Published OnlineFirst May 14, 2013; DOI: 10.1158/1078-0432.CCR-13-0684 Clinical Cancer Human Cancer Biology Research Identification and Selective Degradation of Neopeptide- Containing Truncated Mutant Proteins in the Tumors with High Microsatellite Instability Won Kyu Kim1, Misun Park1, Minhee Park1, Yun Ji Kim1, Nara Shin1, Hyun Ki Kim1, Kwon Tae You2, and Hoguen Kim1 Abstract Purpose: Frameshift mutations in coding mononucleotide repeats (cMNR) are common in tumors with high microsatellite instability (MSI-H). These mutations generate mRNAs containing abnormal coding sequences and premature termination codons (PTC). Normally, mRNAs containing PTCs are degraded by nonsense-mediated mRNA decay (NMD). However, mRNAs containing PTCs located in the last exon are not subject to degradation by NMD (NMD-irrelevant). This study aimed to discover whether genes with frameshift mutations in the last exon generate truncated mutant proteins. Experimental Design: We identified 66 genes containing cMNRs in the last exon by bioinformatic analysis. We found frequent insertion/deletion mutations in the cMNRs of 29 genes in 10 MSI-H cancer cell lines and in the cMNRs of 3 genes in 19 MSI-H cancer tissues. We selected 7 genes (TTK, TCF7L2, MARCKS, ASTE1, INO80E, CYHR1, and EBPL) for mutant mRNA expression analysis and 3 genes (TTK, TCF7L2, and MARCKS) for mutant protein expression analysis. Results: The PTC-containing NMD-irrelevant mRNAs from mutated genes were not degraded. However, only faint amounts of endogenous mutant TTK and TCF7L2 were detected, and we failed to detect endogenous mutant MARCKS. By polysome analysis, we showed that mRNAs from genomic mutant MARCKS constructs are normally translated. After inhibiting 3 protein degradation pathways, we found that only inhibition of the proteasomal pathway facilitated the rescue of endogenous mutant TTK, TCF7L2, and MARCKS. Conclusions: Our findings indicate that cancer cells scavenge potentially harmful neopeptide-containing mutant proteins derived from NMD-irrelevant abnormal mRNAs via the ubiquitin–proteasome system, and these mutant proteins may be important substrates for tumor-specific antigens. Clin Cancer Res; 19(13); 3369–82. Ó2013 AACR. Introduction tions inactivate genes by introducing premature termina- Activating mutations in oncogenes and inactivating tion codons (PTC) in mRNA. If mRNAs bearing PTCs are mutations in tumor suppressor genes are hallmarks of not degraded and allowed to be translated normally, then human cancers (1). The common inactivation pathways of genes with nonsense mutations are expected to generate tumor suppressor genes are deletion of one allele and truncated proteins lacking neopeptides, whereas genes with inactivating mutations of the other allele (2). Of these frameshift mutations are expected to generate truncated pathways, nonsense and frameshift mutations are the most proteins containing neopeptides. Generally, most abnor- critical inactivating mutations (3). These 2 types of muta- mal PTC-containing mRNAs are actively degraded by nonsense-mediated mRNA decay (NMD), thus avoiding the potentially deleterious effects associated with the Authors' Affiliations: 1Department of Pathology and Brain Korea 21 production of truncated proteins (4, 5). NMD is mediated Projects for Medical Science, Yonsei University College of Medicine; and through the recognition of PTC-containing mRNAs, 2School of Biological Science and Center for National Creative Research, Seoul National University, Seoul, Republic of Korea which are recognized by their position relative to the last exon–exon junction. Mammalian transcripts that contain Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). PTCs more than 50 to 55 nucleotides (nt) upstream of the last exon–exon junction are degraded by NMD, which Corresponding Author: Hoguen Kim, Yonsei University College of Med- icine, 134 Shinchon-Dong Seodaemun-Gu, Seoul 120-752, Republic of ensures the degradation of most PTC-containing mRNAs. Korea. Phone: 82-2-2228-1761; Fax: 82-2-363-5263; E-mail: However, PTCs located within 50 to 55 nt or downstream [email protected] of the last exon–exon junction are not recognized by doi: 10.1158/1078-0432.CCR-13-0684 NMD and can potentially lead to the generation of Ó2013 American Association for Cancer Research. mutant proteins (6, 7). www.aacrjournals.org 3369 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst May 14, 2013; DOI: 10.1158/1078-0432.CCR-13-0684 Kim et al. National Research Resource Bank Program of the Korea Translational Relevance Science and Engineering Foundation of the Ministry of Abnormal mRNAs containing premature termina- Science and Technology. Authorization for the use of the tion codon (PTC) derived from frameshift mutation tissues for research was obtained from the Institutional in the last exon are not subject to degradation by Review Board. Conventional pathologic parameters were the nonsense-mediated mRNA decay (NMD) system. examined without prior knowledge of the molecular data These NMD-irrelevant mRNAs are expected to generate (Table 1). neopeptide-containing truncated mutant proteins. We identified mutant genes containing PTC derived from Identification of MSI and mutation analysis the last exon in colon cancers with high microsatellite Genomic DNA and cDNA preparation, analysis of MSI, instability. We showed that the NMD-irrelevant mutant and identification of target gene mutations were conducted mRNAs from the identified genes are normally trans- using a PCR-based assay as described previously (16). lated, but the neopeptide-containing truncated mutant proteins are selectively degraded by the ubiquitin– Semiquantitative RT-PCR and qRT-PCR proteasome system. Our results indicate that mutant The primers for semiquantitative reverse transcription proteins generated from NMD-irrelevant mRNAs can (RT)-PCR and quantitative reverse transcription PCR contribute to the formation of tumor-specific antigens (qRT-PCR) were designed using Primer 3 database and these antigens can be used as specific targets for (http://frodo.wi.mit.edu/primer3/). All RNAs were isolated immunotherapy. from cells using TRIzol (Invitrogen). Reverse transcription was conducted using M-MLV reverse transcriptase (Invitro- gen). For RT-PCR, the reaction was conducted using Ampli- Taq Gold 360 DNA Polymerase (Applied Biosystems). For A subset of colorectal tumors exhibits length alterations qRT-PCR, the reaction was conducted using the ABI PRISM in several coding and noncoding microsatellites, a molec- 7500 Sequence Detector (Applied Biosystems) and SYBR ular phenotype termed high microsatellite instability (MSI- Premix Ex Taq II (TaKaRa). The amount of target mRNA was H; refs. 8, 9). The length alterations in microsatellites of the normalized to that of GAPDH or EGFP mRNA [derived from coding region [coding mononucleotide repeats (cMNRs)] the enhanced GFP (EGFP)-expressing control vector]. The result in frameshift mutations in the affected genes, and sequences of the primers used are listed in Supplementary these mutations are believed to contribute to tumor devel- Table S1. opment and progression (10). Although many reports indicated that numerous genes are frequently mutated in Construction of TTK, TCF7L2, and MARCKS expression their cMNRs in MSI-H cancers, few of these genes have been vectors, RNAi, and transfection reported to express their mutant gene products in MSI-H cDNA expression vectors for TTK [cTTK(WT)], TCF7L2 cancers (11–13). We searched for genes containing cMNRs [cTCF4L2(WT)], and MARCKS [cMARCKS(WT)] were con- in the last exon and analyzed the frequency of mutations in structed by cloning the respective wild-type (WT) genes into these genes. We addressed whether genes with frameshift pcDNA3.1 vectors containing a FLAG tag via amplification mutations generate truncated mutant proteins. We showed of their coding regions using the cDNAs derived from HeLa that mutant proteins are actively translated from genes and WiDr cells. For the genomic DNA form of the MARCKS containing mutations in cMNRs in the last exon but are expression vector [gMARCKS(WT)], all exons and introns rarely detected because these endogenous truncated pro- between the exons of MARCKS were cloned into pcDNA3.1 teins containing neopeptides are extensively degraded by vectors containing a FLAG tag. To generate mutant protein the ubiquitin–proteasome system. expression vectors for TTK [cTTK(À2)], TCF7L2 [cTCF7L2 (À1)], and MARCKS [cMARCKS(À2), gMARCKS(À2)], Materials and Methods deletion mutagenesis was conducted. All transfection Tissue samples and cell lines experiments were carried out using Lipofectamine 2000 For the mutation analysis, 12 cell lines were used. DLD1, (Invitrogen), and a CMV10-EGFP vector was used to con- HCT116, HCT-8, LOVO, LS174T, RKO, SNUC2A, SNUC2B, firm the transfection efficiency. The primers used for cloning SNUC4, and SNU407 cells are MSI-H colorectal carcinoma are listed in Supplementary Table S1. The siRNAs against cell lines, whereas WiDr and HeLa cells are microsatellite- TTK, TCF7L2, and MARCKS used in this study (Bioneer) stable (MSS) cell lines, as determined by previous studies were also transfected into cells using Lipofectamine. (14, 15). Cells were grown in RPMI, minimum essential medium, and Dulbecco’s modified Eagle medium supple- Western blotting and mutant protein-specific antibody mented with 10% FBS (Life Technologies),