Yun Sol Jo

Frequent frameshift mutations in 2 mononucleotide repeats of RNF43 gene and its regional heterogeneity in gastric and colorectal cancers

RNF43, an E3 ligase, inhibits Wnt signaling by removing Wnt receptors and behaves as a candidate tumor suppressor. Recent studies identified that RNF43 gene was frequently mutated in gastric (GC), colorectal (CRC), and endometrial cancers with high microsatellite instability (MSI-H). The aim of this study is to explore whether RNF43 gene is mutated in GC and CRC in Korean patients and whether the mutations show regional intratumoral heterogeneity (ITH). We analyzed 2 exonic repeats (C6 and G7) of RNF43 in 78 GCs and 130 CRCs by single-strand conformation polymorphism and DNA sequencing analyses. Also, we analyzed regional ITH of RNF43 mutation in 16 CRCs. We found RNF43 frameshift mutation in MSI-H (50/118), the incidence of which was significantly higher than that in microsatellite stable/low microsatellite instability (1/90). GCs showed a significantly higher incidence of the mutation than CRCs (66.7% of GC and 32.9% of CRC with MSI-H). Also, we found that all of the 7 CRCs with the mutations harbored mutational ITH. By immunohistochemistry, we observed that loss of RNF43 expression was significantly more common in those with RNF43 frameshift mutation than those with wild-type RNF43. Our data indicate that RNF43 gene harbored not only exceedingly high mutations but also mutational ITH, which together might play a role in tumorigenesis of GC and CRC. We suggest that regional analysis is required for a more comprehensive evaluation of the mutation status in these tumors.

USP9X, a Putative Tumor Suppressor Gene, Exhibits Frameshift Mutations in Colorectal Cancers.

To the Editor: USP9X is a member of the peptidase C19 family that encodes a protein that is similar to ubiquitin-specific proteases. Two opposite activities (a tumor suppressor gene (TSG) and oncogenic) of TMEFF2 have been identified [1–4]. USP9X stabilizes MCL1 in human follicular lymphomas and diffuse large B-cell lymphomas and increases the survival of tumor cells [1]. USP9X inhibition promotes radiation-induced apoptosis in non-small cell lung cancer [2]. These data suggest oncogenic activity of USP9X in cancers. By contrast, USP9X was identified as a TSG in pancreatic ductal adenocarcinoma by ‘sleeping Beauty’ transposon-mediated insertional mutagenesis model [3]. Although previous work had attributed oncogenic roles to USP9X in human tumors [1, 2], this study found instead that loss of USP9x enhances transformation and protects pancreatic ductal adenocarcinoma cells from cell death [3]. In human cancers, low USP9X expression correlates with poor survival and treatment with 5-aza-2-primedeoxycytidine elevates USP9X expression in pancreatic ductal adenocarcinoma cell lines [3]. Also, USP9X downregulation renders breast cancer cells resistant to tamoxifen [4]. These reports [3, 4] suggest that USP9X is a TSG with prognostic and therapeutic relevance in cancers. Collectively, these studies suggest that alteration of USP9X may be tumor typedependent. However, somatic inactivating mutation status of USP9X remains undetermined in most carcinomas. There is a mononucleotide repeat (A7) in the coding sequence of USP9X that could be a target for frameshift mutation in cancers with microsatellite instability (MSI) such as colorectal cancers (CRC) [5]. To see whether USP9X gene harbored frameshift mutations within the repeat in CRC, we analyzed the A7 repeat in exon 43 in 79 CRCs with high MSI (MSI-H) and 45 microsatellite-stable/low MSI (MSS/MSI-L) CRCs by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay as described previously [6]. The MSI evaluation system used five mononucleotide repeats (BAT25, BAT26, NR-21, NR-24 and MONO27), tumoral MSI status of which was characterized as: MSIH, if two or more of these markers show instability, MSI-L, if one of the markers shows instability and MSS, if none of the markers shows instability [7]. In cancer tissues, malignant cells and corresponding normal tissue were selectively procured by microdissection and their DNAs were used in the PCR [8]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, Sanger DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously [6]. In the SSCP, we found aberrantly migrating bands in four CRCs with MSI-H (5.1 %) (Fig. 1), but not in their normal DNA. DNA sequencing analysis confirmed that the aberrant bands represented USP9X somatic mutations, which consisted of frameshift mutations by a deletion of one base (c.7440delA (p.Ala2481Profsx7)) and a duplication of one base (c.7440dupA (p.Ala2481Serfsx17)) within the repeat (Fig. 1). The mutations were detected in CRCs with MSIH, but not in those with MSS/MSI-L. Clinical and histopathological parameters could not distinguish USP9X mutation (+) and (−) cancers, possibly due to the small number of the mutated cases. * Sug Hyung Lee suhulee@catholic.ac.kr

Inactivating frameshift mutation of PBRM1, a putative tumour suppressor gene, in colorectal cancers.

Chromatin remodelling controls not only gene expression but also plays important roles in DNA repair, cell death and cell cycle, which are related to cancers.[1] Alterations in chromatin-remodelling genes, including ARID1A, MLL2 and ATRX, are found in many cancers.[2,3] The chromatin remodellers are subdivided into four families: SWI/SNF, INO80/SWR1, ISWI and CHD families.[1] PBRM1, a member of SWI/SNF family, encodes a subunit of ATP-dependent chromatin-remodelling complexes.[1] Renal cell carcinoma, transitional cell carcinoma and cholangiocarcinoma harbour frequent somatic mutations of PBRM1 gene, many of which are inactivating mutations.[3–5] Functionally, suppression of PBRM1 expression promoted cell proliferation and cell cycle progression,[4,6] indicating that PBRM1 is a candidate tumour suppressor gene (TSG). However to date, it remains unknown whether PBRM1 inactivating mutations are common in colorectal cancer (CRC). In a public genome database (http://genome.cse. ucsc.edu/), we found that human PBRM1 had a mononucleotide repeat in the coding sequence that could be a target for frameshift mutation in cancers with microsatellite instability (MSI). Frameshift mutation of genes containing mononucleotide repeats is a feature of CRC with MSI.[7] In this study, we analysed an A7 repeat in the PBRM1 exon 9 by polymerase chain reaction (PCR)based single strand conformation polymorphism (SSCP) assay. We used methacarn-fixed tissues of 79 CRCs with high MSI (MSI-H) and 53 microsatellite stable (MSS) CRCs. In cancer tissues, malignant cells and normal cells were selectively procured from haematoxylin and eosin-stained slides by microdissection.[8] Radioisotope

Absence of PRKD1 Mutation, a Salivary Tumor-Specific Mutation, in Solid Tumors and Leukemias

To the Editor: A recent genomic study showed that somatic mutations of a serine/threonine kinase-encoding gene PRKD1 were very common in polymorphous low-grade adenocarcinoma (PLGA) of salivary gland [1]. They found that PRKD1 hotspot mutations encoding p.Glu710Asp were recurrent in 72.9 % of PLGAs but not in other salivary gland tumors. PRKD1 plays a role in cell adhesion, migration, vesicle transport and survival [2]. Functional studies demonstrated that this kinase-activating mutations altered glandular structures into larger, coalescent structures with filled lumens and irregular contours not uncommonly displaying infiltrating edges, a phenotype consistent with that induced by the forced expression of other oncogenes in this model system [3]. Because PRKD1 p.Glu710Asp mutations are considered driver mutations and highly recurrent, it may be interesting to know whether the PRKD1 p.Glu710Asp mutations occur in other human tumors besides PLGA. For this, tumor tissues from 2444 Korean patients, including hematologic, epithelial and mesenchymal tumor from various origins, were used for this study (Table 1). The tumors did not include PLGAs where PRKD1 p.Glu710Asp mutations are recurrent, because PLGA tissues were not available in this study. For solid tumors, malignant and normal cells were selectively procured from by microdissection [4]. Approval for this study was obtained from the institutional review board. We analyzed exon 10 of PRKD1 gene that encompassed p.Glu710Asp mutation sites by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP). Genomic DNA each from tumor and normal cells was ampl i f i ed by PCR wi th a pr imer pa i r (5 ′ AGGTTTTAGATGCCACAAAG-3′ (forward) and 5′CCAGCTTACATTGCCATAG-3′ (reverse); product size: 185 base pairs). Other procedures of the PCR-SSCP were described in our previous studies [5]. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts. On the SSCP, all of the PCR products for PRKD1 exon 10 were clearly seen. However, none of the SSCP from the cancers displayed aberrantly migrating bands compared to wildtype bands from the normal tissues, indicating there was no evidence of PRKD1 exon 10 mutations in the tumors. To confirm the SSCP data, we repeated the experiments twice, including tissue microdissection, PCR and SSCP to ensure specificity of the results, and found that the data were consistent. One of the main concerns in cancer genetics is to identify whether anymutation found in a tumor is common in the other tumor types. Based on the earlier data that PRKD1 mutation was recurrent in PLGAs [1], we attempted to determine whether somatic mutation of the recurrent site was present in other tumors in this study. The present study, however, detected no somatic mutations of PRKD1 p.Glu710Asp in 2444 tumors from 24 types. Our data indicate that the PRKD1 p.Glu710Aspmay be specific to PLGA, but not to other tumor development. The discovery of the recurrent PRKD1 mutations offered an opportunity for developing therapeutic and diagnostic tools targeting the mutations in tumors. Our data, however, suggest that such applications mutations should be limited to PLGA tumors.

Somatic mutation of a candidate tumour suppressor MGA gene and its mutational heterogeneity in colorectal cancers

Sir, The MYC protooncogene up-regulates expression of cell growth and cell cycle-related genes and promotes oncogenesis. Heterodimerisation of MAX with MYC activates and mediates transcription activity. MGA binds to MAX and inhibits MYC-dependent cell transformation. Mutational profiling of lung adenocarcinoma genomes revealed somatic loss-of-function mutations of MGA, mostly nonsense mutations. Also, an inactivating mutation of MGA was found in small-cell lung cancers. These data indicate that MGA could be a tumour suppressor gene (TSG) and that its inactivating mutations may contribute to solid tumour development. However, the mutation status of MGA remains unclear in most carcinomas. In colorectal cancer (CRC), one of the major ways in which APC acts as a TSG is by inhibiting degradation of free beta-catenin, which then binds to TCF/ LEF, leading to consequent binding to the MYC promoter and causing up-regulation of MYC. There are mononucleotide repeats in the MGA coding sequence that could be mutational targets for frameshift mutation in cancers with microsatellite instability (MSI) such as CRCs. In this study, we analysed the A7 repeats in exons 11 and 21 in 89 CRCs with high MSI (MSI-H) and 45 microsatellite-stable/low MSI (MSS/MSI-L) CRCs by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay, as described previously. There was no family history of Lynch syndrome in these patients. None of the CRC cases met the minimum requirement for a clinical diagnosis of Lynch syndrome based on the Amsterdam II criteria. The MSI evaluation system used five mononucleotide repeats (BAT25, BAT26, NR-21, NR-24

DAB2IP with tumor-inhibiting activities exhibits frameshift mutations in gastrointestinal cancers

A scaffold protein DAB2 and its interaction partner DAB2IP have putative tumor suppressor gene (TSG) functions. Previous studies identified that both DAB2 and DAB2IP genes were inactivated by promoter hypermethylation in human cancers, but their mutational alterations in cancers remain largely unknown. The aim of our study was to find whether DAB2 and DAB2IP were mutated in gastric (GCs) and colorectal cancers (CRCs) by DNA sequencing. Both DAB2 and DAB2IP have mononucleotide repeats in their coding sequence that could be mutation targets in high microsatellite instability (MSI-H) cancers. We analyzed GC and CRC tissues and found that 8 of 34 GCs (23.5%) and 15 of 79 CRCs (20.0%) with MSI-H harbored DAB2IP frameshift mutations. DAB2 frameshift mutations were found in 2 of 79 CRCs (2.5%) with MSI-H. These mutations were not detected in microsatellite stable (MSS) cancers. We also found intratumoral heterogeneity (ITH) of DAB2IP frameshift mutations in 7 of 16 CRCs (43.8%). Loss of DAB2IP protein expression was found in approximately 20% of GCs and CRCs irrespective of MSI and DAB2IP frameshift mutation status. Our study shows that the TSG DAB2IP harbored frameshift mutations and ITH as well as expression loss. Together these tumor alterations might play a role in tumorigenesis of GC and CRC with MSI-H by down-regulating the tumor-inhibiting activities of DAB2IP.

Inactivating Frameshift Mutations of HACD4 and TCP10L Tumor Suppressor Genes in Colorectal and Gastric Cancers

To the Editor: HACD4 (also known as PTPLAD2; protein tyrosine kinase-like A domain-containing protein 2) belongs to a family of enzymes that catalyze the dehydration step of very long chain fatty acid synthesis [1]. Esophageal squamous cell carcinomas show downregulation of HACD4 expression that is correlated with poor patient survival [2]. Knockdown of HACD4 increases STAT3 phosphorylation and cell proliferation [2]. In glioblastomas, the locus for HACD4 is frequently deleted [3]. TCPL10 encoding T-complex protein 10-like, a nuclear protein with transcription regulation functions, suppresses colony formation, cell cycle progression through G0/G1 phase and cell growth in vivo in liver [4]. Together, these data suggest that both HACD4 and TCP10L possess tumor suppressor gene (TSG) activities. However, the roles of HACD4 and TCP10L in colorectal (CRC) and gastric (GC) cancers are not known. About one third of CRC and GC have defects in mismatch repair that can cause microsatellite instability (MSI). TSGs are often observed to have mutations at monocleotide repeats in high MSI (MSI-H) CRC and GC [5]. There are mononucleotide repeats inHACD4 (A8) and TCP10L (G7) of their coding sequences that could be mutation targets in cancers with MSIH. In addition, it is well known that intratumoral heterogeneity (ITH) plays an important role in cancer development and progression and impedes proper diagnosis and treatment of cancer patients [6, 7]. The present study aimed to find whether HACD4 and TCP10L genes harbored frameshift mutation within the repeats and ITH. For this, we studied the mononucleotide repeats in HACD4 (A8) and TCP10L (G7) in 79 high MSI (MSI-H) CRCs, 45microsatellite stable (MSS) CRCs, 34 MSI-H GCs and 45 MSS GCs by single-strand conformation polymorphism (SSCP) assay. After SSCP, Sanger DNA sequencings were performed in cancers with mobility shifts in the SSCP to confirm the mutations [8]. SSCP and Sanger sequencing identified frameshift mutations of HACD4 in 3 cases of CRC and 1 case of GC, and TCP10L in 3 cases of GC. All of them were detected in CRC or GC with MSI-H, but neither in CRC nor GC with MSS. These mutations were not detected in their matched normal t issues. The HACD4 mutat ions (c.689delA (p.Lys230Argfsx42)) and the TCP10L mutations (c.641delG (p.Gly214Valfsx26)) were recurrent in all mutated cases (Table 1). For ITH of the mutations, we studied 16 cases of CRCs with 4 to 7 regional fragments per CRC. One of the 16 CRCs (6.3%) showed the deletion mutation of HACD4 mutations (c.689delA) in different tissue regions. No ITH of the TCP10L frameshift mutation was observed in the 16 CRCs. Clinical and histopathological parameters, however, could distinguish neither HACD4 frameshift mutation (+) and (−) cancers nor TCP10L frameshift mutation (+) and (−) cancers. The frameshift mutations of HACD4 and TCP10L identified in this study would result in truncation of HACD4 and TCP10L proteins respectively, suggesting that inactivation of their TSG functions would contribute to tumorigenesis in MSI-H GCs and CRCs. Also, one CRC (6.3%) exhibited ITH for the HACD4 frameshift mutation. ITH of the frameshift mutation in the CRC might suggest a possibility that there could be a mixed or ameliorated effect of the HACD4 mutation in the affected CRC. However, we were not able to find any distinguished clinicopathologic features of HACD4 mutation ITH-positive cancer. It * Sug Hyung Lee suhulee@catholic.ac.kr

Candidate Tumor Suppressor Gene EAF2 is Mutated in Colorectal and Gastric Cancers

To the Editor: Fusion of ELL-associated factor 2 (EAF2) with ELL is related to myeloid leukemia development [1]. Loss of expression and deletion of EAF2 is common in prostate cancer and EAF2 expression blocks prostate cancer growth, suggesting that EAF2 might have tumor suppressor gene (TSG) functions [2]. However, the roles of EAF2 in colorectal (CRC) and gastric (GC) cancers are not known. About one third of CRC and GC have defects in mismatch repair that can cause microsatellite instability (MSI). TSGs are often observed to have mutations at monocleotide repeats in high MSI (MSI-H) GC and CRC [3]. We hypothesize that EAF2 gene might harbor frameshift mutations at its A8 repeat that would result in inhibition of the EAF functions and play a role in tumor development. For this, we studied the A8 repeat in 79 high MSI (MSI-H) CRCs, 45 microsatellite stable/low MSI (MSS/MSI-L) CRCs, 34 MSI-H GCs and 45 MSS/MSI-L GCs by single-strand conformation polymorphism (SSCP) assay. After SSCP, Sanger DNA sequencings were done in the cancers with mobility shifts in the SSCP to confirm the mutations [4]. We found a type of somatic frameshift mutation in seven CRCs (7/79, 8.9%) and one GC (1/34, 3.0%) with MSI-H phenotype, but there was none in those with MSS/MSI-L (CRCs (0/45) and GCs (0/45)) (Fisher’s exact test, p = 0.001). The mutation was a deletion (c.334delA (p.Thr112GlnfsX30) in the coding region. We also attempted to find whether there was intratumor heterogeneity (ITH) in the EAF2 frameshift mutation, which is known to play a role in cancer evolution as well as treatment resistance in cancer. We studied 16 cases of CRCs with 4–7 tissue fragments per CRC. Three of the 16 CRCs (18.8%) showed the deletion mutation (c.334delA) in different tissue regions (Table 1), indicating ITH of the EAF2 mutation existed in CRC. In earlier studies, evidence of TSG inactivation in EAF2 was found in other cancers, but neither in CRC nor GC. In the present study, we found EAF2 frameshift mutation, and provided evidence of its inactivation in MSI-H CRC and GC. The EAF2 mutation would produce truncation of EAF2 protein, which resembles a loss-of-function mutation. Truncated EAF2 mutants might inhibit its TSG activities [5] and might contribute to pathogenesis of MSI-H cancers. We identified ITH of EAF2 frameshift mutation in CRCs, suggesting a possibility that EAF2 mutation occurred during tumor progression rather than during tumor development. Although ITH is known to influence on clinical outcome of cancer patients, it was not possible to define clinical feature of the cases with ITH due to small Yun Sol Jo and Sung Soo Kim contributed equally to this work.

Intratumoral Heterogeneity for Inactivating Frameshift Mutation of CYB5R2 Gene in Colorectal Cancers

To the Editor: CYB5R2 is an enzyme involved in drug metabolism such as chemotherapeutic agents. Loss of CYB5R2 expression has been reported in many tumors including prostate, nasopharynx and brain tumors [1, 2] . Functionally, mice with CYB5R2 overexpression showed decreased tumorigenicity [2], suggesting that CYB5R2 might be a tumor suppressor gene (TSG). However, there is no data that have analyzed whether CYB5R2 is a TSG in colorectal cancer (CRC). About 10% of CRC show microsatellite instability (MSI) that has defects in mismatch repair [3]. In addition, intratumoral heterogeneity (ITH) plays an important role in development and progression of cancers and impedes proper diagnosis and treatment of cancers [4]. TSGs are often observed to harbor mutations at monocleotide repeats in high MSI (MSI-H) CRC. There is a mononucleotide repeat (A7) in CYB5R2 coding sequence that could be a mutation target in cancers with MSI-H. This study aimed to find whether CYB5R2 frameshift mutation is common and harbors ITH in MSI-H CRC. For this, we studied the A7 in 79 MSI-H CRCs and 45 microsatellite stable MSI (MSS) CRCs by single-strand conformation polymorphism and Sanger DNA sequencing [5]. We found CYB5R2 somatic frameshift mutations in two CRCs with MSI-H (2/79, 2.5%), but there was none in those with MSS (0/45). They were a same deletion mutation (c.433delA (p.Thr145Hisfsx8)). For ITH of the mutation, we studied 16 cases of CRCs with 4 to 7 regional fragments per CRC. Two of the 16 CRCs (12.5%) showed the c.433delA mutation in different regions, indicating ITH of the CYB5R2 mutation existed in the CRCs (Table 1). Clinical and histopathological parameters, however, could distinguish neither CYB5R2 frameshift mutation (+) and (−) cancers, nor the ITH (+) and (−) cancers. The CYB5R2 frameshift mutation would result in truncation of CYB5R2 protein. Based on the previous known TSG activity of CYB5R2 [1, 2], the frameshift mutations could contribute to cancer development by inhibiting the TSG. Presence of ITH of the frameshift mutation in CRC might suggest a possibility that there could be a mixed or ameliorated effect of CYB5R2 mutations in MSI-H cancers. However, we were not able to find any distinguished clinicopathologic features of CYB5R2-mutated or ITHpositive cancers. It was probably due to small number of CYB5R2mutated cases. Based on our data, further studies are needed to define the clinical implication of CYB5R2 mutation and its ITH in MSI-H cancers.

Frameshift Mutations in Repeat Sequences of ANK3, HACD4, TCP10L, TP53BP1, MFN1, LCMT2, RNMT, TRMT6, METTL8 and METTL16 Genes in Colon Cancers

Diminished ANK3 contributes to cell survival by inhibiting detachment-induced apoptosis. TP53BP1 that interacts with p53 and MFN1 that encodes a mitochondrial membrane protein are considered to have tumor suppressor gene (TSG) functions. HACD4 involving fatty acid synthesis and TCPL10 with transcription regulation functions are considered TSGs. Many genes involved in DNA methylations such as LCMT2, RNMT, TRMT6, METTL8 and METTL16 are often perturbed in cancer. The aim of our study was to find whether these genes were mutated in colorectal cancer (CRC). In a genome database, we observed that each of these genes harbored mononucleotide repeats in the coding sequences, which could be mutated in cancers with high microsatellite instability (MSI-H). For this, we studied 124 CRCs for the frameshift mutations of these genes and their intratumoral heterogeneity (ITH). ANK3, HACD4, TCP10L, TP53BP1, MFN1, LCMT2, RNMT, TRMT6, METTL8 and METTL16 harbored 11 (13.9%), 3 (3.8%), 0 (0%), 5 (6.3%), 1 (1.3%), 2 (2.5%), 4 (5.1%), 3 (3.8%), 2 (2.5%) and 2 (2.5%) of 79 CRCs with MSI-H, respectively. However, we found no such mutations in microsatellite stable (MSS) cancers in the nucleotide repeats. There were ITH of the frameshift mutations of ANK3, MFN1 and TP53BP1 in 1 (6.3%), 1 (6.3%) and 1 (6.3%) cases, respectively. Our data exhibit that cancer-related genes ANK3, HACD4, TP53BP1, MFN1, LCMT2, RNMT, TRMT6, METTL8 and METTL16 harbor mutational ITH as well as the frameshift mutations in CRC with MSI-H. Also, the results suggest that frameshift mutations of these genes might play a role in tumorigenesis through their inactivation in CRC.