Sprouty2 suppresses progression and correlates to favourable prognosis of intrahepatic cholangiocarcinoma via antagonizing FGFR2 signalling

Abstract Fibroblast growth factor receptor 2 (FGFR2) was demonstrated to correlate to the progression and prognosis of intrahepatic cholangiocarcinoma (ICC) by numerous evidences. However, as a well‐recognized suppressor of FGFR2 signalling, the clinical significance of Sprouty (SPRY) family of ICC has not been investigated. In our study, the expressions of SPRY1‐4 in 20 pairs of fresh tumour tissues were detected with qPCR, and in 108 cases of paraffin‐embedded tissues with immunohistochemistry. The prognostic value of SPRY family in ICC was estimated with univariate analysis and multivariate analysis. As a result, SPRY2 was identified as an independent prognostic biomarker predicting favourable prognosis of ICC. High SPRY2 expression was correlated with good differentiation of ICC. With silencing SPRY2 expression, we demonstrated that SPRY2 could suppress FGFR2‐induced ERK phosphorylation, migration, invasion and epithelial‐mesenchymal transition (EMT) under FGF1 stimulation. By overexpressing SPRY2‐wide type or SPRY2‐Y55F, the tyrosine‐55 of SPRY2 was demonstrated to be essential in suppressing ERK phosphorylation, tumour invasion and EMT of ICC cells. In conclusion, SPRY2 was correlated with favourable prognosis of ICC via suppressing FGFR2‐induced ERK phosphorylation, invasion and EMT. The phosphorylation of SPRY2‐Y55 was required in this tumour‐suppressing function of SPRY2.

and FGFR2-CCDC6, [3][4][5] which could activate mitogen-activated protein kinases (MAPK), tumour growth and in vivo tumorigenesis via stimulating the kinase activity of FGFR2. 3 With Integrated genomewide and whole transcriptome sequence analyses, another independent study also demonstrated that FGFR2 downstream signalling pathway was ectopically activated and could be a putative therapeutic target. 6 The autocrine loop of FGFR activation is also demonstrated to promote cholangiocarcinoma progression. 7 In our previous study, we also proved that FGFR4 overexpression correlated with poor prognosis of ICC via promoting proliferation and invasion. 8 Fibroblast growth factor receptors are stimulated by binding with FGFs and function as serine/threonine kinases by activating downstream Ras-MAPK-ERK pathway. During this process, sprouty (SPRY) family, comprising of four isoforms (SPRY 1 -4), are the key feedback inhibitors of FGFR-induced Ras-MAPK-ERK pathway. 9 SPRY can inhibit ERK phosphorylation via modulating different levels of the FGFR-RAS pathway. 10 For example, SPRY2 evidently binds to growth factor receptor-bound protein 2 (GRB2), thereby negatively regulating downstream signalling of FGFR. 11 Deregulation or dysfunction of SPRY was proved to result in pathological conditions such as oncogenesis or cancer progression including breast cancer, hepatocellular carcioma and prostate cancer, etc. [12][13][14][15][16][17][18] Recently, we demonstrated that SPRY2 was correlated with favourable prognosis of gastric adenocarcinoma via suppressing FGFR2-induced ERK phosphorylation and cancer progression. 19 Although SPRY family has a distinct feedback inhibitory effect on FGFR signalling, and FGFR2 was demonstrated to promote ICC progression in numerous previous studies, the expression and clinical significance of SPRY2 family in ICC has never been elucidated. Here we detected the expression of SPRY family in 108 paraffin-embedded ICCs and 20 pairs of fresh ICC tissues, and further evaluated the clinical significance of SPRY2 by analyzing the correlation between SPRY expression, tumour progression and prognosis.

| Tissue microarrays and immunohistochemistry
The tissue microarrays (TMA) of formalin-fixed and paraffinembedded tissue sections were made as to previous report. 21 Before immunohistochemistry (IHC) detection, haematoxylin and eosin staining were performed to confirm the histological characterization of all samples. The protocol of IHC staining was described in detail before. 22 The results of IHC were evaluated independently by two senior pathologists unaware of the clinical information. The IHC results were semi-quantified by the IHC score, which was comprised of the score for staining intensity and the score for percentage of stained cells. The score for staining intensity was defined as negative (0), weak (1), moderate (2) and strong (3). The percentage of stained cells was scored as 1, <10% of cells were positive; 2, 10%-50% of cells were positive; 3, >50% of cells were positive. The final IHC score was the product of the score for staining intensity multiplied by the score for percentage of stained cells, which ranged from 0 to 9. The score with the highest summary of sensitivity and specialty in ROC curve was set as cut-off, which divided the cohort into low and high expression level. The cut-offs of SPRY1,2,3,4 were 3.5, 4.5, 3.5 and 4.5, respectively.

| RNA extraction and RT-PCR
The total mRNA of fresh tissues were extracted by Trizol agent High expression of SPRY2 is significantly associated with low overall survival rates, while the other three SPRY members had no remarkable prognostic significance ATTGAGCGGTTTG-3′ and reverse 5′-GGTCAATGGGTAGGAT GGTG-3′.

| Plasmid construction and transfection
SPRY2-Y55F mutation was generated with a Quikchange mutagenesis kit (Stratagene, La Jolla, CA) as previously report and verified by DNA sequencing. 23

| Expression of SPRY family in ICC
The mRNA levels of SPRY family, including SPRY1-4, were detected with RT-PCR in 20 pairs of ICC tissues and adjacent tissues ( Figure 1A). SPRY2 had relatively higher mRNA level compared with other SPRY members and its mRNA level in adjacent tissues was significantly higher than in tumour tissues, suggesting the potential tumour-suppressing role of SPRY2 in ICC oncogenesis. The expressions of SPRY members were further investigated with IHC in 108 paraffin-bedded specimens. According to the cutoff determined by ROC curve, the cohort was divided into the high expression and low expression group ( Figure 1B-E). In ICC tissues, the expression of SPRY1-4 was mostly observed in cytoplasm.

| Prognostic value of SPRY family in ICC
The prognostic value of SPRY1-4 was evaluated with both univariate analysis and multivariate analysis. Univariate analysis was first performed to screen the prognostic factors with Kaplan-Meier method (Table 1)

| Correlation between SPRY2 and clinicopathological factors
The correlation between SPRY2 and clinicopathological factors was analysed with Chi-Square test to screen the potential tumour progression processes influenced by SPRY2 (Table 2). Low expression of SPRY2 was significantly correlated with poor differentiation (P = 0.036) and positive lymphatic invasion (P = 0.013), which suggested that SPRY2 may be involved in the process of ICC invasion.
TNM stage was also associated with SPRY2 expression, and this may be the secondary consequence because of the correlation between SPRY2 and N stage.

| SPRY2 expression is associated with differentiation of ICC
With Chi-Square test, we demonstrated that poorly differentiated cases appeared to have lower SPRY2 expression ( Figure 3A), so we further stratified the test cohort according to tumour differentiation and compared the SPRY2 expression. Consequently, ICC with good differentiation had significantly higher SPRY2 expression compared with ICC with poor differentiation ( Figure 3B).
Moreover, we compared the SPRY2 mRNA level of different XU ET AL.
| 5603 differentiation in fresh ICC tissues. Patients with poorly differentiated ICC had lower mRNA level of SPRY2 compared with those with well-differentiated ICC. However, the SPRY2 levels in adjacent tissues had no significant difference ( Figure 3C). These results suggested that overexpression of SPRY2 could correlate to better differentiation of ICC.

| SPRY2 suppresses EMT and invasion of ICC
In the clinical analyzation of Table 2, we observed that SPRY2 expression was significantly associated with ICC differentiation.
As tumour differentiation is an important factor determining tumour invasion, we investigated the role of SPRY2 on ICC cell migration and invasion. With wound healing assay, we proved that FGFR2 knockdown notably impaired the tumour cell migration while silencing SPRY2 could promote migration of RBE or HuCCT-1 cells ( Figure 5A EMT is a generally accepted process inducing invasion and migration and SPRY2 has been reported to influence EMT process in ovarian cancer 24 ; therefore, the influence of SPRY2 on EMT process of ICC was investigated. FGFR inhibitor AP24534 could suppress FGF1-induced expression of Slug and Snail, and rescue the decrease in E-cadherin, indicating AP24534 could attenuate the FGF1-induced EMT process of ICC ( Figure 5E). With knocking down FGFR2 or SPRY2, we demonstrated that FGFR2 was necessary for the FGF1induced EMT and SPRY2 could repress the FGFR2-mediated EMT process ( Figure 5F,G).

| Phosphorylation of tyrosine 55 is essential for SPRY2 suppressing ERK phosphorylation and EMT
According to previous studies, The tyrosine55 of SPRY2 is considered to be responsible for binding with Grb2 and recognizing FGF-specific ERK-activating pathway, 25

| DISCUSSION
The breakthroughs in precise classification by molecular profile  25 Due to the redundancy of SPRY family and their multitask mechanism to suppress ERK phosphorylation, the SPRY is generally considered to be cell-and context-specific. Our finding that only SPRY2 in SPRY family correlates to favourable prognosis also supports this suggestion. It is not the main focus to dig out the binding protein of SPRY2 was not identified and the underlying molecular mechanism in ICC, but it is certainly an interesting topic worthy of further study.
In conclusion, we investigated the expression of SPRY family in

CONFLI CT OF INTEREST
None.
F I G U R E 6 Y55 phosphorylation of SPRY2 was essential for repressing FGF1induced ERK phosphorylation and EMT. A, The phosphorylation of SPRY2 increased with FGF1 stimulation. RBE cells were treated with 10 ng/mL FGF1 and/or 1 μmol/L AP24354 for 30 min and lysed. SPRY2 antibody binding with Protein A/G was used to immunoprecipitate SPRY2 and common phosphorylation antibody pY20 was applied to detect the phosphorylation state of SPRY2. B, SPRY2-Y55 was essential for antagonizing ERK phosphorylation. RBE cells were transfected with plasmid carrying wild-type SPRY2 or SPRY2-Y55F 24 h before 10 ng/ mL FGF1 stimulation. ERK phosphorylation of cells transfected with SPRY2-WT was significantly lower than SPRY2-Y55F. C, SPRY2-Y55 was required for suppression of EMT by SPRY2. With FGF1 stimulation, RBE cells transfected with wild-type SPRY2 had lower E-cadherin, higher Snail and Slug expression compared with cells transfected with SPRY2-Y55F. D-E, SPRY2-Y55 was required for SPRY2-repressing migration and invasion of ICC cells. 24 h after transfected with wild-type SPRY2 or SPRY2-Y55F, RBE cells were stimulated with FGF1 for 12 h. Cell migration and invasion was evaluated with would healing assay (D) and transwell assay (E), respectively. *P < 0.05 compared with control group, # P < 0.05