|Year : 2013 | Volume
| Issue : 3 | Page : 376-380
Aberrant expression of trefoil factor 3 is associated with colorectal carcinoma metastasis
You-Guang Huang1, Yun-Feng Li2, Li-Ping Wang3, Yun Zhang1
1 Tumor institue of Yunnan Province, The 3rd Affiliated Hospital of kunming Medical University; The Graduate School of the Chinese Academy of Sciences, Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, China
2 Colorectal Cancer Research Centre, the Tumor Hospital of Yunnan Province, China
3 Department of Pathology, Yan-an Hospital of Kunming, China
|Date of Web Publication||8-Oct-2013|
Kunming Institute of Zoology, Chinese Academy of Sciences, 32 East Jiao Chang Road, Kunming, Yunnan 650223
Source of Support: None, Conflict of Interest: None
Background: Recent evidence has indicated that the trefoil factor family possesses pivotal roles in the progression of human cancer. Aberrant expression of trefoil factor 3 (TFF3) has been reported to correlate with an aggressive tumor phenotype. However, the clinical importance of TFF3 expression in colorectal carcinomas (CRCs) has rarely been addressed.
Purpose: To investigate the putative role of TFF3 in colorectal carcinogenesis and progress, and to clarify whether TFF3 could be a serum marker for CRCs.
Materials and Methods: Fifty-six CRCs were sequenced for TFF3 mutations; subsets of the primary tumors were subjected to real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry analyses and serum TFF3 was detected by enzyme-linked immunosorbent assay (ELISA) for patients with CRCs.
Results: No variants were detected in the code area of TFF3; TFF3 mRNA is increased in CRCs but not up to statistic significance when compared with paired normal colonic mucosa; TFF3 staining by immunohistochemistry in primary CRCs showed that increased expression of TFF3 is associated with lymph node metastases(LNM), and no significant differences were found with respect to patient's sex, cancer cell differentiation and stage. Serum TFF3 is significantly elevated in patients with CRCs, especially CRCs with LNM.
Conclusion: The results indicate that TFF3 point mutations seem to be a rare event in colorectal carcinogenesis; TFF3 expression may play a role in promoting lymph node metastases of CRCs and serum TFF3 may be a potential useful marker for patients with CRCs and their metastases.
Keywords: Trefoil factor 3, colorectal carcinoma, cancer metastasis
|How to cite this article:|
Huang YG, Li YF, Wang LP, Zhang Y. Aberrant expression of trefoil factor 3 is associated with colorectal carcinoma metastasis. J Can Res Ther 2013;9:376-80
| > Introduction|| |
Colorectal carcinoma (CRC) is one of the leading causes of cancer-related deaths worldwide. In 2010, 142,570 new CRC cases and over 513,700 deaths from CRC were estimated in the United States. , Trefoil factors (TFFs), which comprises a group of small peptides with a distinct motif of three-loop structure, was being recognized by different research groups by late 1980 s to early 1990 s.  Since then, their unique biochemical properties and multiple functional effects have been the subject of comprehensive research. The three-loop structure (also trefoil domain) of TFF was formed by highly conserved 6 cysteine residues to form three disulfide bonds in the way of 1-5, 24, 3-6 in a peptide with 42-43 amino acid. Up to the present, three members of the TFF family (TFF1, TFF2 and TFF3) were discovered by several routes in mammals. In human beings, TFFs are generally expressed by secretary cells, such as goblet cells, mucous cells and glandular cells, in various epithelial tissues.  They play important roles in epithelial protection and restitution by their properties of promoting epithelial cell migration, inhibit apoptosis and promote angiogenesis.  Recent evidence has indicated that members of this family also possess pivotal roles in the development and progression of human cancer. Aberrant expression of TFFs in many cancers has been reported, and this has prompted a debate on TFFs as prognostic markers in cancers.
TFF3 is secreted chiefly from the mucus-secreting goblet cells of the gastrointestinal tract. It plays a critical role in mucosal protection and restitution through motogenic and antiapoptotic activities. Aberrant expression of TFF3 has been reported in various human solid tumors. In gastric cancer, 44% and 55% were identified to be positive for TFF3 expression. Increased TFF3 expression is correlated with an aggressive tumor phenotype (advanced stage, infiltrative growth pattern and positive lymph node metastases) in gastric cancer. , The 5-year disease-free survival of patients with advanced gastric cancer was 91.3% for TFF3-negative compared with 42.8% for TFF3-positive.  In prostate cancer, consistently upregulated TFF3 mRNA has been observed from gene expression profiling. , Consistently, patients with advanced prostate cancer have significantly increased plasma concentration of TFF3, and elevated plasma TFF3 was demonstrated to predict bone metastasis.  However, the clinical importance of TFF3 expression in CRC has rarely been addressed. , In the present study, we analyzed TFF3 mutations in CRCs by DNA sequencing, and detected TFF3 expression in mRNA and protein level by real-time quantitative RT-PCR and immunohistochemistry. We also detected TFF3 concentration in the serum of patients with CRC by ELISA to determine whether TFF3 could be a kind of serum markers for patients with CRC and to illuminate its correlation with CRC lymph node metastasis.
| > Materials and Methods|| |
DNA and RNA extraction kits, primer synthesis, PCR amplification reagents and PCR product purify kits were purchased from TIANGEN BIOTH Co. (Beijing, China). RNA enzyme inhibitors, reverse transcriptase PrimerScript RTase, 2 × SYBR Premix Ex Taq™ II, Ex Taq™ and dNTPs were from Takara Biotechnology (Dalian) Co., Ltd. (Takara Dalian, China). TFF3 antibodies for immunohistochenistry analysis were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The ELISA kits for detecting serum TFF3 were from Groundwork Biotechnology Diagnosticate Ltd (San Diego, CA, USA; Catalogue number: T123-9).
Between January 2009 and December 2010, 56 patients with advanced CRC were included in this study. No patient had received preoperative chemo-or radiotherapy. Specimens of CRC and paired normal tissues distal to the tumor tissue were obtained fresh from the operating room. Specimens for DNA and RNA extraction were snapping frozen and then stored at-80˚C till use, and specimens for immunohistochemistry were routinely formalin-fixed and embedded in paraffin. Blood samples from healthy donors and preoperative patients with CRC were obtained from blood routine examination samples. Sera for ELISA detection was separated and kept at-80˚C till use. All patients provided consent for use of tumor tissue for clinical research, and the Ethical Committee of Kunming Medical University approved the research protocol.
Genomic DNA of tumor tissues was extracted according to instructions of the DNA extraction kit. PCR primers for amplification of the complete TFF3 coding sequence were designed by using PubMedline primer design software to amplify each TFF3 exon, including 5`-untranslated sequence, exon/intron boundaries and 3`-untranslated sequence region. Primer sequences for amplifying three exons of TFF3 are listed in [Table 1].
PCR amplification was performed in a 25 μL volume containing 10 pmol of each of the forward and reverse sets of primers, 1 U of pfu TaqDNA polymerase and 2.5 μL of 10× PCR buffer. The reaction was an initial denaturation at 95˚C for 5 min, followed by 30 cycles at 94˚C for 30 s, 56-60˚C for 30 s and 72˚C for 50 s. A final extension was conducted at 72˚C for 7 min, and the products were then maintained at 10˚C. The amplified products were isolated by agarose gel electrophoresis and purified using a gel extraction kit, and then the purified products were sequenced by TIANGEN BIOTH Co.
For real-time quantitative RT-PCR, about 20 mg cancer and matched safety normal colon tissues were ground to powder in liquid nitrogen. Total RNA was extracted according to the RNA kit instructions and first-strand cDNA synthesis (reverse transcription) was performed in 20 μL reaction volume according to the manufacture's instruction. Primers were designed to span across two exons to avoid amplification of possible genomic DNA. Sequences of primers for TFF3 cDNA are: forward primer 5`-GTGCCAGCCAAGGACAG-3` and reverse primer 5`-CGTTAAGACATCAGGCTCCAG-3`, and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA are: forward primer 5'-GAGTCAACGGATTTGGTCGT-3' and reverse primer 5'-GACAAGCTTCCCGTTCTCAG-3'. Quantitative real-time PCR were carried out using SYBR @ Premix-Ex Tag kit (TAKALA) on the iQ5 Bio-rad real-time instrument. PCR reaction was performed according to the kit instruction. Briefly, a 25 μL reaction volume contained 2× SYBR ® Green RT-PCR reaction mix, 2 μL of template cDNA and 10 pmol of each forward and reverse primer. The reaction was first initial denaturation at 95˚C for 5 min, followed by 40 cycles at 95˚C for 10 s, 58˚C for 15 s and 72˚C for 20 s. All reactions were performed in triplicate. For each reaction, melt curve analysis was performed to ensure specificity. The TFF3 mRNA level was normalized to the endogenous GAPDH reference to obtain the relative threshold cycle (ΔC t ). 
For immunohistochemical analysis, 5-μm paraffin-embedded tissue sections were deparaffinized, rehydrated in graded alcohols and placed in phosphate buffer solution (PBS) solution. Antigen retrieval was performed by autoclaving the slides for 10 min in citrate buffer. Endogenous peroxidase activity was quenched by treatment with 3% hydrogen peroxide for 30 min. Nonspecific bindings were blocked by treating slides with 5% normal rates' serum for 30 min. TFF3 primary antibody (1:100 dilution; Santa Cruz, USA) was added and incubated at 4˚C overnight. After rinsing with Tris buffered saline (TBS). slides were incubated with avidin-biotinylated peroxidase complex (AB complex/HRP; Dako, Glostrup, Denmark). Reactions were visualized with diaminobenzidine (DAB) and counterstained with Mayer's hematoxylin solution. TBS alone without primary antibody was used as a negative control of immunostaining. Immunohistochemical staining was assessed semiquantitatively by measuring both the intensity of staining (0, 1, 2 or 3) and extent of staining (0, 0%; 1, <25%; 2, <50%; 3, <75%; 4, <100%). The scores for the intensity and extent of staining were multiplied to give a weighted score for each case. And then, the score of 0 was counted as negative (-), 1-4 was weak (+), 5-8 was moderate (++) and 9-12 was strong (+++). For the statistical analysis, the weighted scores were grouped in two categories where negative to weak (-~ +) positive were in one and moderate to strong positive (++ ~ +++) were in the other.
Serum TFF3 level was measured by the TFF3 ELISA kit according to the manufacture's protocol. Briefly, full-length recombinant human TFF3 protein was diluted with 1% (w/v) BSA in PBST(0.05% v/v Tween 20 in PBS, pH 7.4) to obtain calibrators for the standard curve preparation, and the diluents were used as zero calibrator. Fifty-microliter calibrators or human sera (1:5) in diluents were added and incubated for 2 h at room temperature. After five washes, 50 μL of unconjugated rabbit anti-TFF3 antibody was added to each well and the plates were incubated for 60 min at 37°C then washed five times. Fifty microliter per well of Streptavidin HRP-conjugated goat anti-rabbit IgG (dilution 1:5000) was added and incubated for 60 min at room temperature, followed by five washes. The color reaction was developed by addition of 100 μL substrate solution to each well at room temperature for approximately 30 min. The reactions were terminated with 50 μL/well of stop solution. Absorbance was measured at 450 nm using an automatic plate reader (Spectramax 340 PC; Molecular Devices Corporation, Sunnyvale, CA, ). Each sample was examined in duplicate.
| > Results|| |
Sequence analysis of the entire coding region of TFF3 and adjacent intron borders of three exons was performed for 42 tissue samples. No nucleotide alternations were found in the coding region and, therefore, we ceased sequencing TFF3 on larger scale.
Real-time quantitative RT-PCR was used to investigate the expression of TFF3 mRNA in CRCs and paired normal colonic mucosa. Thirty-two cases with specific amplification of TFF3 and GAPDH cDNAs were included in the study (showed a single melting curve peak). The values of TFF3 mRNA were normalized to that of GAPDH. Using Spearman's rank order correlations, there is no significant difference of TFF3 mRNA in CRCs and paired normal colonic mucosa (P=0.852) [Figure 1]a. However TFF3 mRNA is increased in CRCs with lymph node metastasis (LNM) when compared with non-LNM (P=0.045) [Figure 1]b.
|Figure 1: Box-plot showed the trefoil factor 3 (TFF3) mRNA relative expression in colorectal carcinomas (CRCs) and paired normal colonic mucosa. The CRCs include 20 cases with lymph node metastasis (LNM) and 12 cases with non-LNM. (a) TFF3 mRNA is upregulated but not up to statistic significance when compared with paired normal colonic mucosa (P = 0.852). (b) TFF3 mRNA is elevated in CRCs with LNM when compared with that of non-LNM (P=0.045, Spearman's rank order correlations)|
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In the normal colonic mucosa, TFF3 staining by immunohistochemistry was seen mainly secreted in superficial epithelium [Figure 2]a. However, TFF3 staining was heterogeneous and positive in 95% (54 of 56) of CRCs, and staining of TFF3 was mostly noticed in the cytoplasm [Figure 2]b. As measured by immunohistochemistry scores, TFF3 expression in CRCs with LNM was elevated significantly from that in non-LNM CRCs (P=0.038) [Figure 2]c. And, obviously, five cases with strong TFF3 expression (+++) were all with lymph node metastasis. The correlation between TFF3 expression and clinical pathology is shown in [Table 2]. There was no significant correlation of TFF3 expression with patients' gender, cancer differentiation status and T stage in this study (X 2 test).
|Figure 2: Immunohistochemical staining of trefoil factor 3 (TFF3) in normal colonic mucosa and colorectal carcinoma (CRCs). The slides were viewed by light microscopy (×100). (a) Normal colonic mucosa; TFF3 staining was mainly secreted in superficial epithelia. (b) Cancer cells express diffuse immunoreactivity for TTF3, the specimen of CRC with nonlymph node metastasis (non-LNM) (b-1) and specimen of CRC with LNM (b-2). (c) Scatter plot showed that TFF3 immunohistochemical staining score is elevated in the LNM group when compared with the non-LNM group (P = 0.038, X2 test)|
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|Table 2: Correlation of clinicopathologic findings with TFF3 expression in CRCs|
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TFF3 was measured in serum samples from 56 patients with histologically confirmed CRCs at the Third Affiliate Hospital of Kunming Medical University. The control group consisted of 45 presumably healthy blood donors (ages 44-65 years; median, 57 years). Among CRC patients, 27 cases with had LNM and 29 cases did not (non-LNM). The serum concentration of TFF3 for patients with CRC and healthy individuals are shown in [Figure 3]. The results showed that serum TFF3 concentration in healthy individuals was 908.7±195.6 ng/mL (median±SD), and in CRCs was 2340±1065 ng/mL (median±SD). There was a significantly statistic difference between the two groups (Student's t test: P<0.0001). Compared with the CRC non-LNM group, the LNM CRC group showed a significantly elevated serum level of TFF3 (LNM 3043±1739 ng/mL versus non-LNM 1255±403.6 ng/mL). These results indicated that TFF3 might be a potential serum biomarker for CRCs, especially for CRCs with LNM.
|Figure 3: Box-plot showed trefoil factor 3 (TFF3) concentrations in serum of colorectal carcinoma (CRC) patients and healthy individuals; P values were from Student's t test. (a) Serum TFF3 in healthy individuals was 908.7±195.6 ng/mL, and in CRC patients was 2340 ± 1065 ng/mL. There was an abundantly significant difference between the two groups (P<0.0001). (b) Serum level of TFF3 is elevated in patients with lymph node metastasis (LNM) CRC (3043 ± 1739 ng/mL) compared with non-LNM CRC (1255 ± 403.6 ng/mL), P<0.0001|
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| > Discussion|| |
CRC tumorigenesis appears to be the result of a progressive transformation of colorectal epithelial cells due primarily to the accumulation of mutations in a number of oncogenes as well as tumor suppressor genes.  Recent compelling evidences have emerged from experimental and clinical studies to indicate a pivotal role of TFFs in oncogenic transformation, growth and metastatic extension of common human solid tumors. , Somatic missense mutations of TFF1 have been identified in 16% of gastric cancers from the Korean population  ; these mutations were scattered in exons 1 and 2 with no obvious mutation hot spot. However, detecting TFF3 mutations in CRCs or other cancers has not been reported. In the present study, we performed an analysis encompassing all three TFF3 exons and respective exon/intron boundaries for 42 CRCs in the Chinese population. The DNA sequencing results revealed no alterations in CRCs. For this reason, we gave up for more TFF3 sequencing in a larger scale.
TFFs are found expressed in a site-specific fashion in distinct, often complementary, locations in human beings.  Although they exhibit a number of beneficial effects in mucosa protection and restitution, they may have adverse consequences when expressed in tumors. Previous studies have suggested the possible effects of TFF on cancer cell proliferation, apoptosis, motility and invasion.  TFF3 is normally expressed by intestinal epithelial cells; its expression is maintained in colonic adenomas and carcinomas,  suggesting that it may play a role in neoplasia. However, TFF3 expression in CRCs has not been extensively studied. In the present study, we analyzed the relative level of TFF3 mRNA in CRCs and their paired distal normal tissues by QRT-PCR. The results showed a wide variability of TFF3 mRNA levels in tumors. Comparison of paired distal normal mucosa and carcinoma samples showed no statistic significant difference of TFF3 mRNA level by QRT-PCR. However, increased TFF3 mRNA in CRCs indicates a correlation with lymph node metastasis. The results are consistent with that of immunohistochemistry staining, which showed that among 56 CRCs, 96% of the primary tumors were at least focally positive for the TFF3. And, immunohistochemistry score showed that TFF3 are more positive in CRCs with LNM than that with non-LNM. The relationship between TFF3 expression and CRC metastasis may be explained by its oncogenic potential. Rivat Christine reported that TFF3 produces a migratory and invasive phenotype in HCT8/S11 cells through the STAT3 signaling pathway.  TFF3 has also been demonstrated to enhance colonic carcinoma cell migration through modulation of E-cadherin-catenin complex function.  Yio et al. found that overexpression of TFF3 in nonaggressive colon cancer cells (LP cells) enhanced their ability to migrate, invade and block apoptosis, and these cells exhibited a more aggressive phenotype.  A study revealed that TFF3 was expressed in 100% colonic cancer liver metastasis and in 91% of primary colon cancers at the mRNA and protein level.  These data provide direct evidences that TFF3 contributes to the malignant behavior of colonic cancer cells. Abnormal expression of TFF3 has been reported in various human solid tumors, such as breast, prostate, lung and gastric cancer, and elevated expression of TFF3 in these cancers usually indicate poor outcome. ,,
The trefoil factors (TFF1-3) are mainly expressed in mucin-secreting epithelial cells and secreted to the upper layer of mucosa.  They play a critical role in mucosal protection and repair. It seems that the expression of trefoil peptides by these tissues may mask the protein expressed by the target tumor. In fact, we detected serum TFF3 by the ELISA method, and our results showed that serum TFF3 is significantly increased in the patients with CRC, and that increased serum TFF3 concentration also correlates with LNM of CRCs. This result might be explained by the constitution changes from normal epithelial tissues to epithelial cancer tissues. When epithelial cells transformed to cancer cells, they lost polarity and tight conjunct as well as new angiogenesis in tumor tissues. This may provide an access for the secretary small peptides in epithelial cancers to enter blood circulation more than the normal epithelial tissues.
With the present data, there is clearly sufficient basis for more extensive studies to elucidate the potential clinical value of TFF3 as a marker of CRCs and CRCs with LNM. The serum level of TFF3, however, may better reflect not only the primary tumor but also its associated metastases. Whether serum TFF3 will proves itself as a marker that can be applied in clinical practice remains to be established in prospective studies.
| > Acknowledgement|| |
This work was supported by grants from National Basic Research Program of China (973 Program, 2010CB529800), the Chinese National Natural Science Foundation (U1132601 and 31270835) and CAS grant KJZD-EW-L03 to Yun Zhang.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]