FGF16 promotes invasive behavior of SKOV-3 ovarian cancer cells through activation of mitogen-activated protein kinase (MAPK) signaling pathway

Abstract

Uncontrolled cell growth and tissue invasion define the characteristic features of cancer. Several growth factors regulate these processes by inducing specific signaling pathways. We show that FGF16, a novel factor, is expressed in human ovary, and its expression is markedly increased in ovarian tumors. This finding indicated possible involvement of FGF16 in ovarian cancer progression. We observed that FGF16 stimulates the proliferation of human ovarian adenocarcinoma cells, SKOV-3 and OAW-42. Furthermore, through the activation of FGF receptor-mediated intracellular MAPK pathway, FGF16 regulates the expression of MMP2, MMP9, SNAI1, and CDH1 and thus facilitates cellular invasion. Inhibition of FGFR as well as MAPK pathway reduces the proliferative and invasive behavior of ovarian cancer cells. Moreover, ovarian tumors with up-regulated PITX2 expression also showed activation of Wnt/β-catenin pathway that prompted us to investigate possible interaction among FGF16, PITX2, and Wnt pathway. We identified that PITX2 homeodomain transcription factor interacts with and regulates FGF16 expression. Furthermore, activation of the Wnt/β-catenin pathway induces FGF16 expression. Moreover, FGF16 promoter possesses the binding elements of PITX2 as well as T-cell factor (Wnt-responsive), in close proximity, where PITX2 and β-catenin binds to and synergistically activates the same. A detail study showed that both PITX2 and T-cell factor elements and the interaction with their binding partners are necessary for target gene expression. Taken together, our findings indicate that FGF16 in conjunction with Wnt pathway contributes to the cancer phenotype of ovarian cells and suggests that modulation of its expression in ovarian cells might be a promising therapeutic strategy for the treatment of invasive ovarian cancers.

Keywords: Cell Proliferation; Fibroblast Growth Factor (FGF); Homeobox; Invasion; Ovarian Cancer; β-Catenin.

FIGURE 1.

The expression of FGF16 and PITX2 is up-regulated in human ovarian cancer. Q-PCR analysis was performed in normal ovaries (n = 20) and ovarian cancer (n = 25) samples with primers of FGF16 (a), CTNNB1, AXIN2 (b), and PITX2A/B/C (c). * represents p < 0.05. d and e, immunohistochemical analysis of FGF16 and PITX2 expression was performed in ovarian tissue sections of normal individual and high grade adenocarcinoma patients. Immunostaining of FGF16 (d) and PITX2 (e) followed by Alexa Fluor-488 (green) in normal (A) and cancer (B) tissues was shown. The nuclei stained with DAPI and the merged images were also shown as indicated. The negative control images represent the staining in presence of secondary antibody and DAPI but without primary antibodies. The images were taken at the same exposure time. Scale bar, 10 μm.

FIGURE 2.

PITX2 binds to FGF16 promoter and regulates its expression in human ovarian cancer cells. ChIP with SKOV-3 (a) and OAW-42 (b) cells followed by the PCR showed the amplification of the FGF16 promoter from the chromatin input and from PITX2-IP DNA as indicated in the lanes. The PITX2 antibody used in a and b recognizes all three isoforms of PITX2. c, primers of an unrelated gene did not show amplification from PITX2-IP DNA. d, ChIP with PITX2 isoform-specific antibodies was performed in SKOV-3 cells followed by PCR showed amplification of FGF16-promoter from the IP-DNA. e, upon transient transfection of either PITX2A or -B or -C, SKOV-3 cells were used for ChIP with isoform-specific antibodies followed by PCR with the primers used in a. f, the expression of PITX2A, -B, and -C was checked by Western blot analysis in control (UT) and transfected (T) cells with specific antibodies. g, three isoforms of PITX2 (PITX2A/B/C) were transiently transfected into the cells followed by isolation of RNA. The expression of PITX2 isoforms (g, for SKOV-3; i, for OAW-42) and FGF16 (h, for SKOV-3; j, for OAW-42) were checked in the PITX2-overexpressed cells by Q-PCR using specific primers. k, SKOV-3 cells were transiently transfected with 20 nm non-targeting (NT)-siRNA and PITX2-siRNA followed by Western blot analysis of PITX2 and α-tubulin proteins (top). A Q-PCR assay was also performed from the RNAs isolated from those cells using primers of PITX2 and FGF16 (bottom). Relative gene expression is indicated as -fold change in the y axis (mean ± S.E.). * represents p < 0.05. l, schematic diagram of the FGF16 promoter with the PITX2-specific bicoid (^) and bicoid-like (+) elements (not in scale), which was cloned into pGL3-basic vector. m, CHO cells were transiently co-transfected with the pGL3-FGF16 construct alone or in combination with PITX2 isoforms followed by luciferase assay. The FGF promoter activity was calculated in terms of -fold change in PITX2-overexpressed cells compared with empty vector-transfected cells, after normalization with renilla luciferase activity. The statistical analysis is done as described under “Materials and Methods.”

FIGURE 3.

The expression of FGF16 is induced by activation of the Wnt pathway. 16-h serum-starved SKOV-3 (a) and OAW-42 (b) cells were treated with 20 mm NaCl or LiCl followed by isolation of RNA and Q-PCR analysis of FGF16 and CCND1. c and d, SKOV-3 and OAW-42 cells were transiently transfected with β-catenin-siRNA or non-targeting (NT)-siRNA as the control followed by Western blot analysis of β-catenin and α-tubulin (c, i). RNA isolation and Q-PCR assay was also performed with primers of β-catenin, FGF16 and CCND1 (c,ii, and d). The statistical analysis was done as described under “Materials and Methods.” * represents p < 0.05. e, a schematic representation of 1.4 kb upstream sequence of FGF16 promoter shows the canonical TCF elements (■), PITX2-specific bicoid and bicoid-like elements (not in scale). The solid arrows mark the positions of primers that were used to amplify the region during ChIP-PCR. The positions of respective cis elements were marked with respect to the TSS (plain arrow). f, SKOV-3 and OAW-42 cells were treated with or without 20 mm LiCl and then subjected to ChIP with β-catenin antibody. IP, immunoprecipitation. PCR with the primers of FGF16 shows the amplification from chromatin input (lane B) and from the IP-DNA (lanes C and D). g, ChIP with PITX2 antibody followed by PCR with the primers of FGF16 used in f showed the amplification from the input and the immunoprecipitation-DNA.

FIGURE 4.

The expression of FGF16 is synergistically up-regulated by PITX2, LEF-1, and β-catenin. a and b, 1 μg each of LEF-1, β-catenin, and PITX2A/B/C expression clones were transiently transfected alone or in different combinations in SKOV-3 (a) and OAW-42 (b) cells, and Q-PCR performed with the RNAs isolated from those cells showed the expression level of FGF16 mRNA. The expression of LEF1and β-catenin was checked by Western blot analysis in control (UT) and transfected (T) cells (a). c, SKOV-3 and OAW-42 cells were co-transfected with β-catenin siRNA alone or in combination with LEF-1 and PITX2A, -B, -C expression clones followed by isolation of RNA and Q-PCR with the primer of FGF16. d, cells pretreated with DKK1 were transiently transfected with PITX2A/B/C individually, and Q-PCR performed with the RNAs isolated from those cells showed the expression level of FGF16 mRNA. Relative gene expression is indicated as -fold change in the y axis (mean ± S.E.). e–g, a 1.4-kb sequence of FGF16 promoter was cloned (with primers marked with hollow arrows in Fig. 3e) in pGL3-basic vector. The CHO cells were co-transfected with this construct and LEF-1, β-catenin, and PITX2A/B/C expression clones in different combinations, and luciferase reporter assay was performed. The relative luciferase activities are shown as the mean -fold activation compared with the pGL3-basic vector after normalization with renilla luciferase activity. h, the WT and mutated sequences of PITX2 and TCF binding elements of the FGF16 promoter are shown, and the respective deletion/substitution mutations has been schematically presented for each mutated clone, Mut1, Mut2, Mut3, and Mut4. WRE, Wnt response element. i–m, the -fold change in luciferase reporter activities for each WT and Mut1–4 clone is shown upon transient transfections in given combinations in CHO cells. The -fold change in overexpressed cells was compared with empty vector-transfected cells after normalization with renilla luciferase activity. The statistical analysis was done as described previously. * represents p < 0.05.

FIGURE 5.

FGF16 stimulates proliferation of ovarian cancer cells. 1 μg of FGF16 cDNA clone was transiently transfected in SKOV-3 (a) and OAW-42 (b) cells followed by RNA isolation and Q-PCR with the primers of PCNA and BCL2. c and d, 20 nm FGF16-siRNA was transfected into the SKOV-3 (c) and OAW-42 (d) cells followed by isolation of RNA and Q-PCR with the primers of FGF16, PCNA, and BCL2. Relative gene expression is indicated as -fold change in the y axis (mean ± S.E.). NT, non-targeting. e, lysates of the cells treated with vehicle, rhFGF16, for either 15 or 30 min were immunoblotted with antibodies of p-Rb, Rb, cyclinD1, and -D2. The GAPDH protein expression was used as the loading control. f, BrdU incorporation assay was performed to assess the growth of SKOV-3 and OAW-42 cells after treatment with 0.1% BSA in PBS (vehicle) or rhFGF16 (100 ng/ml). g, Western blot analysis of FGF16 was performed with the lysates of non-targeting-siRNA and FGF16-siRNA-transfected SKOV-3 cells showing the effect of the siRNA (top). BrdU incorporation was measured to assess the proliferation of the respective transfected cells (bottom). h–i, cells treated with rhFGF16 were trypsinized, harvested, and counted with automated cell counter at indicated days. Here, the control cells were incubated with medium containing 1% FBS. The statistical analysis was done as described under “Materials and Methods.” * represents p < 0.05.

FIGURE 6.

FGF16 binds to FGFR and activates ERK1/2 in ovarian cancer cells. Serum-starved SKOV-3 (a) and OAW-42 (b) cells were treated with rhFGF16 (rh; 100 ng/ml) alone, U0126 (U; 50 ng/ml) alone, or both followed by immunodetection of p-ERK1/2 and total ERK1/2 in the cell lysate. α-Tubulin was used as the loading control. The lysates of SKOV-3 (c) and OAW-42 (d) cells treated with DMSO (Control), PD (50 ng/ml), or PD and rhFGF16 were subjected to Western immunoblot with p-ERK1/2, ERK1/2, and α-tubulin antibodies. Densitometric analysis of the respective bands of a–d were calculated with ImageJ software (National Institutes of Health) and are represented as the percent increase in p-ERK compared with total ERK level. e, the confocal images of p-ERK1/2 in SKOV-3 cells treated as mentioned were shown, where the left panel represents the image of cells stained with anti-p-ERK1/2 antibody followed by anti-rabbit Alexa Fluor-488 (green). The right panel shows the nuclei stained with DAPI. The images were taken at the same exposure time. Scale bar, 20 μm. f, the average fluorescence intensity of individual cells treated as in e was plotted. g, cell proliferation was assessed by BrdU incorporation assay in SKOV-3 and OAW-42 cells treated as earlier. h and i, the effect of PD on cell growth at the indicated days was measured by counting. The statistical analysis was done as described under “Materials and Methods.” * represents p < 0.05.

FIGURE 7.

FGF16 stimulates invasion of ovarian cancer cells and regulates the expression of relevant genes through MAPK pathway. Cells were plated in Matrigel-coated membranes in the upper chamber of transwells. rhFGF16 (rh; 100 ng/ml), U0126 (U; 50 ng/ml), PD (50 ng/ml), or in combination of rhFGF16 and inhibitors were added to the medium in the lower chamber. SKOV-3 (a) and OAW-42 (b) cells penetrating the membrane were fixed, stained, and photographed. c, the respective percent values of migrated cells treated as above with respect to control is shown as a histogram. d–g, cells were treated as earlier for 6 h followed by RNA isolation and Q-PCR with the primers of SNAI1, CDH1, MMP2, and MMP9. h, lysates of the SKOV-3 cells treated as indicated were immunoblotted with respective antibodies, and the representative gel image is shown. The statistical analysis was done as described under “Materials and Methods.” * represents p < 0.05.