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Keywords:

  • zinc finger protein 148 (ZNF148);
  • colorectal neoplasms;
  • colon neoplasms;
  • rectal neoplasms;
  • prognosis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

BACKGROUND

It has been speculated that zinc finger protein 148 (ZNF148) is a tumor suppressor. However, to the authors' knowledge, little is known about the clinical significance of ZNF148 expression in patients with colorectal cancer (CRC). The objective of the current study was to clarify the association between ZNF148 expression and the postoperative prognosis of patients with CRC.

METHODS

Tissue microarrays containing 56 normal mucosa, 51 adenoma, 742 CRC (TNM stage I-IV), 16 familial adenomatous polyposis, and 21 metastatic CRC specimens were examined immunohistochemically for ZNF148 expression.

RESULTS

Expression of ZNF148 was found to increase consecutively from normal mucosa to stage I CRC, and then decreased consecutively from stage I to stage IV CRC. Lower expression of ZNF148 in tumors was found to be significantly associated with lymph node metastases, advanced TNM disease stage, poor differentiation, higher rate of disease recurrence, worse overall survival (OS), and shorter disease-free survival. High expression of ZNF148 was also associated with improved OS (P = .025) and disease-free survival (P = .042) in patients with stages II to III CRC. On multivariate Cox analysis, lower ZNF148 expression in tumors, advanced TNM stage, colon cancer, and elevated serum carbohydrate antigen 19-9 (CA19-9) were found to be significant factors for a worse OS. In 16 patients with familial adenomatous polyposis, ZNF148 expression was upregulated at steps toward carcinogenesis. In 21 patients with metastatic CRC, although ZNF148 expression was higher in primary tumors compared with adjacent mucosa, its expression in metastatic tumors was significantly lower than that in primary tumors.

CONCLUSIONS

Although ZNF148 expression is related to colorectal carcinogenesis, high ZNF148 expression in patients with CRC appears to be inversely associated with malignant phenotypes and may serve as a significant prognostic factor after surgery for patients with CRC. Cancer 2013;119:2212–2222. © 2013 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Colorectal cancer (CRC) is the third most common cancer in the world and the second most common cause of cancer-related death.[1] Surgery combined with adjuvant chemoradiotherapy is now the standard treatment of patients with CRC. It is crucial to select high-risk patients for more frequent follow-up and intensive adjuvant chemoradiation.

Although some of the clinicopathological factors such as lymph node metastasis, TNM stage, and differentiation could be used for that purpose, the prognostic power still needs to be improved.[2] The assessment of molecular prognostic factors from initial surgical specimens will be helpful in identifying those patients who are more likely to benefit from adjuvant therapies, thereby leading to improved prognosis.

Recent studies have suggested that the expression level of zinc finger protein 148 (ZNF148), a Kruppel-type zinc finger transcription factor also known as ZBP89 or ZFP148, may play a significant role in the regulation of cell growth, apoptosis, and carcinogenesis.[3-7] ZNF148 is universally expressed at low levels in most tissues,[8] including those in the gastrointestinal tract.[9] ZNF148 is a multifunctional transcription factor associated with signaling pathways upstream of cellular proliferation, embryogenesis, differentiation, growth arrest, and apoptosis.[7, 10] It has been shown that ZNF148 binds to GC-rich DNA elements in a variety of promoters involved in growth regulation.[9] Physiologically, ZNF148 protein potentiates the induction of p21waf1 and leads to growth arrest in cultured colon cancer cells.[11] Elevated expression of ZNF148 inhibits cell proliferation and promotes growth arrest through stabilization of the p53 protein in the nucleus.[7] When overexpressed in vitro, ZNF148 represses growth-promoting genes such as gastrin and ornithine decarboxlyase.[7] The proapoptotic and antiproliferative functions of ZNF148 and its association with 2 known tumor suppressors, p537 and p300,[11] raised the possibility that it might be a tumor suppressor.[12]

It is interesting to note that when upregulated in gastrointestinal cancer cell lines, ZNF148 inhibits cell proliferation and induces apoptosis.[12] The study by Law et al demonstrated that, in the Apc Min/+ mouse, intestinal ZNF148 overexpression would activate intestinal apoptosis, abrogate adenoma development, and extend overall survival (OS).[12] They also suggested that ZNF148 is a therapeutic target to inhibit colon cancer development.[12] In a clinical study focusing on patients with CRC, the messenger RNA (mRNA) expression level of ZNF148 in patients with stage II CRC was found to be significantly higher than that in patients with stage III disease.[13] Moreover, overexpression of ZNF148 mRNA is associated with a lower incidence of disease recurrence.[13] Butyrate, which is produced in the large bowel through bacterial fermentation of undigested carbohydrates and fiber polysaccharides, plays an important role in the maintenance of colonic homeostasis and the suppression of intestinal inflammation. Butyrate can also induce cell growth arrest and stimulate apoptosis and differentiation in colon cancer cell lines, and represses the growth of carcinogen-induced colonic tumors.[14] Butyrate exhibits its antitumor effect through the inducible activation of p21waf1, which can be enhanced by ZNF148.[14, 15] These findings suggest that ZNF148 might act as a tumor suppressor in CRC.

However, to the best of our knowledge, no study published to date has systematically evaluated the expression of ZNF148 in patients with CRC. It is still unknown whether ZNF148 expression is a prognostic factor for local disease recurrence or survival in patients with CRC. To clarify the role of ZNF148 in the development and progression of CRC and the prognostic role of ZNF148 in patients with the disease, we immunohistochemically determined the expression of ZNF148 in a series of patients with different developing stages of CRC (normal mucosa, adenoma, and TNM stage I-IV CRC). In addition, to minimize the interference of hereditary heterogeneity, specimens from 16 patients with familial adenomatous polyposis (FAP) and specimens from 21 patients with metastatic CRC were also evaluated.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Patients

The study population comprised 5 groups. Group A was 56 patients with normal rectal mucosa. Samples were obtained from patients with severe mixed hemorrhoids who underwent the Procedure for Prolapse and Hemorrhoids. All patients had morphologically normal colorectal mucosa that was free of neoplastic or inflammatory diseases as confirmed by preoperative colonoscopy. Group B included 51 patients with colorectal adenomatous polyps. All of these polyps were resected endoscopically and were proved to be adenomas by postoperative pathological examination. Group C was composed of 742 patients with sporadic CRC, including 53 patients with stage I disease, 312 with stage II disease, 322 with stage III disease, and 55 with stage IV disease. Each patient had an available specimen of a resected primary CRC. All patients with CRC were classified according to the TNM staging system using the International Union Against Cancer criteria. Patients who were diagnosed with cancers of any other histotypes and those with a family history of CRC were excluded from the study. We enrolled 344 patients with colon cancer and 398 patients with rectal cancer with a median age of 60 years (range, 20 years-87 years), 423 of whom were male. These patients did not receive preoperative chemotherapy or radiotherapy. However, patients with stage III and stage IV disease received 5-fluorouracil–based systemic chemotherapy after surgery. Postoperative adjuvant radiation was also administered to those patients with stage III and stage IV rectal cancer. Patients were followed until death or December 31, 2011, with a mean postoperative follow-up duration of 56 months. Disease-free survival (DFS) was defined as the duration from the date of surgery to the date of first confirmed disease recurrence or to the date of last follow-up for those without disease recurrence. Group D was composed of 16 patients with FAP and concomitant CRC. Each patient had a set of 3 matched colorectal specimens (adjacent pathologically normal mucosa, adenoma, and carcinoma). Group E was 21 patients with metastatic CRC whose metastatic carcinoma was resected concurrently. Each patient has a set of 3 matched specimens (adjacent pathologically normal mucosa, primary carcinoma, and metastatic carcinoma). Of the 21 metastatic carcinomas, 19 had metastasized to the liver and 2 had metastasized to the greater omentum. The 5 groups were demographically comparable to each other (P > .05). All patients underwent surgery in the Department of Colorectal Surgery at Changhai Hospital, Second Military Medical University in Shanghai, China between December 1999 and December 2009. All patients provided informed consent. This study was approved by the Committee on Ethics of Biomedical Research, Changhai Hospital, Second Military Medical University in Shanghai, China. The ethical approval number is CHEC2011-146.

Tissue Microarray

All CRC cases were histologically reviewed by hematoxylin and eosin staining and representative areas were premarked in paraffin blocks, away from necrotic and hemorrhagic materials. Cylinders measuring 1.5 mm in diameter were then taken from the paraffin blocks. Finally, 6 different tissue microarray (TMA) blocks were constructed, containing a total of 960 cores (56 normal mucosa cores, 51 adenoma cores, 742 CRC cores, 16 × 3 FAP cores, and 21 × 3 metastatic CRC cores). Sections of 4-μm thickness were placed on 3 aminopropyltriethoxysilane-coated slides.

Immunohistochemistry

Immunohistochemistry was performed using a diaminobenzidine-based staining technique (Dako ChemMate Envision Kit; Dako Denmark A/S, Glostrup, Denmark). After deparaffinization and rehydration, antigen retrieval was performed with Tris-ethylenediamine tetraacetic acid buffer (0.01 mol/L [pH 9.0]) using a pressure cooker. The tissue sections were preincubated with 0.3% hydrogen peroxide and 20% normal goat serum to block nonspecific reactions. A monoclonal antibody to ZNF148 (ab69933, dilution 1:100; abcam Inc, Cambridge, Mass) was incubated on the slides for 2 hours in a humidified chamber. A horseradish peroxidase-conjugated secondary antibody was applied neat for 30 minutes. The sections were stained with diaminobenzidine and counterstained with Mayer hematoxylin, washed again, dehydrated in alcohol, cleared in xylene, mounted with Pertex mounting medium (CellPath Ltd, Newtown, Powys, UK), and coverslipped.

All sections were scored blind by 2 investigators (X.H.G. and Q.Z.L.) under a light microscope; in the case of a discrepancy, consensus was always achieved after discussion. The entire tissue section was scanned to assign the score. ZNF148 labeling indices were defined as the percentage of positive nuclei in relation to the entire carcinoma area or entire section for the normal samples. ZNF148 expression was estimated using the criterion given on the ATLAS Web site,[16] based on the percentage and intensity of the stained tumor cells. The staining percentage was graded as 0 (0%-4%), 1 (5%-24%), 2 (25%-49%), 3 (50%-74%), and 4 (≥ 75%) and staining intensity was graded as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The multiplication of the intensity and extent score was used as the final staining score (0-12). Tumors with final staining scores of 0, 1 to 4, 5 to 8, and 9 to 12 were considered to be negative (-), slightly positive (+), moderately positive (++), and strongly positive (+++), respectively.

Statistical Analysis

Associations between ZNF148 expression and clinicopathological variables were analyzed by nonparametric analysis, using the Mann-Whitney U test for dichotomization variables and the Kruskal-Wallis test for the others. The median OS and DFS and their 95% confidence intervals (95% CIs) were estimated by the Kaplan-Meier method. Difference in survivals was analyzed using the log-rank test. The Cox proportional hazards model was used for multivariate analysis of prognostic factors. A P value < .05 (2-sided) was considered to be statistically significant. All statistical analyses were conducted using SPSS 17.0 statistical software (SPSS Inc, Chicago, Ill).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

A flow chart of the enrollment of patients with CRC is shown in Figure 1. Conventional clinicopathologic variables including patient age, patient sex, tumor location, depth of invasion, lymph node metastasis, distant metastasis, TNM stage, differentiation, serum carcinoembryonic antigen (CEA) level, serum carbohydrate antigen 19-9 (CA19-9), postoperative chemoradiotherapy, disease recurrence, and survival status were recorded and are detailed in Table 1.

image

Figure 1. A flow chart of patient selection is shown. The 742 patients with colorectal cancer were selected from 4625 consecutive patients with colorectal cancer, as specified in this flow chart, without regard to clinical or outcome parameters.

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Table 1. Relation Between ZNF148 Expression and Clinicopathological Parameters in 742 Patients With Colorectal Cancer
CharacteristicZNF148 ImmunostainingP
++++++
  1. Abbreviations: −, negative; +, slightly positive; ++, moderately positive; +++, strongly positive; CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; ZNF148, zinc finger protein 148.

  2. a

    Determined using the Mann-Whitney U test.

  3. b

    Determined using the Kruskal-Wallis test.

Gender    .292a
Male235478754 
Female188424940 
Age, y    .703a
60262608752 
≥60161294942 
Tumor location    .001a
Colon cancer217474238 
Rectal cancer206429456 
Invasion depth    .867a
T1-T25172011 
T3-T43728211683 
Lymph node metastasis    .030b
N0213345878 
N114036609 
N27019187 
Distant metastasis    .144a
M03868213386 
M137738 
TNM stage    .026b
I266129 
II174264666 
III186507511 
IV37738 
Tumor differentiation    < .001a
Well, moderate3538013187 
Poor, mucinous70957 
Serum CEA    .085a
5 ng/mL250619160 
≥5 ng/mL172284534 
Serum CA19-9    .103a
37 U/mL3527412385 
≥37 U/mL80151617 
Recurrence    .003a
No2845610181 
Yes95221811 

ZNF148 Expression in Normal Mucosa, Adenoma, and Stage I to IV CRC

ZNF148 was detected mainly in the nucleus of the epithelial cells. The concordance of staining scoring was 94% between the 2 investigators. The expression of ZNF148 in different stages of CRC is presented in Table 2. ZNF148 expression was positive in 37.5%, 47.1%, 50.9%, 44.2%, 42.2%, and 32.7% of normal mucosa, adenoma, stage I CRC, stage II CRC, stage III CRC, and stage IV CRC, respectively. It is interesting to note that the positive expression rate of ZNF148 increased persistently from normal mucosa to adenoma and to stage I CRC, and then decreased persistently from stage I to stage IV CRC. Representative photomicrographs of the staining patterns in different stages of CRC are shown in Figure 2. There were significant differences noted with regard to ZNF148 expression among these 6 groups (P = .013, Kruskal-Wallis test).

Table 2. Expression of ZNF148 in Normal Mucosa, Adenoma, and Stage I to IV Colorectal Cancer
CharacteristicZNF148 ImmunostainingTotalPositive RateMean RankaPb
++++++
  1. a

    The Kruskal-Wallis test was used to determine statistical significance among the 6 groups (P=.013).

  2. b

    The Mann-Whitney U test was used to determine statistical significance between each of the 2 groups.

  3. c

    Compared with the “normal mucosa” group.

  4. d

    Compared with the “adenoma” group.

  5. e

    Compared with the “Stage I” group.

  6. f

    Compared with the “Stage II” group.

  7. g

    Compared with the “Stage III” group.

  8. Abbreviations: −, negative; +, slightly positive; ++, moderately positive; +++, strongly positive; ZNF148, zinc finger protein 148.

Normal mucosa3519205637.5%360.86
Adenoma2715905147.1%420.98.066c
Stage I2661295350.9%471.22.007d
Stage II17426466631244.2%450.77.010e
Stage III18650751132242.2%410.84.072f
Stage IV377385532.7%386.22.823g
image

Figure 2. Representative immunostains of zinc finger protein 148 (ZNF148) expression are shown in different developing stages of colorectal cancer (&× 200). ZNF148 expression was noted mainly in the nucleus of tumor cells. Positive cells were stained brown. (A) The absence of ZNF148 staining in normal mucosa was scored as “−.” (B) Low-intensity ZNF148 expression in adenoma was scored as “+.” (C) High-intensity ZNF148 expression in a stage I tumor was scored as “+++.” (D) Moderate-intensity ZNF148 expression in a stage II tumor was scored as “++.” (E) Moderate-intensity ZNF148 expression in a stage III tumor was scored as “+.” (F) Low-intensity ZNF148 expression in a stage IV tumor was scored as “+”

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Correlation Between ZNF148 Expression and Clinicopathologic Parameters of Patients With CRC

The association between ZNF148 expression and the clinicopathologic parameters is shown in Table 1. Lower expression of ZNF148 was found to be significantly associated with lymph node metastases (P = .030), higher TNM stage of disease (P = .026), poorer differentiation (P < .001), and a higher rate of disease recurrence (P = .003). Spearman correlation coefficients between ZNF148 expression and lymph node metastasis, TNM stage of disease, differentiation, and disease recurrence were all found to be statistically significant (P < .05 for each comparison). In addition, ZNF148 expression in rectal cancer was significantly higher than that in colon cancer (P = .001). No significant association was observed between ZNF148 expression and patient sex, patient age, invasion depth, distant metastasis, serum CEA level, or serum CA19-9 level.

Correlation Between ZNF148 Expression and Survival in Patients With CRC

The OS of those patients with high ZNF148 expression ([++] to [+++]) was significantly higher than that of patients with low ZNF148 expression ([−] to [+]) (P = .001) (Fig. 3A). The DFS of the patients with high ZNF148 expression ([++] to [+++]) was significantly higher than that of patients with low ZNF148 expression ([−] to [+]) (P = .003) (Fig. 3B). In stage II and stage III CRC cases combined, high ZNF148 expression was also found to be significantly associated with improved OS and DFS (OS: P = .025 [Fig. 3C]; DFS: P = .042 [Fig. 3D]).

image

Figure 3. Kaplan-Meier analysis of (A) overall survival (P = .001) and (B) disease-free survival (P = .003) for zinc finger protein 148 (ZNF148) expression in patients with stage I to stage IV colorectal cancer (n = 742) is shown. Kaplan-Meier analysis of (C) overall survival (P = .025) and (D) disease-free survival (P = .042) for ZNF148 expression in patients with stage II and III colorectal cancer (n = 634) is shown. ++ indicates moderately positive; +++, strongly positive; −, negative; +, slightly positive.

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Table 3 presents the factors found to be significantly associated with survival on univariate analysis. Advanced TNM stage (P < .001), colon cancer (P = .003), elevated serum CEA level (P < .001), elevated serum CA19-9 level (P < .001), lower ZNF148 expression (P = .001), and postoperative receipt of chemoradiotherapy (P = .005) were found to be significantly associated with a worse OS. Advanced TNM stage (P < .001), elevated serum CEA level (P = .002), elevated serum CA19-9 level (P < .001), lower ZNF148 expression (P = .004), and postoperative receipt of chemoradiotherapy (P < .001) were found to be significantly associated wit DFS. Multivariate analyses identified advanced TNM stage (P < .001), colon cancer (P = .011), elevated serum CA19-9 level (P = .025), and lower ZNF148 expression (P = .047) as being prognostic of a shorter OS, whereas advanced TNM stage (P < .001) and elevated serum CA19-9 level (P = .016) were found to be significantly predictive of a shorter DFS (Table 4). Compared with patients with negative ZNF148 expression (−), patients with moderately positive (++) (P < .001) and strongly positive (+++) (P < .001) ZNF148 expression were found to have prolonged OS. These results indicated that, in addition to TNM stage, tumor location, differentiation, an elevated CEA level, and an elevated CA19-9 level, ZNF148 expression status was a significant prognostic factor for OS and DFS.

Table 3. Univariate Analyses of Factors Associated With Survival and Disease Recurrence (n=742)
 OSDFS
VariablesHR (95% CI)PHR (95% CI)P
  1. Abbreviations: ++, moderately positive; -, negative; +++, strongly positive; 95% CI, 95% confidence interval; CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; DFS, disease-free survival; HR, hazards ratio; OS, overall survival; ZNF148, zinc finger protein 148.

Sex (male vs female)0.842 (0.577-1.231).3760.879 (0.650-1.189).404
Age (>60 y vs ≤60 y)1.240 (0.854-1.801).2590.919 (0.676-1.249).590
TNM stage (IV/III vs II/I)3.108 (2.057-4.696)<.0012.493 (1.818-3.419)<.001
Differentiation (poor/mucinous vs well/moderate)1.539 (0.929-2.548).0941.293 (0.847-1.975).234
Location (rectal vs colon)0.563 (0.384-0.825).0030.891 (0.662-1.200).449
CEA (>5 ng/mL vs ≤5 ng/mL)1.967 (1.357-2.851)<.0011.609 (1.195-2.166).002
CA19-9 (>37 U/mL vs ≤37 U/mL)2.268 (1.504-3.421)<.0011.898 (1.340-2.689)<.001
ZNF148 [(++)/(+++) vs (-)/(++)]0.454 (0.280-0.736).0010.589 (0.411-0.844).004
Postoperative radiochemotherapy (yes vs no)2.418 (1.298-4.506).0052.820 (1.686-4.717)<.001
 
Table 4. Multivariate Analyses of Factors Associated With Survival and Disease Recurrence (n=742)
VariablesOSDFS
HR (95% CI)PHR (95% CI)P
  1. Abbreviations: ++, moderately positive; +++, strongly positive; -, negative; 95% CI, 95% confidence interval; CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; DFS, disease-free survival; HR, hazards ratio; OS, overall survival; ZNF148, zinc finger protein 148.

TNM stage (IV/III vs II/I)2.822 (1.854-4.296)<.0012.239 (1.624-3.087)<.001
Differentiation (poor/mucinous vs well/moderate)1.344 (0.806-2.240).2571.141 (0.743-1.752).547
Location (rectal vs colon)0.602 (0.407-0.889).0110.887 (0.647-1.216).457
CEA (>5 ng/mL vs ≤5 ng/mL)1.456 (0.971-2.184).0691.284 (0.931-1.772).128
CA19-9 (>37 U/mL vs ≤37 U/mL)1.668 (1.068-2.604).0251.583 (1.090-2.299).016
ZNF148 [(++)/(+++) vs (-)/(++)]0.606 (0.369-0.994).0470.696 (0.483-1.002).051
Postoperative radiochemotherapy (yes vs no)1.014 (0.488-2.110).9701.220 (0.959-1.551).105

ZNF148 Expression in 16 Patients With FAP

As shown in Figure 4, the immunostaining score of ZNF148 in adenomas was significantly higher than that in normal mucosa. In adenocarcinoma specimens, ZNF148 expression was higher than in adenomas. Figure 4E shows that ZNF148 expression in adenocarcinomas was higher than that in adenoma or in normal mucosa specimens (adenocarcinomas vs adenomas: P = .001; adenomas vs normal mucosa: P = .021), suggesting that, in patients with FAP, ZNF148 expression is being upregulated at steps toward carcinogenesis.

image

Figure 4. Zinc finger protein 148 (ZNF148) expression is shown in pathologically normal mucosa, adenoma, and adenocarcinoma specimens from 16 patients with familial adenomatous polyposis. (A) Expression of ZNF148 in normal mucosa is shown (× 200). (B) Expression of ZNF148 in adenoma is shown (× 200). (C) Expression of ZNF148 in adenocarcinoma is shown (× 200). (D) Labeling indices of the 3 specimens from the 16 patients with familial adenomatous polyposis are shown. (E) The mean labeling indices of normal mucosa, adenoma, and adenocarcinoma were compared (*using the Wilcoxon test for paired nonparametric data).

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ZNF148 Expression in 21 Patients With Metastatic CRC

As shown in Figure 5, the immunostaining score of ZNF148 in primary tumors was significantly higher than that in normal mucosa (P = .001). However, the expression level of ZNF148 in metastatic tumors was found to be significantly lower than that in primary tumors (P = .038).

image

Figure 5. Zinc finger protein 148 (ZNF148) expression is shown in pathologically normal mucosa, primary tumor, and metastatic tumor specimens from 21 patients with colorectal cancer. (A) Expression of ZNF148 in normal mucosa is shown (× 200). (B) Expression of ZNF148 in a primary tumor is shown (× 200). (C) Expression of ZNF148 in a metastatic tumor is shown (× 200). (D) Labeling indices of the 3 specimens from the 21 patients are shown. (E) Comparison of the mean labeling indices of normal mucosa, primary tumor, and metastatic tumor is shown (*using the Wilcoxon test for paired nonparametric data).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

In the current study, the expression of ZNF148 protein increased significantly from normal mucosa to stage I CRC, and then decreased persistently from stage I to stage IV CRC. It was also found that lower expression of ZNF148 in CRC was significantly associated with worse clinicopathologic variables, including lymph node metastases, higher TNM stage, poor differentiation, and a higher rate of disease recurrence. These findings are consistent with data from the study by Bandres et al indicating that mRNA expression of ZNF148 in patients with stage II CRC is significantly higher than that in those with stage III CRC and lower mRNA expression of ZNF148 is associated with a higher incidence of disease recurrence.[13] It is interesting to note that the results of the current study demonstrated a significant correlation between ZNF148 overexpression and prolonged OS and DFS in all patients with CRC (stages I-IV) and in those with stage II and stage III CRC (Fig. 3). Univariate and multiple Cox proportional hazards analyses demonstrated that ZNF148 expression as well as TNM stage, tumor location, serum CEA level, and serum CA19-9 level were significant prognostic factors. Taken together, it appears that ZNF148 might be a tumor suppressor in patients with CRC. The antitumor properties of ZNF148 have been reported in several studies of various carcinomas such as CRC,[12, 13, 17] gastric cancer,[18] and hepatocellular carcinoma (HCC).[19] The prognostic value of ZNF148 expression has been reported in studies regarding HCC.[19] However, to the best of our knowledge, there is no published study to date concerning the prognostic value of ZNF148 in patients with CRC. To the best of our knowledge, the current study is the first to investigate the prognostic value of ZNF148 expression in patients with CRC. It is widely believed that lymph node metastases, a higher TNM stage, poorer differentiation, an elevated serum CEA level, and a higher recurrence rate are responsible for the worse prognosis noted among patients with CRC. The results of the current study suggest that patients with CRC with a lower expression of ZNF148 in their tumors have worse OS and DFS, and that ZNF148 expression status is a significant prognostic factor for CRC.

In the current study, we evaluated ZNF148 expression in paired normal mucosa, adenoma, and adenocarcinoma specimens from 16 patients with FAP. Because gene expression in different patients varied significantly due to the interference of hereditary heterogeneity, investigating gene expression in the tissues of different developing stages of disease from the same patients could provide a consistent result. We observed that ZNF148 expression is upregulated at steps toward carcinogenesis in patients with FAP. This finding indicates that ZNF148 expression in cancer cells most likely occurred during the progression of normal mucosa to adenocarcinoma.

Another distinctive feature of the current study was that ZNF148 expression in normal mucosa, primary tumor, and metastatic tumor tissues in 21 patients with metastatic CRC were studied. As shown in Figure 5D, we found that in most cases, ZNF148 expression in the metastatic tumor tissues was lower than that in primary tumors, whereas in a few cases (cases 7, 9, 12, and 15), ZNF148 expression in metastatic tissues was higher than among the primary tumors. This phenomenon may be related to the finding that intratumoral heterogeneity exists in CRC, and that the limited amount of tissue analyzed in TMA technology may not be representative of the whole specimen. As shown in Figure 5E, ZNF148 expression increased from the normal mucosa to carcinoma, but it decreased as the carcinoma metastasized to distant tissues. This finding suggests that ZNF148 expression in cancer cells might inhibit metastasis, and patients with CRC whose tumors overexpress ZNF148 are less likely to develop metastases to distant organs. Further studies are warranted to clarify this hypothesis and, more interestingly, to explore the possible the role of ZNF148 in the treatment of metastatic tumors.

ZNF148 protein expression increased consecutively from normal mucosa to stage I CRC, and then decreased consecutively from stage I to stage IV CRC. Although this appears to be a contradiction, it actually is not because the mechanism of colorectal carcinogenesis (from normal mucosa to carcinoma) might be different from the mechanism of CRC progression (stage I to stage IV) and metastasis. Although to the best of our knowledge the role of ZNF148 in CRC remains unknown, the antitumor property of ZNF148 has been studied in other malignant tumors. In tumor cells, ZNF148 is often overexpressed and exerts its tumor suppressor potential by both arresting cell proliferation and inducing apoptosis. ZNF148 has also been reported to regulate the expression of various molecules involved in tumor growth, invasion, and metastasis.[3] Moreover, ZNF148 is capable of enhancing the killing effects of several anticancer drugs. In many cancer cells, p53 accumulation in the nucleus is associated with an increasing susceptibility to chemotherapy and radiotherapy.[20-22] ZNF148 has been involved in the nuclear retention of p53 in recurrent human HCC.[23] Therefore, overexpression of ZNF148 may define a subgroup of cancer cells that are more sensitive to antitumor treatment. Thus, ZNF148 might also be a predictive factor in patients with CRC.

There are some limitations to the current study. First, this is a retrospective study; the 742 patients with CRC were not randomly selected from 4625 consecutive patients with CRC, and therefore selection bias was inevitable. Second, the incidence of patients being lost to follow-up was as high as 30.9% (810 of 2618 patients) in our CRC database, which would also induce possible biases. Third, to exclude the interference of chemoradiotherapy on gene expression, patients with CRC who were treated with neoadjuvant radiotherapy or chemotherapy were not included in the current study, which may affect OS and DFS rates. Moreover, TMA technology has the advantage that hundreds of specimens are processed simultaneously using identical conditions, thereby conserving reagents, saving time, and decreasing the amount of archival tissue.[24] However, the main limitation of TMA is that the amount of tissue analyzed using this technique is limited and may not be representative of the entire specimen, especially in CRC, in which intratumoral heterogeneity exists.[25]

The results of the current study indicate that, although ZNF148 expression in CRC occurs during the progression of normal mucosa to adenocarcinoma, this expression is consecutively downregulated from stage I to stage III CRC and is inversely associated with CRC metastasis and good postoperative survival. ZNF148 is a significant prognostic factor in patients with CRC, in whom high ZNF148 expression is predictive of a good prognosis. Further studies are needed to clarify the mechanisms of ZNF148 in patients with CRC.

FUNDING SUPPORT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Supported by the National Natural Science Foundation of China (grants 81201936, 30973460, 81272561, and 81025015).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES