 5% CO2 incubator at 37 1C. RNA was extracted as described in figure 1 and 1 mg reversed transcribed with iScript cDNA Synthesis kit (BioRad, Hercules, CA, USA). High molecular weight DNA was isolated using standard methods. (a) CDH1 exon 7 donor splice site sequencing analysis showing a heterozygous G to A transition at nucleotide 1008. (b) Schematic representation of E-cadherin transcripts identified in KATO-III. KATO-III cDNA was PCR-amplified with primers (shown below the schematics as horizontal bars) flanking the 1008G>A mutation, the PCR product was cloned and individual colonies sequenced. (c) E-cadherin transcript-specific expression in KATO-III after treatment with protein synthesis inhibitors cycloheximide (100 mgmlÀ1 4 h) and puromycin (300 mgmlÀ1 4 h). Flanking primers specific to KATO-III CDH1 transcripts were end-labeled with [g32P]-ATP using T4 polynucleotide kinase (New England BioLabs, Beverly, MA, USA). Low-cycle (18) E-cadherin [g32P] end-labeled PCR was performed in no treated, CHX- and puro-treated samples and products were run on 10% acrylamide gels. (d) Quantification of three independent E-cadherin [g32P] end-labeled PCRs. Quantification was performed in a STORM 820 phosphoimager (Amersham Biosciences, Piscataway, NJ, USA). (e and f) Knockdown of the NMD factors UPF1 and eIF4AIII, and the luciferase control. 50 nM of specific siRNA was transfected using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) protocol. siRNA oligonucleotides were purchased from Ambion, TX, USA. Protein and RNA isolation was performed 48 h after transfection. RNA was reverse transcribed as described earlier. siRNA-mediated depletion of NMD factors was evaluated by real-time PCR (e) and western blot (f). (g) Real-time PCR evaluation of E-cadherin expression in KATO-III cells depleted of NMD factors. *t-tests with P-value o0.05 were considered significant. Note: Every primer and siRNA sequence will be available upon request. Experiments performed in triplicate. CHX, cycloheximide; puro, puromycin; FBS, fetal bovine serum.

inactivation of the CDH1 wild-type allele by a second-hit molecules lacking the COOH-terminal region retain event, NMD causes haploinsuffiency by decreasing the partial function (Sasaki et al., 2000). level of transcripts carrying PTCs in the NMD competent In the future, it will be important to determine whether region of the gene, since such transcripts (especially the truncated E-cadherin proteins generated by PTCs found in more COOH-terminal truncations) probably encode CDH1 mutation carriers retain partial tumour suppressor mutated but still functional E-cadherin proteins that function. This information will be critical to determine the retain tumour suppressor activity. In agreement with this, value of inhibiting NMD in HDGC patients as a means to it has previously been shown that truncated E-cadherin treat or reduce the risk of diffuse gastric carcinoma.

Oncogene NMD downregulates aberrant E-cadherin mRNA R Karam et al 4259

K374 N638 F32§ K357 F561 F355 F417 F215 F604 N24 F556§ NORMAL N699 N95 STOP N20 S209 F249 K351 F393 N598 N503 F547§ F783 883 N64 S349§ F587 F688 F815 N335 N1 N63 N196 S239 S281 F536§ F583 S736 F770 PTC S386§ F481 S653 F806

E-cadherin NMD-Competent Region NMD-Inc.

EXON 1 23 45 6 7 8 9 10 11 12 13 14 15 16

(Codon) (16) (55) (130) (177) (229) (278) (336) (379) (440) (522) (571) (646) (722) (765) (813)

7 60 6 50

23) 5

=241) 40 =

n n 4 30 3 20 2

Patients (

Patients ( 10 1 0 0 0 20406080100 0 20 40 60 80 100 Age of onset NMD-comp Age of onset NMD-inc (mean 42.9+/-14.9 years old) (mean 50.0+/-19.2 years old)

P=0.03 Figure 3 Distribution of CDH1 PTCs and age of onset of gastric cancer in carriers of CDH1 truncating mutations. (a) Distribution of predicted PTCs produced by published truncating mutations in HDGC families. The figure is a schematic representation of an E-cadherin transcript. PTCs are described on the top of the figure; letters indicate the truncating event: N (nonsense), F(frameshift), splicing (S); K indicates the PTCs described in KATO-III cell line; numbers indicates the stop codon position; yindicates PTC produced by more than one mutation; arrows indicate PTCs analyzed in experiments showed in Figures 1 and 2. Respective exons and exon-junction codons are at the bottom. NMD Inc.: E-cadherin NMD-incompetent region. (b) Age of onset distribution in carriers of CDH1 truncating mutations. To compare the mean age of onset in patients carrying PTCs at the NMD competent (left panel) and incompetent regions (right panel) of the CDH1 gene, we used unpaired t-test analysis with 95% of confidence interval. P-values o0.05 were considered significant. Dashed lines indicate mean age of the onset of gastric cancer. HDGC, hereditary diffuse gastric cancer; NMD, nonsense-mediated decay; PTC, premature termination codon.

Acknowledgements Nijmegen Medical Center, NL, for providing patients’ material. We also thank G.A.B.B.A. Program at We thank the family members who collaborated in this study. University of Porto, FLAD, CAPES and GRICES. This We thank Dr Marjolijn Ligtenberg from Department of work was supported by grants from FCT (POCTI/SAU- Human Genetics, University Medical Centre Nijmegen, OBS/58111/2004 and SFRH/BD/15241/2004) and NIH NL and Dr Han van Krieken from Radboud University (5-RO1GM058595-08).

References

Carrel L, Willard HF. (2005). X-inactivation profile reveals extensive mRNAs in mismatch repair deficient colorectal cancers. Hum Mol variability in X-linked gene expression in females. Nature 434: Genet 14: 2435–2442. 400–404. Frebourg T, Oliveira C, Hochain P, Karam R, Manouvrier S, Carter MS, Doskow J, Morris P, Li S, Nhim RP, Sandstedt S et al. Graziadio C et al. (2006). Cleft lip/palate and CDH1/E-cadherin (1995). A regulatory mechanism that detects premature nonsense mutations in families with hereditary diffuse gastric cancer. J Med codons in T-cell receptor transcripts in vivo is reversed by protein Genet 43: 138–142. synthesis inhibitors in vitro. J Biol Chem 270: 28995–29003. GuilfordP,HopkinsJ,HarrawayJ,McLeodM,McLeodN,HarawiraP Chang YF, Imam JS, Wilkinson MF. (2007). The nonsense-mediated et al. (1998). E-cadherin germline mutations in familial gastric decay RNA surveillance pathway. Annu Rev Biochem 76: 51–74. cancer. Nature 392: 402–405. Dussaulx-Garin L, Blayau M, Pagenault M, Le Berre-Heresbach N, Holbrook JA, Neu-Yilik G, Hentze MW, Kulozik AE. (2004). Raoul JL, Campion JP et al. (2001). A new mutation of E-cadherin Nonsense-mediated decay approaches the clinic. Nat Genet 36: gene in familial gastric linitis plastica cancer with extra-digestive 801–808. dissemination. Eur J Gastroenterol Hepatol 13: 711–715. Kaurah P, MacMillan A, Boyd N, Senz J, De Luca A, Chun N et al. El-Bchiri J, Buhard O, Penard-Lacronique V, Thomas G, Hamelin R, (2007). Founder and recurrent CDH1 mutations in families with Duval A. (2005). Differential nonsense mediated decay of mutated hereditary diffuse gastric cancer. JAMA 297: 2360–2372.

Oncogene NMD downregulates aberrant E-cadherin mRNA R Karam et al 4260 Knudson Jr AG. (1971). Mutation and cancer: statistical study of Sasaki CY, Lin H, Morin PJ, Longo DL. (2000). Truncation retinoblastoma. Proc Natl Acad Sci USA 68: 820–823. of the extracellular region abrogrates cell contact but retains Oda T, Kanai Y, Oyama T, Yoshiura K, Shimoyama Y, Birchmeier W the growth-suppressive activity of E-cadherin. Cancer Res 60: et al. (1994). E-cadherin gene mutations in human gastric carcinoma 7057–7065. cell lines. Proc Natl Acad Sci USA 91: 1858–1862. Suriano G, Oliveira MJ, Huntsman D, Mateus AR, Ferreira P, Oliveira C, de Bruin J, Nabais S, Ligtenberg M, Moutinho C, Casares F et al. (2003). E-cadherin germline missense mutations Nagengast FM et al. (2004). Intragenic deletion of CDH1 as the and cell phenotype: evidence for the independence of cell inva- inactivating mechanism of the wild-type allele in an HDGC tumour. sion on the motile capabilities of the cells. Hum Mol Genet 12: Oncogene 23: 2236–2240. 3007–3016. Oliveira C, Seruca R, Carneiro F. (2006). Genetics, pathology, and Wilkinson MF. (2005). A new function for nonsense-mediated mRNA- clinics of familial gastric cancer. Int J Surg Pathol 14: 21–33. decay factors. Trends Genet 21: 143–148.

Oncogene