Nuclear localization of β-catenin and expression of target genes are associated with increased Wnt secretion in oral dysplasia
Reyes Rojas, Montserrat
Peña Oyarzún, Daniel
Maturana Ramírez, Andrea
Torres Gómez, Vicente
Cita de ítem
Oral Oncology 94 (2019) 58–67
Objectives: To evaluate the localization of β-catenin in oral dysplastic cells, the expression of target genes upregulated
in oral dysplasia, and the role of Wnt ligands in these events.
Materials and methods: Subcellular localization of total and non-phosphorylated (transcriptionally active) β-
catenin was evaluated by immunofluorescence and biochemical fractionation in dysplastic oral keratinocytes
(DOK), non-dysplastic oral keratinocytes (OKF6), oral squamous carcinoma cells (CAL27) and primary oral
keratinocytes. Tcf/Lef-dependent transcription was measured by luciferase reporter assays. Expression of target
genes, survivin and cyclin D1, was evaluated by RT-qPCR and Western blotting. Wnt secretion was inhibited
with the inhibitor of porcupine, C59. Wnt3a and β-catenin were evaluated in biopsies by tissue immunofluorescence.
Results: Immunofluorescence and fractionation experiments showed augmented nuclear β-catenin (total and
transcriptionally active) in DOK, when compared with OKF6 and CAL27 cells. Intriguingly, conditioned medium
from DOK promoted nuclear accumulation of β-catenin and Tcf/Lef-dependent transcription in OKF6 and primary
oral keratinocytes, suggesting the participation of secreted factors. Treatment of DOK with C59 decreased
Wnt3a secretion, nuclear β-catenin and the expression of survivin and cyclin D1 at both mRNA and protein
levels. Accordingly, DOK secreted higher Wnt3a levels than OKF6, and inhibition of Wnt3a secretion prevented
DOK-induced Tcf/Lef-dependent transcription in OKF6. These observations were confirmed in clinical samples,
since tissue immunofluorescence analysis showed simultaneous expression of Wnt3a and nuclear β-catenin in
oral dysplasia, but not in healthy mucosa biopsies.
Conclusion: These data indicate that secretion of Wnt ligands is critical for β-catenin nuclear localization and
expression of target genes in oral dysplasia.