Background mutation frequency in microsatellite-unstable colorectal cancer

Microsatellite instability (MSI) is observed in approximately 12% of colorectal cancers. Genes containing a mononucleotide microsatellite in the coding sequence are particularly prone to inactivation in MSI tumorigenesis, and much work has been conducted to identify genes with high repetitive tract mutation rates in these tumors. Much less attention has been paid to background mutation frequencies, and no work has focused on nontranscribed regions. Here, we studied 114 nontranscribed intergenic A/T and C/G repeats 6 to 10 bp in length, located distant from known genes, to examine background mutation frequencies in MSI colorectal cancers. A strong correlation with tract length was observed, and mutation frequencies of up to 87% were observed in 8 to 10 bp tracts. Subsequently, to compare the background mutation rate in transcribed and nontranscribed noncoding repeats, we screened nine randomly selected intronic C/G8 repeats. In addition, the coding repeats of seven suggested MSI target genes, and nine previously published intronic A8 and G8 repeats were analyzed. Intronic repeats seemed to mutate less frequently than nontranscribed intergenic repeats. Our results show that strand slippage mutations in mismatch repair-deficient cells are as abundant in short intergenic repeats as in many proposed MSI target genes. However, under mismatch repair deficiency, strand slippage mutations in transcribed sequences seem to be repaired more efficiently than in intergenic nontranscribed sequences. The mechanisms causing these differences are not yet understood and should be a subject for further studies. For MSI target gene identification, repeats in transcribed sequences seem to be the most appropriate reference group for coding region repeat mutations.     

Mutations in two short noncoding mononucleotide repeats in most microsatellite-unstable colorectal cancers

DNA mismatch repair (MMR)-deficient cells typically accumulate mutations in short repetitive DNA tracts. This microsatellite instability (MSI) facilitates malignant transformation when affecting genes with growth-related and caretaker functions. To date, several putative MSI target genes have been proposed mainly based on high mutation frequency within their coding regions. However, some intronic repeat mutations have also been suggested to associate with MSI tumorigenesis, indicating the need for additional analyses on noncoding repeats. Here we have analyzed an intronic T9 repeat of semenogelin I (SEMG1) and report mutation frequencies of 51% (75 of 146) and 62% (8 of 13) in MMR-deficient primary colorectal cancers and cell lines, respectively. The putative effect of the SEMG1 mutations was assessed by RNA and protein level analyses, but no differences were detected between colorectal cancer cell lines with different SEMG1 status. Subsequently, the general background mutation frequency of MSI colorectal cancers was assessed by screening for intergenic T9 repeat alterations. One of 10 examined repeats was mutated in 70% (102 of 145) of the colorectal cancers evaluated. The frequencies observed here are notably higher than previously published in noncoding repeats shorter than 10 bp in MMR-deficient primary tumors. Our results indicate that high mutation frequencies, similar or higher than those observed in proposed and approved target genes, can be detected in repeat tracts of MSI tumors without any apparent selection pressure. These data call for urgent and thorough large-scale evaluation of mutation frequencies in neutral short repetitive sequences in MMR-deficient tumors.     

High frequency of TTK mutations in microsatellite-unstable colorectal cancer and evaluation of their effect on spindle assembly checkpoint

Frameshift mutations frequently accumulate in microsatellite-unstable colorectal cancers (MSI CRCs) typically leading to downregulation of the target genes due to nonsense-mediated messenger RNA decay. However, frameshift mutations that occur in the 3' end of the coding regions can escape decay, which has largely been ignored in previous works. In this study, we characterized nonsense-mediated decay-escaping frameshift mutations in MSI CRC in an unbiased, genome wide manner. Combining bioinformatic search with expression profiling, we identified genes that were predicted to escape decay after a deletion in a microsatellite repeat. These repeats, located in 258 genes, were initially sequenced in 30 MSI CRC samples. The mitotic checkpoint kinase TTK was found to harbor decay-escaping heterozygous mutations in exon 22 in 59% (105/179) of MSI CRCs, which is notably more than previously reported. Additional novel deletions were found in exon 5, raising the mutation frequency to 66%. The exon 22 of TTK contains an A(9)-G(4)-A(7) locus, in which the most common mutation was a mononucleotide deletion in the A(9) (c.2560delA). When compared with identical non-coding repeats, TTK was found to be mutated significantly more often than expected without selective advantage. Since TTK inhibition is known to induce override of the mitotic spindle assembly checkpoint (SAC), we challenged mutated cancer cells with the microtubule-stabilizing drug paclitaxel. No evidence of checkpoint weakening was observed. As a conclusion, heterozygous TTK mutations occur at a high frequency in MSI CRCs. Unexpectedly, the plausible selective advantage in tumourigenesis does not appear to be related to SAC.     

Frameshift Mutations at Mononucleotide Repeats in RAD50 Recombinational DNA Repair Gene in Colorectal Cancers with Microsatellite Instability

To identify additional genes targeted for microsatellite instability (MSI), we search for human genes which contain mononucleotide repeats in their coding region, selected 7 genes ( RAD50, DNA-PKcs, FLASH, Apaf-1, XPG, CtIP , and MLSN1 ), and analyzed frameshift mutations in them. Here we report that 60% (3 out of 5) of human colorectal cancer cell lines exhibiting a high frequency of MSI (MSI-H) and 46% (6 out of 13) of MSI-H primary colorectal tumors had mutations in the (A)9 repeat of RAD50 recombinational repair gene. In contrast, no frameshift mutations were found in any of the 5 MSI-negative colorectal cancer cell lines, 8 colorectal tumors exhibiting a low frequency of MSI (MSI-L), or 28 MSI-negative colorectal tumors. No mutations were found in the mononucleotide repeats of 6 other genes, even in MSI-H cancers. These results suggest that RAD50 frameshift mutations may play a role in the tumorigenesis of MSI-H colorectal cancers.     

Frameshift mutations at mononucleotide repeats in RAD50 recombinational DNA repair gene in colorectal cancers with microsatellite instability.

To identify additional genes targeted for microsatellite instability (MSI), we search for human genes which contain mononucleotide repeats in their coding region, selected 7 genes (RAD50, DNA-PKcs, FLASH, Apaf-1, XPG, CtIP, and MLSN1), and analyzed frameshift mutations in them. Here we report that 60% (3 out of 5) of human colorectal cancer cell lines exhibiting a high frequency of MSI (MSI-H) and 46% (6 out of 13) of MSI-H primary colorectal tumors had mutations in the (A)9 repeat of RAD50 recombinational repair gene. In contrast, no frameshift mutations were found in any of the 5 MSI-negative colorectal cancer cell lines, 8 colorectal tumors exhibiting a low frequency of MSI (MSI-L), or 28 MSI-negative colorectal tumors. No mutations were found in the mononucleotide repeats of 6 other genes, even in MSI-H cancers. These results suggest that RAD50 frameshift mutations may play a role in the tumorigenesis of MSI-H colorectal cancers.     

Synchronous multiple primary gastrointestinal cancer exhibits frequent microsatellite instability

Colorectal (CRC) and gastric cancers (GC), the most common gastrointestinal malignancies, have been known to develop occasionally in a same patient. Previous studies have focused on the etiology of patients with multiple primary gastric and colorectal cancer (MPGCC); however, the carcinogenic process of MPGCC remains unclear. In this study, we have examined the genetic alterations in MPGCC in order to clarify the carcinogenic pathway. Twenty patients with sporadic MPGCC were examined for microsatellite instability (MSI) and frameshift mutations of target genes such as TGFbetaRII, BAX and IGFIIR. In 10 (50%) of 20 patients with MPGCC, MSI was present at least at 1 lesion of GC or CRC. Four (50%) of 8 cases with synchronous MPGCC displayed MSI in both GC and CRC, while only 1 (8%) of 12 cases of metachronous MPGCC exhibited MSI in both organs. Carcinogenic process of MPGCC was fairly associated with the MSI pathway, particularly in cases of synchronous MPGCC. MSI was found in 5 (25%) of 20 GCs and in 10 (50%) of 20 CRCs. MSI was involved more closely in CRC than in GC among MPGCC. Although most frameshift mutations at target genes were found in the MSI-positive MPGCC, infrequent mutations were observed in the genes. Frameshift mutation was found in only 1 of 5 cases of MSI-positive GC at TGFbetaRII. Only 2 of 10 cases of CRC with MSI showed mutation at TGFbetaRII, and 1 case also showed mutation at BAX and IGFIIR. Our findings suggest that TGFbetaRII, BAX and IGFIIR are not the main target genes for carcinogenesis in MSI-positive MPGCC.     

Mutations of the transforming growth factor β type II receptor gene and microsatellite instability in gastric cancer

Forty-three sporadic gastric cancers were analyzed with regard to whether mutations of simple repeated sequences in the transforming growth factor β type II receptor (TβR-II) gene are associated with microsatellite instability (MSI) and gastric carcinogenesis. In 12 of the 43 cancers (28%), MSI was observed at least at 1 of the 2 microsatellite loci. Frameshift mutations of the TβR-II gene, all of which were 1 base deletion of 10 adenine repeats, were detected in 3 of 6 cancers, with MSI at 2 loci. However, mutations were not detected in 6 cancers, with MSI only at 1 locus and 31 cancers without MSI. Moreover, micro-analysis in these cases revealed that the mutant-type alleles of TβR-II were invariably common in different areas within the tumor, in contrast to the markedly variable alleles of microsatellite loci. Our results suggest that frameshift mutation of the TβR-II gene may be a critical event associated with MSI and may contribute to carcinogenesis of the stomach. One of the possible mechanisms of escape from growth control by TGFβ during gastric carcinogenesis could involve frameshift mutations of the TβR-II gene caused by DNA replication errors. © 1996 Wiley-Liss, Inc.     

Simplified MSI marker panel for diagnosis of colorectal cancer

Background: Colorectal cancers (CRCs) tumors are diagnosed by microsatellite instability (MSI) due to accumulation of insertion/deletion mutations in tandem repeats of short DNA motifs (1–6 bp) called microsatellites. Microsatellite instability (MSI) is not only a hallmark marker for screening of hereditary nonpolyposis colorectal cancer (HNPCC), but also a prognostic and predictive marker for sporadic colorectal cancer. Our objective was to determine and study of five mononucleotide microsatellite markers status among Iranian patients with HNPCC and sporadic colorectal cancer. Material and Methods: In the current investigation 80 sporadic CRC and 80 HNPCC patients were evaluated for MSI. The pentaplex panel including 5 quasimonomorphic mononucleotide repeats (NR-21, BAT-26, BAT-25, NR-27 and NR-24) was used. Results: Our findings showed that the NR-21 was the most frequent instable marker among the other markers. 53% and 25.6% specimens had instability in sporadic CRC and HNPCC, respectively. Furthermore, the frequencies of instability BAT-25 was determined in 20% sporadic CRC and 23% HNPCC samples. Interestingly our results demonstrated that the frequency of instability NR-24 was similar 20% sporadic CRC and 20.5% HNPCC. Moreover, percentage of NR-27 in HNPCC was 19.2 and 0% in sporadic CRC. Finally, BAT-26 was instable in 21.8% HNPCC patients while we could find 6.6% instability for BAT-26 in sporadic cases. Conclusion: It seems that among 5 mononucleotides markers NR-21 was the most useful marker for diagnosis HNPCC and sporadic cancer. Following NR-21, BAT-25 and NR-24 are the most reliable markers. Therefore using a triplex panel including 3 aforementioned MSI markers should be more promising markers for identifying MSI status in both patients with HNPCC and/or sporadic colorectal cancer.     

Chk1 frameshift mutation in sporadic and hereditary non-polyposis colorectal cancers with microsatellite instability.

AIM: Protein kinase Chk1 (hChk1) is essential in human cells for cell cycle arrest in response to DNA damage, and has been shown to play an important role in the G2/M checkpoint. The BRAF mutations have been suggested to be linked with defective mismatch repair in colorectal cancers. The aim of this study was to investigate whether a frameshift mutation within the Chk1 gene contribute to the development or progression of eastern sporadic and hereditary non-polyposis colorectal cancer (HNPCC) with microsatellite instability (MSI). METHODS: We analyzed MSI using the 6 microsatellite markers and a frameshift mutation in the BRAF gene and in poly(A)9 within the Chk1 gene in 51 sporadic colorectal cancer and 14 HNPCC specimens. RESULTS: Eleven of the 51 sporadic colorectal cancers and all of the 14 HNPCCs were MSI-positive. Chk1 frameshift mutations were observed in 2 and 3 sporadic colon cancers and HNPCC, respectively, whereas no BRAF mutations were detected in these samples. Interestingly, all cases with the Chk1 frameshift mutation had high-frequency MSI. CONCLUSION: These results suggest that the Chk1 gene is a target of genomic instability in MSI-positive colorectal cancers and that the Chk1 framshift mutations might be involved in colorectal tumourigenesis through a defect in response to DNA damage in a subset of sporadic colorectal cancers and HNPCCs.     

PTEN gene mutations in colorectal cancers displaying microsatellite instability.

To determine the relationship between the mutation of the PTEN gene and genomic instability in human colorectal cancer, we screened the PTEN gene in 32 colorectal cancers (eight cell lines and 24 tissues) displaying microsatellite instability (MSI) and 32 colorectal cancers (six cell lines and 26 tissues) displaying microsatellite stability (MSS). Of 64 samples, six frameshift mutations were identified in six MSI colorectal cancers (two cell lines and four tumor tissues) but not in MSS colorectal cancers. Of the six MSI colorectal cancers containing PTEN mutation, two case (one cell line and one tumor tissue) showed biallelic mutation of PTEN. Furthermore, to compare the frequency of PTEN mutation with that of other MSI target genes, the mononucleotide repeat sequences of TGF-betaRII, BAX, IGFIIR, hMSH6 and hMSH3 were analyzed. The frequencies of frameshift mutation were revealed 78% in TGF-betaRII, 56% in BAX, 46.9% in hMSH3, 37.5% in hMSH6, 21.9% in IGFIIR, and 18.8% in PTEN. These results suggest that the PTEN gene is a target of genomic instability in MSI colorectal tumorigenesis.     

Histone Demethylase Gene PHF2 Is Mutated in Gastric and Colorectal Cancers

Alterations of genes involved in histone modification are common in cancers. A histone demethylase-encoding gene PHF2 is considered a putative tumor suppressor gene (TSG). PHF2 is essential for p53-mediated TSG functions such as chemotherapy-mediated cancer cell killing. However, inactivating mutations of PHF2 that could inactivate its functions are not reported in cancers. In a genome database, we observed that the PHF2 gene possessed mononucleotide repeats, which could be mutated in cancers with high microsatellite instability (MSI-H). For this, we analyzed 124 colorectal cancers (CRCs) and 79 gastric (GCs) cancers for the mutations and their intratumoral heterogeneity (ITH). Twenty-two of 79 CRCs (27.8 %) and 7 of 34 GCs (20.6 %) harboring MSI-H exhibited frameshift mutations. However, we found no such mutations in microsatellite stable/low MSI (MSS/MSI-L) cancers. Also, we studied ITH for the detected frameshift mutations in 16 cases of CRCs and detected ITH in two (12.5 %) cases. Our data reveal that TSG gene PHF2 harbors mutational ITH as well as the frameshift mutations in CRC and GC with MSI-H. Based on this, it is suggested that frameshift mutations of PHF2 may play a role in tumorigenesis through its TSG inactivation in CRC and GC.     

Frequent alteration of DNA damage signalling and repair pathways in human colorectal cancers with microsatellite instability

Accumulation of frameshift mutations at genes containing coding mononucleotide repeats is thought to be the major molecular mechanism by which mismatch repair-deficient cells accumulate functional alterations. These mutations resulting from microsatellite instability (MSI) can affect genes involved in pathways with a putative oncogenic role, but may also arise in genes without any expected role in MSI carcinogenesis because of the high mutation background of these tumours. We here screened 39 MSI colorectal tumours for the presence of mutations in 25 genes involved in DNA damage signalling and repair pathways. Using a maximum likelihood statistical method, these genes were divided into two different groups that differed significantly in their mutation frequencies, and likely represent mutations that do or do not provide selective pressure during MSI tumour progression. Interestingly, the so-called real-target mutational events were found to be distributed among genes involved in different functional pathways of the DNA metabolism, for example, DNA damage signalling (DNA-PKcs, ATR), double-strand break (DSB) repair (DNA-PKcs, RAD50), mismatch repair (MSH3, MSH6, MBD4) and replication (POLD3). In particular, mutations in MRE11 and/or RAD50 were observed in the vast majority of the tumours and resulted in the concomitant loss of immunohistochemical expression of both proteins. These data might explain why MSI colorectal cancers (CRC) behave differently in response to a wide variety of chemotherapeutic agents, notably those targeting DNA. More generally, they give further insights into how MSI leads to functional changes with synergistic effects in oncogenic pathways.     

Frameshift Mutations of <Emphasis Type="Italic">AKAP9</Emphasis> Gene in Gastric and Colorectal Cancers with High Microsatellite Instability

A-kinase-anchoring protein 9 (AKAP9) coordinates the cellular location and function of protein kinase A. AKAP9 plays an important role in centrosome duplication, cell cycle progression and maintenance of cell membrane integrity, alterations of which contribute to tumorigenesis. Somatic mutations of AKAP9 gene have been detected in many cancers including gastric (GC) and colorectal cancers (CRC), but the mutation status with respect to microsatellite instability (MSI) has not been reported. In a public database, we found that AKAP9 gene had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with MSI. 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. Overall, we found AKAP9 frameshift mutations in 4 (11.7 %) GCs and 20 (17.7 %) CRCs with MSI-H (24/113), but not in MSS/MSI-L cancers (0/90) (p < 0.001). In addition, we analyzed intratumoral heterogeneity (ITH) of AKAP9 frameshift mutations in 16 CRCs and found that five CRCs (31.3 %) harbored regional ITH of the AKAP9 frameshift mutations. Our data indicate that AKAP9 gene harbors not only somatic frameshift mutations but also mutational ITH, which together may be features of GC and CRC with MSI-H. Our results also suggest that regional mutation analysis is needed for a better evaluation of mutation status in these tumors to overcome ITH.     

Frameshift mutations and a length polymorphism in the hMSH3 gene and the spectrum of microsatellite instability in sporadic colon cancer.

Mutations in the hMVSH3 gene in sporadic colon cancer with microsatellite instability (MSI) were investigated, since several mismatch repair genes were known to be mutated in cancers with MSI, but only deletions in the (A)8 region in the hMSH3 gene have been reported. We also analyzed the relationships between hMSH3 mutations and the spectrum of MSI. We screened MSI in 79 sporadic colon cancer samples using mono- and dinucleotide repeat markers and the samples with MSI were further analyzed for tri- and tetranucleotide repeat instability and mutations in the hMSH3 gene by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Five (6%) out of 79 tumors were MSI-H and 15 (19%) were MSI-L. Two MSI-H tumors showed insertion in the (C)8 region in the hMSH6 gene and one tumor showed insertion and deletion in the (A)8 region in the hMSH3 gene, and two of the three above tumors showed MSI in tri-and tetranucleotide repeats. One MSI-L tumor showed somatic alteration in a 9-bp repeat sequence in hMSH3. No frameshift mutations were found in the (A)7 and (A)6 regions in hMSH3. Thus, we confirmed that the (A)8 region in hMSH3 is a hot spot and mutations in the (A)7 and (A)6 regions in hMSH3 are not common. The hMSH3 mutation may enhance genomic instability in some colorectal cancers.     

Microsatellite instability and MLH1 and MSH2 germline defects are related to clinicopathological features in sporadic colorectal cancer

Clinical and pathological features were evaluated to predict tumor microsatellite instability (MSI) and germline mutations in MLH1 and MSH2 DNA mismatch repair genes in two patient groups with sporadic colorectal cancer (CRC): 38 young patients (age /=60 years). Nine (25.7%) young patients out of 35 and five (16%) old patients out of 31 exhibited MSI in their cancers. MSI+ cancers were related to proximal cancer and mucinous carcinoma independently of the age at cancer onset. Three (7.9%) out of 38 young patients had mutations in MLH1 and MSH2 genes that led to truncated protein products; they were all at age <35 years and showed MSI in their tumors, with mucinous histotype in two cases. In conclusion, histopathological and clinical features of CRC allow identification of cancers showing DNA microsatellite instability. MSI in CRC at very early onset (age <35 years) appears useful to predict germline MMR gene defects.     

Variable mutation frequencies in coding repeats of TCF-4 and other target genes in colon, gastric and endometrial carcinoma showing microsatellite instability

Frameshift mutations in genes containing mononucleotide repeats are often observed in cancers exhibiting a high frequency of microsatellite instability (MSI-H). Several tumor types, including colorectal, gastric, and endometrial carcinomas, display this phenotype in a significant proportion of cases. We recently showed in a large series of MSI-H colorectal tumors that approximately 40% of them exhibited frameshift mutations in an (A)9 tract within the coding region of the TCF-4 gene, a crucial member of the APC/beta-catenin/TCF pathway. In the present study, we have examined MSI-H cancers from other primary tumor sites for mutations in this new target gene. Two of 22 (9%) MSI-H primary gastric cancers and none of 23 MSI-H endometrial primary tumors and cell lines were found to have a 1 bp deletion in the TCF-4 repeat. In the same series of tumors we also looked for frameshift mutations in other coding repeats localized within the TGF beta-RII, BAX, IGFIIR, hMSH3 and hMSH6 genes. Our results suggest that the TCF-4 gene, in a similar manner to some of these latter genes, is differentially altered in MSI-H tumors from different primary sites.     

Genes involved in DNA repair are mutational targets in endometrial cancers with microsatellite instability

Microsatellite instability (MSI) is observed in a subset of endometrial cancers (ECs) and is attributed to defects in mismatch repair. Mismatch repair deficiency allows for accumulation of mutations in the coding repeats of key target genes, which may be involved in the initiation and progression of MSI+ EC. We examined genes implicated in DNA repair pathways in 38 MSI-high (MSI-H), 10 MSI-low, 25 microsatellite stable ECs, and a selected panel of associated premalignant hyperplasias. Genetic alterations were correlated to histopathological data, including tumor grade and stage. Somatic frameshift mutations were observed in hMLH3, hMSH3, hMSH6, CHK1, and BAX genes in MSI-H endometrial hyperplasias and cancers, whereas mutations in ATR and CDC25C were observed only in MSI-H ECs. Increased mutation frequency in DNA damage response pathway genes including ATR, CHK1, and BAX demonstrated a significant trend with advancing tumor grade (P < 0.05). Our observations of the same mutations at short coding mononucleotide repeats in both premalignant lesions and tumors and association of increased frequency of mutation accumulation with advancing tumor grade suggest that these alterations may play a role in the development and progression of MSI+ EC.