WNT5A expression in ameloblastoma and its roles in regulating enamel epithelium tumorigenic behaviors

Abstract

Odontogenic tumors originate from the remains of migrating enamel epithelium after the completion of normal tooth genesis. These enamel epithelium remnants exhibit the ability to recapitulate the events that occur during tooth formation. Several lines of evidence suggest that aberrance in the signaling pathways similar to the ones that are used during tooth development, including the WNT pathway, might be the cause of odontogenic tumorigenesis and maintenance. In this study we demonstrated that WNT5A expression was intense in both the epithelial component of ameloblastomas, the most common epithelial odontogenic tumor, and in this tumor's likely precursor cell, the enamel epithelium located at the cervical loop of normal developing human tooth buds. Additionally, when WNT5A was overexpressed in enamel epithelium cells (LS-8), the clones expressing high levels of WNT5A (S) exhibited characteristics of tumorigenic cells, including growth factor independence, loss of anchorage dependence, loss of contact inhibition, and tumor formation in immunocompromised mice. Moreover, overexpression of WNT5A drastically increased LS-8 cell migration and actin reorganization when compared with controls. Suppression of endogenous WNT5A in LS-8 cells (AS) greatly impaired their migration and AS cells failed to form significant actin reorganization and membrane protrusion was rarely seen. Taken together, our data indicate that WNT5A signaling is important in modulating tumorigenic behaviors of enamel epithelium cells in ameloblastomas.

Figure 1

Expression of WNT5A in ameloblastoma and during human tooth development. A: Ameloblastomas were immunostained with anti-WNT5A antibody (1:200) and images were captured under ×100 magnifications. Sections incubated with normal horse serum and recombinant WNT5A (rWNT5A) (2 μg/ml) blocked antibody were used as negative controls. Scale bar = 100 μm; n = 52. B: At the cap stage, WNT5A was localized in both outer (OE) and inner enamel epithelium (IE) but weakly detected in dental mesenchyme (DM). During the early bell stage, WNT5A remained in IE and presecretory ameloblasts (PAM) with intense expression around enamel knots (EK) and cervical loops (CL). At the late bell stage, WNT5A expression remained in EK and CL but was absent from mature or secretory ameloblasts (MAM), as well as dental papilla (DP) and developing odontoblasts (OD). Scale bar = 50 μm; n = 3. Arrows identify structures and cells defined by abbreviations.

Figure 2

Level of WNT5A production from different transfection. A: Secreted Wnt5a in conditioned medium was partitioned in detergent phase (De) but not aqueous phase (Aq) after Triton X-114 phase separation. The 42 kDa band of endogenous WNT5A (asterisks) was faintly visible in wild-type (WT), overexpressed (S) and empty vector (EV) transfections. The 47 kDa band of WNT5A fusion protein (arrow) was only detected in S transfected clones while WNT5A production was suppressed in AS transfections. B: Stable clones established from S transfection were selected and categorized according to level of expression in to high (SH), moderate (SM), and low (SL) overexpressing WNT5A clones. C: The endogenous Wnt5a (42 kDa) in EV clones was comparable with wild-type, while it was suppressed in AS stable clones. Cell lysate from NIH3T3 cell was used as a negative control. Three independent clones from each S group, together with EV and AS clones were selected for further experiments. D: Quantification of Wnt5a expression in selected stable clones by real-time PCR. Selected stable clones from S transfection showed increase in Wnt5a expression compared with wild-type and EV clones, 2.37-, 4.5-, and 7.97-fold increase in SL, SM, and SH respectively. Wnt5a expressions in stable clones from AS transfection were decreased by 0.13-fold.

Figure 3

Cell morphology and proliferation assay of stable clones. A: No significantly different morphology was observed among wild-type (WT), EV, S, and AS clones. When recombinant WNT5A (rWNT5A) (0.25 μg/ml) was added to the culture medium, WNT5A treated LS-8 showed similar phenotype to wild-type. B: 1 × 10⁵ cells WNT5A from each clone were plated and supplemented with medium containing 10% FBS. Proliferation rates were similar among all groups. C: When the same number of cells were cultured in 1% FBS medium, sense clones had a dramatically decreased proliferation rate but still survived in serum-reduced condition up to 7 days of culture. Wild-type, EV, and AS clones could not survive under these conditions. Original magnification, ×200. Scale bar = 100 μm.

Figure 4

WNT5A promoted loss of contact inhibition and anchorage-independent growth in enamel epithelium. A: SH, SM, and SL formed multilayered foci on culture dishes after being cultured for 3 weeks while few foci formations were found in wild-type (WT), EV, or AS clones. B: Numbers of foci formation were correlated with the level of WNT5A expression in overexpressed clones suggesting dose dependent effect. C: S clones formed visible colonies in soft agar within 2 weeks after seeding, whereas wild-type, EV, and AS clones failed to form visible colonies by bare eyes. D: The number of colonies formed by S clones corresponded to the level of WNT5A expression. Original magnification ×40. Scale bar = 200 μm. *P < 0.05 vs EV, **P < 0.05 vs. SH.

Figure 5

Tumor formation in nude mice. WNT5A overexpressing (SH) or EV-LS-8 cells (2 × 10⁶) were subcutaneously transplanted into nude mice (n = 8 per group). A: SH cells formed tumors within 4 days after injection. EV groups also exhibited tumor growth at 18 days. In average, SH cells exhibited tumors within 18 days post implantation compared with 32 days in the EV group. B: SH groups also showed faster tumor growth rates when total tumor volume was calculated. Tumor formation was monitored for up to 83 days. C: H&E staining of tumors formed in nude mice. Tumors histology appeared similar in both groups. The majority of the tumor mass comprised of mesenchyme with epithelium that randomly formed a duct-like structure (arrows). Scale bar = 50 μm; n = 8.

Figure 6

WNT5A increased cell migration in enamel epithelium cells. A: 2 × 10⁶ of cells were seeded onto the fibronectin coated 60-mm dish and supplemented with 2.5% FBS in DMEM. A scratch was made and cell migration into the space was monitored at 17 and 24 hours. WNT5A overexpressing cells (S) showed enhanced migration compared with EV control. In contrast, WNT5A underexpressing cells (AS) had greatly decreased cell migration. B: The number of migrated cells in to the space was quantified from five random fields. Original magnification ×100; scale bar = 100 μm. *P < 0.05 vs EV. C: Actin cytoskeleton was stained by using fluorescein isothiocyanate phalloidin (bright) and examined by using confocal microscopy. WNT5A overexpressing (S) cells showed a greatly increased density of stress fiber formation when compared with EV and AS cells. Filopodia and lamellipodia (arrows) were generally seen in WNT5A-S. D: The number of filopodia/lamellipodia was counted and shown as mean ± SD value from 20 cells/group. Original magnification ×600; scale bar = 20 μm.