Sprouty4 correlates with favorable prognosis in perihilar cholangiocarcinoma by blocking the FGFR-ERK signaling pathway and arresting the cell cycle

Abstract

Background: Perihilar cholangiocarcinoma (PHCC) is the most common subtype of cholangiocarcinoma(CCA). We previously investigated the expression pattern of Sprouty(SPRY) in intrahepatic cholangiocarcinoma(ICC), but the expression and clinical significance of SPRY family members in PHCC are still unknown.

Methods: The expression of SPRY family members(SPRY1-4) was detected in different subtypes of CCA and corresponding adjacent tissues. SPRY4 expression in 142 cases of PHCC was detected by immunohistochemistry, and its clinical significance was evaluated using univariate and multivariate analyses. The functions of SPRY4 in the FGFR-induced proliferation and migration of PHCC cells were investigated through in vitro and in vivo experiments. We further investigated the effects and mechanisms of SPRY4 on FGFR-induced ERK phosphorylation and cell cycle distribution in the presence of FGFR and ERK inhibitors.

Findings: SPRY4 was the only SPRY family member associated with PHCC prognosis, and it was identified as an independent factor predicting favorable prognosis. In PHCC, SPRY4 expression was extensively associated with FGFR2, and its expression can be induced by ectopic FGFR2 activation. Through in vitro and in vivo experiments, we demonstrated that SPRY4 suppressed FGFR-induced proliferation and migration by inhibiting ERK phosphorylation. Moreover, SPRY4 knockdown was shown to decrease the percentage of cells in the G1 phase and promote the percentage of cells in the S and G2/M phases by increasing cyclin D1 expression, which also required FGFR-induced ERK phosphorylation.

Interpretation: High expression of SPRY4 was an independent biomarker of favorable prognosis in PHCC. SPRY4 expression can be induced by ectopic FGFR2 activation in PHCC. SPRY4 arrested the cell cycle at G1 phase and suppressed FGFR-induced proliferation and migration by inhibiting ERK phosphorylation, indicating that SPRY4 may be a potential therapeutic target in PHCC.

Keywords: Cell cycle; Perihilar cholangiocarcinoma; Prognosis; Proliferation; Sprouty 4.

Fig. 1

Expression of SPRY family members in ICC, PHCC, DCC and adjacent normal tissues. (a) Relative mRNA levels of SPRY family members in ICC (n = 12), PHCC (n = 24) and DCC (n = 12) tissues and adjacent normal tissues were detected by qRT-PCR. Results were analyzed using the 2^−∆CT method with GAPDH as an internal control.* and ** represent P < 0.05 and P < 0.01, respectively, calculated by paired t-test between the indicated subgroups. (b) Relative mRNA levels of SPRY family members between PHCC tumor and adjacent tissues (n = 24) compared with paired t-tests. Results were analyzed using the 2^−∆CT method with GAPDH as an internal control. Results were analyzed with paired t-test. (c) Representative IHC images of SPRY family members in human PHCC tissues. Scale bar: 50 μm.

Fig. 2

Correlation between overall survival rates, SPRY members and clinicopathological factors. (a)–(c) Expression of SPRY1 (a), SPRY2 (b) and SPRY3 (c) displayed no prognostic significance. (d) Low expression of SPRY4 was significantly correlated with low overall survival rates in PHCC patients. (e)–(h) Advanced patient age (e), positive lymph node metastasis (f), positive distant metastasis (g) and advanced TNM stage (h) were all associated with poor prognosis. Results were analyzed using the Kaplan–Meier method, and the statistical significance between groups was assessed using the log-rank test.

Fig. 3

SPRY4 expression was associated with FGFR2 in PHCC (a) Representative IHC images of FGFR family members in human PHCC tissues. Scale bar: 50 μm. (b) Correlations between overall survival rates and FGFR members in PHCC patients. The statistical significance between groups was assessed using the log-rank test. (c) The correlation of IHC score between SPRY4 expression and FGFR family. The linear regression was analyzed with Pearson method. (d) The IHC score of SPRY4 in patients with low and high expression of FGFR members. P value was analyzed with student's t-test.

Fig. 4

SPRY4 suppressed the proliferation and migration of PHCC cells. (a) Left: SPRY4 protein expression in 8 pairs of PHCC and adjacent tissues were detected by western blotting. Right: quantification of western blots. Results were analyzed with paired t-test and shown with ±S.D. (b) SPRY4 expression was detected in ICC cell lines RBE and HCCC-9810, and PHCC cell lines QBC939 and FRH0201. (c) Knockdown of SPRY4 in QBC939 cells, and overexpression of SPRY4 in FRH0201 cells with lentivirus infection was verified by western blotting. (d) and (e) Proliferation of QBC939 cells was detected with CCK-8 (d) and colony formation assays (e) after silencing SPRY4 in QBC939 cells and overexpressing SPRY4 in FRH0201 cells in normal medium with 10% FBS. (f) and (g) Migration of QBC939 cells was assessed with transwell assay (g). After SPRY4 knockdown, cells were seeded in the upper transwell chamber and incubated for 24 h, with FBS in the lower chamber. (g) Wound healing assay was applied to evaluate migration of QBC939. 24 h after a scratch in the cell monolayer, the wound size was measured again. Wound closure percentage was calculated by (1 – [final wound size / initial wound size]) × 100. In d, e, f and g, ** and *** represented P < 0.01 and P < 0.001 by Student t-test, between scrambled/vector groups and SPRY4-overexpressing/silencing groups (h) Xenografts were established in nude mice with stable QBC939 cells with SPRY4 knockdown or control cells. (i) and (j) SPRY4 knockdown increased the volume(i) and weight (j) of xenograft tumors. Tumor diameter was measured every 3 day. In d, e, f, g, i and j, ** and *** represented P < 0.01 and P < 0.001 by Student's t-test, between the indicated groups.

Fig. 5

Down-regulation of SPRY4 expression promoted FGF2-mediated proliferation and migration. (a) Proliferation of QBC939 cells was measured with CCK-8 assay in medium containing FGF2 (50 ng/ml) and 1% FBS after SPRY4 knockdown. (b) After starvation in serum-free medium for 6 h, QBC939 cells were incubated with FGF2 (50 ng/ml), 1%FBS and/or 100 nM AP24534 for 1–4 days, and cell proliferation was detected with CCK-8 assay. (c) Left panel: representative images of migrated cells with/without 100 nM AP24534 for 24 h. Lower chamber contained 50 ng/ml FGF2 and 5% FBS. Right: statistical analysis of cells undergoing different stimulation. (d) Xenografts were established in nude mice with stable QBC939 cells infected with shSPRY4 or scrambled shRNA in the presence or absence of AP24534 at 10 mg/kg/day via intraperitoneal injection. (e and f)AP24534 decreased the volume and weight of xenograft tumors, which had been promoted by SPRY4 knockdown. In a, c, e and f, *, ** and *** represent P<0.05, P<0.01 and P<0.001, respectively. The statistical significance was analyzed with Student's t-tests or one-way ANOVA.

Fig. 6

SPRY4 knockdown promoted the progression of PHCC by enhancing FGF2-mediated ERK phosphorylation. (a) Left: SPRY4 expression was silenced, and ERK phosphorylation was detected with western blotting. Right: quantification of p-ERK expression in the left panel. (b) ERK phosphorylation in QBC939cells peaked after 5 min of stimulation of 10 ng/ml FGF2. (c) Left: the FGFR inhibitor AP24534 antagonized ERK phosphorylation induced by SPRY4 knockdown. QBC939 cells were incubated in serum-free medium with AP24534 (100 nM) for 6 h and then stimulated with FGF2 (10 ng/ml) for 10minutes. Right: quantification of p-ERK levels in the left panel. (d) Proliferation was detected after QBC939 cells were starved in serum-free medium for 6 h and cultured in medium containing 50 ng/ml FGF2 and 1%FBS, in the presence or absence of 100 nM ulixertinib. (e) QBC939 cells were starved and incubated with 1 µM ulixertinib and 50 ng/ml FGF2 for 24 h. Left: representative images of migrated cells. Right: statistical analysis of cells undergoing different stimulation. (f) SPRY4-overexpressing QBC939 cells were injected subcutaneously for xenografts with 3 mg/kg/day FGF2 via intraperitoneal injection. (g) and (h) SPRY4 overexpression decreased tumor volume and weight, while FGF2 addition reversed the effect. In a, c, d, e, g and h, ** and *** represent P < 0.01 and P < 0.001, respectively, between the indicated subgroups. The significance was calculated by Student's t-test.

Fig. 7

SPRY4 arrested PHCC cells in the G1 phase by decreasing Cyclin D1 expression. (a) SPRY4 expression did not influence apoptosis in QBC939 in response to 50 ng/ml FGF2. Left: representative images of apoptotic cells detected using flow cytometry. Right: proportions of apoptotic cells. (b) SPRY4 expression had no effect on the expression of BAX, Bcl-2 and Caspase-3. Left: expression levels of BAX, Bcl-2, Caspase-3 and cleavage Caspase-3 were detected by western blotting after SPRY4 knockdown and 50 ng/ml FGF2 stimulation. Right: quantification of cleaved Caspase-3 in QBC939 cells. In A and B, N.S. represents not significant. (c) Left: proportions of QBC939 cells in different phases of the cell cycle detected using flow cytometry in the presence of 50 ng/ml FGF2. Right: FGF2 stimulation decreased the proportion of cells in the G1 phase. (d) Left: proportions of QBC939 cells in different phases of the cell cycle were detected in the presence of 50 ng/ml FGF2 and/or 100 nM AP24534. Right: proportion of cells in the G1 phase was decreased by SPRY4 knockdown and increased by 100 nM AP24534 incubation, and that of cells in the S and G2/M phases displayed the opposite tendency. (e) Expression levels of Cyclin A2, B1 and D1 in QBC939 cells were detected with/without AP24534 incubation after SPRY4 knockdown. (f) Left: QBC939 cells were incubated in 1 μM ulixertinib for 24 h, and the cell cycle distribution was detected. Right: ulixertinib increased the percentage of cells in the G1 phase and decreased the percentage of cells in the S and G2/M phases. (g) Expression levels of Cyclin A2, B1 and D1 in QBC939 cells were detected after incubation in 1 μM ulixertinib for 24 h. (h) and (i) mRNA (h) and protein (i) levels of cell cycle-related proteins in subcutaneous xenograft tumors established with stable SPRY4 knockdown cells were detected using qRT-PCR and western blotting. (j) Left: levels of cell cycle-related proteins in subcutaneous xenograft tumors treated with AP24534 were detected. Right: Cyclin D1 expression was increased by SPRY4 knockdown and decreased by AP24534 treatment. In D, F, H and J, *, ** and *** represent P < 0.05, P < 0.01 and P < 0.001, respectively, between the indicated subgroups. Significance was calculated with Student's t-test.