Frameshift Mutations of HSPA4 and MED13 in Gastric and Colorectal Cancers.

Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). In the public genome database, we found that human HSPA4 gene encoding a heats hock protein 70 protein (HSP70-4) and MED13 gene had mononucleotide repeats in the coding sequences that could be targets for frameshift mutation in cancers with MSI. HSP70-4 is a member of HSP70 that is known to play a role in cell survival. MED13 is a member of MED genome-wide transcription regulators that function as a regulator for diverse biological processes. In this study, we analyzed the mutations in 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases by single-strand conformation polymorphism analysis and DNA sequencing. We found frameshift mutations of HSPA4 gene in two cancers (one GC and one CRC) and MED13 gene in the other two cancers (one GC and one CRC). The frameshift mutations were deletions of one base (c.2396delA (p.Asn799MetfsX50)) in HSPA4 and (c.2175delA (p.Lys725AsnfsX4)) in MED13. Each of HSPA4 and MED13 mutations were detected in GC with MSI-H (1/34: 2.9 %) and CRC with MSI-H (1/79: 1.3 %), but not in those with MSS. Our data show that unconventional HSPA4 and MED13 genes harbored frameshift mutations in GC and CRC with MSI. These mutations might possibly inactivate their functions and could be a feature of GC and CRC with MSI-H.

Frameshift mutations in mammalian target of rapamycin pathway genes and their regional heterogeneity in sporadic colorectal cancers.

Mammalian target of rapamycin (mTOR) pathway is known to be involved in cancer pathogenesis. The aim of our study was to find whether mTOR-related genes were mutated and expressionally altered in colorectal cancers (CRCs). Through public database searching, we found that PIK3CB, insulin receptor substrate 1/2 (IRS1), RPS6, EIF4B, RPS6KA5, and PRKAA2 that were known as mTOR-related genes possessed mononucleotide repeats in DNA coding sequences that could be mutated in cancers with microsatellite instability (MSI). We analyzed 124 CRCs by single-strand conformation polymorphism analysis and DNA sequencing and found 7 (8.9%), 8 (10.1%), and 3 (3.8%) of 79 CRCs with high MSI that harbored IRS1, EIF4B, and RPS6KA5 frameshift mutations, respectively. These mutations were not identified in stable MSI/low MSI (0/45). In addition, we analyzed intratumoral heterogeneity (ITH) of PIK3CB, IRS1, RPS6, EIF4B, RPS6KA5, and PRKAA2 frameshift mutations in 16 CRCs and found that IRS1, EIF4B, and RPS6KA5 mutations had regional ITH in 2, 2, and 1 CRCs, respectively. We also analyzed IRS1 expression in the CRCs by immunohistochemistry. Loss of IRS1 expression was identified in 31% of the CRCs. The loss of expression was more common in those with IRS1 mutation than those with wild-type IRS1. Our data indicate mTOR-related genes harbored not only somatic mutations but also mutational ITH and loss of expression, which together might play a role in tumorigenesis of CRC, especially with high MSI. Our data also suggest that mutation analysis in multiregional areas is needed for a precise evaluation of mutation status in CRC with MSI-H.

Regional Bias of Intratumoral Genetic Heterogeneity of Apoptosis-Related Genes BAX, APAF1, and FLASH in Colon Cancers with High Microsatellite Instability.

BACKGROUND: Apoptosis inactivation and intratumoral heterogeneity (ITH) are common features of cancers, including colorectal cancer (CRC). Inactivation of apoptosis prolongs cancer cell survival, and ITH may contribute to CRC progression. AIM: To examine the presence and extent of mutational ITH in the pro-apoptotic genes APAF1, BAX, and FLASH and the association of mutational ITH with pathologic parameters of CRC. METHODS: The ITH of mutations in the mononucleotide repeats of APAF1, BAX and FLASH in different tumors were analyzed in 16 cases of CRC with high microsatellite instability (MSI-H) and 41 cases of CRC with stable MSI/low MSI (MSS/MSI-L) by single-strand conformation polymorphism and DNA sequencing analyses. RESULTS: Frameshift mutations of APAF1, BAX, and FLASH were identified in 19, 31, and 6 % of CRC with MSI-H, respectively, but also in cases of CRC with MSS/MSI-L. All but one CRC with a mutation (8/9) harbored regional ITH of the APAF1, BAX and FLASH frameshift mutations. ITH, however, was not associated with histopathologic features of CRC with MSI-H, suggesting that ITH might not be related to development of the MSI-H phenotype itself, but rather to disease progression. CONCLUSIONS: Our results indicate that the APAF1, BAX, and FLASH genes not only harbor frameshift mutations but also demonstrate mutational ITH, which together might play a role in the tumorigenesis of CRC with MSI-H by affecting the apoptosis of cancer cells. Our data also suggest that multiregional mutation analysis is needed for a better evaluation of the mutation status in CRC.

Laminin gene LAMB4 is somatically mutated and expressionally altered in gastric and colorectal cancers.

Laminins are important in tumor invasion and metastasis as well as in maintenance of normal epithelial cell structures. However, mutation status of laminin chain-encoding genes remains unknown in cancers. Aim of this study was to explore whether laminin chain genes are mutated and expressionally altered in gastric (GC) and colorectal cancers (CRC). In a public database, we found that laminin chain genes LAMA1, LAMA3, LAMB1 and LAMB4 had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with microsatellite instability (MSI). We analyzed the genes in 88 GC and 139 CRC [high MSI (MSI-H) or stable MSI/low MSI (MSS/MSI-L)] by single strand conformation polymorphism analysis and DNA sequencing. In the present study, we found LAMB4 (11.8% of GC and 7.6% of CRC with MSI-H), LAMA3 (2.9% of GC and 2.5 of CRC with MSI-H), LAMA1 (5.9% of GC with MSI-H) and LAMB1 frameshift mutations (1.3% of CRC with MSI-H). These mutations were not found in MSS/MSI-L (0/114). We also analyzed LAMB4 expression in GC and CRC by immunohistochemistry. Loss of LAMB4 expression was identified in 17-32% of the GC and CRC. Of note, the loss expression was more common in the cancers with LAMB4 mutation or those with MSI-H. Our data show that frameshift mutations of LAMA1, LAMA3, LAMB1 and LAMB4, and loss of LAMB4 may be features of GC and CRC with MSI-H.

Mutational and expressional analysis of ERBB3 gene in common solid cancers.

ERBB3 is a member of EGFR family receptor tyrosine kinases, genetic alterations of which are common and therapeutically targeted in human cancers. Recently, somatic mutations of ERBB3 gene, including recurrent mutation in exon 3 altering Val104, were reported in gastric cancers (GC) and colorectal cancers (CRC), strongly suggesting its role in the development of GC and CRC. To examine whether the recurrent ERBB3 mutations of exon 3 occur in GC and CRC, and other malignancies as well, we analyzed the ERBB3 in 1677 cancer tissues by a single-strand conformation polymorphism (SSCP) assay. We identified ERBB3 mutations altering the Val104 mutations in GC (0.5%) and CRC (2.2%). However, we did not find the ERBB3 mutations in the other cancers besides GC and CRC. We observed that an increased intensity of phosphorylated ERBB3 (pERBB3) in GC and CRC. Of note, all of the cancers with ERBB3 mutations displayed an increased intensity of pERBB3 immunostaining. Our data indicate that the recurrent ERBB3 mutations altering Val104 occur predominantly in GC and CRC. Also, the data suggest that ERBB3 is altered in GC and CRC by various ways, including somatic mutations and increased expression that might play roles in tumorigenesis.

Frameshift mutation of a histone methylation-related gene SETD1B and its regional heterogeneity in gastric and colorectal cancers with high microsatellite instability.

Histone methyltransferase (HMT), which catalyzes a histone methylation, is frequently altered in cancers at mutation and expression levels. The aims of this study were to explore whether SETD1B, SETDB2, and SETD2, SET domain-containing HMT genes, are mutated and expressionally altered in gastric (GC) and colorectal cancers (CRC). In a public database, we found that SETD1B, SETDB2, and SETD2 had mononucleotide repeats in coding sequences that might be mutation targets in cancers with microsatellite instability (MSI). We analyzed the mutations in 76 GCs and 93 CRCs and found SETD1B (38.7% of GC and 35.6% of CRC with high MSI [MSI-H]), SETDB2 (11.1% of CRC with MSI-H), and SETD2 frameshift mutations (6.7% of CRC with MSI-H). These mutations were not found in stable MSI/low MSI. In addition, we analyzed intratumoral heterogeneity (ITH) of SETD1B mutation in 6 CRCs and found that 2 CRCs harbored regional ITH of SETD1B. We also analyzed SETD1B expression in GC and CRC by immunohistochemistry. Loss of SETD1B expression was identified in 15% to 55% of the GC and CRC with respect to the MSI status. Of note, the loss of expression was more common in those with SETD1B mutations than those with wild-type SETD1B. We identified alterations of SET domain-containing HMT at various levels (frameshift mutations, genetic ITH, and expression loss), which together might play a role in tumorigenesis of GC and CRC with MSI-H. Our data suggest that mutation analysis in multiple regions is needed for a better evaluation of mutation status in CRC with MSI-H.

Frameshift mutations of axon guidance genes ROBO1 and ROBO2 in gastric and colorectal cancers with microsatellite instability.

AIMS: Several lines of evidence indicate that axon guidance genes are involved not only in neural development but also in cancer development. ROBO1 and ROBO2, crucial regulators of axon guidance, are considered potential tumour suppressor genes. The aim of this study was to explore whether ROBO1 and ROBO2 genes are somatically mutated and expressionally altered in gastric (GC) and colorectal cancers (CRC). METHODS: In a public database, we observed that both ROBO1 and ROBO2 had mononucleotide repeats in their coding exons that could be mutation targets in cancers with microsatellite instability (MSI). We analysed mutations of these repeats in 77 GC and 88 CRC either with high MSI (MSI-H) or low MSI/microsatellite stability (MSI-L/MSS) by single-strand conformation polymorphism (SSCP) and DNA sequencing. We analysed ROBO1 and ROBO2 expressions in GC and CRC by immunohistochemistry as well. RESULTS: Overall, we found five ROBO1 and five ROBO2 frameshift mutations in the repeats. They were detected exclusively in the cancers with MSI-H (10/70, 14.2%), but not in MSI-L/MSS (0/95, 0%) (p=0.018). In the immunohistochemistry, loss of ROBO2 expression was identified in 22 (29%) and 17 (19%) of GC and CRC, respectively, while increased expression of ROBO2 was found in 15 (20%) and 22 (25%) of GC and CRC, respectively. There were co-occurrences of mutation and loss of expression in both ROBO1 (4/5, 80% mutated cases, p<0.001) and ROBO2 (5/5, 100% mutated cases, p<0.05) genes. CONCLUSION: This is the first report of ROBO1 and ROBO2 frameshift mutations in GC and CRC. Frameshift mutations of ROBO1 and ROBO2 genes and alteration of ROBO2 expression in GC and CRC suggest that both genes might play roles in the pathogenesis of GC and CRC.