Genes with dual proto-oncogene and tumor suppressor gene activities are frequently altered by protein losses in colon cancers.

Cancer genes are largely categorized into tumor suppressor gene (TSG) and proto-oncogene, but many have dual activities depending on the cellular context. In the present study, we analyzed DYRK1B, ESRP1, MTSS1, ADAMTS1, and INPP5F genes known to possess the dual activities in sporadic colon cancers (CCs). By the mutation analysis, we identified DYRK1B, ESRP1, MTSS1, ADAMTS1, and INPP5F frameshift mutations in 2, 2, 3, 3, and 1 CCs in instability-high (MSI-H) cases (1.1-3.2% of MSI-H CCs), respectively, but not microsatellite stable (MSS) cases. One CC showed regional heterogeneous mutations (RHM) of ESRP1 mutation. Immunohistochemistry identified protein expression of ESRP1, MTSS1, and ADAMTS1 in the CCs, revealing that approximately 30% of CCs lost the protein expression irrespective of the MSI status. Our study showed that dual TSG and proto-oncogene genes DYRK1B, ESRP1, MTSS1, ADAMTS1, and INPP5F harbored low incidences of inactivating mutations, but that the protein losses were frequent in CCs. Our study suggests a possibility that the dual-function genes could be altered mainly at the expression level, which might contribute to CC pathogenesis.

Concurrent inactivating mutations and expression losses of RGS2, HNF1A, and CAPN12 candidate tumor suppressor genes in colon cancers.

Microsatellite instability-high (MSI-H) colorectal cancer (CRC) is different from microsatellite stable (MSS) CRC concerning biological, and clinical features. In MSI-H CRCs, defects of mismatch repair genes produce increased mutation accumulation in repetitive DNA sequences. To see whether candidate tumor suppressor genes (TSGs) are altered in MSI-H CRC, we studied frameshift mutation and protein expression of candidate TSGs of RGS2, HNF1A, HNF1B, CAPN12, RCBTB2, ATE1, PKNOX1, and USP19. We found frameshift mutations of RGS2 in 5 (5%), HNF1A in 6 (6%), HNF1B in 2 (2%), CAPN12 in 3 (3%), RCBTB2 in 4 (4%), ATE1 in 2 (2%), PKNOX1 in 2 (2%), and USP19 in 2 (2%) MSI-H CRCs. However, we found no such mutations in MSS CRCs. RCBTB2, CAPN12, HNF1A, and HNF1B frameshift mutations revealed the regional difference in the same tumors. In addition, we identified loss of RGS2, HNF1A, and CAPN12 protein expression irrespective of MSI phenotype in 13-29% of CRCs. The results indicate that many TSGs harbor concurrent inactivating mutations and protein loss in MSI-H CRCs with intratumoral mutational heterogeneity, and that MSS CRCs are altered by protein losses. These alterations could contribute to CRC development and underlying mechanisms and consequences of the TSG alterations remain to be clarified.

Mutational Alterations of DNA Methylation-related Genes CTCF, ZFP57, and ATF7IP Genes in Colon Cancers.

Deregulations of DNA-methylation-related genes are common in cancers, but frameshift mutation status in colon cancer (CC) is unknown. Our study aims to assess whether CTCF, ZFP57, and ATF7IP genes in this category are mutated in CC. CTCF, ZFP57, and ATF7IP genes have repeat coding sequences, which are frequently deleted or duplicated in CC, harboring the phenotype of unstable or high microsatellite instability (MSI-H). We studied 140 CCs [95 MSI-H CCs and 45 stable MSI (MSS) CCs], and found 7 CCs with MSI-H (6/95: 6.3%) harbored frameshift mutations within the repeats, whereas those with MSS did not. Of note, the CTCF frameshift mutations showed the regional difference in the 2 (12.5%) of 16 MSI-H CCs, indicating there was intratumoral heterogeneity. In the immunohistochemistry for ATF7IP, the MSI-H CC showed low intensity compared to MSS CC. Together, CTCF, ZFP57, and ATF7IP genes, despite the low incidence of the mutations, are altered in several ways (mutation, expression, and intratumoral heterogeneity) and could contribute to MSI-H CC development.

Somatic Mutation of NLRP Genes in Gastric and Colonic Cancers.

Nucleotide-binding and leucine-rich repeat protein (NLRP) genes are involved in inflammasome formation that plays a role in inflammation/host defense and cell death. Both cell death and inflammation are crucial for cancer development, but the roles of NLRPs in cancer are partially known. In this study, we analyzed mononucleotide repeats in coding sequences of NLRP1, NLRP2, NLRP4 and NLRP9, and found 1, 1, 1 and 8 frameshift mutation (s) in gastric (GC) and colonic cancers (CRC), respectively. Five of the 32 high microsatellite instability (MSI-H) GCs (15.5%) and 6 of 113 MSI-H CRCs (5.5%) exhibited the frameshift mutations. There was no NLRP frameshift mutations in microsatellite stable (MSS) GCs and CRCs. We also discovered that 2 of 16 CRCs (12.5%) harbored intratumoral heterogeneity (ITH) of the NLRP9 frameshift mutations in one or more areas. In both GC and CRC with MSI-H, NLRP9 expression in NLRP9-mutated cases was significantly lower than that in NLRP9-non-mutated cases. Our data indicate that NLRP9 is altered at multiple levels (frameshift mutation, mutational ITH and loss of expression), which together could contribute to pathogenesis of MSI-H GC and CRC.

Brief Research Report Regional Difference in TRAF2 and TRAF3 Gene Mutations in Colon Cancers.

TRAF2 and TRAF3 genes of tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family are involved in diverse cell signaling, and function as both tumor suppressor gene and oncogene. Alterations of TRAF2 and TRAF3 in colon cancer (CC) along with their regional difference and microsatellite instability (MSI) are largely unknown. In the present study, we analyzed TRAF2 and TRAF3 frameshift mutations in 168 sporadic CCs (100 high MSI (MSI-H) and 68 microsatellite-stable (MSS) CCs). We identified TRAF2 and TRAF3 frameshift mutations in 4 (4%) and 3 CCs (3%) with MSI-H, respectively, but none in 68 cases of MSS CCs. Of the 168 CCs, we analyzed the mutations in multi-regions for 39 CCs (16 MSI-H and 23 MSS CCs), and discovered that 12.5% (2/16) and 6.3% (1/16) of MSI-H CCs exhibited regional difference in TRAF2 and TRAF3 mutations, respectively. In the multi-region samples of 23 MSS CCs, neither TRAF2 nor TRAF3 frameshift mutation was found. In 40% of CCs, both TRAF2 and TRAF3 expressions were increased compared to normal colon cells. Our data indicate that TRAF2 and TRAF3 frameshift mutations and their regional difference as well as altered expressions are present in MSI-H CCs, which could contribute to MSI-H cancer development.

Expression and Mutation Alterations of ZMYM4 Gene in Gastric and Colonic Cancers.

ZMYM4 is a zinc finger protein, whose cancer-related functions are partially known (cell shape maintenance and cell death). In this study, we analyzed 4 sites of mononucleotide repeats in the coding sequence of ZMYM4 in gastric (GC) and colonic cancers (CC). Seven of the 32 high microsatellite instability (MSI-H) GCs (21.9%) and 23 of 113 MSI-H CCs (20.4%) harbored ZMYM4 frameshift mutations with no significant difference between the 2 organs (P>0.05). There was no ZMYM4 frameshift mutations in microsatellite-stable GCs and CCs. We also identified that 6 of 16 MSI-H CCs (37.5%) exhibited intratumoral heterogeneity of the ZMYM4 frameshift mutations. In both GC and CC with MSI-H, ZMYM4 expression in ZMYM4-mutated cases was significantly lower than that in ZMYM4-nonmutated cases. Our study indicates that ZMYM4 is altered at multiple levels (frameshift mutation, mutational intratumoral heterogeneity, and loss of expression), suggesting their relations with MSI-H GC and CC.

Mutation and expression alterations of histone methylation-related NSD2, KDM2B and SETMAR genes in colon cancers.

Epigenetic dysregulation is a hallmark of cancers, and examples of its cancer-associated expression and mutation alterations are rapidly growing. Histone methylation, a process by which methyl groups are transferred to amino acids of histone proteins, is crucial for the epigenetic gene regulation. NSD2 (nuclear receptor-binding SET domain protein 2) and SETMAR are epigenetic regulators for histone methylation. KDM2B, also known as FBXL10, is a histone demethylase that targets histone methylation processes. They are known to be altered in many cancers, but somatic frameshift mutation and expression of these genes remain undetermined in many other subsets of cancers, including high microsatellite instability (MSI-H) colon cancer (CC). In this study, we analyzed mononucleotide repeats in coding sequences of NSD2, KDM2B and SETMAR genes, and found frameshift mutations in 10 %, 2 % and 1 % of CCs with MSI-H, respectively. Of note, there was no frameshift mutation of these genes in microsatellite stable (MSS) CCs. In addition, we discovered that 2 and 2 of 16 CRCs (12.5 % and 12.5 %) harbored intratumoral heterogeneity (ITH) of the NSD2 and KDM2B frameshift mutations, respectively. In the immunohistochemistry for NSD2, intensity of NSD2 immunostaining in MSI-H CC is decreased compared to that in MSS. These results suggest that NSD2 might be altered at multiple levels (frameshift mutation, mutational ITH and expression) in MSI-H CCs, and could be related to MSI-H cancer pathogenesis.

Cancer-related SRCAP and TPR mutations in colon cancers.

Current information suggests that SRCAP, TPR and CEACAM5 genes have cancer-related activities, but their alteration status is not well identified in colon cancer (CC). In this study, we analyzed frameshift mutations of these genes in CCs according to the microsatellite instability (MSI) status (high MSI (MSI-H) and microsatellite stable (MSS) CCs). In addition, regional difference in frameshift mutations of SRCAP, TPR and CEACAM5 genes were studied in CCs. In this study, we detected frameshift mutations (deletion or duplication of one or two bases) of SRCAP in 12 (12 %), TPR in 3 (3%) and CEACAM5 in 2 (2%) CCs with MSI-H. However, there was no such mutations in MSS cancers (P < 0.001). 18.8 % and 6.3 % of 16 CCs showed the regional difference in the SRCAP and TPR mutations, respectively. Approximately in 60 % of the CCs, SRCAP expression was increased compared to normal colon cells. Our study shows that SRCAP, TPR and CEACAM5 frameshift mutations and their regional difference as well as altered SRCAP expression are present in MSI-H CCs, which could contribute to CC development with MSI-H.

Inactivating mutations of class II transactivator (CIITA) gene in gastric and colorectal cancers.

Expression of MHC class II, which is important against cancer immunity, depends on the transcactivator CIITA. These data suggest a possibility that CIITA gene might be inactivated in cancers. In this study, we studied inactivating mutation status of CIITA gene in gastric (GC) and colorectal (CRC) cancers by analyzing the C7 repeat in the CIITA (exon 11) gene. We found frameshift mutations in 3 GCs and 6 CRCs in cancers with high microsatellite instability (MSI-H) (9/113), but not in those with microsatellite-stable (MSS) (0/90) (P = 0.004). They were all deletion or duplication of one base in the C7 repeat that would result in truncation of amino acid synthesis. Immune therapy is now a major option in cancer therapy and our results on the genetic alterations of MHC II-related CTIITA in MSH-H GC and CRC might provide useful information for the treatment of MSI-H cancers.

Frameshift Mutations and Loss of Expression of CLCA4 Gene are Frequent in Colorectal Cancers With Microsatellite Instability.

Chloride channel calcium-activated (CLCA) genes encode regulators for chloride transport across the cell membrane. As for cancer development, some CLCA genes are considered putative tumor suppressor genes. The aim of this study was to explore whether CLCA4 gene would have mutations in its nucleotide repeats in colorectal cancer (CRC). In a public database, we found that CLCA4 gene had mononucleotide repeats in the coding sequences that might be mutational targets in the cancers with microsatellite instability. For this, the current study studied 146 CRCs for mutation and expression analyses by single-strand conformation polymorphism analysis, DNA sequencing, and immunohistochemistry. Overall, we found CLCA4 frameshift mutations in 12/101 (11.8%) CRCs with high-microsatellite instability (MSI-H), but none in microsatellite stable CRCs (0/45) (P<0.01). In addition, we analyzed intratumoral heterogeneity of the CLCA4 frameshift mutations and found that 1 CRC harbored regional intratumoral heterogeneity of the CLCA4 frameshift mutation. Loss of CLCA4 protein expression was identified in 50% of CRCs. Also, cancers with MSI-H harboring CLCA4 frameshift mutations showed lower CLCA4 immunostaining than those with the wild-type. Our data indicate that the CLCA4 gene harbors alterations both in somatic mutation and expression, suggesting their roles in tumorigenesis of CRC with MSI-H.

Tight Junction-Related CLDN5 and CLDN6 Genes, and Gap Junction-Related GJB6 and GJB7 Genes Are Somatically Mutated in Gastric and Colorectal Cancers.

Tight junction and gap junction are major cell junctions that play important roles in cellular communication and structural integrity. Alterations of these junctions are known to be involved in cancer pathogenesis. Claudins and connexins are major tight and gap junction proteins, but genetic alterations of these genes have not been reported in gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). Claudin genes CLDN5 and CKDN6, and connexin genes GJB6 and GJB7 have mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with MSI. We analyzed 79 GCs and 145 CRCs, and found CLDN5 frameshift mutations in 3 (3%) CRCs and 1 (2.9%) GC, CLDN6 frameshift mutations in 6 (6%) CRCs, GJB6 frameshift mutations in 5 (5%) CRCs and GJB7 frameshift mutation in one CRC (1%) with high MSI (MSI-H). We also analyzed intratumoral heterogeneity (ITH) of the frameshift mutations in 16 CRCs and found that CLDN6 and GJB6 frameshift mutations showed regional ITH in 2 (12.5%) and 2 (12.5%) cases, respectively. Our results show that CLDN5, CLDN6, GJB6 and GJB7 genes harbor not only frameshift mutations but also mutational ITH, which together may be features of GC and CRC with MSI-H. Based on the roles of cellular junctions in cancers, frameshift mutations of tight junction and gap junction genes might contribute to tumorigenesis by altering their functions in GC and CRC.