Upregulated FFAR4 correlates with the epithelial-mesenchymal transition and an unfavorable prognosis in human cholangiocarcinoma

Abstract

BACKGROUND:

Free fatty acid receptor 4 (FFAR4) is associated with the epithelial mesenchymal transition (EMT) and is involved in the progression of several types of cancer. However, the role of FFAR4 in cholangiocarcinoma (CCA) remains unclear.

OBJECTIVE:

The present study evaluated the diagnosis and prognosis of CCA using FFAR4 as a biomarker.

METHODS:

Immunohistochemistry was employed to detect expression of FFAR4 in 98 samples of CCA tissues and adjacent tissues. In addition, expression of E-cadherin, vimentin, Snail-1, CK7 and CK19 in the 98 samples of CCA tissues was detected, and relationships with FFAR4 were analyzed. Correlation between FFAR4 and clinical pathological factors and prognosis was also analyzed.

RESULTS:

FFAR4 was highly expressed in 72.4% (71/98) of CCA tissues and 29.6% (29/98) of adjacent tissues, with a statistically significant difference between the two tissue types ( $P<$ 0.05). A negative correlation between high expression FFAR4 and E-cadherin expression in CCA tissues was also observed ( $r=$ $-$ 0.445, $P<$ 0.001), and high expression of FFAR4 was positively correlated with vimentin ( $r=$ 0.354, $P<$ 0.001), Snail-1( $r=$ 0.496, $P<$ 0.001), CK7( $r=$ 0.494, $P<$ 0.001) and CK19 ( $r=$ 0.532, $P<$ 0.001). Moreover, the degree of FFAR4 expression was associated with aggressive clinicopathological characteristics, such as histological grade, perineural invasion (PNI), lymph node metastasis (LNM), advanced TNM stage and preoperative serum CA19-9 level ( $P<$ 0.05). In terms of prognosis, CCA patients with high FFAR4 expression showed shorter disease-free survival (DFS) ( $P<$ 0.05) and overall survival (OS) ( $P<$ 0.05) than did patients with low FFAR4 expression.

CONCLUSIONS:

FFAR4 overexpression may mediate the process of CCA EMT. In addition, FFAR4 is promising as a new diagnostic molecule and therapeutic target for CCA.

Keywords

Immunohistochemistry FFAR4 cytokeratin7 cytokeratin19 E-cadherin vimentin Snail-1 prognosis

1. Introduction

Cholangiocarcinoma (CCA) is an epithelial malignant tumor that originates from different locations on the biliary tree [1]. This tumor type can be divided according to the anatomic location into intrahepatic cholangiocarcinoma (iCCA), hilar cholangiocarcinoma (pCCA) and distal cholangiocarcinoma (dCCA), which have frequencies of 10%–20%, 50% and 30%–40%, respectively [2]. CCA is one of the most aggressive malignant gastrointestinal diseases and has a poor prognosis [1, 2]. Because conventional chemotherapy and radiotherapy have a limited impact on improving long-term survival, radical surgery is currently the only option for CCA patients to be cured or to achieve long-term survival. Unfortunately, due to severe local invasion and distant metastases, many patients when diagnosed with CCA have already missed the best option of radical surgery [3, 4]. Therefore, to improve the early diagnosis of CCA patients and postoperative quality of life, more in-depth studies of the occurrence and progression of CCA are needed.

As an important source of energy and nutrition, free fatty acids (FFAs) act as extracellular signaling molecules, primarily through binding to FFA receptors (FFARs), which belong to a family of G-protein-coupled transmembrane receptors (GPCRs) [5, 6, 7]. Disorders of GPCR signaling and expression have been identified as one of the hallmarks of cancer [8]. Free fatty acid receptor 4 (FFAR4), which is also called GPR120, is a new member of the GPCR family that has attracted considerable attention for its role as a medium-and long-chain FFA receptor in diabetes. Increasing evidence shows that FFAR4 also plays a role in tumorigenesis, migration and metastasis. In contrast to its contributions in diabetes, FFAR4 appears to have opposing functions in cancer biology [9]. The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signaling pathway is necessary to activate the FFAR4 signal transduction process and is an important pathway involved in the epithelial mesenchymal transition (EMT) process. Accordingly, this study investigates expression of FFAR4 in human CCA tissues and adjacent tissues; we also analyzed the relationship between FFAR4 expression and that of epithelium markers including E-cadherin, vimentin, snail-1, CK7 and CK19 in CCA tissues. At the same time, we assessed the relationship between FFAR4 expression and clinicopathological characteristics and correlations between FFAR4 expression and prognosis. We found that the mechanism of FFAR4 invasion and metastasis may be related to the EMT. Overall, our results suggest that by enhancing the invasion and metastasis of CCA, FFAR4 is a potential new target for clinical therapeutic drug research and is expected to be an effective marker for the early diagnosis of CCA.

2. Materials and methods

2.1 Samples and clinicopathological data

A total of 98 patients who underwent curative intent surgery for CCA at An Hui Provincial Hospital between January 2007 and January 2015 were enrolled in this study. All patients underwent radical surgical resection, and the pathology was diagnosed as CCA. The tumor tissue and adjacent normal tissue were fixed in formaldehyde and embedded in paraffin. Adjacent normal tissue was defined as bile duct tissue more than 2 cm from the edge of the tumor and confirmed by two experienced pathologists. The study was approved by the research medical ethics committee of An Hui Provincial Hospital. Informed consent was obtained from each patient. In addition, clinicopathological variables, including age, sex, tumor size, tumor location, preoperative serum carbohydrate antigen19-9 (CA19-9) concentrations, histological grade, LNM, PNI and stage of the tumor were collected. The cohort included 51 males and 47 females with an average age of 58.7 years (range 38–78 years). The histological classification of tumor differentiation was based on the WHO classification. The TNM stage was determined in accordance with the seventh edition of the Cancer Staging Manual of the American Cancer Commission (AJCC). The follow-up period was 5–62 months (median 19).

2.2 Immunohistochemistry

Sections (4 $\mu$ m thick) were cut, heated at 60 ${}^{\circ}$ C for 15 min, de-waxed in xylene, and rehydrated. The slides were treated by pressure cooking for 3 min in citrate buffer (PH $=$ 6) and immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Next, the sections were incubated overnight with polyclonal rabbit anti-human FFAR4 (Abcam, Inc, Cambridge, UK), polyclonal rabbit anti-human snail-1 (Abcam, Inc, Cambridge, UK), monoclonal mouse anti-human vimentin (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), monoclonal mouse anti-human E-cadherin (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), monoclonal mouse anti-human CK7 (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) or monoclonal mouse anti-human CK19 (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) antibodies in a humid chamber at 4 ${}^{\circ}$ C (the dilution ratio of all primary antibodies was 1:100). Subsequently, the sections were incubated with a secondary antibody (Zhongshan Golden Bridge Biotechnology) for 20 minutes at 37 ${}^{\circ}$ C, stained with 3, 3’-diaminobenzid-ine and counterstained with hematoxylin before mounting. Negative controls without the primary antibody were processed simultaneously. Semi-quantitative assessment was based on the product of intensity and proportion of positively stained cells. The proportion of positively stained cell scores (0, no staining; 1, $<$ 10%; 2, 10%–30%; 3, $>$ 30%) and the staining intensity scores (0, negative; 1, weak; 2, moderate; 3, strong) were aggregated. A final total score $\leqslant$ 3 was considered to be a low expression, and $>$ 3 was defined as a high expression [10, 11, 12]. The results of staining were assessed by two independent pathologists in the blinded manner.

2.3 Statistical analysis

Categorical patient characteristics were compared using the $\chi^{2}$ test. Associations between FFAR4, E-cadherin, CK7, CK19, Snail-1 and vimentin were evaluated by Spearman’s rank correlation. Kaplan-Meier survival curves were used to analyze CCA patients’ OS and DFS. Differences were assessed by a log-rank test. Cox proportional hazard regression models were used to obtain unadjusted and adjusted hazard ratio (HR) and 95% confidence interval (CI) for survival in univariate and multivariate analyses. A 2-sided $P<$ 0.05 was considered to be statistically significant. SPSS 21.0 software (SPSS, Inc., Chicago, IL, USA) and R3.2.3 (http://www.R-project.org) were used for the statistical analysis.

Table 1
Differential expression of FFAR4 in CCA tissues and corresponding paracarcinoma tissues ( $n=$ 98)

Tissue	FFAR4 expression		$P$ -value
	Low (%)	High (%)
CCA tissues	27 (27.6%)	71 (72.4%)	$<$ 0.001
Paracarcinoma tissues	69 (70.4%)	29 (29.6%)

3. Results

3.1 FFAR4 expression in CCA tissues and paracancerous tissue

To identify expression patterns of FFAR4 in CCA, FFAR4 mRNA expression profiles were obtained from the Ualcan database [13], indicating that FFAR4 was significantly up-regulated in tumors compared with adjacent non-tumor tissues of CCA. Although FFAR4 was often overexpressed in most cancers, down-regulation was observed in some types. Therefore, the role of FFAR4 should be assessed in a cancer-specific manner. Immunohistochemistry was performed to validate and evaluate FFAR4 expression as well as localization in CCA, which was mainly distributed in the cytoplasm of tumor cells (Fig. 1A1 and A2). The results indicate obviously higher ( $P<$ 0.05) FFAR4 expression in tumor specimens (71/98, 72.4%; Table 1) than in paracarcinomatous specimens (29/98, 29.6%; Table 1).

Table 2
Correlations of FFAR4 expression with E-cadherin, vimentin, Snail-1, CK7 and CK19 in CCA

Immunoreactivity	FFAR4 expression		$r$ -value	$P$ -value
	Low	High
E-cadherin expression
Low	12	62	$-$ 0.445	$<$ 0.001
High	15	9
Vimentin expression
Low	14	12	0.354	$<$ 0.001
High	13	59
Snail-1 expression
Low	19	13	0.496	$<$ 0.001
High	8	58
CK7 expression
Low	21	17	0.494	$<$ 0.001
High	6	54
CK19 expression
Low	19	11	0.532	$<$ 0.001

3.2 Relationship of FFAR4 with CK7, CK19, E-cadherin, Snail-1 and vimentin in CCA tissues

In 98 cases of cholangiocarcinoma, 74 exhibited low expression of E-cadherin; the proportion was 75.5% (Fig. 1D1 and D2). However, in 98 samples of CCA tissue, high expression of CK7, CK19, Snail-1 and vimentin was observed in 60, 68, 66, and 72 cases, accounting for 61.2%, 69.4%, 67.3% and 73.5%, respectively (Fig. 1B1, B2, C1, C2, E1, E2, F1, F2). Expression of FFAR4 in CCA tissues was positively correlated with that of CK7 ( $r=$ 0.494, $P<$ 0.001; Table 2), CK19 ( $r=$ 0.532, $P<$ 0.001; Table 2), Snail-1 ( $r=$ 0.496, $P<$ 0.001; Table 2) and vimentin ( $r=$ 0.354, $P<$ 0.001; Table 2) but negatively correlated with that of E-cadherin ( $r=$ $-$ 0.445, $P=$ 0.00; Table 2).

Figure 1.

Immunohistochemical staining for FFAR4, CK7, CK19, E-cadherin, Snail-1 and vimentin. FFAR4 is principally localized in cytoplasm of tumor cells; CK7 and CK19 are mainly localized in the bile duct cell membrane and tumor cell membrane; E-cadherin is mainly localized in the bile duct cell membrane; Snail-1 is mainly localized in the nucleus and cytoplasm of tumor cells; vimentin is mainly localized in the cytoplasm of tumor cells. A1: Positive FFAR4 expression in CCA tissue. A2: Negative FFAR4 expression in adjacent tissue. B1: Positive CK7 expression in CCA tissue. B2: Negative CK7 expression in adjacent tissue. C1: Positive CK19 expression in CCA tissue. C2: Negative CK19 expression in adjacent tissue. D1: Negative E-cadherin expression in CCA. D2: Positive E-cadherin expression in adjacent tissue. E1: Positive Snail-1 expression in CCA tissue. E2: Negative Snail-1 expression in adjacent tissue. F1: Positive vimentin expression in CCA tissue. F2: Negative vimentin expression in adjacent tissue. All images were taken at 400 $\times$ magnification.

Figure 2.

Kaplan-Meier analysis of disease-free survival (DFS) and overall survival (OS) in patients with CCA according to FFAR4 staining. Patients with high FFAR4 expression had a shorter OS and DFS than did those with low FFAR4 expression ( $n=$ 98; $P<$ 0.05; log rank test).

3.3 Association of FFAR4 expression and clinicopathological factors

Experimental data show that FFAR4 expression in cholangiocarcinoma was markedly related to histological grade, perineural invasion, LNM, advanced TNM stage and preoperative serum CA19-9 ( $P<$ 0.05) (Table 3). However, FFAR4 expression was not significantly associated with other factors, such as age, gender, tumor size and tumor location (Table 3).

Table 3
Correlations between FFAR4 expression and clinicopathological characteristics in CCA

Parameters	Number of case	FFAR4 expression		$\chi^{2}$	$P$ -value
		Low	High
Age
$\leqslant$ 60	39	11	28	0.014	0.906
$>$ 60	59	16	43
Gender
Male	51	11	40	1.907	0.167
Female	47	16	31
Tumor size
$\leqslant$ 30	60	13	47	2.684	0.101
$>$ 30	38	14	24
Tumor location
Intrahepatic	28	9	19	0.433	0.805
Perihilar	36	9	27
Distal	34	9	25
PNI
Absent	57	21	36	5.892	0.015
Present	41	6	35
LNM
Absent	63	22	41	4.800	0.028
Present	35	5	30
Histological grade
Moderate-poor	73	14	59	10.051	0.002
Well	25	13	12
TNM stage
I–II	64	22	42	4.304	0.038
III–IV	34	5	29
Serum CA19-9, U/ml
$\leqslant$ 37	18	10	8	8.664	0.003
$>$ 37	80	17	63

Table 4

Univariate analysis of factors associated with DFS and OS

	HR	95% CI	$P$ -value
Variable	OS
Age, years ( $>$ 60 and $\leqslant$ 60)	0.955	0.528–1.507	0.821
Gender (male and female)	1.163	0.732–1.845	0.537
Tumor size, cm ( $\geqslant$ 3 and $<$ 3)	0.732	0.523–1.174	0.314
Tumor location (intrahepatic and perihilar/distal)	0.814	0.558–1.616	0.536
Histologicalgrade (moderate/poor and well)	2.627	1.567–4.765	$<$ 0.001
LNM (absent vs present)	0.432	0.243–0.546	$<$ 0.001
PNI (absent vs present)	0.649	0.463–1.241	0.241
Serum CA199, U/mL ( $\leqslant$ 37 and $>$ 37)	1.621	0.813–2.878	0.257
TNM stage (I–II vs III–IV)	24.562	11.537–57.572	$<$ 0.001
FFAR4 expression (low and high)	0.543	0.313–0.914	0.014
Variable	DFS
	HR	95% CI	$P$ -value
Age, years ( $>$ 60 and $\leqslant$ 60)	1.014	0.714–1.549	0.961
Gender (male and female)	1.264	0.789–2.004	0.372
Tumor size, cm ( $\geqslant$ 3 and $<$ 3)	0.797	0.497–1.299	0.341
Tumor location (intrahepatic and perihilar/distal)	0.976	0.673–1.534	0.759
Histological grade (moderate/poor and well)	2.377	1.438–4.478	0.003
LNM (absent vs present)	0.544	0.279–0.688	0.001
PNI (absent vs present)	0.769	0.474–1.239	0.351
Serum CA199, U/mL ( $\leqslant$ 37 and $>$ 37)	1.469	0.824–2.765	0.279
TNM stage (I–II and III–IV)	26.232	10.142–49.364	$<$ 0.001
FFAR4 expression (low and high)	0.638	0.236–0.843	0.016

Table 5

Multivariate analysis of factors associated with DFS and OS

Variable	OS
	HR	95% CI	$P$ -value
FFAR4 expression (low and high)	0.416	0.181–0.923	0.029
LNM (absent vs present)	0.197	0.145–0.367	$<$ 0.001
TNM stage (I–II vs III–IV)	21.656	8.352–47.867	$<$ 0.001
Histological grade (moderate/poor and well)	1.893	0.879–3.143	0.243
Variable	DFS
	HR	95% CI	$P$ -value
FFAR4 expression (low and high)	0.578	0.239–0.817	0.023
LNM (absent vs present)	0.373	0.149–0.458	$<$ 0.001
TNM stage (I–II vs III–IV)	18.345	7.671–42.679	$<$ 0.001
Histological grade (moderate/poor and well)	1.456	0.717–2.534	0.432

3.4 Relationship between expression of FFAR4 and prognosis of cholangiocarcinoma patients

Kaplan-Meier survival curves (Fig. 2) were generated to analyze DFS and OS for high and low expression of FFAR4 in cholangiocarcinoma patients. The log-rank test showed that patients with high FFAR4 expression (29.0 months) had a significantly decreased DFS compared to those with low FFAR4 expression (37.0 months; $P<$ 0.05). Similarly, high FFAR4 expression (34.0 months) was associated with a shorter OS than was low FFAR4 expression (39.0 months; $P<$ 0.05) in patients with CCA.

Univariate analysis suggested that FFAR4 expression, histological grade, LNM, and TNM stage had significant prognostic influence on DFS and OS (Table 4). Multivariate survival analysis revealed FFAR4 expression as an independent predictive factor for DFS [hazard ratio (HR) 0.578; 95% confidence interval (CI) 0.239-0.817; $P<$ 0.05] and OS [hazard ratio (HR) 0.416; 95% confidence interval (CI) 0.181-0.923; $P<$ 0.05]. In addition, histological grade, LNM and TNM stage were also independent predictive factors for prognosis (Table 5).

4. Discussion

There is increasing evidence that dysregulation of FFAR4 plays an important role in tumorigenesis, migration and invasion. Fukushima et al. [14] found that enhanced FFAR4 expression in pancreatic cancer cells may play a role in stimulating pancreatic cancer cell motility, invasion and tumorigenesis by inhibiting expression of MMP-2. Wu et al. [15] reported that increased FFAR4 expression significantly induced tumor-associated progression of colorectal cancer tissue and cell lines and showed that FFAR4 signaling promotes angiogenic transition and motility in human colorectal cancer (CRC) cells. Moreover, activation of FFAR4 enhances the viability of CRC cells, inducing epithelial-mesenchymal transition. Notably, FFAR4 expression was also significantly increased in paraffin-embedded colorectal cancer tissues derived from patients, correlating with poor pathologic differentiation. Kleemann et al. [16] reported significant expression of the FFAR4 protein in 114 malignant melanoma tissue samples. A study by Serna-Marquez et al. [17] suggested that LA induces the migration and invasion of breast cancer cells through FFAR4 (GPR120)-EGFR and PI3K-/Akt-dependent pathways in the human breast cancer MDA-MB-231b cell line. However, Liu et al. [18] presented evidence that activation of FFAR4 in human prostate cancer cells initiates a pathway that inhibits growth factor-induced signaling and provides a novel mechanism for inhibiting cancer cell proliferation, and Kita et al. [19] observed that stimulation of GPR120 in lung cancer cells led to lower cell motility. These inconsistent findings suggest that FFAR4 may play different roles in different tumors.

Based on the present FFAR4 study, a preliminary analysis of FFAR4 expression in CCA and adjacent normal bile duct tissue was performed to determine whether there is a difference in expression and whether clinically and pathologically related features are associated. To the best of our knowledge, this study is the first to confirm FFAR4 expression in CCA and its clinical significance. FFAR4 is overexpressed in CCA tissues compared to adjacent tissues. Statistical analysis suggested that FFAR4 expression levels were higher in tumors with aggressive clinical and pathological features, such as poor tissue differentiation, neural invasion, lymph node metastasis, TNM staging, and preoperative serum CA19-9 levels.

EMT refers to a specific program of epithelial cell transition to a mesenchymal phenotype. Epithelial cells lose polarity via EMT and connection with the basement membrane. In addition, these cells obtain the capacity for higher invasion, migration, anti-apoptosis and degradation of the extracellular matrix [20]. This is the molecular basis of tumorigenesis, invasion and metastasis. Both E-cadherin, cytokeratin 7, cytokeratin 19, Snail-1 and vimentin are important protein indicators reflecting the process of EMT. The main markers of EMT transformation in tumors are low expression of E-cadherin and the high expression of Snail-1 and vimentin. E-cadherin is a cell adhesion molecule present in the epithelium that accumulates through organization of intracellular catenin and peptide proteins linked to the cytoskeletal actin. In addition, E-cadherin participates in a series of processes, such as cell adhesion, growth and proliferation, and maintains epithelial cell morphology and structure integrity. Snail-1 is a most important marker in the process of primary epithelial tumor cell transformation into mesenchymal cells with migratory ability. In the process of EMT, inhibition of E-cadherin expression by Snail-1 promotes tumor invasion and metastasis [21]. Vimentin, a major intermediate filament protein present in stromal cells, participates in the adhesion, migration, invasion and cell signaling of tumor cells by regulating interaction between cytoskeletal proteins and cell adhesion molecules. Cytokeratin (CK) is also an intermediate filament protein that is mainly expressed in epithelial cells. Due to the high tissue specificity and differentiation specificity of CK, this protein is widely used as a marker for various epithelial and tumor cells. CK7 and CK19 are called the CK of the bile duct because of their specific expression in bile duct epithelial cells [22]. Recent studies have shown that CKs, which are expressed in bile duct cells, undergo certain changes in bile duct injury and bile duct diseases. Bile duct cells and cholangiocarcinoma may additionally express either type of bile duct CK (CK19 or CK7) [23], and abnormal expression of CKs may be related to EMT. In this retrospective study, FFAR4 expression was observed to be negatively correlated with E-cadherin expression in CCA. In contrast, FFAR4 expression was positively correlated with CK7, CK19, Snail-1 and vimentin expression in CCA. These results suggest that FFAR4 overexpression may be closely related to expression of important target proteins in EMT. FFAR4 may also be involved in EMT, affecting the invasion and metastasis of CCA.

In terms of prognosis, our results show that patients with high FFAR4 expression and low FFAR4 expression have significantly shorter survival times. These findings suggest that high FFAR4 expression is associated with poor prognosis in patients with CCA and is promising for use as a valuable prognostic biomarker.

5. Conclusions

We acknowledge several limitations to this study. This study is a single retrospective study with a relatively small sample size; therefore, expression of FFAR4 in CCA was detected at a superficial level. Overexpression of FFAR4 may be related to the malignancy of CCA. However, the underlying mechanism by which FFAR4 promotes the invasion and metastasis of CCA cells is unclear. It is necessary to expand sample sizes to further confirm our findings and perform in vitro cytology and verification in animal models. In summary, CCA, a malignant tumor with strong invasion and metastasis properties, is usually diagnosed late, which contributes to a poor prognosis. As a newly discovered important G protein-coupled receptor, FFAR4 upregulation may play an important role in the development of CCA. In addition, our study showed that FFAR4 is associated with the EMT process in CCA. Moreover, FFAR4 may be an independent biomarker of poor prognosis of CCA. The mechanism of FFAR4 needs to be further researched, and FFAR4 is a promising new target for the early diagnosis of CCA and a biomarker for clinical drug treatment.

Footnotes

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No 81501354), the Natural Science Foundation of Anhui Province (No 1608085QH197), and the Programs for Science and Technology Development of Anhui Province in 2016 (No 1606c08234).

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary data

The supplementary files are available to download from https://dx-doi-org.web.bisu.edu.cn/10.3233/CBM-181358.

References

Rizvi

Khan

S.A.

Hallemeier

C.L.

Kelley

R.K.

and Gores

G.J.

, Cholangiocarcinoma – evolving concepts and therapeutic strategies, Nat Rev Clin Oncol 15 (2018), 95–111.

Valle

J.W.

Borbath

Khan

S.A.

Huguet

Gruenberger

Arnold

and Committee

E.G.

, Biliary cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up, Ann Oncol 27 (2016), v28–v37.

Weber

S.M.

Ribero

O’Reilly

E.M.

Kokudo

Miyazaki

and Pawlik

T.M.

, Intrahepatic cholangiocarcinoma: Expert consensus statement, HPB (Oxford) 17 (2015), 669–80.

Mansour

J.C.

Aloia

T.A.

Crane

C.H.

Heimbach

J.K.

Nagino

and Vauthey

J.N.

, Hilar cholangiocarcinoma: Expert consensus statement, HPB (Oxford) 17 (2015), 691–9.

Ichimura

Hasegawa

Kasubuchi

and Kimura

, Free fatty acid receptors as therapeutic targets for the treatment of diabetes, Front Pharmacol 5 (2014), 236.

Hara

Kashihara

Ichimura

Kimura

Tsujimoto

and Hirasawa

, Role of free fatty acid receptors in the regulation of energy metabolism, Biochim Biophys Acta 1841 (2014), 1292–300.

Hara

Hirasawa

Ichimura

Kimura

and Tsujimoto

, Free fatty acid receptors FFAR1 and GPR120 as novel therapeutic targets for metabolic disorders, J Pharm Sci 100 (2011), 3594–601.

D.Y.

and Olefsky

J.M.

, Omega 3 fatty acids and GPR120, Cell Metab 15 (2012), 564–5.

Houthuijzen

J.M.

, For Better or worse: FFAR1 and FFAR4 signaling in cancer and diabetes, Mol Pharmacol 90 (2016), 738–743.

10.

Zhai

L.L.

Cai

C.Y.

Huang

and Tang

Z.G.

, High-level expression and prognostic significance of matrix metalloprotease-19 and matrix metalloprotease-20 in human pancreatic ductal adenocarcinoma, Pancreas 45 (2016), 1067–72.

11.

Zhai

L.L.

Cai

C.Y.

and Tang

Z.G.

, Upregulated matrix metalloproteinase-2 and downregulated tissue factor pathway inhibitor-2 are risk factors for lymph node metastasis and perineural invasion in pancreatic carcinoma, Onco Targets Ther 8 (2015), 2827–34.

12.

Zhai

L.L.

Cai

C.Y.

and Tang

Z.G.

, Correlation and prognostic significance of MMP-2 and TFPI-2 differential expression in pancreatic carcinoma, Int J Clin Exp Pathol 8 (2015), 682–91.

13.

Chandrashekar

D.S.

Bashel

Balasubramanya

S.A.H.

Creighton

C.J.

Ponce-Rodriguez

Chakravarthi

and Varambally

, UALCAN: A portal for facilitating tumor subgroup gene expression and survival analyses, Neoplasia 19 (2017), 649–658.

14.

Fukushima

Yamasaki

Ishii

Tomimatsu

Takahashi

Hirane

Fukushima

Honoki

and Tsujiuchi

, Different roles of GPR120 and GPR40 in the acquisition of malignant properties in pancreatic cancer cells, Biochem Biophys Res Commun 465 (2015), 512–5.

15.

Wang

Zhao

Shi

Jin

Wan

Cheng

and Zhang

, Identification of G-protein-coupled receptor 120 as a tumor-promoting receptor that induces angiogenesis and migration in human colorectal carcinoma, Oncogene 32 (2013), 5541–50.

16.

Kleemann

Hrgovic

Ter-Nedden

Kleimann

Steinhorst

Harle

Muller

Kaufmann

Meissner

and Kippenberger

, Fatty acid receptor GPR120: A novel marker for human melanoma, Melanoma Res (2018).

17.

Serna-Marquez

Diaz-Aragon

Reyes-Uribe

Cortes-Reynosa

and Salazar

E.P.

, Linoleic acid induces migration and invasion through FFAR4- and PI3K-/Akt-dependent pathway in MDA-MB-231 breast cancer cells, Med Oncol 34 (2017), 111.

18.

Liu

Hopkins

M.M.

Zhang

Quisenberry

C.B.

Fix

L.C.

Galvan

B.M.

and Meier

K.E.

, Omega-3 fatty acids and other FFA4 agonists inhibit growth factor signaling in human prostate cancer cells, J Pharmacol Exp Ther 352 (2015), 380–94.

19.

Kita

Kadochi

Takahashi

Fukushima

Yamasaki

Uemoto

Hirane

Fukushima

Honoki

and Tsujiuchi

, Diverse effects of G-protein-coupled free fatty acid receptors on the regulation of cellular functions in lung cancer cells, Exp Cell Res 342 (2016), 193–9.

20.

De Craene and Berx

, Regulatory networks defining EMT during cancer initiation and progression, Nat Rev Cancer 13 (2013), 97–110.

21.

Solanas

Porta-de-la-Riva

Agusti

Casagolda

Sanchez-Aguilera

Larriba

M.J.

Pons

Peiro

Escriva

Munoz

Dunach

de Herreros

A.G.

and Baulida

, E-cadherin controls beta-catenin and NF-kappaB transcriptional activity in mesenchymal gene expression, J Cell Sci 121 (2008), 2224–34.

22.

Nakanuma

Harada

Ishikawa

Zen

and Sasaki

, Anatomic and molecular pathology of intrahepatic cholangiocarcinoma, J Hepatobiliary Pancreat Surg 10 (2003), 265–81.

23.

Liu

L.Z.

Yang

L.X.

Zheng

B.H.

Dong

P.P.

Liu

X.Y.

Wang

Z.C.

Zhou

Fan

Wang

X.Y.

and Gao

, CK7/CK19 index: A potential prognostic factor for postoperative intrahepatic cholangiocarcinoma patients, J Surg Oncol (2018).

Upregulated FFAR4 correlates with the epithelial-mesenchymal transition and an unfavorable prognosis in human cholangiocarcinoma

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Samples and clinicopathological data

2.2 Immunohistochemistry

2.3 Statistical analysis

Table 1 Differential expression of FFAR4 in CCA tissues and corresponding paracarcinoma tissues ( n = 98)

3.1 FFAR4 expression in CCA tissues and paracancerous tissue

Table 2 Correlations of FFAR4 expression with E-cadherin, vimentin, Snail-1, CK7 and CK19 in CCA

Table 3 Correlations between FFAR4 expression and clinicopathological characteristics in CCA

4. Discussion

5. Conclusions

Footnotes

Acknowledgments

Conflict of interest

Supplementary data

References

Table 1
Differential expression of FFAR4 in CCA tissues and corresponding paracarcinoma tissues ( $n=$ 98)

Table 2
Correlations of FFAR4 expression with E-cadherin, vimentin, Snail-1, CK7 and CK19 in CCA

Table 3
Correlations between FFAR4 expression and clinicopathological characteristics in CCA