Are there tumor suppressor genes on chromosome 4p in sporadic colorectal carcinoma?

Abstract

AIM: To study the candidate tumor suppressor genes (TSG) on chromosome 4p by detecting the high frequency of loss of heterozygosity (LOH) in sporadic colorectal carcinoma in Chinese patients.

METHODS: Seven fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. The same procedure was performed by the other six microsatellite markers spanning D4S3013 locus to make further detailed deletion mapping. Comparison between LOH frequency and clinicopathological factors was performed by χ² test.

RESULTS: Data were collected from all informative loci. The average LOH frequency on 4p was 24.25%, and 42.3% and 35.62% on D4S405 and D4S3013 locus, respectively. Adjacent markers of D4S3013 displayed a low LOH frequency (< 30%) by detailed deletion mapping. Significant opposite difference was observed between LOH frequency and tumor diameter on D4S412 and D4S1546 locus (0% vs 16.67%, P = 0.041; 54.55% vs 11.11%, P = 0.034, respectively). On D4S403 locus, LOH was significantly associated with tumor gross pattern (11.11%, 0, 33.33%, P = 0.030). No relationship was detected on other loci compared with clinicopathological features.

CONCLUSION: By deletion mapping, two obvious high frequency LOH regions spanning D4S3013 (4p15.2) and D4S405 (4p14) locus are detected. Candidate TSG, which is involved in carcinogenesis and progression of sporadic colorectal carcinoma on chromosome 4p, may be located between D4S3017 and D4S2933 (about 1.7 cm).

Key Words: Loss of heterozygosity; Colorectal carcinoma; Chromosome 4p; Tumor suppressor gene

INTRODUCTION

Colorectal cancer (CRC) constitutes the second most common neoplasm in Western countries and is the third leading cause of cancer-related death, the overall 5-year survival rate is approximately 45%[1]. Improvement in its prognosis can not be achieved without a better understanding of its etiology and tumor molecular biology. In recent years, the genetic basis of human tumors has been increasingly elucidated. As a model for both multistep and multipathway carcinogenesis, colorectal neoplastic progression provide paradigms of both oncogenes and tumor suppressor gene in epithelial tumors[23]. The latter changes predominate. In addition to the allelic loss on chromosome 5q, 17p and 18q, many other chromosome losses can be observed in colorectal carcinoma. Regions on chromosome 1q, 4p, 6p, 6q, 8p, 9p and 22q were lost in 25%-50% of the colorectal tumor cases studied previously[2].

Chromosome losses in colorectal tumor were first detected by cytogenesis, later, by probes of restriction fragment length polymorphisms (RFLP) and now by loss of heterozygosity (LOH) in analyzing allelic loss. The loss of tumor suppressor genes is believed to be one of the key steps to carcinogenesis of colorectal cancer[4]. The loss of one allelic at specific locus is caused by deletion mutation or loss of a chromosome from a chromosome pair[5]. When this occurs at a tumor suppressor gene locus where one of the allelics is already abnormal, it can result in neoplastic transformation. The LOH analysis based on polymorphic microsatellite DNA has become an effective and powerful tool currently to find informative loci and candidate tumor suppressor genes[67]. Most investigations concentrated on defining the minimal regions of loss of specific chromosomes in various cancers in an effort to identify the putative tumor suppressor genes targeted by the loss[8].

In this study, we first analyzed the LOH events on chromosome 4p using seven microsatellite markers and made further refined deletion mapping analysis spanning D4S3013 locus in 83 sporadic colorectal carcinoma cases in an attempt to identify additional candidate tumor suppressor genes involved in colorectal tumorigenesis.

MATERIALS AND METHODS

Patient sample and DNA extraction

This study was based on consecutively collected tumors in 83 patients with colorectal cancer, including 40 males and 43 females, treated at the surgical department in Shanghai First People’s Hospital, China. The patients’ ages ranged from 31 to 84 years with a median of 66. The cancerous tissue and adjacent normal control tissue (> 10 cm) were freshly frozen. The tissues were cut into cubes of approximately 2 mm³ and immediately frozen in liquid nitrogen. Each patient gave his or her informed consent for the use of his or her tissue in this study. DNA was extracted using standard methods with proteinase K digestion and phenol/chloroform purification[9]. All patients were confirmed by pathology and were staged by Duke’s criteria.

Microsatellite markers and PCR

Initially, 83 cases of colorectal cancer were analyzed by PCR using seven microsatellite markers (Shanghai Biology Technology Company, China) which map to chromosome 4p. DNA samples were analyzed as matched normal and tumor pairs using primers of the following microsatellite loci (hereditary location/heterozygote ): pter-D4S412 (4p16.3/76)-D4S2935 (4p16.1/62)-D4S1599 (4p16.1/81)-D4S303 (4p15.33/76)-D4S3013 (4p15.2/84)-D4S391 (4p15.2/85)-D4S405 (4p14/85). The average hereditary distance was 8.65 cm[10] (Figure 1A). As the D4S3013 locus showed high LOH frequency (35.62%), six additional microsatellite markers map to chromosome 4p15 were employed to further investigate LOH. The same DNA samples were then analyzed as matched pairs for the following microsatellite markers (location/heterozygote): pter-D4S2926 (4p15.32/80)-D4S1546 (4p 15.31 /77)

Figure 1 A: Microsatellite markers and candidate tumor suppressor genes on 4p (http://http://www.gdb.org); B: Microsatellite markers on D4S3013 (http://http://www.gdb.org).

-D4S3017 (4p 15.31/82)-D4S2933 (4p 15.31/60)-D4S2948 (4p 15.2 /81)-D4S1551 (4p 15.2/78). The average hereditary distance was restricted within 1.03 cm[10] (Figure 1B).

LOH result analysis

A portion of each PCR product (0.5 &mgr;L) was combined with 0.1 &mgr;L Genescan 500 size standard (PE Applied Biosystems Foster City, CA, USA) and 0.9 &mgr;L formamide loading buffer. After denaturation at 96°C for 5 min, products were eletrophoresed on 5% polyacrylamide gels using an ABI 377 DNA sequencer (PE Applied Biosystems Foster City, CA, USA) for 2.5 h. Genotype 3.7 software display individual gel lanes as electropherograms with a given size, height, and area for each detected fluorescent peak. Stringent criteria were used to score the samples. Alleles were defined as the two highest peaks within the expected size range. A ratio of T1:T2/N1:N2 of less than 0.67 or greater than 1.50 was scored as a loss of heterozygosity (Figure 2). Most amplified normal DNA produced two PCR products indicating heterozygosity. A single fragment amplified from normal DNA (homozygosity) and those PCR reactions, in which fragments were not clearly amplified, were scored as not informative. The LOH frequency of a locus is equal to the ratio of the number between allelic loss and informative cases. The average LOH frequency of chromosome 4p is the average value of each locus.

Figure 2 LOH Demonstration. A: Classic LOH peak: Allele ratio = (T1/T2)/(N1/N2) = (612/357)/(305/332) = 1.87 > 1.5; B: Heterozygosity retention: Allele ratio = (T1/T2)/(N1/N2) = (348/280)/(361/391) = 0.67 < 1.34 < 1.5. T: Tumor; N: Normal

Statistical analysis

Comparisons between LOH and clinicopathological data were performed by χ² test. P < 0.05 was considered as statistically significant.

RESULTS

LOH analysis of colorectal cancer on 4p

Eighty-three colorectal cancers were analyzed for LOH at the seven marker loci spanning chromosome 4p. All loci got informative messengers. The average LOH frequency on 4p was 24.25%. Sixty-three samples (75.90%) showed at least one LOH event. Two distinct regions of frequent allelic loss at D4S3013 (4p15.2) and D4S405 (4p14) locus on chromosome were detected (Table 1). The LOH frequency was 35.62% and 42.3%, respectively. This suggested that putative tumor suppressor genes may be located near D4S3013 and D4S405 loci.

Table 1 LOH result and hereditary distance/location of chromosome 4p.

Locus	Location	LOH cases	Normal cases	Informative rate	Distance (cm)	LOH rate(%)
D4S412	4p16.3	5	57	74.7	-	8.06
D4S2935	4p16.1	24	55	95.18	7.4	30.38
D4S1599	4p16.1	9	28	44.58	10.8	24.32
D4S403	4p15.33	3	34	44.58	5.1	8.1
D4S3013	4p15.2	26	47	86.9	7.7	35.62
D4S391	4p15.2	13	46	71.84	7.4	22.03
D4S405	4p14	22	30	62.65	13.4	42.3

LOH deletion mapping results on 4p15 encompassing D4S3013

The chromosome region spanning D4S3013 locus on 4p15 was investigated using a saturation mapping strategy with another 6 microsatellite markers that are closely located within this region (Figure 2). To detect putative tumor suppressor genes easily, we limited average hereditary distance to 1.03 cm. Forty samples (48.19%) showed at least one LOH event. The average LOH frequency spanning D4S3013 was 24.2% (Table 2). We found that adjacent markers of D4S3013 displayed a low LOH frequency (< 30%), especially on D4S2933 locus, much less information was obtained because of more homozygosity.

Table 2 LOH result and hereditary distance/location of detailed deletion mapping spanning D4S3013.

Locus	Location	LOH cases	Normal cases	Informative rate	Distance (cm)	LOH rate (%)
D4S2926	4p15.32	7	38	54.22	-	15.56
D4S1546	4p15.31	9	38	56.63	1.6	19.15
D4S3017	4p15.31	13	46	71.08	0.5	22.03
D4S3013	4p15.2	26	47	87.95	1.2	35.62
D4S2933	4p15.31	2	14	19.28	0.5	12.5
D4S2948	4p15.2	15	49	77.11	1.9	23.44
D4S1551	4p15.2	12	64	91.57	0.5	21.05

Relationship between clinicopathological features and LOH on 4p

On D4S412 locus, none LOH was detected in patients with tumor larger than 5 cm in diameter (0/27), while in patients with tumor less than 5 cm in diameter, LOH frequency was 14.29% (5/35, P = 0.041). On the contrary, on D4S1546 locus, LOH frequency was 35.29% (6/17) in the former; and only 10% (3/30) in the latter locus (P = 0.030). Notably, on D4S403 locus, LOH was significantly associated with tumor gross pattern. In tumor of the massive, ulcerative and encroaching pattern, the LOH frequency was 10%, 0%, 33.33%, respectively (P = 0.030). No significant relationship was found between clinicopathological features and LOH on other loci (data not shown).

DISCUSSION

Inactivation of tumor suppressor genes appears to be one of the genetic mechanisms involved in the development of colorectal cancer[1112]. Deletion of tumor suppressor genes occur frequently in human malignances. Such events can be detected using markers from the region of genome that include a tumor suppressor gene. Allelic deletions detected as LOH have been proved useful for mapping regions of DNA that contains tumor suppressor genes, i.e., LOH at specific chromosomal regions strongly suggests the existence of tumor suppressor genes at the relevant segment.

A great deal of evidence supported the presence of tumor suppressor genes in the short arm of chromosome 4. These include the reversion of the immortal phenotype by chromosome 4 transfer[13] and the frequent occurrence of losses in or near the 4p14-4p16 region in bladder cancer[14]. LOH has been observed at distal 4p in sporadic neuroblastoma with an incidence raging from 20% to 29%[1516]. Using array comparative genomic hybridization, Hurst et al[17] reported the loss frequency of 4p to be 52% in bladder cancer. More importantly, Shirapurkar et al[18] observed the loss frequency of > 50% at 4p15.1-4p15.3 in malignant mesothelioma and lung carcinoma. LOH on 4p was 21% and > 30% in differentiated adenocarcinoma of stomach as well[1920]. Head and neck squamous cell carcinoma, invasive cervical cancer and acinic cell carcinoma also showed a high allelic loss frequency[21–23].

In colorectal tumors, previous alletic typing[24], cytogenetic[25–27] and comparative genomic hybridization[28] studies have reported moderate losses(0%-30%) of chromosome 4. These data have not raised special interest in this chromosome as a candidate to harbor a tumor suppressor gene, therefore, colorectal cancer investigations have not included a detailed analysis of loss in this chromosome. Choi et al[29] reported a LOH frequency of 24%-30% at just several loci on chromosome 4 in colorectal cancer. Later, Arribas et al[3031], used AP-PCR method and suggested chromosome 4p14-4p16 may contain tumor suppressor gene, because LOH frequency on D4S2397 was as high as 35%. These reports indicate that 4p14-4p16 region displayed frequent loss in a couple of cancers, so 4p14-4p16 region is of important value for TSG screening.

D4S3013 locus region, 4p15.2, was concordant with several reports in other tumors before[14162021]. In this study, we investigated the LOH on 4p in 83 sporadic cases of colorectal cancer. The results showed putative tumor suppressor gene may harbor adjacent to D4S405 and D4S3013 locus. We made further detailed deletion mapping spanning D4S3013 locus, and found that the surrounding markers of D4S3013 displayed a low LOH frequency (< 30%). Therefore, we speculate that the candidate TSG may be located between D4S3017 and D4S2933, about 1.7 cm in hereditary distance.

We found several loci were significantly associated with clinicopathological features. On D4S412 locus, no LOH was detected in patients with tumor larger than 5 cm in diameter, while in patients with tumor less than 5 cm in diameter, the LOH frequency was 14.29% (P = 0.041). On the contrary, on D4S1546 locus, the LOH frequency showed opposite phenomenon. On D4S403 locus, LOH was significantly associated with tumor gross pattern. Similarly, Arribas et al[31] found solely at the D4S2397 locus was indicative of a shorter disease-free survival (P = 0.027). Choi et al[29] found 4p loss was significantly associated with early onset of colorectal cancer. The effect of 4p loss on the early-onset disease is unlikely to be the result of tumor aggressiveness, because 4p loss was not found to be correlated with cancer-related death. Nishizuka et al[20] found 4p LOH had an essentially similar frequency in early and advanced differentiated adenocarcinoma. The differential behavior of LOH at different markers suggested that distinct mechanisms and/or selection presses participate in the mutational event that affect this chromosomal region during the tumorigenic process.

Regarding allelic loss at 4p, cholecystokinin type A receptor (CCK-AR) gene maps near D4S2397[3233]

(Figure 1). Recent reports have suggested that cholecystokinin receptor may function as a tumor suppressor gene[3435].

In summary, we investigated LOH on 4p in sporadic colorectal carcinoma in Chinese patients and detected two high deletion regions encompassing D4S3013 (4p15.2) and D4S405 (4p14). Candidate TSG, involved in sporadic colorectal carcinoma on chromosome 4p, may be located between D4S3017 and D4S2933 (about 1.7 cm). Further related gene screening and functional studies may contribute to the identification of the tumor suppressor gene in these regions.

COMMENTS

Background

Cancer arises from the accumulation of inherited polymorphism (i.e. SNPs) and mutation and/or sporadic somatic polymorphism (i.e. non-germline polymorphism) in cell cycle, DNA repair, and growth signaling genes. Neoplastic progression is generally characterized by the accumulation of multiple somatic-cell genetic alterations as the tumor progresses to advanced stages. The classic mechanism of tumor suppressor gene inactivation is described by two-hit modes in which one allele is mutated (or promoter hypermethylation or a small intragenic deletion) and the other allele is lost through a number of possible mechanisms, resulting in loss of heterozygosity at multiple loci. Loss of heterozygosity is the most common molecular genetic alteration observed in human cancers. In the model of colorectal tumorigenesis, mutational inactivation of tumor suppressor genes predominates.

Research frontiers

Most genome-wide scans for loss of heterozygosity (LOH) have been conducted at low resolution with a relatively small number of polymorphic markers. For example, an average of 120 microsatellites have been used to determine the allelotype of multiple different human neoplasms in a series of studies since 1995, and the highest density microsatellite allelotype was about 280 polymorphic markers before the year 2000. SNPs are the most common form of sequence variation in human genome, occurring approximately every 1200 base pairs (bps). High density mapping of genetic losses reveals potential tumor suppressor loci and might be useful for clinical classification of individual tumors. SNP array has been introduced recently for genome-wide screening of chromosome imbalance. Higher density SNP array can effectively detect small regions of chromosomal changes and provide more information regarding the boundaries of loss regions.

Innovations and breakthroughs

A great deal of evidence supported the presence of tumor suppressor genes in the short arm of chromosome 4. Much less studies have been reported in colorectal cancer. Previous allelotyping analysis of cancer by many groups was used with a relatively low density of markers. By deletion dense markers mapping, we detected two obvious high frequency LOH regions spanning D4S3013 and D4S405 locus in colorectal cancer. Candidate TSG, might be located between D4S3017 and D4S2933 (about 1.7 cm).

Applications

We used this method to detect some major allelic loss regions in genome-wide scans of LOH in patients with colorectal cancer.

Terminology

LOH is caused by a variety of genetic mechanisms, including physical deletion of chromosome non-disjunction and mitotic non-disjunction followed by republication of the remaining chromosomes, mitotic recombination and gene conversion. The mechanisms of LOH are remarkably chromosome-specific. Some chromosomes display a complete loss. However, more than half of the losses are associated with a only partial loss of a chromosome rather than a whole chromosome. LOH is also a common form of allelic imbalance and the detection of LOH has been used to identify genomic regions that harbor tumor suppressor genes and to characterize different tumor types, pathological stages and progression.

Peer review

This is a report that describes the LOH events in sporadic colorectal cancer in Chinese patients, further studies must benefit from this paper. The data presented is clear and concise in the text.

Footnotes

Supported by The National Natural Science Foundation of China, No. 30080016 and No. 30470977

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