http://informahealthcare.com/bmk ISSN: 1354-750X (print), 1366-5804 (electronic) Biomarkers, 2014; 19(1): 81–85 ! 2014 Informa UK Ltd. DOI: 10.3109/1354750X.2013.876555

RESEARCH ARTICLE

Fibroblast growth factor receptor 4 as a potential prognostic and therapeutic marker in colorectal cancer Chen-Sheng Li1*, Shu-Xiang Zhang2*, Hong-Jun Liu1, Yu-Long Shi1, Le-Ping Li1, Xiao-Bo Guo1, and Zhen-Hai Zhang3 1

Department of Gastrointestinal Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong Province, China, Department of Critical-Care Medicine, Qianfoshan Hospital Affiliated to Shandong University, Jinan, Shandong Province, China, and 3Department of Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong Province, China Abstract

Keywords

The purpose of the study was to explore the significance of FGFR4 protein expression in colorectal cancer. Immunohistochemistry showed 46.8% (148/316) tumors positive for FGFR4 and 7.3% (23/316) for adjacent normal specimens. FGFR4 positivity was correlated with shortened disease free survival (DFS) and overall survival (OS). Multivariate analysis revealed that FGFR4 was an independent prognostic factor. FGFR4 silencing markedly reduced the migration and invasion capacity of colorectal cancer cell lines. These results suggest FGFR4 is a potential prognostic and therapeutic marker for colorectal cancer.

Colorectal cancer, FGFR4, marker, prognosis

Introduction Colorectal carcinoma (CRC) is seen with increasing frequency in the Asia-Pacific region, and it is one of the most important causes of cancer mortality worldwide (Kimman et al., 2011; Ku et al., 2012). Despite surgical resection of the primary tumor and adjuvant chemotherapy, nearly half of the patients ultimately die of local recurrence and metastases (Punt & Tol, 2009). Tumor growth and metastasis comprise complex biological processes. Therefore, understanding the key factors in these processes is crucial in the search of the new treatment modalities. Although many molecular markers, including carcinoembryonic antigen, have been exploited for detecting CRC, these factors lack sensitivity and specificity for evaluating the prognosis of CRC patients (Siena et al., 2009). Thus, there is an urgent demand for novel biomarkers that can serve as good prognostic indicators for colorectal cancer. The fibroblast growth factor receptor (FGFR) family plays an important role in a wide range of biological processes, including cell differentiation, proliferation, motility and angiogenesis (Powers et al., 2000). Deregulation of FGFR signaling pathway has been implicated in multiple malignant diseases (Jeffers et al., 2002). Functional studies demonstrated that FGFR4 promotes normal cell adhesiveness and invasive *These authors contributed equally to this work. Address for correspondence: Dr. Zhen-Hai Zhang, Department of Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, No. 324 Road Jingwuwei 7, Jinan 250021, Shandong, China. Tel: +860531 87938911. E-mail: [email protected]

History Received 13 November 2013 Revised 11 December 2013 Accepted 14 December 2013 Published online 13 January 2014

properties of pituitary tumors (Ezzat et al., 2002). Nevertheless, to our knowledge, it remains yet to explore the prognostic significance of FGFR4 in colorectal cancer. The present study evaluated the FGFR4 protein expression and its possible correlations with clinicopathological features and clinical outcome.

Materials and methods Patient samples In the present study, we investigated 316 colorectal cancer cases during the period of January 2002 to December 2007. The clinical follow-up was performed at 3-month interval after surgery. Previous surgical treatment, no removal of cervical lymph nodes and positive surgical margins were exclusion criteria. Clinical data including the cause of death were obtained through medical record review. Formalin-fixed paraffin-embedded tissue blocks were collected at Provincial Hospital Affiliated to Shandong University at the time of surgical procedures performed. Written informed consent was obtained from all participants. This study was reviewed and approved by Shandong University Ethics Committees.

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Immunohistochemistry and evaluation Haematoxylin and eosin-stained sections were examined by two independent pathologists blinded to clinical data on the condition that the concordance was reached. Tumor differentiation was categorized as well-moderate or poor (450% versus 50% gland formation). The expression of FGFR4 protein was analyzed by immunohistochemistry using anti-FGFR4

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antibody as described previously (Giovannucci et al., 2011). Absence of primary antibody served as a negative control. Immunohistochemical staining intensity was evaluated using the scoring system in the previous report (Dutra et al., 2012). Considering the percentage of FGFR4 immunepositive tumor cells, a score of 1 was given when 10% of cells were positive; 2 when 10–50% and 3 when 50% of cells were positive. Signal intensity was scored as negative (0), weak (1), moderate (2) and strong (3). Both scores were multiplied and the resulting score was used to categorize FGFR4 expression as negative (3), and positive (43).

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Cell culture and transfection Three colorectal cancer cell lines DLD-1, SW480 and HT29 were purchased from the American Type Culture Collection. Cells were maintained in RPMI-1640 (Thermo Scientific Hyclone, Waltham, MA) in standard conditions. FGFR4 siRNA and the control scrambled siRNA (both from Santa Cruz, CA) were transfected by using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Cell migration and invasion assay About 12 h after transfection, the mixed media containing Lipofectamine 2000 were discarded and cells were maintained in serum-free RPMI–1640 overnight. After starvation, the ability of cell migration and invasion was assessed using 24-well Cell Migration and Invasion Assay (BD Biosciences, San Jose, CA) with 8 mm pores according to the manufacturer’s protocol. For the migration assay, cells were incubated for 24 h; for the invasion assay, cells were incubated for 72 h. For the invasion assay, the upper compartment was coated with 50 mg Matrigel (BD Biosciences, San Jose, CA) and the lower compartment was filled with medium containing 10% fetal calf serum as chemoattractant. Spectrophotometry was conducted on a microtiter plate reader at 490 nm. All assays were carried out 3 times in triplicates.

Biomarkers, 2014; 19(1): 81–85

(Millipore, Bedford, MA). The membrane was immunoblotted overnight at 4  C with primary antibodies against human FGFR4 (1:1000 dilution; Sigma, St. Louis, MO) or antibody against b-actin (1:2000 dilution; CST, USA). A horseradish peroxidase-conjugated secondary antibody (1:2000 dilution; Santa Cruz) was incubated with the membrane for 2 h. Signals were detected with ECL detection reagent (Pierce Biotechnology, Rockford, IL). Statistical analysis All statistical analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL). The chi-squared test was performed for categorical data. Kaplan–Meier method and log-rank test were used for survival analyses. Multivariate Cox proportional hazards regression models were also used. p50.05 was defined as significant.

Results Correlations of FGFR4 with clinicopathological parameters Clinicopathological features of colorectal cancer patients are summarized in Table 1. There were 46.8% (148/316) cancer samples positive for FGFR4 by immunohistochemistry. Representative staining images are shown in Figure 1. In contrast, among all the 316 matched adjacent colorectal specimens, 7.3% (23/316) was positive for FGFR4. Correlation analysis showed that FGFR4 positivity was significantly correlated with poor differentiation (p ¼ 0.0015) and disease stage (p ¼ 0.010) as shown in Table 1.

Table 1. Clinicopathological characteristics according to FGFR4 status in colorectal cancer. FGFR4

Quantitative real-time PCR Total RNA from cells was extracted with RNA Extraction Kit and reversely transcribed into cDNA using the Reverse Transcription Kit (Invitrogen, Carlsbad, CA). cDNA (5 ng) was used for SYBR Green PCR (Roche Diagnostics Corporation, Indianapolis, IN). Specific primers used were as follows: FGFR4: forward primer 50 -GACCGCAGCAGCGCCCGA GGCCAG-30 and reverse primer 50 -AGAGGGAAGAGGGAGAGCTTCTG-30 ; b-actin: forward primer 50 -CCTGTACGCCAACACAGTGC-30 and reverse primer 50 -ATACTCCTGCTTGCT GATCC-30 . Samples were normalized to b-actin RNA and fold change of expression was calculated using the comparative Ct method. Western blot Proteins were extracted with Protein Extraction Kit (Invitrogen, Carlsbad, CA) from cells and concentrations were measured by the BCA assay kit (Invitrogen, Carlsbad, CA). Total protein was separated by SDS–PAGE using a 12.5% polyacrylamide gel and transferred onto a polyvinylidenefluoride membrane

Features All cases Sex Male Female Mean age ± SD Family history () (+) Tumor location Proximal colon Distal colon Rectum Stage I II III IV Tumor differentiation Well to moderate Poor

Total N

Negative

Positive

316

168

148

129 (41%) 187 (59%) 64.5 ± 9.2

72 (43%) 96 (57%) 63.5 ± 8.7

57 (39%) 91 (61%) 65.1 ± 9.6

245(78%) 71 (22%)

126 (75%) 42 (25%)

119(80%) 29 (20%)

135 (43%) 89 (28%) 92 (29%)

79 (47%) 52 (31%) 37 (22%)

56 (38%) 37 (25%) 55 (37%)

35 50 47 36

(21%) (30%) (28%) (21%)

36 (24%) 38 (26%) 52 (35%) 22(15%)

27 (16%) 141(84%)

33 (22%) 115 (78%)

p 0.44 0.71 0.62

0.67

0.010 71 88 99 58

(22%) (29%) (31%) (18%)

0.0015 60 (19%) 256(81%)

The chi-squared test was performed for categorical data. p50.05 was defined as significant.

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DOI: 10.3109/1354750X.2013.876555

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Figure 1. Immunohistochemical expression patterns of FGFR4 in colorectal tissues. FGFR4 expression pattern in colorectal cancer was determined by immunohistochemical analysis. (a and b) Specimens (score 1) with 10% staining (200), (c and d) specimens with 10–50% staining (score 2), and (e and f) specimens with 450% staining (score 3). 200.

Figure 2. Kaplan–Meier curves for patient survival according to FGFR4 status in colorectal cancer. FGFR4 positivity correlated with disease free survival (A) and overall survival (B) in colorectal cancer patients.

Table 2. Univariate and multivariate analysis for overall survival rate (Cox proportional hazards regression model). Univariate analysis

Multivariate analysis

Factors

RR

95% CI

p Value

HR

95% CI

p Value

Age (years) (755/75) Gender (male/female) Disease stage Tumor size (305/30) Lymphatic invasion (present/absent) Venous invasion (present/absent) FGFR4 expression (positive/negative)

1.23 2.05 22.0 3.66 4.49 3.10 4.33

0.80–2.39 0.87–6.70 4.78–100.55 1.76–16.15 1.73–13.83 1.21–7.53 1.45–18.59

0.475 0.055 50.001 50.001 0.0014 0.029 0.007

25.9 3.04 0.72 1.68 3.96

3.57–215.84 1.18–13.62 0.20–2.95 0.61–4.53 1.09–19.00

0.001 0.017 0.637 0.301 0.012

HR, hazard risk; CI, confidence interval.

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FGFR4 positivity correlated with OS of colorectal cancer patients In the Kaplan–Meier analysis, FGFR4 positivity was significantly correlated with shorter disease free survival (DFS) (log-rank p ¼ 0.0033) and OS (log-rank p50.001) (Figure 2). Cox univariate analysis confirmed the association between FGFR4 and OS (HR ¼ 4.53; 95% CI 1.46–17.33, p ¼ 0.007) (Table 2). This association remained significant in multivariate analysis adjusted for age, gender, disease stage, lymphatic or vascular invasion (HR ¼ 3.86; 95% CI 1.09–16.90, p ¼ 0.012).

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FGFR4 siRNA significantly reduced the migration and invasion of colorectal cancer cell lines First of all, we analyzed the expression level of FGFR4 in three colorectal cancer cell lines DLD-1, SW480 and HT29. We observed that FGFR4 expression was relatively higher in SW480 and HT29 cells than that in DLD-1 cells (Figure 3A). Based on this expression pattern, we therefore chose SW480 and HT29 cells for the following functional studies. The silencing effect was confirmed by quantitative realtime PCR and Western blot (Figure 3B). As a result, FGFR4 knockdown in vitro significantly reduced the migration (118.21 ± 23.03 versus 63.37 ± 17.61 for SW480, p50.01; 106.46 ± 19.28 versus 53.49 ± 17.8 for HT29, p50.01) and invasion (111.24 ± 22.16 versus 52.19 ± 18.43 for SW480, p50.01; 100.29 ± 28.93 versus 48.70 ± 23.15 for HT29, p50.01) of the colorectal cancer cell lines (Figure 3C and D).

Discussion Here, we report our data on FGFR4 status in a relatively large population of colorectal cancer patients (n ¼ 316) in relation to clinicopathological features as well as survival. We found significant relationships between FGFR4 positivity, poor differentiation and increased mortality, independent of other patient characteristics. Our results indicate that FGFR4 positivity may be considered as a good prognostic marker for colorectal cancer. In the current study, FGFR4 positivity was observed in 46.8% of colorectal cancers, while normal colorectal epithelial cells were stained negatively for FGFR4. Thus, FGFR4 positivity in biopsy specimens may provide a valuable clue in the diagnosis of colorectal cancer. We believe that the identification of FGFR4 may represent a key advancement in search of valuable markers for colorectal cancer that have the potential to be translated into clinical applications including diagnosis and prognosis. Bange et al. (2002) reported that elevated levels of FGFR4 transcripts in several mammary carcinoma cell lines were associated with cancer progression and tumor cell motility. Another study demonstrated that FGFR4 single nucleotide polymorphism as predictive factors for nodal metastasis in oral squamous cell carcinoma (Choi et al., 2012). Functional studies demonstrated that FGFR4 promotes normal cell adhesiveness and invasive properties of pituitary tumors (Ezzat et al., 2002). FGFR4 Gly388Arg polymorphism acts as a cancer prognostic factor (Frullanti et al., 2011). Although past data suggest that the FGFR4 polymorphism may not be relevant in predicting nodal involvement of breast cancer or

Figure 3. FGFR4 siRNA significantly reduced the migration and invasion of the colorectal cancer cell lines. (A) FGFR4 expression in DLD-1, SW480 and HT29 cells by Western blot. (B) Silencing effect of FGFR4 by siRNA was confirmed by real-time PCR and Western blot. (C) FGFR4 siRNA inhibited the migration of SW480 and HT29 cells. (D) FGFR4siRNA inhibited the invasion of SW480 and HT29. Data were presented as mean ± SD for three independent experiments (*p50.01).

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DOI: 10.3109/1354750X.2013.876555

colon cancer patients (Spinola et al., 2005), FGFR4 Gly388Arg genotype and expression could act as a novel marker of prognosis in squamous cell carcinoma of the mouth and oropharynx (Dutra et al., 2012). A recent report has shown that Mechanistic study shows that FGFR4 plays crucial roles in TAF-induced epithelial-to-mesenchymal transition (EMT) in colorectal cancer cell lines, suggesting that FGFR4 promotes stroma-induced EMT in colorectal cancer (Liu et al., 2013). Consistently, data also support a crucial role for FGFR4 in tumorigenesis, invasion and survival in colorectal cancer and FGFR4 targeting demonstrated its applicability for colorectal cancer therapy (Pela´ez-Garcı´a et al., 2013). Considering the prognostic association of FGFR4 overexpression might be related to biological effects on invasion and metastasis, we also performed in vitro study via gene silencing in colorectal cancer cell line DLD-1, SW480 and HT29. As a result, cell migration and invasion were significantly inhibited after transfection with FGFR4 siRNA, indicating that FGFR4 is not only a prognostic biomarker, but also a potential therapeutic target. Taken together, FGFR4 is a promising prognostic marker for colorectal cancer and its expression is independently associated with poorer prognosis. Our findings may promote the translation of FGFR4 as a therapeutic target. Further investigation is warranted to elucidate the underlying mechanisms through which FGFR4 affects the biological behaviors of colorectal cancer.

Declaration of interest None. This study was supported by grants from the Young and Middle-Aged Scientists Research Award Fund of Shandong Province of China (No. BS2010YY060), Natural Science Foundation of Shandong Province of China (No. ZR2012HM079) and the National Youthful Science Foundation of China (81101858).

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Fibroblast growth factor receptor 4 as a potential prognostic and therapeutic marker in colorectal cancer.

The purpose of the study was to explore the significance of FGFR4 protein expression in colorectal cancer. Immunohistochemistry showed 46.8% (148/316)...
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