Clinicopathologic significance of serum response factor expression in colorectal adenocarcinomas

Authors


Correspondence
Dr Seung Sam Paik, MD, Department of Pathology, Hanyang University College of Medicine, 17 Haengdang-dong, Seongdong-ku, Seoul 133-792, Korea. Email: sspaik@hanyang.ac.kr

ABSTRACT

Background and aim: The purpose of this study was to investigate the role of serum response factor (SRF) expression and to evaluate its correlation with various clinicopathological parameters in colorectal adenocarcinomas. Methods: We used tissue microarrays consisting of 24 normal mucosa, 50 tubular adenomas, 496 adenocarcinomas, and 128 metastatic lesions. Results: The expression of SRF was rare in normal colonic mucosa with a mean expression score of 0.67 ± 0.17. Tubular adenomas had a mean expression score of 2.48 ± 0.31, adenocarcinomas 2.82 ± 0.13 and lymph node metastases 2.82 ± 0.36. Interestingly, SRF expression was high in distant metastases with a mean expression score of 4.83 ± 0.43. The mean SRF expression was increased significantly early in the normal-adenoma-carcinoma sequence and again in distant metastases. The positive SRF expression was strongly correlated with non-mucinous tumor type (P < 0.001). Moderately and poorly differentiated adenocarcinomas had higher mean expression scores than well-differentiated adenocarcinomas (2.88 ± 0.13 vs 1.33 ± 0.32) (P= 0.015). There were significant associations between SRF expression and expression of p53 (P= 0.034) and Ki-67 (P= 0.001). Conclusions: Our results suggest that SRF expression may be the early event of normal-adenoma-carcinoma sequence of colorectal cancer, especially in adenomatous change of colonic mucosa and may play an important role in distant metastasis of colorectal cancers.

INTRODUCTION

Although the overall incidence and death rates for colorectal cancer (CRC) have slightly decreased, CRC still remains a serious cause of morbidity and mortality in the United States and throughout the world.1,2 There have been remarkable advances in understanding of the molecular basis of colorectal carcinogenesis and its cancer biology; however, treatment remains problematic.3 Except for the advanced stage, most patients with CRC undergo curative resection, and stage II patients with adverse prognostic factors, as well as stage III patients, receive adjuvant chemotherapy after curative surgical excision.4 Molecular indicators of prognosis would be of great help in the case of patients who are likely to benefit from adjuvant therapies.5

The transition from normality to malignancy through the adenoma-carcinoma sequence is accompanied by alterations in the expression of a number of genes associated with maintenance of cellular homeostasis.6 Among the known carcinogenesis-related molecular factors, serum response factor (SRF) has recently been highlighted, and studies of this factor have grown exponentially over the last two decades. SRF is a member of the MADS-box family of transcription factors. It is encoded by the SRF gene, which is located at chromosome 6p21.1, and several groups have reported a correlation between human cancers and elevated SRF expression.7 Since its initial discovery as a response to serum, SRF has been shown to be activated by several other stimulants, including specific oncogenes.8

Serum response factor regulates a number of genes involved in cellular activities such as cell growth, proliferation, differentiation, migration, angiogenesis, and apoptosis. In the gastrointestinal tract, SRF expression has been detected in mucosal epithelial cells and smooth muscular tissue such as muscularis mucosae and muscularis propria.9 Some workers have suggested that SRF plays positive roles in the development of various gastrointestinal cancers, emphasizing in particular its role in angiogenesis, Helicobacter pylori infection and metaplasia, and in promoting metastasis by disrupting cell adhesion.10–12 However, it is not known whether levels of SRF expression differ in the different steps of CRC development.

Our aim of this study was to investigate changes in SRF expression over the normal-adenoma-carcinoma-metastasis sequence and to evaluate the relation between SRF expression and clinicopathological parameters as well as survival. In addition, we evaluated the relations between SRF expression and p53 and Ki-67 expression.

METHODS

Patients and tissue samples

A consecutive series of 496 patients with colorectal adenocarcinomas was enrolled. All the patients were diagnosed and treated in Hanyang University Hospital (Seoul, Korea) between January 1991 and August 2001. There were 281 male and 215 female patients. The mean age was 58 years. The tumors consisted of 473 non-mucinous adenocarcinomas and 23 mucinous adenocarcinomas. They were located in the cecum (n= 17), ascending colon (n= 71), hepatic flexure (n= 10), transverse colon (n= 25), splenic flexure (n= 4), descending colon (n= 22), sigmoid colon (n= 105), and rectum (n= 242). The mean tumor size was 5.7 cm and the mean follow-up interval was 5.8 years. Of the patients, 171 (34.5%) died and 325 (65.5%) survived. We selected at random 24 cases of normal mucosae, 50 cases of tubular adenoma, 65 lymph node metastases, and 63 distant metastases to evaluate the role of SRF in multistep carcinogenesis.

Tissue microarray construction

Tissue microarray (TMA) were constructed from archived formalin-fixed, paraffin-embedded tissue blocks using a manual tissue arrayer (Quick-Ray Manual Tissue Microarrayer; Unitma Co. Ltd., Seoul, Korea). As described previously,13 for each sample an area rich in tumor cells was identified by light microscopic examination of hematoxylin and eosin-stained sections. Tissue cylinders with a diameter of 2 mm were punched from the previously marked tumor areas (donor blocks) and transferred to a recipient paraffin block. The arrays consisted of 5 × 10 samples.

Immunohistochemical staining

For immunohistochemical staining, multiple 4-μm sections were cut with a Leica microtome and transferred to adhesive-coated slides. These TMA slides were dewaxed by heating at 55°C for 30 min and washed three times for 5 min each with xylene. The tissues were rehydrated by a series of 5-min washes in 100, 90, and 70% ethanol, followed by phosphate-buffered saline (PBS). Antigen retrieval was performed by heating the samples in a microwave for 4 min 20 s at full power in 250 mL of 10 mM sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase for 20 min. The primary polyclonal rabbit anti-SRF antibody (G-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted 1:500 using goat serum. The primary mouse monoclonal p53 and Ki-67 antibodies (Novocastra Laboratories, Newcastle upon Tyne, UK) were also diluted 1:100 using goat serum, and then incubated at room temperature for 1 h. After three washes of 2 min each with PBS, the sections were incubated with biotinylated goat anti-mouse secondary antibody for 30 min (Dako, Carpinteria, CA, USA). After three more washes with PBS, horseradish peroxidase-streptavidin (Dako) was added to the sections for 30 min, followed by another three washes with PBS. The samples were developed with 3,3′-diaminobenzidine substrate (Vector Laboratories, Burlington, ON, Canada) for 1 min and counterstained with Mayer's hematoxylin. Then the slides were dehydrated by the standard procedure, and sealed with coverslips. Negative controls were performed by omitting the SRF, p53, and Ki-67 antibodies during the primary antibody incubation.

Interpretation of SRF, p53 and Ki-67 immunostainings

Serum response factor, p53, and Ki-67 expression was evaluated semi-quantitatively by two independent pathologists (Jang SM and Paik SS) without prior knowledge of the clinical follow-up data. SRF immunostaining was scored semi-quantitatively by multiplying the intensity of staining by the fraction of stained nuclei, as described previously.12,14 The intensity of staining was graded according to the following scale: 0, no staining; 1+, mild staining of nuclei; 2+, moderate staining of nuclei; 3+, strong staining of nuclei. The fraction of stained nuclei was evaluated using the following scale: 0, < 10% of the tumor cells positive; 1+, 10–30% of the tumor cells positive; 2+, 31–70% of the tumor cells positive; 3+, > 71% of the tumor cells positive. The maximum combined score was therefore 9, and the minimum was 0. Each sample was assigned to one of four expression groups according to its combined score as follows: 0, negative; 1–3, low; 4–6, moderate; 7–9, high. For statistical analysis, a cut-off value of score 1 was used, and each tissue section was classified as either negative (score 0) or positive (score 1 to 9). Expression of p53 and Ki-67 was evaluated according to the percentage of positively-stained cells as described previously.15 If the nuclei of > 10% of the neoplastic cells were stained, the sample was considered positive for p53 and Ki-67. In cases where the assessment of the two scorers differed, the slides were reinvestigated by both pathologists with a multi-head microscope and agreement was reached.

Statistical analysis

Statistical analysis was performed using SPSS (version 15.0; SPSS Inc., Chicago, IL, USA). The χ2 test for linear trend, Fisher's exact test, and one-way anova were used to examine the association between SRF expression and clinicopathological parameters. Univariate survival analysis with the log-rank test was used to compare the survival rates of the patient subgroups. Multivariate survival analysis with the Cox proportional hazards regression model was used to evaluate independent prognostic factors. The Kaplan–Meier method was used to calculate overall survival and disease-free survival curves. A difference of P < 0.05 between groups was considered significant.

RESULTS

The pattern of SRF expression

Representative photomicrographs of the SRF immunostaining are shown in Fig. 1. SRF expression was negative in 13 (54.2%) of 24 normal mucosa and low in 11 cases (45.8%) (mean, 0.67 ± 0.17). Expression was negative in eight (16%) of 50 tubular adenomas, low in 32 cases (64%), and moderate to high in 10 cases (20%) (mean, 2.48 ± 0.31). Of 496 adenocarcinomas, 144 (29%) were negative, 190 (38.3%) had low expression, and 162 (32.7%) had moderate to high expression (mean, 2.82 ± 0.13). Eighteen (27.7%) of 65 lymph node metastases were negative, 27 (41.5%) had low expression, and 20 (30.8%) had moderate to high expression (mean, 2.82 ± 0.36). However, 13 (20.6%) of 63 distant metastases were negative, 12 (19%) had low expression, and 38 (60.3%) had moderate to high expression (mean, 4.83 ± 0.43). There was a significant difference in the level of SRF expression between the normal mucosa and tubular adenomas (P < 0.001) and between the adenocarcinomas and distant metastases (P < 0.001) (Table 1, Fig. 2).

Figure 1.

Representative photomicrographs of serum response factor (SRF) immunostaining in colorectal adenocarcinomas. (a) Negative. (b) Low. (c) Moderate. (d) High. SRF is present in the nuclei of the tumor cells.

Table 1.  Serum response factor expression in normal mucosa, tubular adenomas, adenocarcinomas, lymph node metastases and distant metastases (n= 698)
Tissue samplesnExpression of SRF
Negative (n= 196)Low (n= 272)Moderate (n= 150)High (n= 80)P-valueMean ± SEMP-value
  1. LN, lymph node; SEM, standard error of the mean; SRF, serum response factor.

  2. χ2 test for linear trend, One-way anova.

Normal mucosae 24 13 (54.2) 11 (45.8)  0 (0.0) 0 (0.0) 0.67 ± 0.17 
Tubular adenoma 50  8 (16.0) 32 (64.0)  8 (16.0) 2 (4.0) 2.48 ± 0.31 
Adenocarcinoma496144 (29.0)190 (38.3)109 (22.0)53 (10.7)0.0042.82 ± 0.13< 0.001
LN metastasis 65 18 (27.7) 27 (41.5) 13 (20.0) 7 (10.8) 2.82 ± 0.36 
Distant metastasis 63 13 (20.6) 12 (19.0) 20 (31.7)18 (28.6) 4.83 ± 0.43 
Figure 2.

Mean serum response factor (SRF) expression scores in normal mucosa, tubular adenomas, adenocarcinomas, lymph node metastases, and distant metastases.

Correlation between SRF expression and clinicopathologic parameters in colorectal adenocarcinomas

We found that positive SRF expression was strongly correlated with non-mucinous tumor type (P < 0.001). Also non-mucinous tumors had higher mean SRF expression scores than mucinous tumors (2.94 ± 0.13 vs 0.26 ± 0.54) (P < 0.001). In addition, moderately and poorly differentiated adenocarcinomas had higher mean expression scores than well-differentiated ones (2.88 ± 0.13 vs 1.33 ± 0.32) (P= 0.015). There was no significant correlation between SRF expression and age, tumor location, tumor size, T category, N category, and American Joint Committee on Cancer (AJCC) stage (Table 2).

Table 2.  Correlation between expression of serum response factor (SRF) and clinicopathologic factors, p53 and Ki-67 in colorectal adenocarcinomas
FactorsnExpression of SRF
Negative (%) (n= 144)Positive (%) (n= 352)P-valueMean ± SEMP-value§
  1. AJCC, American Joint Committee on Cancer; SEM, standard error of the mean.

  2. Fisher's exact test, χ2 test for linear trend, §One-way anova.

Age (years)
 <58237 75 (31.6)162 (68.4)0.1302.84 ± 0.200.875
 ≥58259 69 (26.6)190 (73.4) 2.80 ± 0.17 
Gender
 Male281 71 (25.3)210 (74.7)0.0222.71 ± 0.160.327
 Female215 73 (34.0)142 (66.0) 2.96 ± 0.21 
Tumor location
 Colon254 82 (32.3)172 (67.7)0.0622.67 ± 0.180.235
 Rectum242 62 (25.6)180 (74.4) 2.98 ± 0.18 
Tumor size (cm)
 <5.7275 75 (27.3)200 (72.7)0.1942.86 ± 0.170.731
 ≥5.7221 69 (31.2)152 (68.8) 2.77 ± 0.20 
Tumor type
 Non-mucinous473126 (26.6)347 (73.4)< 0.0012.94 ± 0.13< 0.001 
 Mucinous 23 18 (78.3)  5 (21.7) 0.26 ± 0.54 
T category
 Tis, T1 17  7 (41.2) 10 (58.8)0.5192.24 ± 0.680.666
 T2 33  9 (27.3) 24 (72.7) 3.24 ± 0.52 
 T3434124 (28.6)310 (71.4) 2.82 ± 0.14 
 T4 12  4 (33.3)  8 (66.7) 2.43 ± 0.70 
N category
 N0217 57 (26.3)160 (73.7)0.1583.03 ± 0.200.339
 N1128 37 (28.9) 91 (71.1) 2.59 ± 0.24 
 N2151 50 (33.1)101 (66.9) 2.71 ± 0.23 
AJCC stage
 0, I 41 14 (34.1) 27 (65.9)0.4132.78 ± 0.460.314
 IIA, IIB174 42 (24.1)132 (75.9) 3.13 ± 0.23 
 IIIA, IIIB, IIIC262 81 (30.9)181 (69.1) 2.65 ± 0.17 
 IV 19  7 (36.8) 12 (63.2) 2.32 ± 0.56 
Differentiation
 Well 21  8 (38.1) 13 (61.9)0.2401.33 ± 0.320.015
 Moderate/Poor475136 (28.6)339 (71.4) 2.88 ± 0.13 
p53
 Negative243 76 (31.3)167 (68.7)0.0342.65 ± 0.180.045
 Positive184 42 (22.8)142 (77.2) 3.21 ± 0.22 
Ki-67
 Negative317106 (33.4)211 (66.6)0.0012.55 ± 0.150.002
 Positive176 35 (19.9)141 (80.1) 3.36 ± 0.23 

Correlation between SRF expression and expression of p53 and Ki-67 in colorectal adenocarcinomas

There was a positive correlation between p53 and Ki-67 expression and positive versus negative SRF expression (P= 0.034 and P= 0.001, respectively). p53 positive colorectal adenocarcinomas also had higher mean SRF expression scores than p53 negative ones (3.21 ± 0.22 vs 2.65 ± 0.18, P= 0.045), and Ki-67 positive colorectal adenocarcinomas had higher mean SRF expression scores than Ki-67 negative ones (3.36 ± 0.23 vs 2.55 ± 0.15, P= 0.002) (Table 2).

Correlation between SRF expression and overall survival and disease-free survival

We examined the impact of SRF expression on patient survival. However, positive versus negative SRF expression was not significantly correlated with overall survival (P= 0.291, log-rank test) or disease-free survival (P= 0.198, log-rank test) in univariate analysis. In multivariate survival analysis with the Cox proportional hazards model, SRF expression was also not an independent prognostic factor for overall survival or disease-free survival (P= 0.371 and P= 0.251, respectively) (Table 3). Similarly Kaplan–Meier survival curves showed no difference of patient survival as a function of SRF expression status (data not shown).

Table 3.  Effect of variables on overall and disease-free survival in colorectal adenocarcinomas (n= 496)
VariablesSignificance univariateSignificance multivariateHazard ratio95% CI
  1. AJCC, American Joint Committee on Cancer; CI, confidence interval; SRF, serum response factor

  2. Log-rank test; Cox proportional hazards model.

Overall survival
 SRF expression (negative vs positive) 0.291 0.3710.8610.620–1.195
 Patient age (< 58 years vs ≥ 58 years)<0.001<0.0011.9451.418–2.669
 Differentiation (low vs high)<0.001<0.0011.8541.333–2.578
 AJCC stage (0, I, II vs III, IV)<0.001<0.0012.6491.850–3.794
 Vascular invasion (absent vs present) 0.007 0.0202.6611.169–6.056
Disease-free survival
 SRF expression (negative vs positive) 0.198 0.2510.8450.634–1.127
 Patient age (< 58 years vs ≥ 58 years) 0.004 0.0031.5091.151–1.978
 Differentiation (low vs high)<0.001 0.0021.6161.199–2.177
 AJCC stage (0, I, II vs III, IV)<0.001<0.0012.9782.176–4.075
 Vascular invasion (absent vs present) 0.056 0.1021.9770.873–4.479

DISCUSSION

In the present study, we assessed SRF expression in TMAs consisting of 24 normal colon mucosa, 50 tubular adenomas, 496 adenocarcinomas, 65 lymph node metastases and 63 distant metastases by immunohistochemistry. Our data suggest that SRF expression may be the early event of adenoma-carcinoma sequence of colorectal cancer, especially in adenomatous change of colonic mucosa, and also plays an important role in distant metastasis. There were significant associations between expression of SRF and non-mucinous tumor type and poor differentiation, as well as a significant relation between expression of SRF and p53 and Ki-67 expression. However, there was no significant association between expression of SRF and the survival of patients with adenocarcinomas.

Recently, Choi et al.12 suggested that SRF overexpression is associated with modulation of E-cadherin/β-catenin expression. E-cadherin is a mediator of cell to cell adhesion implicated in tumor invasion and metastasis.16β-catenin is a submembranous cytosolic protein that connects E-cadherin to actin filaments,17 and altered expression of the E-cadherin/β-catenin complex is associated with de-differentiation, invasion, and metastasis of CRC.18 Choi et al.12 reported that the increased expression of SRF in metastatic CRC is associated with decreased expression of E-cadherin and increased nuclear translocation of β-catenin. In our study, expression of SRF was rare in normal colonic mucosa, with a mean expression score of 0.67 ± 0.17. Tubular adenomas had a mean expression score of 2.48 ± 0.31, adenocarcinomas 2.82 ± 0.13 and lymph node metastases 2.82 ± 0.36. Interestingly, the frequency of SRF expression was high in distant metastases, with a mean expression score of 4.83 ± 0.43. Our results thus suggest that SRF expression plays a role at the early event of adenoma-carcinoma sequence of colorectal cancer, especially in adenomatous change of colonic mucosa. Immunohistochemical detection of a high level of SRF in biopsies of colorectal cancers may be useful as an indicator of aggressive behavior, especially as a predictor of distant metastasis.

Patten et al.19 have documented the existence in human colon cancer cell lines of SRFΔ5, an alternatively spliced isoform of SRF lacking the exon-5-encoded sequence. Because it lacks the SRF transactivation domain, SRFΔ5 may act as dominant-negative inhibitor of full-length SRF.20 The same authors demonstrated higher expression of SRFΔ5 in colon cancer cell lines derived from poorly differentiated tumors than in cell lines derived from normal colonic mucosa or well-differentiated colonic adenocarcinomas. Park et al.21 have examined SRF expression in hepatocellular carcinomas. They observed that there was no SRF expression in hepatocytes in regions of normal or cirrhotic liver outside tumors, but that SRF expression gradually increased with grade of tumor differentiation from low to high, especially in sarcomatoid carcinomas. We determined the correlation between SRF expression and various clinicopathologic factors, but found no statistically significant association between SRF expression and patient age, tumor location, tumor size, T category, N category, and AJCC stage. Tumor type and differentiation were correlated with SRF expression. Non-mucinous tumors had a higher SRF expression rate and mean SRF expression score than mucinous tumors (P < 0.001), and moderately and poorly differentiated adenocarcinomas had a higher mean SRF expression score than well-differentiated ones (P < 0.015).

p53 has established itself as a key tumor suppressor, potent apoptosis-inducer, and prognostic marker in cancer. Translocation of p53 to mitochondria promotes apoptosis through transcription-independent mechanisms.22 We found a significant correlation between SRF expression and expression of p53 (P= 0.034). Lee et al.23 demonstrated suppression of transforming growth factor-β (TGF-β)-induced cell cycle arrest by SRF. TGF-β is a potent inhibitor of cell cycle progression in epithelial cells, and they suggested that SRF acts as a nuclear repressor of Smad3-mediated TGF-β1 signaling, and so activates expression of various genes involved in cell proliferation. Ki-67 protein is a nuclear and nucleolar protein that is tightly associated with somatic cell proliferation.24 We found that overexpression of SRF was linked to a high Ki-67 labeling index (P= 0.001).

Until now, the relation between SRF expression and clinical outcome has not been investigated in CRC patients. Park et al.15 demonstrated that overall cumulative survival of patients with cholangiocarcinomas was significantly worse in the SRF-positive group than in the negative group on univariate analysis; however, SRF expression was not a significant prognostic factor when they used multivariate analysis. We found that SRF expression was not significantly correlated with overall survival or disease-free survival in univariate analysis with the log-rank test or in multivariate analysis with the Cox proportional hazards model.

In conclusion, the pattern of SRF expression pointed to an important role of SRF in CRC development, especially early in the normal-adenoma-carcinoma sequence, and in distant metastasis. SRF may be a useful marker for predicting the risk of distant metastasis and a target molecule for anticancer therapy in colorectal cancer.

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