Cancer-associated fibroblasts secrete FGF-1 to promote ovarian proliferation,migration,and invasion through the activation of FGF-1/FGFR4 signaling

Abstract

Ovarian cancer is the most lethal gynecologic malignancy, due to its high propensity for metastasis. Cancer-associated fibroblasts, as the dominant component of tumor microenvironment, are crucial for tumor progression. However, the mechanisms underlying the regulation of ovarian cancer cells by cancer-associated fibroblasts remain little known. Here, we first isolated cancer-associated fibroblasts from patients’ ovarian tissues and found that cancer-associated fibroblasts promoted SKOV3 cells’ proliferation, migration, and invasion. Fibroblast growth factor-1 was identified as a highly increased factor in cancer-associated fibroblasts compared with normal fibroblasts by quantitative reverse transcription polymerase chain reaction (~4.6-fold, p < 0.01) and ELISA assays (~4-fold, p < 0.01). High expression of fibroblast growth factor-1 in cancer-associated fibroblasts either naturally or through gene recombination led to phosphorylation of fibroblast growth factor receptor 4 in SKOV3 cells, which is followed by the activation of mitogen-activated protein kinase/extracellular signal–regulated protein kinase pathway and epithelial-to-mesenchymal transition–associated gene Snail1 and MMP3 expression. Moreover, treatment of SKOV3 cell with fibroblast growth factor receptor inhibitor PD173074 terminated cellular proliferation, migration, and invasion, reduced the phosphorylation level of fibroblast growth factor receptor 4, and suppressed the activation of mitogen-activated protein kinase/extracellular signal–regulated protein kinase pathway. In addition, the expression level of Snail1 and MMP3 was reduced, while the expression level of E-cadherin increased. These observations suggest a crucial role for cancer-associated fibroblasts and fibroblast growth factor-1/fibroblast growth factor receptor 4 signaling in the progression of ovarian cancer. Therefore, this fibroblast growth factor-1/fibroblast growth factor receptor 4 axis may become a potential target for the treatment of ovarian cancer.

Introduction

Ovarian cancer (OC) is a relatively scarce malignancy, accounting for only 1.5%, but it is the fifth most common cause of cancer deaths among women. ¹ Because of their anatomic location (within the peritoneal cavity), the lack of typical early symptoms, and detectable pre-invasive methods, over 60% of OCs are diagnosed at an advanced stage, and the 5-year survival rate is only about 45%.^1,2–4 According to the American Cancer Society estimates, there will be 22,280 new cases of OC diagnosed in US women and 14,240 deaths in 2016. ¹

Tumor microenvironment is biologically heterogeneous and contains various cell types including fibroblasts, immune and inflammatory cells, adipocytes, and endothelial cells, along with many cytokines and extracellular matrix (ECM). ⁵ Among these, cancer-associated fibroblasts (CAFs) are the most prominent cell types in the tumor microenvironment. Emerging evidence has highlighted the role of CAFs in promoting carcinogenesis and cancer progression in different cancer cell types, including OC.^6–9 However, the mechanism of CAF in OC pathogenesis and progression remains to be unraveled.

Moreover, recent studies have shown that CAF autocrine secretes a lot of growth factors and chemokines into the tumor microenvironment, such as stromal-cell-derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF), which may stimulate adhesion, motility, and escaping from the local growth control.^10–14 FGF is one of the growth factors secreted by CAFs, which execute diverse functions by binding to and activating members of the fibroblast growth factor receptor 4 (FGFR4) family, such as cell survival, proliferation, and migration in a variety of cell types.^15,16 To date, 22 FGF genes have been identified in human, which are differentially expressed in many tissues. ¹⁵ Among these FGFs, FGF-1 is a multifunctional factor involved in tumorigenesis, epithelial-to-mesenchymal transition (EMT), as well as migration and invasion. ¹⁷ However, the importance of FGF-1 in the interaction between CAFs and OC cell, as well as the mechanisms underlying the regulation of OC cells by CAFs, is yet unclear.

In this study, we show evidence that CAFs can induce phosphorylation of FGFR4 in ovarian carcinoma through secreting FGF-1, promoting tumor cellular proliferation, migration, and invasion. In addition, we delineated the FGF-1/FGFR4 and mitogen-activated protein kinase/extracellular signal–regulated protein kinase (MAPK/Erk) signaling pathways involved in ovarian tumor progress. Therefore, our studies suggest that FGF-1/FGFR4 axis may become a potential target for the treatment of OC patients.

Materials and methods

Human OC cell line SKOV3 was purchased from Cell Bank of Type Culture Collection, Chinese Academy of Science. Human recombinant FGF-1 protein was purchased from Sigma. PD173074 was ordered from Selleck. Anti–smooth muscle actin (α-SMA) and anti–fibroblast activation protein (FAP) were purchased from Abcam. IgG-FITC and IgG-TR were purchased from Santa. 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Genview. Fluoromount-G was purchased from Southern Biotech. 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazo-lium bromide (MTT) was purchased from Genview.

Isolation and culture of primary fibroblast cells

For this study, primary normal ovarian fibroblasts (NFs) and ovarian tumor tissues (CAFs) were isolated from OC patient tumor tissues following an approved procedure as described. ¹⁸ Briefly, human OC specimens and adjacent normal tissues, which are 5 cm far away from cancer lesions, were obtained. After several washings with sterile phosphate-buffered saline (PBS) (1.5 mM KH₂PO₄, 135 mM NaCl, 2.7 mM KCl, and 8 mM K₂HPO₄, pH 7.4), a 1 mm × 1 mm × 1 mm piece of tissues was minced with scissors and incubated on an orbital shaker with 10 mL of PBS and 10 mL of 0.25% trypsin/25 mM ethylenediaminetetraacetic acid (EDTA) at 37°C for 30 min. The solution containing cells in suspension was centrifuged at 1500 r/min for 5 min, and then NFs and CAFs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Scientific), at 37°C in a 5% CO₂ and 80% humidity incubator. Primary fibroblast cells at early passages (about three passages) were used for the experiments to minimize dedifferentiation and modification of the original phenotype.

SKOV3 cells were maintained in DMEM/F12 supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C, 5% CO₂. SKOV3 cells were treated with the culture medium (CM) from NFs and CAFs (cultured 3 days), as well as an equal volume of complete culture medium. A total of 100 ng/mL FGF-1 was added to the media to further confirm the function of FGF-1 which was secreted from CAFs cells, whereas 10 µM PD173074 was added to conditional medium treatment to block FGF-1 signaling in OC cells.

Immunocytochemistry analysis

Primary OC cell purification was verified by positive immuno-histochemical staining for α-SMA and FAP. First, the primary cell of CAFs and NFs was spread on slide glasses and fixed in 4% ice-cold paraformaldehyde plus 0.1% Triton X-100 for 20 min, followed by blocking with 2% bovine serum albumin in PBS for 1 h. Cells were incubated with anti-α-SMA (1:50) or anti-FAP (1:50) and then treated with secondary antibodies IgG-FITC (1:150) or IgG-TR (1:150), respectively. And then, coverslips mounted with Fluoromount-G. Immunofluorescent images were taken using a OLYMPUS IX71-F22FL/PH microscope (Japan, magnification ×200).

Quantitative real-time polymerase chain reaction analysis

Total RNAs were extracted with Trizol (Invitrogen) according to the manufacture’s instruction. Reverse transcription was conducted by using random primers in TaKaRa system (Dalian, China). Quantitative real-time polymerase chain reaction analysis (qRT-PCR) was performed by the SYBR green method using the iQTM SYBR^® Green Supermix (BioRad). The expression of target genes was calculated based on the cycle threshold (Ct) values comparative with a reference gene using formula 2^−ΔΔCt and normalized against the amount of U6 control in each sample. The following primers were used: α-SMA-forward: 5′-AGCTACCCGCCCAGAAACTA-3′, α-SMA-reverse: 5′-ATGATGCCGTGCTCGATAGG-3′; FAP-forward: 5′-TGTGCATTGTCTTACGCCCT-3′, FAP-reverse: 5′-GAGTATCTCCAAAGCATGGTTCTA-3′; FGF-1-forward: 5′-CAATGTTTGGGCTAAGACCTG-3′, FGF-1-reverse: 5′-GGCTGTGAAGGTGGTGATTT-3′; U6-forward: 5′-CTCGCTTCGGCAGCACA-3′, U6-reverse: 5′-AACGCTTCACGAATTTGCGT-3′. Three biological replicates were included in each experiment, and the data were statistically analyzed using Student’s t-test.

Detection of extracellular FGF-1 levels by ELISA

The conditioned media of CAFs, NFs, and SKOV3 cells were collected and the FGF-1 levels in cell medium were determined using a specific ELISA kit against FGF-1 according to the manufacturer’s instructions. The data are expressed as the target molecule (pg) per total protein (mg) for each sample, and each sample was repeated three times.

Western blot analysis

Total cell lysates were prepared with Mammalian Cell Lysis/Extraction Reagent (Sigma; 1% Triton X-100 and 1% protease inhibitor Cocktail were added) followed by incubating the plate on ice for 30 min. The lysates were collected and centrifuged at 12,000g for 10 min at 4°C, and the supernatant was recovered and placed into a fresh tube. Samples were boiled and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (12% acrylamide), and then proteins were blotted onto nitrocellulose membranes (Millipore) using electrotransfer system (mini-protein Tetra System; BioRad) in a buffer solution containing 193 mM glycine, 25 mM Tris (pH 8.3), and 20% methanol. The blots were then probed with P-tyrosine, FGFR4, ERK1/2, phospho-ERK1/2, p38, phosphor-p38, c-jun, phospho-c-jun, Ecad, Snail, MMP3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primary antibodies. Bound primary antibodies were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies and the bound conjugate was then detected using the enhanced chemiluminescence (ECL) detection system. Equal protein loading was assessed by the expression of GAPDH.

Cell proliferation, migration, and invasion assay

Cell proliferation assay

Cell proliferation was detected by MTT. First, SKOV3 and primary fibroblast cells were dispensed into 96-well plates (10³–10⁴cells/well in 200 µL medium), repeating nine wells. Then, the cells were incubated in an incubator with 5% CO₂ at 37°C for 12–24 h, and 20 µL MTT solution (5 mg/mL) was added. Incubation was continued for extra 4 h, and 150 µL dimethyl sulfoxide (DMSO) (Sigma) was added into every well. The samples were slightly shaken for 10 min to speed up the dissolution of crystals. The absorbance value of each well was detected by MTT at time points of days 0, 1, 2, and 3, and then the absorbance at 490 nm was detected. Growth was calculated as percent (%) = [((A/B) − 1) × 100], where A and B are the absorbance of treatment and control cells, respectively. All procedures were repeated at least three times.

Cell migration assay

To assess the effect of CAFs and inhibitor PD173074 on cell migration, we took a series of scratch wound assay. Briefly, 10⁵–10⁶ cells/well of SKOV3 were plated in a six-well plate and cultured under standard condition. When the cells reached confluent monolayer, a wound was made with a 100-µL pipette tip on cell monolayer. Then, cell debris was washed away with 1× PBS and cultured with DMEM supplemented with 5% FBS. Photographs were taken at time points of 0, 24, and 48 h. A total of four to six images were captured per time point per plate. Triplicate plates were completed at each time point.

Cell invasion assay

The invasive behaviors of the SKOV3 cells were evaluated by Transwell Matrigel invasion assay. A total of 10⁵ cells/well of SKOV3 cell in serum-free medium were seeded in the upper chamber coated with Matrigel and bottom wells were filled with CAFs-CM or NFs-CM or CM+FGF-1 or CAFs-CM+PD173074. After 24 h, invaded cells on the bottom surface were fixed with 4% paraformaldehyde and stained with crystal violet. For quantification, five fields (up, down, median, left, and right ×100) per filter were counted under a microscope. Results presented are representative of three independent experiments with duplicates.

Statistical analysis

Each experiment was repeated at least three times, and all the values were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using SPSS 12.0 statistical software. The differences between the groups were compared using Student’s t-tests. The value of p < 0.05 was considered statistically significant.

Results

Isolation and characterization of primary NFs and CAFs

To better understand the involvement of CAFs in the OC, we isolated fibroblasts from the ovarian tumor tissues (CAFs) and normal tissues (NFs). The fibroblast cell population was first verified by cell morphology. As shown in Figure 1(a), the CAFs and NFs cells exhibited typical fibroblast characteristics: a spindle-like shape morphology. Moreover, we carried out qRT-PCR and immunofluorescence staining assays to compare the expression levels of two CAF-specific biomarkers, including FAP and α-SMA. CAFs showed higher expression levels of FAP and α-SMA, both at messenger RNA (mRNA) level (Figure 1(b)) and protein level (Figure 1(a) and (c)), compared with NFs. The detailed contents of FAP and αSMA in CAFs and NFs are shown in Table 1. Altogether, these results indicated that we successfully isolated fibroblast cells from ovarian tumor tissues and normal tissues, and subsequent experiments were performed using CAFs and NFs cells.

Figure 1.

Isolation of fibroblast from human epithelial ovarian tissues. (a) Characterization of fibroblasts: primary human fibroblast isolated from tumor and normal issues was characterized by immunolabeling for myofibroblast marker fibroblast activation protein (FAP, red) and α-smooth muscle actin (α-SMA, green). FAP and α-SMA expression were upregulated in CAFs cells compared to NFs. Scale, 200 µm. (b) FAP and α-SMA mRNA levels measured by qRT-PCR (relative to NFs as a control). The data are presented as the mean ± SD. *p < 0.05 compared to control group. (c) Western blotting analysis of FAP and α-SMA protein in CAFs and NFs cells. Total actin was used for normalization. Data were representative of three independent experiments.

Table 1.

The detailed contents of FAP and SMA in CAFs and NFs.

Case	FAP/actin		α-SMA/actin
	NFs	CAFs	NFs	CAFs
1	0.59	1.16	0.34	0.86
2	0.71	1.13	0.26	0.91
3	0.65	0.9	0.19	0.77
4	0.41	0.84	0.51	1.01
5	0.67	1.06	0.4	1.08

FAP: fibroblast activation protein; α-SMA: α-smooth muscle actin; NFs: normal fibroblasts; CAFs: cancer-associated fibroblasts.

CAFs promoted the proliferation, migration, and invasion of OC cells

In order to investigate the effect of CAFs on OC cell proliferation, migration, and invasion in vitro, we cultured SKOV3 in culture medium, NF- and CAF-conditioned media (CM, NF-CM, CAF-CM), respectively. MTT assay showed that the cell numbers significantly increased for SKOV3 cells incubated with supernatants from CAF-CM but not from CM or NF-CM (Figure 2(a)). In addition, scratch wound assay was performed to examine whether conditioned media from CAFs and NFs could affect the cell migration rate. As shown in Figure 2(b), NF-CM had no significant effect on wound-healing time in comparison with culture medium. However, CAF-CM treatment greatly accelerated cell migration rates compared with culture medium. Transwell Matrigel invasion assay was then used to evaluate cell invasion. CAF-CM increased cell migration more than twofolds in SKOV3 compared with NF-CM and CM (Figure 2(c) and (d)). To summarize, these results elucidated that CAFs isolated from human OC tissue significantly promoted the proliferation, migration, and invasion of SKOV3 cells.

Figure 2.

Cancer-associated fibroblasts (CAFs) promote the proliferation, migration, and invasion of SKOV3 cells. (a) The effect of CAFs on SKOV3 cell growth was detected using MTT assay. (b) An increase in migration ability was measured using the migration wound-healing assay in SKOV3 cells upon treatment with CAFs medium for 0, 24, or 48 h. Two million cells were incubated in NF, CAF-conditioned media for 24 h and were then scraped with a plastic tip. Photomicrographs were taken at 0, 24, and 48 h after wound formation. (c) Crystal violet staining of SKOV3 cells that crossed the Matrigel-coated polycarbonate membrane of the Transwell chamber to detect the invasion of cells. (d) Number of cells that crossed the Transwell invasion chamber in CM, NFs, and CAFs.

CAFs promoted tumor growth through the paracrine effect of FGF-1

Billottet et al. ¹⁹ suggested that FGF-1 was involved in the cell invasion and tumorigenic behavior of carcinoma cells, which was associated with tyrosine phosphorylation ofseveral signaling molecules. However, the function of FGF-1 in the interaction between CAFs and OC cell is still unclear. Here, we first detected the mRNA and the protein levels of FGF-1 in CAFs, NFs, and OC cell lines (SKOV3). The results showed that CAFs exhibited a higher expression of FGF-1 both at mRNA level (~4.6-fold, p < 0.001) and at protein level (~4-fold, p < 0.001), in contrast to NFs (Figure 3(a) and (b)). To further confirm the function of FGF-1, we cultured SKOV3 cell with human recombinant FGF-1 and then examined the effect of FGF-1 on tumor progression. Compared to control medium, treatment with FGF-1 significantly increased cell proliferation (Figure 3(c)), as well as cell migration (Figure 3(d)) and invasion (Figure 3(e) and (f)) of SKOV3 cells. Altogether, these results suggest that CAFs derived from OC tissue may promote tumor growth through secretion of FGF-1.

Figure 3.

Cancer-associated fibroblasts (CAFs) predominantly secrete FGF-1 and can upregulate the MAPK/Erk pathway through the activation of FGFR4. (a) The relative mRNA levels of FGF-1 in NFs, CAFs, and SKOV3 cells. *p < 0.05. (b) CAFs, NFs, or SKOV3 cell culture conditioned medium was assessed for levels of FGF-1 by ELISA. Columns: mean (n = 3); bars: SEM. *p < 0.05 compared to controls (NFs and SKOV3). (c) Cell viability was detected by MTT assay for SKOV3 cells treated with human recombination FGF-1 for different incubation times. *p < 0.05 compared to the conditioned medium group. (d) The migration of cells cultured with FGF-1 protein was analyzed by a wound-healing assay. (e) Transwell invasion assays of SKOV3 cells cultured with human recombination FGF-1 protein. (f) Quantitative analysis of SKOV3 invasion. The graph shows the summarized data. Columns: mean (n = 3); bars: SEM. *p < 0.05 compared to controls. (g) The total and phosphorylation levels of FGFR4, Erk, p38, and c-Jun proteins in SKOV3 were analyzed by western blot.

Effects of FGF-1 on signaling transductions in the SKOV3 cells

Because FGFR4 is a receptor of FGF-1 ²⁰ and FGF-1 is highly expressed in CAFs, we assumed that the CAF-promoted cell proliferation, migration, and invasion are dependent on FGF-1/FGFR4 pathway. Here, we cultured SKOV3 cells with CM, NF-CM, CAF-CM, and CM+FGF-1, respectively, to measure the expression of FGFR4. The western blotting analysis showed that FGF-1 did not affect total FGFR4 expression. Interestingly, FGFR4 phosphorylation was obviously increased in CAF-CM groups, as well as in CM+FGF-1 groups, compared with cells incubated in CM and NF-CM (Figure 3(g)). Meanwhile, although no apparent differences were observed in total Erk/p38/c-Jun expression, the phosphorylation level of Erk/p38/c-Jun was enhanced in SKOV3 cells culture in CAF-conditioned media or conditional media containing recombinant FGF-1 compared with cells incubated in NF-conditioned media or condition media (Figure 3(g)).

PD173074 suppresses FGF-1-induced proliferation, migration, and invasion abilities of ovarain cancer cells

To examine the utility of PD173074 as a FGFR inhibitor and a therapeutic drug for OC, we treated SKOV3 cells with 10 µM PD173074 and counted the number of cells at 1, 2, and 3 days after treatment. The data showed that PD173074 suppressed the cell proliferation of SKOV3 in vitro (Figure 4(a)). Furthermore, using migration and Matrigel invasion assays, we found that PD173074 substantially reduced the ability of SKOV3 cells to invade and migrate (Figure 4(b)–(d)). Meanwhile, the phosphorylation level of FGFR4, detected by western blotting, was inhibited by PD173074 (Figure 4(e)). Subsequently, the levels of P-Erk, P-p38, and P-c-Jun decreased, but it does not change the levels of total Erk, p38, or c-Jun (Figure 4(e)).

Figure 4.

PD173074 suppresses FGF-1-induced proliferation, migration, and invasion abilities of ovarian cancer cells. (a) The effect of PD173074 on cancer cell growth was detected using MTT assay. All experiments were repeated three times, and representative results are shown here. *p < 0.05. (b) Scratch wound assay of SKOV3 cells was treated or not treated with 10 µM PD173074. (c and d) A decrease in invasive ability was measured using the Matrigel invasion assay in SKOV3 cells upon treatment with 10 µM PD173074. (e) PD173074 suppressed the MAPK/Erk pathway. The total proteins were extracted and the phosphorylation levels of FGFR4, Erk, p38, and c-Jun proteins in SKOV3 were analyzed by western blot.

Taken together, these data further indicate that CAFs promote tumor growth through FGF-1/FGFR4 and MAPK/Erk signaling pathway.

FGF-1 induced EMT in epithelial OC cell lines (SKOV3)

EMT has been reported to be related to cancer cell invasion and metastasis. Western blotting analysis (Figure 5(a)) showed that CAFs-CM or human recombinant FGF-1 led to the decrease in E-Cadherin expression (epithelial cell marker) along with the increase in Snail1 expression (EMT-associated transcription factor) in epithelial OC cell lines (SKOV3). In addition, invasion-related MMP3 genes also increased in CAF-CM and CM+FGF-1 groups (Figure 5(a)). Meanwhile, the expression of E-cadherin, Snail1, and MMP3 was analyzed upon treatment with 10 µM PD173074. The result showed that PD173074 treatment reduced Snail1 and MMP3 expression but induced E-cadherin expression (Figure 5(b)). These data implied that EMT was a potential mechanism for cell migration and invasion induced by CAF-CM.

Figure 5.

CAFs induced the MET. The expression of major epithelial markers (E-cadherin), MMP3, MMP7, and EMT master genes (Snail1) was analyzed by western blotting in SKOV3 cells upon treatment with (a) CM, NFs-CM, CAFs-CM, and CM+FGF-1 after 2 days and (b) treatment without or with 10 µM PD173074 for 2 days. Anti-β-actin expression was used as loading control.

Discussion

Recent studies have revealed that CAFs are the dominant component of tumor microenvironment and are found to be crucial for tumor progression. ⁶ Herein, we isolated CAFs from patients’ ovarian tissues and characterized by two specific biomarkers: α-SMA and FAP. Moreover, we demonstrated that CAFs can promote cell proliferation, migration, and invasion of OC cell line SKOV3. However, the mechanisms underlying the regulation of cancer cells by CAFs remain little known.

The role of CAFs in ovarian tumorigenesis has not fully been clarified yet. However, different types of growth factors and cytokines, such as SDF-1, HGF, epidermal growth factor (EGF), interleukin (IL)-6, and transforming growth factor (TGF)-β, implicated as autocrine and paracrine mediators of stromal–epithelial interactions are involved in tumor progression.^12,13,21,22 In this study, we observed that the CAFs secrete FGF-1 at a higher level than NFs. The function of FGF-1 was subsequently investigated in our study. We found that SKOV3 cells, treated with either CAFs medium or exogenous human combination FGF-1, showed an increase in tumor proliferation, migration, and invasion. These results suggested FGF-1 acts as a primary paracrine factor of CAFs in the OC progression in vitro. These findings were consistent with the result of Birrer et al. ²³

The FGF/FGFR signaling system plays critical roles in normal developmental and physiological processes, and it has also shown that dysregulation of this signaling axis plays significant roles in tumor development and progression. ²⁴ Several reports indicate that FGFR4, one of the key receptors for FGF-1, is highly expressed in cancer cells and has a crucial role in promoting cancer growth and metastasis. ²⁵ So, we hypothesized that the deregulation of the FGFR4 pathway is responsible for the FGF-1-mediated ovarian tumorigenesis. To test this hypothesis, we first detected the protein level of FGFR4 by western blotting. The results indicated that FGF-1 increased the expression of phosphorylation FGFR4 but not FGFR4. It is likely that several parallel pathways contribute to FGFR4-mediated tumorigenesis. MAPKs are evolutionary conserved enzymes, and accumulating evidence suggested that MAPK/ERK is a classical pathway in promoting the growth, proliferation, and migration of cancers. ²⁶ Klint and Claesson-Welsh ²⁷ reported MAPK pathways are likely downstream signal transducers of the FGFRs. Therefore, we tested some key factors by western blotting assay to determine whether the MAPK pathways were involved. The phosphorylation level of ErK, p38, and Jun was significantly upregulated, indicating that the MAPK signaling pathway was activated by FGF-1/FGFR4 signaling and it participated in ovarian tumorigenesis. Thus, FGF-1/FGFR4 may suggest a novel therapeutic target and its inhibition may be beneficial for the treatment of patients with OC.

The function of FGF-1/FGFR4 in ovarian was further explored by treatment of cells with PD173074, a small-molecule inhibitor of FGFR4. Western blotting analysis indicated that PD173074 treatment reduced the phosphorylation of FGFR4, ErK, p38, and Jun. What’s more, disruption of FGF-1/FGFR4 signaling byPD173074 suppressed cell proliferation, migration, and invasion. This result strongly indicated that PD173074 may decrease the malignant degree of ovarian cells. Other investigators have recently found that PD173074 inhibited glioblastoma cell proliferation by reducing phosphorylation of AKT and MAPK signaling. ²⁸ This is consistent with our results.

EMT is thought to have a major role in tumor progression. And CAFs crosstalk with cancer cells promoted tumor progression by establishing a favorable microenvironment for cell proliferation, migration, and invasion via the EMT. ¹⁰ In addition, it was reported that FGFR1 induced EMT through the transcription factor AP-1 in HOC313 cells. ²⁹ In this study, CAFs activated the expression of Snail1 and MMP3, as well as reduced the expression of E-cadherin. Moreover, PD173074 treatment reduced the expression of Snail and MMP3, but induced the expression of E-cadherin.

In summary, we propose that CAFs promote OC cell proliferation, migration, and invasion through paracrine FGF-1 factor. In addition, we demonstrated that FGF-1/FGFR4 signaling axis is likely to be one of many coordinate sets of factors that regulate the complex biology of a reactive stroma microenvironment in carcinomas. Our findings might facilitate the development of diagnostics and therapeutics against OC. Understanding the molecular mechanism of CAF may help to explore new promising therapeutic strategy for the treatment of OC. However, the specific mechanism for the downstream of FGF-1/FGFR4 remains to be further studied. We will continue to work on it to address these problems.