Clinicopathological significance of microsatellite instability and mutated RIZ in colorectal cancer

A. Emterling1, Å. Wallin1, G. Arbman2 and X.-F. Sun1,*

1 Department of Oncology, Linköping University, Linköping; 2 Department of Surgery, Vrinnevi Hospital, Norrköping, Sweden

Received 29 May 2003; revised 21 August 2003; accepted 4 September 2003


    ABSTRACT
 Top
 ABSTRACT
 Introduction
 Patients and methods
 Results
 Discussion
 REFERENCES
 
Background:

Several studies have shown that microsatellite instability (MSI) is related to favourable survival in colorectal cancer patients but there are controversial results. Tumour suppressor gene RIZ is a susceptible mutational target of MSI. However, its clinicopathological significance has not been investigated. We investigated the prognostic significance of MSI in Swedish colorectal cancer patients and the clinicopathological significance of RIZ mutations.

Patients and methods:

We analysed 438 colorectal adenocarcinomas for MSI by microsatellite analysis. Among them, 29 MSI and 28 microsatellite stable (MSS) tumours were examined for RIZ mutations by DNA sequencing.

Results:

MSI (13% of 438 cases) was not associated with survival (rate ratio = 0.97, 95% confidence interval = 0.57–1.64, P = 0.90), although it was related to proximal tumour (P <0.001), poor differentiation and mucinous carcinomas (P <0.001), multiple tumours (P = 0.01) and negative/weak expression of hMLH1 (P = 0.03). RIZ mutations were detected in 31% of 29 MSI tumours but in none of the 28 MSS tumours. The mutations were related to female (P = 0.01), proximal tumour (P = 0.01), stage B (P = 0.01) and poor differentiation (P = 0.047).

Conclusions:

MSI was not a prognostic factor in the Swedish patients included in this study. Clinicopathological variables associated with RIZ mutations might be a consequence of the MSI characteristics.

Key words: colorectal cancer, microsatellite instability, RIZ


    Introduction
 Top
 ABSTRACT
 Introduction
 Patients and methods
 Results
 Discussion
 REFERENCES
 
Microsatellite instability (MSI) is recognised as genome-wide alterations in repetitive DNA sequences and is caused by defects in mismatch repair (MMR) genes. The most common affected genes are hMLH1 and hMSH2. MSI is found in about 90% of hereditary non-polyposis cancer-associated tumours and in about 15% of sporadic colorectal cancer [13].

Colorectal cancer with MSI has a distinct phenotype, characterised by MMR deficiency, proximal location, poor differentiation and mucinous carcinoma, dense lymphocytic infiltration, increased number of tumours, older onset of the disease, preponderance amongst females and, paradoxically, by a favourable prognosis [1, 2]. However, some studies did not find a correlation between MSI and better survival [4, 5].

The tumour suppressor gene Retinoblastoma-interacting zincfinger (RIZ) harbours several microsatellites within its coding region [6], and is a candidate for a mutational target in MSI-mediated carcinogenesis. RIZ gene consists of at least 10 exons and is located at 1p36 [7, 8]. RIZ is a member of a small gene family defined by the PR domain, a region of approximately 100 amino acids [9]. The PR family is involved in cancers through an unusual ‘yin-yang’ fashion [10]. The two alternative protein products of RIZ are RIZ1, which contains a PR domain at the N-terminus, that is involved in tumour suppressor function, and RIZ2, which is lacking this domain, may have a positive role in oncogenesis [7]. RIZ1 is a 280 kDa protein and the majority is encoded by the large exon 8. The PR domain, which is encoded by three small exons 4–6, can interact with a PR-binding domain at the C-terminus of RIZ. Several observations have shown that RIZ1, but not RIZ2, is underexpressed in human cancers of the breast, liver, pancreas, stomach and colorectum [6, 9, 11]. RIZ1 may act as a transcription repressor, and an inducer of G2–M cell cycle arrest and/or apoptosis [11, 12]. The protein binds to GC-rich elements in the DNA. Many promoters of genes, especially those of growth-regulated genes, are characterised by GC-rich elements. This may indicate that RIZ1 is involved in the transcriptional regulation of a wide spectrum of genes.

Both hereditary and sporadic gastrointestinal cancers, as well as endometrial cancers that harbour MSI have shown a high incidence of somatic mutations within the coding area of RIZ [13, 14]. The mutations are located at two polyadenosine tracts; the (A)8 tract and the (A)9 tract, within the coding C-terminal region of RIZ, containing the PR-binding motif. These mutations generate truncated RIZ1/2 proteins that lack the C-terminal PR-binding domain and are expected to have serious deleterious effects on the PR domain-specific function of RIZ1.

In this study, the microsatellite status of 438 colorectal tumours was investigated. Of them, 29 MSI tumours and 28 microsatellite stable (MSS) tumours were screened for mutations in the (A)8 and (A)9 tracts of the RIZ gene. The aim was to identify relationships between MSI and clinicopathological variables, especially survival. Furthermore, we examined relationships between RIZ mutations and clinicopathological variables.


    Patients and methods
 Top
 ABSTRACT
 Introduction
 Patients and methods
 Results
 Discussion
 REFERENCES
 
Patients
For MSI analysis, frozen tumour was obtained from 438 colorectal adenocarcinoma patients diagnosed at the Department of Pathology at Vrinnevi Hospital between 1975 and 2000. Proximal tumours include tumours in the caecum, ascending and transverse colon, and distal tumours include ones in the descending colon, sigmoid colon and rectum. The classification of growth pattern was based on the patterns of growth and invasiveness. The data on hMLH1 were taken from previous studies carried out at our laboratory: 33 tumours showed strong expression and 59 were weak/negative. The patients with colon cancers in Dukes’ stage C/advanced tumour received chemotherapy, which had <20% influences on survival. Some patients with rectal cancers received adjuvant preoperative radiotherapy, which has a marginal influence on survival. The patients were followed up until the end of 2001; the mean and median follow-ups were 47 and 25 months. During the follow-up, 119 had died from colorectal cancer and the rest were considered as alive. There was no information available on age in seven cases, tumour location in 12, Dukes’ stages in 27, growth pattern in 197, differentiation in 17, numbers of tumours in 43 and hMLH1 in 346 cases.

For RIZ analysis, we randomly chose 29 MSI and 28 MSS cases from 438 patients. During the follow-up period, 15 had died from the cancer. There was no information available on stage in one case and differentiation in two cases.

Methods
Microsatellite analysis. The BAT-26 locus was amplified by a primary and a secondary PCR using primers: 5'-TGA CTA CTT TTG ACT TCA GCC-3' and 5'-AAC CAT TCA ACA TTT TTA ACC C-3' (Life Technologies). The primary PCR was carried out in a mixture containing 20 ng DNA, 1 x magnesium-free buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 2 µM of each primer, 0.5 units Taq polymerase (Promega) and water to a final volume of 19.5 µl. The PCR was carried out with initiating denaturation at 94°C for 4 min, 40 cycles of 94°C for 1 min, 52°C for 45 s and 72°C for 45 s, extension at 72°C for 10 min. A negative control was included in each run.

To incorporate [{alpha}-33P]dATP (Amersham Pharmacia Biotech, Bucks, UK) into the samples a secondary PCR was carried out. The secondary PCR was carried out under the same conditions as the primary PCR except that the cycles were reduced to 15. After the PCR the DNA products were denatured by adding 15 µl Blue Juice (containing formamide, xylene cyanol FF, bromophenol blue and EDTA) and incubated at 90°C for 5 min. The DNA products were separated on a denaturing 6% polyacrylamide gel containing 8 M urea by electrophoresis. The gel was dried and detection was carried out by autoradiography.

RIZ mutation analysis. Two DNA sequences including RIZ(A)8 and RIZ(A)9 tract in exon 8 of RIZ were amplified by PCR using the primers: RIZ(A)8: 5'-GGA CAG CCC AAA AGG CTT A-3' and 5'-TTC AAG TCG GCC TTC TGC-3', RIZ(A)9: 5'-GAA TAA ACA CGC CGC CTT CA-3' and 5'-GAT GAG TGT CCA CCT TTC TTA GAT GA-3'. The primary PCR of the RIZ(A)8 tract was carried out in a mixture containing 20 ng DNA, 1 x magnesium-free buffer, 3.0 mM MgCl2, 0.2 mM dNTP, 2 µM of each primer, 0.5 units Taq polymerase and water to a final volume of 19.5 µl. The PCR was carried out with initiating denaturation at 94°C for 4 min, 40 cycles of 94°C for 1 min, 54°C for 45 s and 72°C for 45 s, and extension at 72°C for 10 min. A negative control was included in each run. The primary PCR of the RIZ(A)9 tract was run under the same conditions as for RIZ(A)8 except for a lower primer concentration at 1 µM and an annealing temperature of 56.5°C. The PCR products were confirmed on a 2% agarose gel containing ethidium bromide.

The PCR products were purified from the primers using a GFXTM PCR DNA and Gel Band Purification Kit (Amersham). To incorporate [{gamma}-33P]ddNTP into the samples a secondary PCR was carried out using a Thermo Sequenase Radiolabelled Terminator Cycle Sequencing Kit (Amersham), with 30 cycles of amplification under the same conditions as in the primary PCR. After PCR the DNA products were denatured by adding 15 µl Blue Juice and incubated at 90°C for 5 min. The secondary PCR and sequencing process of the RIZ(A)9 were carried out under the same conditions as for RIZ(A)8 except for the use of RIZ(A)9 forward primer and an annealing temperature of 56.5°C. The DNA products were then separated on a denaturing 6% polyacrylamide gel containing 8 M urea by electrophoresis. The gel was dried and detection was carried out by autoradiography.

Statistical analysis
The chi-square or Fisher exact test was used to investigate the associations of microsatellite status or RIZ status with other variables. Cox’s proportional hazard model was used to test whether microsatellite status or RIZ mutation was related to survival. Survival curves were computed according to the Kaplan–Meier method.


    Results
 Top
 ABSTRACT
 Introduction
 Patients and methods
 Results
 Discussion
 REFERENCES
 
Microsatellite analysis
Among 438 patients, 59 cases (13%) showed MSI in at least one allele and 379 (87%) were MSS (Figure 1). Relationships between microsatellite status and clinicopathological variables are presented in Table 1. MSI was related to proximal tumours (P <0.001), poor differentiation and mucinous/signet-ring cell carcinoma (P <0.001), patients who had multiple tumours (P = 0.01) and negative/weak hMLH1 expression (P = 0.03). MSI showed a very slight trend towards an older onset of the disease (P = 0.14), and Dukes’ B and C (P = 0.16). The microsatellite status was not associated with patient survival (rate ratio = 0.97, 95% confidence interval = 0.57–1.64, P = 0.90, Figure 2).



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Figure 1. Two microsatellite instability cases with altered BAT-26 allelic size are indicated by the arrows; the other three cases are microsatellite stable.

 

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Table 1. Microsatellite instability (MSI) in relation to clinicopathological variables in colorectal cancer patients
 


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Figure 2. Microsatellite status in relation to survival in colorectal cancer patients. MSI, microsatellite instability; MSS, microsatellite stable.

 
RIZ mutation analysis
Among 29 MSI and 28 MSS cases analysed for RIZ mutation, nine of 29 MSI cases (31%) were mutated, of which one (11%) was in the (A)8 tract and eight (89%) in the (A)9 tract. All of the mutations were 1-bp deletions and heterozygous since the normal sequence was retained (Figure 3). We did not detect any mutation in 28 MSS cases (P = 0.001).



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Figure 3. DNA sequencing analysis of RIZ mutations. (A) Wild-type (A)8 tract, (B) wild-type (A)9 tract and (C) an adenine deletion in the (A)9 (indicated by the arrow), which results in double-band patterns downstream of the deletion due to the shifted allele. The deletion was heterozygous since the normal sequence was retained.

 
As shown in Table 2, RIZ mutations showed an overrepresentation in females (P = 0.01), proximal location (P = 0.01), stage B (P = 0.01) and poor differentiation (P = 0.047). The RIZ was not correlated with age, number of tumours or survival (P >0.05). There were too few cases with hMLH1 expression to perform a statistical analysis.


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Table 2. RIZ in relation to clinicopathological variables in colorectal cancer patients
 

    Discussion
 Top
 ABSTRACT
 Introduction
 Patients and methods
 Results
 Discussion
 REFERENCES
 
Several studies have found that MSI is related to improved survival in colorectal cancer patients [1, 2]. However, we did not find such a relationship in this large series of 438 patients with a long follow-up period. Some studies including a Swedish study in colorectal cancer patients showed a similar result where there was non-association between MSI and survival either in the whole group of patients [4, 5] or in patients with stage B [1518]. Another study showed a week association between MSI and better survival in the whole group of the patients with Dukes’ C colorectal cancer. The association was even stronger in the chemotherapy group but no association was seen in the non-chemotherapy group [19]. The reasons for the non-association between MSI and survival are unclear. The theory behind a better prognosis in patients with MSI is that MSI tumours usually have an increased lymphocytic infiltration that enhances the host immune response to the tumour [2]. The extent of lymphocytic infiltration in the tumours included in our and others’ studies where there was non-association between MSI and survival is unknown. Recently, a study [20] showed that colorectal cancers with MSI harboured mutations in the promoter of matrix metalloproteinase (MMP)-3. Consequently, the lower levels of MMP-3 decrease the activation of pro-MMP-9 that in turn may inhibit the development of an invasive tumour. The involvement of matrix MMPs in the progression and invasiveness of tumours has been proposed before and is here applicable as a new theory behind lesser aggressiveness in MSI colorectal tumours. It is clear, due to the uncertainty that exists about the clinical and biological significance of the MSI phenotype, that more extensive research has to be done to reveal the features of MSI in colorectal tumours, which may contribute to the understanding of tumorigenesis. It is necessary to identify patient characteristics such as adjuvant therapy and tumour features such as stage when we investigate a relationship between MSI and survival.

In the current study, we detected MSI in 13% of 438 patients using the marker BAT-26, and the rate was the same or similar to most previous studies in unselected or sporadic colorectal cancers [2, 3]. Observations have shown that the use of this marker alone may constitute a sensitive and specific tool for identifying tumours with MSI with a certainty of 86–99.4% [2123]. In agreement with previous studies [2, 3], we found the associations between MSI and proximal tumour, poor differentiation and mucinous/signet-ring cell carcinoma, multiple tumours and decreased expression of hMLH1. Our results indicated that the method of microsatellite analysis by using the marker BAT-26 in the present study was reliable.

Mononucleotide repeat sequences within coding regions in a number of different genes such as RIZ, TGF-ßRII, IGF-RII, BAX, MSH6, MSH, TCF-4, CHK1, MBD4, BLM, Caspase-5, PTEN, FAS, APAF-1, BCL-10, RAD50, CDX2, AXIN2 and WISP-3 have been demonstrated to be targets of somatic frameshift mutations with oncogenic potential in tumour cells with defective DNA repair [1, 3, 24]. Through loss of cellular growth regulation, escape from apoptosis and defective repair, somatic frameshift mutations in these genes are thought to provide a selective growth advantage during MSI tumour progression. In the present study, we found that 31% of MSI tumours had frameshift mutations of the RIZ gene in the (A)8 (one case) or (A)9 (eight cases) tract. Our results correlated well with previous observations where the (A)8 and (A)9 tract in the RIZ gene were mutated in 25–38% of colorectal MSI tumours [6, 13, 14]. Overall, the (A)9 tract seems to be the most affected one. The deletion of one adenine in the (A)8 tract produces a stop codon two residues from the deletion that predicts termination of translation that results in protein lacking the C-terminus, including one of the zinc fingers [6, 13]. The frameshift caused by the deletion of one adenine at the (A)9 tract, that is located 30 nucleotides past the same zinc finger, causes a fusion of truncated RIZ1 and RIZ2 lacking the C-terminus. Frameshift mutations in either tract are therefore predicted to lead to the loss of the C-terminal domain of the RIZ protein that is involved in PR binding. Inactivation of the normal function of RIZ would probably have serious effects on the capacity of the protein to induce G2–M cell cycle arrest, apoptosis or both, and tumour suppression [11, 13]. Furthermore, we, to the best of our knowledge, are the first to investigate the clinicopathological significance of RIZ mutations, and found that the RIZ mutated cases showed female overrepresentation, proximal tumour location, Dukes’ stage B and poor differentiation, but not related to patient survival.

In conclusion, in this large series of 438 patients with colorectal cancer patients, MSI was not associated with survival. Together with other groups [4, 5, 1519], the results conflict with a great number of previous studies in which MSI was related to a better prognosis. Further investigations have to be carried out to clarify the prognostic significance of MSI. This may answer the question whether there is any up to now unknown factor that may be involved in the prognostic significance of MSI. Furthermore, the result of the present study is additional evidence that RIZ is a mutational target in colorectal tumours with MSI. The clinicopathological features of the RIZ mutations may be the reflection of MSI tumours and did not seem to be characteristic for the RIZ mutations.


    FOOTNOTES
 
* Correspondence to: Professor X.-F. Sun, Department of Oncology, Linköping University, S-581 85 Linköping, Sweden. Tel: +46-13-222066; Fax: +46-13-222846; E-mail: xiasu{at}ibk.liu.se Back


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 Patients and methods
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 Discussion
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